Mood, Personality, and Behavior Changes During Treatment with Statins: A Case Series

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ABSTRACT

Psychiatric adverse drug reactions (ADRs) have been reported with statin use, but the literature regarding statin-associated mood/behavioral changes remains limited. We sought to elicit information germane to natural history and characteristics of central nervous system/behavioral changes in apparent connection with statin and/or cholesterol-lowering drug use, and delineate mechanisms that may bear on an association. Participants (and/or proxies) self-referred with behavioral and/or mood changes in apparent association with statins completed a survey eliciting cholesterol-lowering drug history, character and impact of behavioral/mood effect, time-course of onset and recovery in relation to drug use/modification, co-occurrence of recognized statin-associated ADRs, and factors relevant to ADR causality determination. Naranjo presumptive ADR causality criteria were assessed. Participants (n = 12) reported mood/behavior change that commenced following statin initiation and persisted or progressed with continued use. Reported problems included violent ideation, irritability, depression, and suicide. Problems resolved with drug discontinuation and recurred with rechallenge where attempted. Eight met presumptive criteria for “probable” or “definite” causality; others had additional factors not considered in Naranjo criteria that bear on casual likelihood. (1) Simvastatin 80 mg was followed in 5 days by irritability/depression culminating in suicide in a man in his 40s (Naranjo criteria: possible causality). (2) Simvastatin 10 mg was followed within 2 weeks by depression in a woman in her 50s (probable causality). (3) Atorvastatin 20 mg was followed in ~1 month by depression and irritability/aggression in a male in his 50s (probable causality). (4) Atorvastatin 10 mg was followed in several months by aggression/irritability and depression culminating in suicide in a man in his 40s (possible causality). (5) Fenofibrate + rosuvastatin (unknown dose), later combined with atorvastatin were followed in 1 month by aggression/irritability in a male in his 30s (probable causality). (6) Lovastatin (unknown dose and time-course to reaction) was followed by depression, dyscontrol of bipolar disorder, and suicide attempts in a male in his 40s (possible causality). (7) Atorvastatin 20 mg was followed within 2 weeks by cognitive compromise, and nightmares, depression, and anxiety culminating in suicide in a man in his teens (definite causality). (8) Simvastatin 10 mg was followed (time-course not recalled) by depression, aggression/irritability culminating in suicide in a man in his 60s (possible causality). (9) Simvastatin 20 mg then atorvastatin 10 mg were followed (time-course not provided) by irritability/aggression in a man in his 60s (definite causality). (10) Atorvastatin 10 then 20 then 40 mg were followed shortly after the dose increase by violent ideation and anxiety in a man in his 30s (probable causality). (11) Atorvastatin 20 mg and then simvastatin 20 mg were followed in 2 weeks by aggression/irritability in a man in his 50s (definite causality). (12) Lovastatin, rosuvastatin, atorvastatin, and simvastatin at varying doses were followed as quickly as 1 day by aggression, irritability, and violent ideation in a man in his 40s (definite causality). Most had risk factors for statin ADRs, and co-occurrence of other, recognized statin ADRs. ADRs had implications for marriages, careers, and safety of self and others. These observations support the potential for adverse mood and behavioral change in some individuals with statin use, extend the limited literature on such effects, and provide impetus for further investigation into these presumptive ADRs. Potential mechanisms are reviewed, including hypothesized mechanisms related to oxidative stress and bioenergetics.

KEY POINTS

Psychiatric adverse effects, altering mood, personality, and behavior, sometimes arise in patients receiving statins.

Statin psychiatric effects can include irritability/aggression, anxiety or depressed mood, violent ideation, sleep problems including nightmares, and possibly suicide attempt and completion.

INTRODUCTION

Most adverse drug reaction (ADR) reporting focuses on non-behavioral health risks to the medication-taking individual; however, attention is increasingly given to drug-induced behavioral and mood changes that may affect self or others. Drugs and medications with behavioral concerns include alcohol (best recognized) [], but also varenicline [], loratadine [], mefloquine [], tramadol [], isotretinoin [], tricyclics [], benzodiazepines [], and selective serotonin reuptake inhibitors (SSRIs) [], among others []. Emerging evidence suggests such problems may occasionally arise with cholesterol-lowering drugs []. These drugs are widely prescribed and most prominently include statins (3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors), which held the place of best-selling class of prescription drugs in the world and include the best-selling prescription drug in history [].

Neuropsychiatric ADRs of statins, including suicide and aggression, have been reported in pharmacovigilance databases [] and in adverse event reports and series []. Moreover, adverse behaviors have been reported in settings of low cholesterol []; and of lower omega-3 fatty acid levels (the omega-3 to omega-6 ratio is reportedly reduced with statins) []. Both naturally low cholesterol and randomized assignment to cholesterol reduction in the pre-statin era have been reported to be linked to increased violent deaths [], though statin randomization has not []. Recent randomized controlled trial (RCT) evidence indicating that statins can have bidirectional effects on aggression may be germane here: different mediating factors, including increased sleep problems and perhaps reduced testosterone, appear to drive effects in differing directions, and bidirectional effects on oxidative stress may also be speculated to do so. Nonetheless, the literature relating mood and behavioral changes to cholesterol-lowering drugs, and depicting the character and potential implications of adverse psychiatric effects with statins, remains relatively sparse []. Here we present 12 cases of mood and behavioral change apparently associated with lipid-lowering agents.

METHODS

A total of 12 subjects and/or their family members (if the subject was deceased) who reportedly experienced mood and behavioral changes while receiving one or more statin, as identified by the subject and/or by family members, contacted our study group and provided survey information. Written informed consent was obtained from each participant (or proxy for deceased subjects) for inclusion of their case in this case series. These 12 represent a convenience sample, chosen because the neuropsychiatric problem was the primary complaint, because the nature or severity appeared to warrant representation in the literature, because the participant or proxy was amenable to inclusion (with proper de-identification), and because the aggregate number, 12, was small enough to allow inclusion of some individual detail, yet sufficient to illustrate a suite of potential issues.

Elicited information included demographic characteristics, drug(s) used (statin and concomitant medications), dose(s), time-course of mood and behavior change relative to drug use (onset, duration, recovery), character of symptoms, and open-ended narrative of the impact of behavior, mood, and/or personality changes. We inquired whether a modification to the treatment regimen occurred—such as changes in dose, drug discontinuation, and drug rechallenge—and the impact on symptoms. Recognized risk factors for statin ADRs, and development of other recognized statin ADRs, were also elicited. Information for each case is presented in tabular form.

Cases were assessed for adherence to presumptive causality criteria using the published Naranjo drug ADR causality classification. This employs a point system of positive and negative causality points to estimate the probability of an ADR, with a score ≥9 deemed to indicate “definite” ADR causality, 5–8 “probable”, 1–4 “possible”, and ≤0 “doubtful” []. For all participants, adverse event causality was at least “possible”, since psychiatric and behavioral reactions to statins or cholesterol reduction bear biological plausibility and prior reports. “Probable” causality was limited to those who experienced occurrence following drug initiation, had the drug discontinued, and improved following drug discontinuation. “Definite” causality assignments were limited to those who, in addition to recovery with discontinuation, were rechallenged with the drug, and experienced symptom recurrence.

RESULTS

Information on drug, drug dose, reported mood and behavioral effect, factors supporting a causal connection, other statin symptoms, family history of psychiatric problems associated with statin use, and presence of risk factors for statin adverse effects are listed in Table 1. Cases commonly involved more than one psychiatric element (Table 2). An expanded description of each case is provided (Table 3). Marked change in mood and/or behavior was commonly noted, in some instances leading to tragic consequences. In some cases, proximal mood or behavioral changes arising with cholesterol-lowering agents led to the addition of psychiatric medications, and a role for these psychotropic medications in behavioral sequelae cannot be excluded. Behavioral/mood findings often accompanied muscle, cognitive, or other better recognized statin adverse effects []. Some participants had family members who also had experienced psychiatric adverse effects attributed to statins. Several participants exhibited compelling on-off-on reproducibility of findings.

Based on Naranjo presumptive criteria for ADR causality, eight cases qualified as bearing a “probable” or “definite” causality designation. The four cases designated “possible” are included because of factors not considered in Naranjo criteria that bear on likelihood of a causal connection. (As was said for a drug bearing a similar spectrum of behavioral adverse behaviors, “the clear temporal relationship, lack of prior history of this behavior, and unusual nature of these events strengthens the accumulating scientific evidence” []). Each patient exhibited persistent absence of the symptom prior to administration of the statin, followed by persistent presence while receiving the statin (days to years). One possible exception was a man with bipolar disorder; however, he had manifested years of stability and good control since initiation of lithium until statin initiation, which resulted in loss of psychiatric stability persisting for the years he was receiving statins until his death. Prospects for a causal connection were buttressed by an adverse behavioral change while receiving statins in a first-degree relative who also experienced dechallenge–rechallenge support (family history and genetics are risk factors for statin problems) []. A total of 75 % of cases were accompanied by other symptoms with a confirmed relation to statin use, including muscle symptoms [], cognitive problems [], and dermatologic reactions []. Additionally, 50 % of cases had factors previously shown to be linked to an elevated risk of statin ADRs, such as thyroid conditions [].

Discussion

Summary

Behavioral and psychiatric changes in the cases presented range from violent nightmares to aggression, mood/personality change, violent or homicidal ideation (in some instances culminating in suicide), each in apparent association with statin use. The temporal association between the drug initiation and mood and behavior change, and again between drug discontinuation and resolution of symptoms where this occurred, suggests a causal connection in a number of these cases. Notable mood and behavioral changes for all patients or introduction of serious psychiatric events began after drug initiation. The latency profile is consistent with that for other statin ADRs that share common mechanisms [], and bear RCT support []. Symptoms persisted or progressed with continued use in all cases. Those able to discontinue the drug experienced resolution of symptoms. For those in whom rechallenge was possible, symptoms recurred. The presence of ADRs and risk factors with a confirmed relation to statin ADRs is consistent with common pathophysiological factors, hypothesized to underlie many statin adverse effects, extending to behavioral effects []. (12 cases are included; for those interested in knowledge of other cases of this kind, a 13th case, involving a physician with behavioral changes while receiving statins and leading to professional review, is planned for inclusion in a separate manuscript on statin adverse effects in physicians).

Explanation of Findings; Comparison/Contrast with Other Findings in Literature

Though a relation of statins to instances of behavior alteration may seem counterintuitive, it fits with a body of existing literature. Observationally, lower cholesterol has been linked to greater violence (including aggression, suicide, homicide, violence) in many studies, including prospective longitudinal studies []. Behavioral consequences of lipid-lowering medications have been reported for non-statin treatments, including fibrates [] (implicated in case 5), with support extending to meta-analyses of RCTs showing significantly increased violent death []. RCT meta-analysis in the statin era did not support an increase in violent death on average (indeed the point estimate was lower, though not significantly) []. RCT patient selection may be one factor: excess violent deaths on pre-statin lipid drugs were preferentially evident in those with risk factors for violence—e.g., alcohol, psychiatric history, and non-compliance []. Of note, there was no evidence of more patients with these characteristics in the cholesterol-lowering group, but the excess of events emphasized these patients. Psychiatric histories, alcohol/substance use, and low conscientiousness are risk factors for lower compliance, so may lead to exclusion with compliance run-ins, but are also risk factors for adverse behaviors, so vulnerable individuals might be preferentially excluded []. Relative exclusion may affect detection of risk in two ways. First, the same fractional (relative) risk change will lead to a greater number affected, and more power to show a change, in those at higher risk (a doubling of nothing is still nothing) []. Second, the fractional risk change itself may be greater in a behaviorally vulnerable subset (effect modification): illustrating this point, the odds ratios risk of psychiatric events with mefloquine use was reported to be 3.8 among those without a psychiatric history, versus apparently double that (8.0) in those with a psychiatric history [].

Other factors may also explain this. Randomized trial evidence examining statin effects on aggression provides potential insights: statins (vs. placebo) promoted both average significant increases and reduction in aggression, in different groups []. Typical effects in men (particularly in men who were both younger and less aggressive at baseline) were toward reduced aggression [], with older age and female sex shifting the distribution. Simvastatin has been shown to both significantly lower testosterone [] and worsen sleep problems on average []. (It may also promote sleep apnea in some []). For men receiving simvastatin, the magnitude of each of these effects significantly predicted the change in aggression, in opposite directions []. Sleep problems and sleep apnea are elsewhere linked to irritability [], as well as aggression and violence []. Lower testosterone in some settings is linked to lower aggression and violence [].

Other mechanisms may be theorized. Low central serotonin has been linked to low cholesterol and to aggression []. However, whole blood serotonin (which correlates inversely to central serotonin) did not predict aggression in mediation analysis within a randomized trial []. We also hypothesize that effects on aggression, and perhaps mood problems on statins, may be linked to oxidative stress [] and inter-related mitochondrial dysfunction (which has been linked to temper disorders) []. The explanation accords with our finding that behavioral adverse effects commonly co-occur with better known statin adverse effects, with the documented relation of better known statin adverse effects to oxidative stress and mitochondrial dysfunction [], and with the documented connection of mitochondrial dysfunction to a range of psychiatric problems [].

Other hypothetical mechanisms of potential relevance, such as the role of cholesterol in synapse formation or membrane function, and myelin production have been reviewed elsewhere [], but in contrast to mechanisms cited above, triangulating evidence for a role is currently lacking.

The explanation accords with our finding that behavioral adverse effects commonly co-occur with better known statin adverse effects, for which a relation to oxidative stress and mitochondrial dysfunction has been elucidated []. Mitochondrial dysfunction has a documented connection to a range of psychiatric problems [], and may also contribute to behavioral change with statin-induced oxidative damage.

More lipophilic statins have better brain penetration [], though all statins have some ability to cross the blood–brain barrier []. Whether this is important for central effects of the kinds described is unknown, since peripheral effects can have central consequences—and since prooxidant effects of statins, which are linked to the occurrence of statin adverse effects [], also raise blood–brain barrier penetrability []. Each of the patients in our report experienced problems while receiving at least one of simvastatin, atorvastatin, or lovastatin, which are lipophilic statins. However, most involved atorvastatin and simvastatin, which have also historically been the most frequently prescribed statins. This, and the lack of a defined base population in which statin prescribing rates are known, obviates our ability to comment on whether lipophilicity is relevant to these effects.

Limitations

This study has the limitations inherent to all case series: data are observational, which constrains causal inference. However, a profound change in state while receiving a drug, particularly with dechallenge–rechallenge support, strengthens the case for causality. There is no defined base population or control group, precluding calculation of rates and risk ratios, and occurrence of adverse neuropsychiatric effects in these cases, even if presumed causal, has no inherent implications for usual effects of statins on the outcomes reported—nor do usual effects have necessary implications to individual ones. Rather, this case series underscores that statins may in specific individuals promote adverse behavioral, mood, and personality changes, irrespective of whether behavioral (or mood) effects are on average favorable, adverse, or neutral. Even reporting rates relative to other ADRs may be a challenge to gauge meaningfully for neuropsychiatric problems, because these may be particularly sensitive, and may go unreported. In an analysis of emails abstracting statin ADRs mentioned spontaneously, mood or personality problems were spontaneously related as part of the ADR complex in a minority, but in a survey directly asking about each of a list of symptoms the participant attributed to statins, among those self-selected for having at least one such symptom, a majority cited a neuropsychiatric effect. (However, in the latter, there is no gauge of severity; and physical problems can themselves contribute to some level of psychic distress).

Some of our participants had underlying conditions (such as a psychiatric disorder) that can themselves lead to an adverse outcome; however, as above, the odds ratios for risk of psychiatric events due to a drug can be magnified in the presence of a psychiatric history []. Consistent with this, most of the excess cases of violent death arising in the active treatment, cholesterol-lowering arms in a pre-statin-era analysis of randomized trials had other risk factors for violent death []; since those risk factors themselves were not shown to be increased in the active treatment group, this underscores that individuals with such issues may represent a vulnerable subgroup (as with mefloquine). As a further analogy, an analysis of rhabdomyolysis cases in the San Diego area showed that typical instances involved the confluence of more than one risk factor—consistent again with an increase in the odds of an event “due to” each agent, in the presence of the other []. In our cases, factors such as the temporal relation to statin use, and concurrence of other statin-compatible ADRs increases the prospects for causality with statins.

Data rely on self-report. However, patient self-reports of ADRs can be a valuable and reliable tool [], and—if heeded—may hasten recognition of important ADRs []; such benefits have led to standardized implementation of patient reporting for EU-based pharmacovigilance databases []. Self-selection of participants is inherent to studies with volunteer patients; however, for studying those with ADRs, whether or not they reflect typical effects, observed effects are important.

Conclusions

Though statins are widely tolerated, they may be among the growing list of prescription agents that, in some participants, may increase the risk of serious psychiatric events and/or behavioral changes. In the cases cited here, these adverse experiences posed risks to the safety of self and others—sometimes, tragically, adversely affecting marriages and careers, or culminating in death. The possibility of such ADRs, even if rare, should be recognized by physicians who prescribe cholesterol-lowering drugs, such that if personality or behavior changes arise, the drug can be included in considerations of etiology and treatment. This series extends the modest literature on behavior and psychiatric changes apparently associated with cholesterol-lowering treatment. These findings further the evidence that cholesterol-lowering drugs should be added to the list of agents that bear consideration when new irritability, or aggressive or violent behavioral changes arise.

Candida albicans-Endothelial Cell Interactions: a Key Step in the Pathogenesis of Systemic Candidiasis

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Candida albicans is a normal commensal organism of the oral cavity, gastrointestinal tract, and vagina. Under certain circumstances, C. albicans is capable of causing host damage (or disease) leading to oral, vaginal, cutaneous, or systemic candidiasis. The latter is a serious infection with a high mortality range of 33% to 54% and high morbidity in those who survive. In fact, in recent years, systemic candidal infections have become the fourth most frequent cause of nosocomial bloodstream infections in the United States, giving rise to an enormous associated personal and economic cost. Systemic candidiasis involves the hematogenous spread of C. albicans to multiple organs, including the brain, kidneys, heart, liver, and lungs. Histologically, infection of these organs is characterized by ramifying candidal hyphae and accompanying yeast forms that produce multiple necrotic nodules or abscesses that result in extensive organ damage leading to organ failure. Risk factors for systemic candidiasis include neutropenia, intravascular catheters, hemodialysis, total parenteral nutrition, abdominal surgery, burns, broad-spectrum antibiotics, and corticosteroids. Systemic innate immune responses by phagocytic cells, particularly neutrophils and macrophages, appear to play a critical role in the host defense against systemic Candida infections, and consequently, the majority of candidal infections occur in patients with neutropenia or defects in neutrophil or macrophage function.

MORPHOGENETIC CONVERSIONS AND C. ALBICANS VIRULENCE

C. albicans is a polymorphic organism that is capable of converting between yeast, pseudohyphal, and hyphal forms. The conventional view was that yeast forms were associated with commensal carriage, whereas hyphal forms were associated with disease. This was based on evidence showing that mutant forms of C. albicans that were locked into the yeast form were avirulent. However, this notion was challenged by Braun et al., who found that a tup1-deficient C. albicans strain that was constitutively pseudohyphal was avirulent in a murine model of systemic candidiasis. Although the precise nature of the association between fungal morphogenesis and host invasion is a hotly debated topic, it is now widely accepted that it is the ability to undergo morphogenetic conversion, rather than the morphological form itself, that is the primary determinant of pathogenicity.

The dissemination of fungal organisms in systemic candidiasis starts with their entry into the bloodstream. Given the known risk factors for systemic candidiasis, this is most likely to occur in susceptible individuals by seeding from contaminated intravascular devices, by persorption of C. albicans across the gastrointestinal mucosa, by invasion of epithelially denuded surfaces, or via trauma or surgically related inoculation. Exit from the circulation is thought to occur by adhesion and then penetration into the endothelial lining of blood vessels, except possibly in the kidney, where direct adhesion to exposed extracellular matrix components within glomerular regions may occur. Animal studies suggest that candidal trafficking from the circulation into the tissues occurs rapidly. This review discusses the two critical steps in the migration of C. albicans cells from the circulation into the tissues, which are (i) candidal adhesion to endothelial cells lining the blood vessels and (ii) transmigration of C. albicans across the endothelium into the tissues.

ADHESION OF C. ALBICANS TO ENDOTHELIAL CELLS

During hematogenous dissemination of C. albicans, organisms must first adhere to the endothelial lining of blood vessels before transmigrating across the endothelium to invade the tissues. However, little is known about the mechanisms involved in either process. What is known is complicated further by conflicting evidence concerning the roles played by yeast, pseudohyphal, and hyphal forms of C. albicans and the role of morphogenetic change in the adhesion and transmigration processes.

There are currently two different theories as to how C. albicans adheres to the endothelium. The first theory proposes that cells must first undergo morphogenetic conversion to hyphal forms, which then bind to and damage the endothelial lining of blood vessels before undergoing transmigration from the circulation into the tissues. However, more recent data indicate a second possibility in which morphogenetic change is not necessary for C. albicans invasion of the tissues. In this scenario, yeast cells adhere to the endothelium and then transmigrate into the tissues without undergoing morphogenetic conversion.

The basis of the first theory is morphogenetic conversion of C. albicans to the hyphal form, and there are many lines of evidence to support this hypothesis. These include the observations that germination of C. albicans is necessary for the organism to damage endothelial cells and that substances that inhibit germination block C. albicans-induced endothelial cell damage. Moreover, the time course of candidal germination and germ tube elongation on endothelial cells parallels the time course of endothelial cell damage. Further evidence has come from experiments using genetically engineered forms of C. albicans with filamentation defects. The ability of these organisms to damage and invade endothelial cells is severely impaired compared to that of wild-type parent strains.

Studies showing that germinated C. albicans cells exhibit much greater adherence to epithelial cells than do yeast forms prompted suggestions that C. albicansadherence to endothelial cells might also be hypha dependent. Indeed, there is some evidence to suggest that germinated candidal forms exhibit greater endothelial cell adhesion than do yeast forms of C. albicans. However, it is also possible that yeast forms adhere to the endothelial surface, germinate there, and then penetrate and damage the endothelium during transmigration or that yeast forms adhere and are then endocytosed before germinating within the endothelial cell to cause damage. Taken together, the data suggest that morphogenetic transformation is involved in endothelial cell adhesion but, more particularly, in the subsequent trans-endothelial cell migration.

Conversely, there is also evidence to suggest that morphogenetic change may not be necessary for C. albicans invasion, and this is the basis for the alternative hypothesis. In animal studies in which mice were intravenously inoculated with different mutant strains, Bendel et al. found that cells from a C. albicans mutant strain locked into the yeast form were able to leave the circulation and enter the tissues in greater numbers than those of the wild-type control. However, once cells were in the tissues, the ability of the wild-type strain to undergo hyphal transformation was associated with higher mortality, despite the lower fungal burden in the tissues than that with mutant yeast forms. Further evidence to support this theory has come from in vivo experiments investigating tissue invasion and damage, performed by Saville et al. using a genetically engineered strain of C. albicans (SSY50-B). This study demonstrated that yeast cells are capable of extravasating from blood vessels into the tissues without undergoing morphogenetic change. However, once cells were in the tissues, morphogenetic conversion from yeast to hyphal forms was crucial in causing tissue damage leading to death.

Such observations have led to a hypothesis in which circulating yeast cells bind to the endothelium and then transmigrate into the tissues before undergoing the hyphal transformation that results in tissue damage. In support of this, C. albicans migration from the circulation is very rapid (80 to 90% migration within 5 min), whereas hyphal transformation and endothelial cell damage may take several hours. Furthermore, because of their more compact shape and size, yeast cells may be better adapted for free dissemination within the circulation. In addition, the emergence of C. glabrata and C. parapsilosis as contenders for the second most common cause of disseminated candidiasis, after C. albicans, indicates that the ability to form true hyphae may not be essential for tissue adhesion, invasion, and pathogenesis among Candida species.

CANDIDATE CANDIDA ADHESINS AND THEIR ENDOTHELIAL LIGANDS

The cell wall of C. albicans is composed primarily of an inner structural layer of β1,3- and β1,6-glucans and chitin (a β1,4-linked polymer of N-acetylglucosamine) and a matrix primarily consisting of proteins (mannoproteins) that are heavily glycosylated with mannose-containing polysaccharides, sometimes called mannans. These take the form of short, linear, O-linked mannan side chains that stabilize the protein in the cell wall and large, highly branched N-linked mannans. It is this outermost layer that represents the first point of contact between C. albicans and the endothelium, although not at bud scars, where the components of the inner layers of the cell wall are exposed. Proteins and carbohydrates in these outer layers may have a number of functions, including the ability to act as adhesion molecules, and over recent years several C. albicans cell wall components with the potential to mediate adhesion to the endothelium have been identified. These include proteins with integrin-like properties, Candida agglutinin-like sequence (ALS) gene products, and mannans.

Cell wall protein adhesin candidates. (i) Integrin αMβ2-like adhesin.

Integrin analogues first gained interest in 1991, when Gustafson et al. found that adhesion of yeast forms of C. albicans to cultured monolayers of human endothelial cells was mediated in part by a candidal protein antigenically and structurally related to the leukocyte integrin αMβ2 (Mac-1, CD11b/CD18, CR3, or iC3b receptor) (). They demonstrated the expression of the αMβ2-like molecule on yeast forms of C. albicans and showed that expression was increased by growth in 20 mM d-glucose, as opposed to 20 mM l-glutamine (). Furthermore, the adhesion of yeast forms of C. albicans to endothelial cells was significantly reduced by anti-αMβ2 antibodies or pretreatment of the Candida cells with purified iC3b. Expression of this ligand may be altered at different temperatures and in different morphogenetic forms of C. albicans (), and this may affect the ability of C. albicans to adhere to endothelium (). αMβ2 has many different ligands, including iC3b, fibrinogen, factor X, urokinase receptor, CD14, CD23, CD54 (ICAM-1), CD102 (ICAM-2), CD242 (ICAM-4), heparin, haptoglobin, kininogen, and various microbial proteins (). Of these molecules, only ICAM-1 and -2 are widely expressed on endothelial cells, although CD14 was recently identified on primary, but not passaged, cultures of human umbilical vein endothelial cells (). There are no data on the role of CD14, CD102, or CD242 as a possible endothelial ligand for C. albicans adhesion, but Yokomura et al. () have shown that anti-CD54 monoclonal antibodies can partially inhibit the adhesion of yeast forms of C. albicans to rat pulmonary artery endothelial cells in vitro and significantly prolong the survival of rats injected intravenously with C. albicans. In certain circumstances, it is also possible that αMβ2 ligands such as fibrinogen, heparin and iC3b could in turn bind to endothelial cells and act as an intermediary in Candida-endothelial cell adhesion.

(ii) Integrin αvβ3– and αvβ5-like adhesins.

Two other integrin-like adhesins that may play a role in candidal adhesion to endothelium have been identified. They are homologs of the vitronectin-binding integrins αvβ3 (CD51/CD61) and αvβ5 (). Spreghini et al. () reported the expression of both of these adhesins on yeast forms of C. albicans, while Santoni et al. () showed that transformation to germ tubes was associated with a marked reduction in αvβ5-like integrin expression and an increase in αvβ3-like integrin expression. They also showed that adhesion of C. albicans germ tubes to endothelium was partially inhibited by anti-αvβ3 antibodies or an RGD sequence peptide. Heparin also inhibited germ tube adhesion, and when heparin treatment was combined with either anti-αvβ3 antibody or RGD peptide, the reduction in adhesion was greater still (). More recently, it was shown that a candidal focal adhesion kinase-like protein may be involved in regulating yeast cell adhesion to endothelium via the αvβ3– or αvβ5-like adhesins () or in mediating intracellular signaling following ligand binding, much as focal adhesion kinase proteins are involved in integrin-mediated signaling in mammalian cells (). Like its human counterpart, the candidal αvβ3-like adhesin has been shown to bind to vitronectin (), but other ligands for αvβ3 include CD31 (PECAM-1), fibronectin, fibrinogen, thrombospondin, von Willebrand factor, and RGD sequence peptides (). CD31 is expressed by endothelial cells and could act as a direct ligand for Candida adhesion (), while in certain circumstances it is possible that other ligands could act as a bridge in Candida-endothelial cell binding. Like its human counterpart, the αvβ5-like adhesin on C. albicans also binds vitronectin and RGD peptides (), but αvβ5 lacks a known endothelial cell target ligand and thus may not be involved directly with adherence to the endothelium.

(iii) ALS gene family.

The ALS (agglutinin-like sequence) gene family encodes a group of large glycosylphosphatidylinositol-linked cell surface glycoproteins (). To date, eight ALS genes have been identified, including ALS1 to ALS7 and ALS9, all of which appear to have differing roles in adhesion and transmigration. These genes have gained particular interest recently, and evidence shows that ALS1-transformed Saccharomyces cerevisiae exhibits up to 100-fold greater adherence to endothelial cells (), while Als1-deficient C. albicans hyphae exhibit reduced adhesion to endothelial cells (). Similarly, S. cerevisiae transformed with ALS3 shows increased adhesion (), while Als3-deficient hyphal forms of C. albicans exhibit defective adhesion to endothelial cells (). The loss of Als9 from yeast forms of C. albicans () or the loss of Als4 and decreased expression of Als2 from 1-hour-old germ tubes () also inhibit the adhesion of mutant C. albicans strains to endothelial cells. In contrast, mutational analysis has shown that deletion of ALS5ALS6, or ALS7 results in increased adhesion of yeast forms of C. albicans to endothelial cells, suggesting an antiadhesive role for these proteins (). On the other hand, the protein Als5 has been found to mediate adhesion, along with Als1, when expressed in S. cerevisiae (). To date, the only ligand for the ALSgene products that has been found on endothelial cells is N-cadherin, which binds to Als3 on C. albicans hyphae ().

(iv) C4BP.

The complement protein regulator C4b binding protein (C4BP) is able to bind to both yeast and hyphal forms of C. albicans and is predominantly localized at the tip of the germ tube on hyphae (). This binding is normally regarded as a survival mechanism that inhibits complement activation and the attachment of opsonins to the microbial surface. However, it may also enhance the adhesion of yeast forms of C. albicans to endothelial cells. It is not clear if this enhancement of adhesion by the C4BP coating occurs by activating other Candidaadhesins or by acting as a bridge.

Cell wall carbohydrate adhesin candidates.

The outer cell wall proteins of Candida are heavily glycosylated with N- or O-linked mannosyl residues and have been found to be strongly involved in the recognition of C. albicans by the innate immune system. Indeed, some of these sugar residues provide conserved Candida-associated chemical signatures, known as pathogen-associated molecular patterns, by which the host is able to recognize the presence of the pathogen via host pattern recognition receptors (PRRs). In recent years, it has become apparent that specific host PRRs bind to and recognize specific mannosyl residues on C. albicans. For example, the mannose receptor (MR) recognizes and binds to N-linked mannosyl residues, while Toll-like receptor 4 (TLR-4) binds O-linked mannosyl residues. Similarly, TLR-2 recognizes and binds phospholipomannan, and galectin-3 binds β-mannosides. As these mannosyl residues are part of the structure of the cell wall, they are expressed on all three different morphological forms of C. albicans. However, there is evidence to suggest that there are differences in the recognition of yeast and hyphae by TLR-2 and TLR-4.

Although these PRRs are principally involved in the recognition of C. albicans by components of the host immune response, it is also possible that they are used by C. albicans to adhere to and transmigrate across the endothelial lining of blood vessels. Indeed, several studies have demonstrated the important role of the TLRs in experimental models of disseminated candidiasis. Netea et al. showed that TLR-4-defective C3H/HeJ mice have an increased susceptibility to disseminated candidiasis, and mice deficient in the universal TLR adaptor protein myeloid differentiation factor 88 (MyD88) are extremely susceptible to C. albicans infection. However, it has also been shown that TLR-4-deficient mice are more resistant to disseminated Candida infection. This is also the case for TLR-2-deficient mice, which have also been shown to be more resistant to disseminated candidiasis. However, the majority of the literature on knockout mice and disseminated candidiasis looks at susceptibility to infection and correlates it with the immune response without focusing on receptor expression on endothelial cells. To date, endothelial cells have been shown to express a number of PRRs, including the MR, TLRs, and galectins. The MR was the first receptor on the surfaces of macrophages to be described as a mannan receptor, and it recognizes oligosaccharides that terminate in mannose, fucose, and N-acetylglucosamine. It is also expressed on subtypes of dendritic cells and endothelial cells from certain vascular beds, including human dermal microvascular endothelial cells but not human umbilical vein endothelial cells. So far, 10 TLRs have been found, of which 7 or 8 are expressed on unstimulated endothelial cells. However, upon stimulation with proinflammatory cytokines, all 10 TLRs are expressed. Perhaps most importantly for interactions with C. albicans, endothelial cells express TLR-2 and TLR-4. TLR-4 is expressed constitutively at a higher level than that of TLR-2 by endothelial cells. However, the expression of both is significantly upregulated by stimulation with gamma interferon or bacterial lipopolysaccharide. It is also notable that the expression of TLR-2 on endothelial cells is strongly affected by the effects of flow on the endothelial cells. The galectins are a family of 15 carbohydrate binding proteins with high affinities for β-galactosides, extracellular glycoproteins, and glycolipids. So far, expression of galectin-1, -3, and -9 has been found on cultured endothelial cells, but only galectin-3 has been found to recognize C. albicans. Other PRRs that have been found to be involved in the recognition of C. albicans include DC-SIGN, αMβ2, FcγR, and dectin-1, but so far these receptors have not been found to be expressed on endothelial cells.

With so many different cell wall components having the potential to mediate adhesion of C. albicans to the endothelium, it seems that there could be a number of different mechanisms of adhesion. This may have consequences for the development of therapies aimed at blocking adhesion, because with so many molecules potentially playing a role, blocking only one could simply result in its role being taken up by other molecules. However, to investigate this further, more research is needed on the molecules involved in adhesion of C. albicans to the endothelial lining of blood vessels.

STATIC VERSUS FLOW ADHESION ASSAYS

The majority of the above studies that have directly explored candidal adhesion to endothelium were performed by using static in vitro assays where C. albicans was left in prolonged contact with cultured monolayers of endothelial cells. This is very different from the fleeting interactions C. albicans has with endothelial cells under the conditions of shear stress and flow that occur in blood vessels in vivo. Numerous studies with other cells and microorganisms have shown that static assays do not replicate the dynamic interactions that occur with endothelium under conditions of flow and are poor at elucidating the contributions of specific adhesion molecules. Only a few studies have attempted to study candidal adhesion to synthetic substrata under conditions of flow. These have shown that there are significant differences in the adhesion of Candida to the same substrata when the assays are performed under static and flow conditions. To date, only one study has attempted to examine the adhesion of Candida to endothelium under conditions of flow. Glee et al. found that under shear flow, C. albicans formed rapid, tight adhesions in less than 67 ms. This is much quicker than in static assays and is comparable to the rapid adhesion interactions that occur between leukocytes and endothelial cells. In view of this, it is difficult to fully evaluate the contributions of the mechanisms and adhesion molecules discussed above to the adhesion of C. albicans to endothelium in vivo, as none have been studied under conditions of flow.

TRANSMIGRATION

After adhesion of C. albicans to the endothelial lining of blood vessels, the second step in the migration of C. albicans from the circulation into the tissues is transmigration across the endothelial barrier. This step may involve some of the same molecules used for adhesion but could involve others. Transmigration is hard to research in isolation, which explains why there is little information on specific methods of Candida-endothelial cell transmigration. Even so, there are several proposed mechanisms for Candida transmigration across the endothelium. The first mechanism proposes that endothelial cells endocytose adherent organisms and allow their passage through to the abluminal surface of the endothelial cell layer. It is this mechanism that has gained the most interest and for which a model has evolved to explain how candidal hyphae adhere to and then induce endothelial cells to endocytose them. In this model, C. albicanshyphae bind to N-cadherin and other, as yet unidentified proteins on the endothelial cell surface via the candidal protein Als3. This adhesive interaction induces tyrosine phosphorylation of unidentified intracellular endothelial cell proteins, causing microfilament rearrangement to produce pseudopods, which initiate the endocytosis of adherent hyphal forms of C. albicans. However, endothelial endocytosis of C. albicans is not restricted to hyphal forms, and strains that do not undergo hyphal change and cause little endothelial cell damage are endocytosed to a significant degree. Since the Als3 protein is predominantly expressed on candidal hyphae, this could involve other adhesin-endothelial ligand pairs. There is also evidence that suggests that adherent yeast forms could penetrate endothelial cells, damaging them in the process, without undergoing morphogenetic change allowing them to cross the endothelial barrier. Another proposed mechanism of trans-endothelial cell migration of adherent C. albicans involves the extension of penetrating hyphal processes through the endothelial cells, likely destroying them in the process, much as fungal hyphae ramify through other tissues. Alternatively, a further proposal suggests that adherent C. albicans cells may pass between adjacent endothelial cells as a result of translocation and cyclical switching of adhesion molecules at the junction between endothelial cells, in a manner similar to that of leukocyte and tumor cell trans-endothelial cell migration.

Two alternative methods of transmigration across the endothelium that may not require prior adhesion of C. albicans to the endothelial cell surface have also been proposed. The first mechanism proposes that organisms phagocytosed by leukocytes are transported across the endothelial barrier inside the leukocytes. It is well known that leukocytes are able to cross the endothelium, between adjacent endothelial cells, by diapedesis and cyclical switching of adhesion molecules. Furthermore, there is evidence of C. albicans being found inside circulating leukocytes in systemic candidiasis. However, it is unlikely that this represents the only mechanism for candidal transmigration, since neutropenia is a major risk factor for invasive disease. The second mechanism, which may or may not require prior adhesion, suggests that circulating Candida cells simply pass through endothelial fenestrations between adjacent endothelial cells in vascular beds such as the kidney.

Some of these mechanisms may operate only for the yeast, pseudohyphal, or hyphal form of C. albicans, some may work for all forms, and others may require morphogenetic change for transmigration to occur. As with C. albicans adhesion to endothelial cells, there is clearly much more research required in order to elucidate the precise mechanism by which C. albicans migrates across the endothelium and into the tissues. Additionally, as with leukocyte and tumor cell transmigration, the validity of these mechanisms may become apparent only when transmigration is studied in vivo or in situations where the endothelium is subject to conditions of flow.

CONCLUSION

In summary, the interaction of C. albicans with the endothelial lining of blood vessels and its invasion of the tissues involve a complex series of processes that is further complicated by the role played by the morphogenetic conversion of C. albicans. There is still a large amount of work required to clarify these processes. Furthermore, it is important that this work be performed under conditions that replicate the fleeting contacts of C. albicans with the endothelium and the dynamic conditions of flow that occur in vivo. Nonetheless, understanding these mechanisms may be critical in identifying a means for preventing Candida invasion of the tissues and its lethal sequelae in systemic candidiasis.

Statin Therapy and Decreased Incidence of Positive Candida Cultures Among Patients With Type 2 Diabetes Mellitus Undergoing Gastrointestinal Surgery

~ Content Source

OBJECTIVE

To assess whether statin therapy decreases the incidence of cultures positive for Candida species among high-risk hospitalized patients with type 2 diabetes mellitus (DM).

PATIENTS AND METHODS

We performed a retrospective cohort study analyzing the records of all patients with type 2 DM who were admitted to Massachusetts General Hospital for lower gastrointestinal tract surgery between January 1, 2001, and May 1, 2008. We defined statin exposure as the filling of at least 1 prescription of statins during the 6 months before hospitalization or during the current hospital stay. The primary outcome was a culture positive for Candida species during hospitalization. Clinical information on a wide range of covariates was collected. Logistic regression analysis was used to adjust for possible confounders.

RESULTS

Of the 1019 patients who were eligible for the study, 493 (48%) were receiving statins. A total of 139 patients (14%) had at least 1 culture positive for Candida species during hospitalization. An adjusted multivariate model based on a backward stepwise elimination procedure showed that statin therapy significantly decreased the incidence of cultures positive for Candida species (odds ratio, 0.60; 95% confidence interval [CI], 0.38-0.96; P=.03) with a statistically significant prolonged time to event compared with no statin therapy (adjusted hazard ratio, 0.62; 95% CI, 0.44-0.88; P=.01). The benefit of statins was more prominent in patients with type 2 DM who had greater comorbidities (Charlson Comorbidity Index ≥2) (adjusted odds ratio, 0.47; 95% CI, 0.27-0.79; P=.01).

CONCLUSION

Among patients with type 2 DM who underwent gastrointestinal surgery, use of statins correlated with a decreased incidence of cultures positive for Candida species.

Cancer as a metabolic disease: implications for novel therapeutics

Content Source ~ NIH

Abstract: Emerging evidence indicates that cancer is primarily a metabolic disease involving disturbances in energy production through respiration and fermentation. The genomic instability observed in tumor cells and all other recognized hallmarks of cancer are considered downstream epiphenomena of the initial disturbance of cellular energy metabolism. The disturbances in tumor cell energy metabolism can be linked to abnormalities in the structure and function of the mitochondria. When viewed as a mitochondrial metabolic disease, the evolutionary theory of Lamarck can better explain cancer progression than can the evolutionary theory of Darwin. Cancer growth and progression can be managed following a whole body transition from fermentable metabolites, primarily glucose and glutamine, to respiratory metabolites, primarily ketone bodies. As each individual is a unique metabolic entity, personalization of metabolic therapy as a broad-based cancer treatment strategy will require fine-tuning to match the therapy to an individual’s unique physiology.

Introduction: Cancer is a disease involving multiple time- and space-dependent changes in the health status of cells and tissues that ultimately lead to malignant tumors. Neoplasia (abnormal cell growth) is the biological endpoint of the disease. Tumor cell invasion into surrounding tissues and their spread (metastasis) to distant organs is the primary cause of morbidity and mortality of most cancer patients (). A major impediment in the effort to control cancer has been due in large part to the confusion surrounding the origin of the disease. Contradictions and paradoxes continue to plague the field (). Much of the confusion surrounding cancer origin arises from the absence of a unifying theory that can integrate the many diverse observations on the nature of the disease. Without a clear understanding of how cancer arises, it becomes difficult to formulate a successful strategy for effective long-term management and prevention. The failure to clearly define the origin of cancer is responsible in large part for the failure to significantly reduce the death rate from the disease (). Although cancer metabolism is receiving increased attention, cancer is generally considered a genetic disease (,). This general view is now under serious reevaluation (,). The information in this review comes in part from our previous articles and treatise on the subject (,).

Provocative question: does cancer arise from somatic mutations?

Most of those who conduct academic research on cancer would consider it a type of somatic genetic disease where damage to a cell’s nuclear DNA underlies the transformation of a normal cell into a potentially lethal cancer cell (,,,). Abnormalities in dominantly expressed oncogenes and in recessively expressed tumor suppressor genes have been the dogma driving the field for several decades (,). The discovery of millions of gene changes in different cancers has led to the perception that cancer is not a single disease, but is a collection of many different diseases (,,,). Consideration of cancer as a ‘disease complex’ rather than as a single disease has contributed to the notion that management of the various forms of the disease will require individual or ‘personalized’ drug therapies (,). Tailored therapies, unique to the genomic defects within individual tumors, are viewed as the future of cancer therapeutics (,). This therapeutic strategy would certainly be logical if the nuclear somatic mutations detected in tumors were the drivers of the disease. How certain are we that tumors arise from somatic mutations and that some of these mutations drive the disease? It would therefore be important to revisit the origin of the gene theory of cancer.

The gene theory of cancer originated with Theodor Boveri’s suggestion in 1914 that cancer could arise from defects in the segregation of chromosomes during cell division (,). As chromosomal instability in the form of aneuploidy (extra chromosomes, missing chromosomes or broken chromosomes) is present in many tumor tissues (,), it was logical to extend these observations to somatic mutations within individual genes including oncogenes and tumor suppressor genes (,). Boveri’s hypothesis on the role of chromosomes in the origin of malignancy was based primarily on his observations of chromosome behavior in nematodes (Ascaris) and sea urchins (Paracentrotus) and from his consideration of von Hansemann’s earlier observations of abnormal chromosome behavior in tumors (,,). In contrast to Boveri’s view of aneuploidy as the origin of cancer, von Hansemann considered the abnormal chromosome behavior in tumors as an effect rather than as a cause of the disease (). Although Boveri’s hypothesis emerged as the foundation for the somatic mutation theory of cancer, it appears that he never directly experimented on the disease (,,). The reason for the near universal acceptance of Boveri’s hypothesis for the origin of cancer is not clear but might have been linked to his monumental achievement in showing that Gregor Mendel’s abstract heredity factors resided on chromosomes (). Boveri’s cancer theory was also consistent with the gradual accumulation of evidence showing that DNA abnormalities are abundant in cancer cells.

In his 2002 review, Knudson stated that, ‘considerable evidence has been amassed in support of Boveri’s early hypothesis that cancer is a somatic genetic disease’ (). The seeds of the somatic mutation theory of cancer might have been sowed even before the work of von Hansemann and Boveri. Virchow considered that cancer cells arose from other cancer cells (). Robert Wagner provided a good overview of those early studies leading to the idea that somatic mutations give rise to cancer (). It gradually became clear that almost every kind of genomic defect could be found in tumor cells whether or not the mutations were connected to carcinogenesis (,,,,). The current somatic mutation theory involves a genomic landscape of incomprehensible complexity that also includes mysterious genomic ‘Dark Matter’ (,,,). Although massive evidence exists showing that genomic instability is present to some degree in all tumor cells, it is unclear how this phenotype relates to the origin of the disease. It appears that almost every neoplastic cell within a naturally arising human tumor is heterogeneous in containing a unique genetic architecture ().

Inconsistencies with a nuclear gene origin of cancer

The distinguished British geneticist, C.D.Darlington (), was one of the first to raise concerns regarding the nuclear genetic origin of cancer. Based on several inconsistencies in the association of mutagens with cancer, Darlington argued persuasively that nuclear genomic defects could not be the origin of cancer. Rather, he was convinced that cancer cells arose from defects in cytoplasmic elements, which he referred to as ‘plasmagenes’. Although Darlington did not specifically characterize the nature of the plasmagene, several characteristics of the plasmagenes suggested that they were mitochondria. It was unclear, however, if the radiation damage to the plasmagenes acted alone in causing cancer or also acted in conjunction with mutations in nuclear genes.

Inconsistencies regarding the somatic nuclear gene theory of cancer also come from nuclear/cytoplasmic transfer experiments between tumorigenic and non-tumorigenic cells. Several investigators showed that tumorigenicity is suppressed when cytoplasm from non-tumorigenic cells, containing normal mitochondria, is combined with nuclei from tumor cells (). Moreover, the in vivotumorigenicity of multiple human and animal tumor types is suppressed when the nucleus from the tumor cell is introduced into the cytoplasm of a non-tumorigenic cell (). Tumors generally did not form despite the continued presence of the tumor-associated mutations. The nuclear gene mutations documented in mouse brain tumors and melanomas were also detected in the normal embryonic tissues of the mice derived from the tumor nuclei (,). Some embryos derived from tumor nuclei, which contained major chromosomal imbalances, proceeded through early development forming normal appearing tissues before dying. Despite the presence of tumor-associated aneuploidy and somatic mutations, tumors did not develop from these tumor-derived nuclei (). Boveri also found that sea urchin embryos with chromosomal imbalances developed normally to gastrulation but then aborted (,). Hochedlinger et al. () showed that nuclei derived from melanoma cells were unable to direct complete mouse development due presumably to the chromosomal imbalances and irreversible tumor-associated mutations in the melanoma nucleus. Tumors did not arise in the embryos derived from the melanoma nuclei. These findings suggest that the nuclear genomic defects in these tumor cells have more to do with directing development than with causing tumors.

More recent mitochondrial transfer experiments support the general findings of the nuclear transfer experiments (,). The tumorigenic phenotype is suppressed when normal mitochondria are transferred to the tumor cell cytoplasm. On the other hand, the tumorigenic phenotype is enhanced when tumor mitochondria are transferred to a normal cell cytoplasm. These findings further suggest that tumorigenesis is dependent more on mitochondrial function than on the types of mutations in the nucleus.

In contrast to the suppressive effects of normal mitochondria on tumorigenicity, tumorigenicity is enhanced when nuclei of non-tumorigenic cells are combined with cytoplasm from tumor cells (,). These observations are consistent with the original view of Darlington that tumor cells arise from defects in the cytoplasm rather than from defects in the nucleus (). Wallace et al. () also showed that introduction of mitochondrial DNA mutations into non-tumorigenic cybrids could reverse the anti-tumorigenic effect of normal mitochondria leading to the conclusion that cancer can be best defined as a type of mitochondrial disease. The nuclear transfer studies are summarized in Figure 1, highlighting the role of the mitochondria in suppressing tumorigenesis. These studies also raise questions regarding the role of somatic mutations as drivers of tumorigenesis. Further studies will be needed to determine whether tumors arise from defects in the nuclear genome alone or in the mitochondria alone, or require defects in both the mitochondria and the nuclear genome. Such studies will provide evidence for or against the nuclear gene driver hypothesis of cancer initiation.

Respiratory insufficiency as the origin of cancer and the ‘Warburg effect’

Otto Warburg (,) first proposed that all cancers originate from dysfunctional cellular respiration. Warburg stated,

Just as there are many remote causes of plague, heat, insects, rats, but only one common cause, the plague bacillus, there are a great many remote causes of cancer-tar, rays, arsenic, pressure, urethane- but there is only one common cause into which all other causes of cancer merge, the irreversible injuring of respiration.

The key points of Warburg’s theory are (i) insufficient respiration initiates tumorigenesis and ultimately cancer, (ii) energy through glycolysis gradually compensates for insufficient energy through respiration, (iii) cancer cells continue to ferment lactate in the presence of oxygen and (iv) respiratory insufficiency eventually becomes irreversible (). Efraim Racker () was the first to describe the increased aerobic glycolysis seen in cancer cells as the ‘Warburg effect’. Warburg, however, referred to the phenomenon in cancer cells as ‘aerobic fermentation’ to highlight the abnormal production of lactate in the presence of oxygen (). As lactate production is widely recognized as an indicator of respiratory insufficiency in biological systems (), Warburg also viewed the aerobic production of lactate in tumor cells as an indicator of respiratory insufficiency.

A deficiency in oxidative phosphorylation (OxPhos) energy is responsible for lactate production in most cases (,). For example, muscle cells significantly increase their metabolic rate during intense exercise and as a result oxygen becomes limiting. The oxygen deficiency causes a lack of energy through OxPhos prompting lactate production in an effort to provide compensatory energy from fermentation (glycolytic energy) (). A competing argument would be that OxPhos is not insufficient during intense exercise and that aerobic fermentation is needed to provide more energy and growth metabolites in response to the increased work demand. This would be similar to the suggestion of Weinhouse and others for the increased aerobic glycolysis in tumor cells (,). Indeed, Kopennol et al. () suggest that the increased lactate production in tumor cells arises from damage to the regulation of glycolysis and not to insufficient respiration. However, the competing argument is inconsistent with the observation that the lactate made by muscle cells during intense exercise falls significantly after oxygen is restored to the muscle tissue. This would indicate that the lactate was made primarily because O2 was unavailable for robust OxPhos (). In addition, oxygen deprivation or hypoxia causes all known cultured mammalian cells to increase lactate production (). An increase in lactate is also seen in adequately oxygenated cells when respiration is inhibited either by respiratory poisons or null mutations in key respiratory enzymes (). It is therefore clear from established bioenergetic principles that the excess lactate made by most mammalian cells is needed to sustain fermentation energy in order to compensate for insufficient energy from respiration. It is our view that tumor cells are not an exception to this general principle and that their lactate production results in part from insufficient respiratory activity. It is expected that an upregulation of glycolytic genes would be needed to facilitate compensatory energy production through glycolysis when cellular respiration is deficient for protracted periods (). The reduction of pyruvate to lactate is needed to enhance the glycolytic pathway when respiration becomes insufficient.

It is important to recognize that pyruvate is produced through aerobic glycolysis in most normal cells of the body that use glucose for energy. The reduction of pyruvate to lactate distinguishes the tumor cells from most normal cells, which fully oxidize pyruvate to CO2 and water for adenosine triphosphate (ATP) production through the tricarboxylic acid (TCA) cycle and the electron transport chain (). Aerobic glycolysis with lactate production can occur in normal retina though more ATP is produced through respiration than through glycolysis, as is the case in most respiring tissues (). On the other hand, enhanced aerobic glycolysis without significant lactate production or energy through fermentation can occur in normal cardiac and brain tissues under conditions of increased activity (). The slight transient increase in lactate production under these conditions is not associated with a significant increase in total energy production. As enhanced aerobic glycolysis does not produce significant lactate in normal cells under well-oxygenated conditions, a phenotype of enhanced aerobic glycolysis is therefore not synonymous with a Warburg effect.

Lactate will be produced in normal tissues under low oxygen conditions. Tumor cells also produce lactate under hypoxia through anaerobic glycolysis. Although many investigators of tumor cell energy metabolism use the term ‘aerobic glycolysis’ in referring to the Warburg effect, we consider the term ‘aerobic fermentation’ as a more accurate description of the Warburg effect since aerobic glycolysis occurs in most normal cells of the body. A key issue is whether the lactate produced in tumor cells under aerobic conditions results from insufficient respiration as Warburg proposed or is due to some other phenomenon. The origin of the Warburg effect is an issue of controversy that persists today despite Warburg’s data showing that it arose from insufficient respiration.

According to Warburg and Burk respiratory insufficiency together with lactate production are the key features of tumor cell energy metabolism (,,,). Respiratory insufficiency as the origin of tumorigenesis has remained controversial, however, due to observations of high oxygen consumption rates in many tumor cells (,,). It is generally assumed that oxygen consumption rate is a good indicator of cellular respiration and OxPhos. Although low oxygen consumption rate seen together with high lactate production can be indicative of insufficient respiration, high oxygen consumption might not be indicative of sufficient respiration especially if lactate is also produced. It is now recognized from numerous studies that oxygen consumption rates are not always linked to a normally coupled oxidative phosphorylation (). It can be difficult to determine the degree to which mitochondrial ATP production arises from coupled respiration or from TCA cycle substrate level phosphorylation (). The origin of mitochondrial ATP production in tumor cells requires further clarification in light of these issues.

Mitochondrial structure is intimately connected to mitochondrial function. This fact cannot be overemphasized. We have reviewed substantial evidence of morphological, proteomic, and lipidomic abnormalities in mitochondria of numerous types of cancer cells (,,). Tumor cells can have abnormalities in both the content and composition of their mitochondria. The work of Arismendi-Morillo and Oudard et al. showed that the ultrastructure of tumor tissue mitochondria differs markedly from the ultrastructure of normal tissue mitochondria (,). In contrast to normal mitochondria, which contain numerous cristae, mitochondria from tumor tissue samples showed swelling with partial or total cristolysis (Figure 2). Cristae contain the proteins of the respiratory complexes and play an essential structural role in facilitating energy production through OxPhos (). The structural defects in human glioma mitochondria are also consistent with lipid biochemical defects in murine gliomas (,).

More recent electron micrographic studies from Elliott et al. showed that mitochondria ultrastructure was abnormal to some degree in 778 patients with breast cancer (). Remarkably, mitochondria were severely reduced in number or were undetectable in the tumor tissue from over 80% of the patients. These findings together with the evidence from the Pedersen () review would support Warburg’s central hypothesis that respiration is insufficient in tumor cells. It is obvious that mitochondrial function or OxPhos sufficiency cannot be normal in tumor cells that contain few if any mitochondria. Glycolysis and lactate fermentation would need to be upregulated in these tumor cells in order to compensate for the absence of OxPhos. Furthermore, the degree of malignancy in these breast tumors was correlated directly with the degree of mitochondrial structural abnormality (). The high glycolytic activity and lactate production seen in the most malignant tumors were also linked to the mitochondrial structural abnormalities seen in the tumors (,). In contrast to inherited mitochondriopathies, where glycolysis might not compensate completely for mitochondrial energy failure, fermentation energy appears capable of compensating completely for the respiratory insufficiency in tumor cells (,). Further studies will be needed to distinguish the differences in glycolytic and respiratory energy metabolism in tumor cells and in cells with mitochondriopathies ().

Pedersen () presented massive evidence showing that mitochondria in tumor cells are abnormal compared with mitochondria from normal cells. His review provides a comprehensive discussion of mitochondrial bioenergetics and dysfunction in cancer cells. It was clearly shown that the mitochondria of cancer cells contain numerous qualitative and quantitative abnormalities compared with mitochondria from tissue specific control cells. Summarized here are just a few of the conclusions from the Pedersen review. (i) Tumor mitochondria are abnormal in morphology and ultrastructure and respond differently to changes in growth media than mitochondria from normal cells. (ii) The protein and lipid composition of tumor mitochondria are markedly different from that of normal mitochondria. (iii) Proton leak and uncoupling is greater in tumor mitochondria than in normal mitochondria. (iv) Calcium regulation is impaired in tumor mitochondria. (v) Anion membrane transport systems are abnormal or dysregulated in mitochondria from many tumors. (vi) Defective shuttle systems are not responsible for elevated glucose fermentation in tumor cells. (vii) Pyruvate is not effectively oxidized in tumor mitochondria. (viii) Tumor mitochondria contain a surface-bound, fetal-like hexokinase. (ix) A deficiency in some aspect of respiration can account for excessive lactic acid production in tumor cells. Clearly, substantial evidence exists showing that mitochondrial structure, function and respiratory capacity is defective to some degree in all types of tumor cells. This information should be addressed in discussions of tumor cell energy metabolism.

Besides a generalized defect at the level of the mitochondrial electron transport chain in most tumor cells, numerous other mitochondrial abnormalities do exist that would diminish respiratory function (,). Interestingly, Warburg never stated that a generalized defect in electron transport was responsible for the origin of cancer despite suggestions from others (,). Rather, Warburg stated that insufficient respiration was responsible for aerobic fermentation and the origin of cancer (,,,,). We know from the work of numerous investigators that electron transport may not be coupled to ATP synthesis in cancer cells (,). Any mitochondrial defect that would uncouple electron transport from OxPhos could reduce respiratory sufficiency and thus contribute to lactate formation or a Warburg effect.

Influence of unnatural growth environment on cellular energy metabolism

Much of the evidence arguing against Warburg’s central theory that respiratory insufficiency is the origin of the aerobic fermentation seen in cancer cells (Warburg effect) was derived from investigations of tumor cells grown in vitro(,,,). In contrast to the structural defects, reduced numbers or the absence of mitochondria observed in human cancerous tissues, such mitochondrial abnormalities are not generally seen in many human and animal tumor cells when they are grown in the in vitro environment. It is interesting that oxygen consumption rate can be similar or even greater in cultured tumor cells than in non-tumorigenic cells (,,). The presence of mitochondria and robust oxygen consumption rates in tumor cells grown in vitro suggested to some that mitochondria are normal in tumor cells and that Warburg’s central theory was incorrect (,,). As mentioned above, however, oxygen consumption rate is not always an indicator of coupled respiration. Some tumor cells consume oxygen while importing and hydrolyzing glycolytically derived ATP through the mitochondrial adenine nucleotide transporter 2 in order to maintain the proton motive gradient (). We also showed that the growth of tumorigenic and non-tumorigenic cells in typical cell culture media changes the content and fatty acid composition of lipids especially cardiolipin, the signature phospholipid of the inner mitochondrial membrane that regulates OxPhos (). No tumor cells have yet been described with a normal content and composition of cardiolipin (,,). Cells cannot respire effectively if the content or composition of their cardiolipin is abnormal (,,). This point cannot be overemphasized.

It is not clear why mitochondria might appear functionally normal in many types of cultured tumor cells but appear structurally abnormal when evaluated in the tumor cells of many primary malignant cancers. Cultured cell lines are usually derived from only a single cell or a few cells of a heterogeneous tumor. Is it possible that only those tumor cells with some level of mitochondrial function are capable of growing in vitro? Also the in vitro environment forces many cells into a state of aerobic fermentation whether or not they are tumorigenic. We showed that the typical culture environment produces immature cardiolipin in non-tumorigenic glial cells, which reduces the activity of mitochondrial respiratory chain complexes (). Further studies are needed on the structure and function of mitochondria in tumor tissue and their derived cell lines.

Lactate production should be minimal in adequately oxygenated cells that have the capacity to respire normally. However, significant lactate production is often observed in proliferating non-tumorigenic cells grown in well-oxygenated cultures (,,). It is not likely that the high aerobic fermentation seen in normal cells grown in culture is due to deregulated glycolysis, as suggested for tumor cells (). Enhanced glycolysis in tumor cells cannot be considered only as deregulated but can also be considered as necessary to compensate for respiratory insufficiency.

Some investigators consider lactate production as necessary for normal cell proliferation (,). It is important to consider the differences in the metabolic requirements of tumorigenic and non-tumorigenic cells when grown in the in vivoand in vitro environments (,). In contrast to what is seen in cultured cells, no lactate production is seen in the rapidly growing embryonic chorion under aerobic conditions (). Moreover, lactate production is minimal in rapidly growing hepatocytes during liver regeneration (,). Instead, regenerating liver cells use fatty acids rather than glucose to fuel proliferation. Fatty acid metabolism produces mostly water and CO2, but not lactate. In contrast to hepatomas, which have abnormal cardiolipin composition, the content and composition of cardiolipin is similar in resting liver cells and in proliferating liver cells during regeneration (,). These findings suggest that respiration can occur normally in rapidly proliferating liver cells during liver regeneration. Viewed together, these findings indicate that lactate production is not required for rapid cell proliferation in vivo. Tumor cells are an exception in this regard, as lactate production in these cells arises as a consequence of abnormal respiration, which can be linked to either the structural defects seen in tumor tissue mitochondria or to reduced number of mitochondria. If lactate production is not required for rapid cell growth, why are significant amounts of lactate produced in many types of rapidly growing tumorigenic and non-tumorigenic cells when grown in culture?

The ‘Crabtree effect’ can confound the interpretation of energy metabolism in cultured cells. The Crabtree effect involves a glucose-induced suppression of respiration leading to lactate production whether or not mitochondria are damaged (,,,). The Crabtree effect differs from the Warburg effect, which involves lactate production arising from insufficient respiration. In other words, the aerobic lactate produced under the Crabtree effect arises from a suppressed respiration rather than from insufficient respiration as occurs in the Warburg effect. It can be difficult to determine with certainty, however, whether the aerobic fermentation (aerobic glycolysis) observed in cultured cells arises from a Crabtree effect, a Warburg effect or some combination of these effects (). We consider the Crabtree effect as an artifact of the in vitro environment that causes some non-tumorigenic mammalian cells to ferment lactate even in the presence of oxygen. It would therefore be important for investigators to exclude the influence of a Crabtree effect on the assessment of energy measurements in cultured cells. Although a Crabtree effect might suppress OxPhos, the TCA cycle should remain functional and produce ATP through substrate level phosphorylation (). Under certain conditions (hypoxia), the tumor TCA cycle can work in both forward and reverse (reductive) directions (,). Although some tumor cells can have a functional TCA cycle linked to insufficient respiration, sufficient respiration is unlikely to occur without a functional TCA cycle. Support for this comes from findings that some rare cancers can arise from inherited mutations in TCA enzymes, e.g. fumarate hydratase and succinate dehydrogenase, which impede the TCA cycle (,). Based on the data presented over many years by numerous investigators, we consider that OxPhos is universally insufficient to some degree in all tumor cells. However, the Crabtree effect and the unnatural conditions of the in vitro environment can obscure this insufficiency. Although respiratory insufficiency might be more profound in some tumor cells than in others, most if not all tumor cells will express some degree of OxPhos insufficiency compared with appropriate controls matched for species, age and tissue type.

Besides the confounding influence of the in vitro environment on energy metabolism, abnormalities and misinformation can be obtained when human tumor cells are grown in non-syngeneic hosts (). This is especially relevant with respect to the mouse xenograft models including the ‘patient-derived xenografts’. We found that human U87MG brain cancer cells express mouse carbohydrates on their surface when grown as a xenograft in immune deficient mice (). Over 65% of the sialic acid composition on the U87MG tumor cells consisted of the nine-carbon sugar, N-glycolylneuraminic acid. Humans, however, are unable to synthesize N-glycolylneuraminic acid due to a mutation in the gene that encodes a common mammalian hydroxylase enzyme (,). The hydroxylase mutation occurred in the human genome sometime after our evolutionary split with the great apes (). The acquisition of murine carbohydrates and lipids will likely occur in any human tumor cell grown in the body of a mouse or rat. N-glycolylneuraminic acid alters the characteristics of human embryonic stem cells when grown on non-human feeder cells (). The influence of the murine host on gene expression in human tumor cells is a confounding variable that can create difficulties for data interpretation in tumor cells. Few investigators address these issues.

Expression of mouse carbohydrates and lipids on human tumor cells when grown as xenografts can alter gene expression patterns and growth behavior of the tumor cells, thus altering their response to changes in the microenvironment. It might be reasonable to view the human xenograft tumor models as a type of human-mouse centaur (). In addition, the basal metabolic rate of the mouse is 7- to 8-fold greater than that of humans (,). The basal metabolic rate is the energy needed for the maintenance of all physiological processes under rest. Little attention is given to differences in metabolic rate when comparing metabolism among human and animal tumors (). The difference in metabolic rate could cause the human tumor cells to grow slow or not at all in xenografts due to competition for energy metabolites with mouse host stromal cells that have a higher metabolic rate than the human tumor cells. This could account in part for the low incidence of systemic metastasis seen in xenograft models implanted with tumor cells taken from human metastases. Solid tumors that do not metastasize or are not invasive are generally considered benign (). Further studies will be needed to determine if the human tumor cells that are selected to grow in the mouse have a metabolic rate more similar to that of the mouse than to that of the human.

Many human tumor cells or tissues are grown in mice that are Non-Obese Diabetic and have Severely Compromised Immuno-Deficiency (NOD-SCID) (). These mice not only have a compromised innate and/or adaptive immune system but also express characteristics of both type-1 diabetes and type-2 diabetes (). This is not a usual situation for most cancer patients. Despite some limited success, it is naive to assume that the growth behavior and response to therapies of human tumors grown as xenografts would be similar to the situation in the natural host. The evaluation of cancer drugs against tumor cells grown in unnatural environments together with the misunderstanding on the origin of cancer is responsible in large part for widespread failure in developing new cancer therapies (). The use of syngeneic mouse tumor models will be more representative of the natural physiological state in humans than will the xenograft models.

Connecting the links from respiratory insufficiency to cancer origin

The path from normal cell physiology to malignant behavior, where all major cancer hallmarks are expressed, is depicted in Figure 3. Any unspecific condition that damages a cell’s respiratory capacity but is not severe enough to kill the cell can potentially initiate the path to a malignant cancer. Reduced respiratory capacity could arise from damage to any mitochondrial protein, lipid or mtDNA. Some of the many unspecific conditions that can diminish a cell’s respiratory capacity thus initiating carcinogenesis include inflammation, carcinogens, radiation (ionizing or ultraviolet), intermittent hypoxia, rare germline mutations, viral infections and age. The evidence supporting this statement also addresses Szent Giorgy’s ‘oncogenic paradox’, as was described in a recent treatise on the subject (). The paradox addresses the difficulty in knowing how a plethora of disparate carcinogenic agents might produce cancer through a common mechanism. Some of the rare germline mutations that increase risk for cancer through an effect on cellular respiration include p53BRACA1RB and xeroderma pigmentosum (). Cancer-causing viruses can be linked to mitochondrial dysfunction (). If respiratory damage is acute, the cell will die. On the other hand, if damage is mild and protracted, the cell will elevate lactate or amino acid fermentation in order to compensate for insufficient OxPhos. Recent evidence also shows that mitochondrial dysfunction is the initial event in the path to tumorigenesis induced by the mutated Ras oncogene and is closely linked to the action of the BRAF oncogene (,,). Cells will enter their default state of proliferation following loss of respiratory control (,). Several cancer hallmarks can be linked to the transition from quiescence to proliferation (Figure 3). Unbridled proliferation is linked to fermentation, which was the dominant form of energy metabolism during the oxygen deficient α period of earth’s history (). OxPhos insufficiency in fusion hybrids between immune cells (mostly macrophages) and cancer stem cells can underlie the ability of tumor cells to intravasate the circulation locally and to extravasate the circulation at distant sites (,). As macrophages are already mesenchymal and naturally capable of systemic tissue dispersion, it is not necessary to explain the phenomenon of metastasis in terms of complicated gene-linked epithelial to mesenchymal and mesenchymal to epithelial transitions. Metastasis in our view would arise from the dysregulation of normal macrophage functions in fusion hybrids including intravasation and extravasation (). All major hallmarks of cancer including genomic instability can be linked directly or indirectly to the respiratory dysfunction and the compensatory fermentation of the tumor cell.

Are mutations in the P53 and the Ras genes primary or secondary causes of cancer?

Although germline or somatic mutations in the P53 tumor suppressor gene and somatic mutations the Ras oncogene occur frequently in many tumor cells and cancers (,), it is not clear if these genes or their products are primary or secondary causes of cancer. Hwang et al. showed that p53 regulates mitochondrial respiration through its transcriptional target gene Synthesis of Cytochrome c Oxidase 2 (SCO2) (). In these studies the Warburg effect was linked directly to impaired respiration. Singh et al. showed that mitochondrial energy metabolism is impaired in human cancer cells containing defects in p53 (). Huang et al. recently showed that the common K-RasG12V mutation causes a metabolic switch from OxPhos to glycolysis (Warburg effect) due to mitochondrial dysfunction (Figure 4). Lee et al. showed that transfection of human diploid cells with V12Ras significantly increased damaging oxygen species in mitochondria (), whereas Weinberg et al. () showed that mitochondrial reactive oxygen species (ROS) generation and damage to complex III was essential for K-Ras-induced cell proliferation and tumorigenesis. Moreover, Yang et al. () showed that H-Ras transformation of mouse fibroblasts damaged respiration, thus forcing the cells into a glycolytic metabolism. This is notable since activated RAS has been proposed to induce MYC activity and to enhance non-hypoxic levels of HIF-1α (). As MYC and HIF-1 drive glycolysis, their upregulation would be necessary to prevent senescence following respiratory impairment (,). As constitutive Rasactivation is incompatible with prenatal development, a disruption of mitochondrial energy metabolism could underlie tumor formation in mice cloned from melanoma nuclei following the inadvertent expression of the Ras oncogene (). Viewed collectively, these and other observations are consistent with Warburg’s theory and suggest that mutations in the P53 and Ras genes initiate cancer through their adverse effects on respiratory function. It will be up to each investigator to determine whether they consider these mutations as primary or secondary causes of cancer according to Warburg’s central theory (,,). It is our view that all roads to the origin and progression of cancer pass through the mitochondria (Figure 3).

Can tumor somatic mutations arise as a downstream epiphenomenon of abnormal energy metabolism?

How might protracted respiratory insufficiency cause somatic mutations and the nuclear genomic instability seen in tumor cells? The integrity of the nuclear genome is dependent to a large extent on the efficiency of mitochondrial respiratory function (). Evidence indicates that a persistent retrograde response or mitochondrial stress response leads to abnormalities in DNA repair mechanisms and to the upregulation of fermentation pathways (,). Oncogene upregulation becomes essential for increased glucose and glutamine metabolism following respiratory impairment (,). The metabolic waste products of fermentation can destabilize the morphogenetic field of the tumor microenvironment thus contributing to inflammation, angiogenesis and progression (). Normal mitochondrial function is necessary for maintaining intracellular calcium homeostasis, which is required for chromosomal integrity and the fidelity of cell division. Aneuploidy can arise during cell division from abnormalities in calcium homeostasis (). In this general picture, the abnormal genomic landscape seen in tumor cells is considered a downstream epiphenomenon of dysfunctional respiration and protracted oncogene-driven fermentation. In other words, the somatic mutations arise as effects rather than as causes of tumorigenesis. The nuclear transfer experiments support this view (Figure 1). In light of this perspective, it would be important for those working in cancer genomics field to justify the logic of their experimental approach to the cancer problem (,).

Cancer progression is more consistent with Lamarckian than Darwinian evolution

When viewed as a mitochondrial metabolic disease cancer progression is more in line with the evolutionary theory of Lamarck than with the theory of Darwin (). Many investigators in the cancer field have attempted to link the Darwinian theory of evolution to the phenomenon of tumor progression (). The attempt to link cancer progression to Darwinian evolution is based largely on the view that nuclear somatic mutations are drivers of the disease. According to Lamarck’s theory, it is the environment that produces changes in biological structures (). Through adaptation and differential use, these changes lead to modifications in the structures. The modifications of structures would then be passed on to successive generations as acquired traits. Lamarck’s evolutionary synthesis was based on his belief that the degree of use or disuse of biological structures shaped evolution along with the inheritance of acquired adaptability. Lamarck’s ideas could also accommodate a dominant role for epigenetics and horizontal gene transfer as factors that could facilitate tumor progression (,). In addition to nuclear epigenetic events involving acetylation and phosphorylations, mitochondria are also recognized as a powerful extra nuclear epigenetic system (,). Other epigenetic phenomena such as cytomegalovirus infection, cell fusion and horizontal gene transfer can also contribute to cancer progression and metastasis (,,).

Considering the dynamic behavior of mitochondria involving regular fusions and fissions, abnormalities in mitochondrial structure can be rapidly disseminated throughout the cellular mitochondrial network and passed along to daughter cells somatically, through cytoplasmic inheritance (,). The capacity for mitochondrial respiratory function becomes progressively less with each cell division as adaptability to substrate level phosphorylation increases (Figure 3). The somatic progression of cancer would therefore embody the concept of the somatic inheritance of an acquired trait. The acquired trait in this case is alteration to mitochondrial structure. The most malignant cancer cells would sustain the near-complete replacement of their respiration with fermentation. This is obvious in those tumor cells with quantitative and qualitative abnormalities in their mitochondria (Figure 2). The somatic inheritance of mitochondrial dysfunction in tumor cells could contribute in part to the appearance of a clonal origin, but not directly involving the nuclear genome. However, the degree of nuclear genomic instability can be linked to mitochondrial dysfunction and both defects together can contribute to tumor progression. A Lamarckian view can account for the non-uniform accumulation of mutations and drug resistance seen during cancer progression. Drug resistance is linked to enhanced lactate fermentation, which is acquired during tumor progression (,). It is our opinion that the evolutionary concepts of Lamarck can better explain the phenomena of tumor progression than can the evolutionary concepts of Darwin. We encourage further research on this perspective of tumor progression.

Exploiting mitochondrial dysfunction for the metabolic management of cancer

If cancer is primarily a disease of energy metabolism, then rational strategies for cancer management should be found in those therapies that specifically target tumor cell energy metabolism. These therapeutic strategies should be applicable to the majority of cancers regardless of tissue origin, as nearly all cancers suffer from a common malady, i.e. insufficient respiration with compensatory fermentation (,,,). As glucose is the major fuel for tumor energy metabolism through lactate fermentation, the restriction of glucose becomes a prime target for management. However, most normal cells of the body also need glycolytic pathway products, such as pyruvate, for energy production through OxPhos. It therefore becomes important to protect normal cells from drugs or therapies that disrupt glycolytic pathways or cause systemic reduction of glucose. It is well known that ketones can replace glucose as an energy metabolite and can protect the brain from severe hypoglycemia (). Hence, the shift in energy metabolism associated with a low carbohydrate, high-fat ketogenic diet administered in restricted amounts (KD-R) can protect normal cells from glycolytic inhibition and the brain from hypoglycemia.

When systemic glucose availability becomes limiting, most normal cells of the body will transition their energy metabolism to fats and ketone bodies. Ketone bodies are generated almost exclusively in liver hepatocytes largely from fatty acids of triglyceride origin during periods of fasting (,). There are no metabolic pathways described that can produce ketone bodies from carbohydrates despite suggestions to the contrary (). A restriction of total caloric intake will facilitate a reduction in blood glucose and insulin levels and an elevation in ketone bodies (β-hydroxybutyrate and acetoacetate). Most tumor cells are unable to use ketone bodies for energy due to abnormalities in mitochondria structure or function (,). Ketone bodies can also be toxic to some cancer cells (,). Nutritional ketosis induces metabolic stress on tumor tissue that is selectively vulnerable to glucose deprivation (). Hence, metabolic stress will be greater in tumor cells than in normal cells when the whole body is transitioned away from glucose and to ketone bodies for energy.

The metabolic shift from glucose metabolism to ketone body metabolism creates an anti-angiogenic, anti-inflammatory and pro-apoptotic environment within the tumor mass (,). The general concept of a survival advantage of tumor cells over normal cells occurs when fermentable fuels are abundant, but not when they are limited (). Figure 5 illustrates the changes in whole body levels of blood glucose and ketone bodies (β-hydroxybutyrate) that will metabolically stress tumor cells while enhancing the metabolic efficiency of normal cells. This therapeutic strategy was illustrated previously in cancer patients and in preclinical models ().

Implications for novel therapeutics

Once the whole body enters the metabolic zone described in Figure 5, relatively low doses of a variety of drugs can be used to further target energy metabolism in any surviving tumor cells (). It is interesting that the therapeutic success of imatinib (Gleevec) and trastuzumab (Herceptin) in managing BCR-ABL leukemia cells and ErbB2-positive breast cancers, respectively, is dependent on their ability to target signaling pathways linked to glucose metabolism (,). In contrast to these drugs, which target energy metabolism primarily in those individuals with mutations in specific receptors linked to the IGF-1/PI3K/Akt pathway, calorie-restricted KDs will target similar pathways in any cancer cell regardless of the mutations involved (,). Dietary energy reduction will simultaneously target multiple metabolic signaling pathways without causing adverse effects or toxicity (). Non-toxic metabolic therapies might also be a preferable alternative to toxic immunotherapies for cancer management especially if both therapies target the same pathways. It must be emphasized that the therapeutic efficacy of the KD is strongly dependent on restricted intake, as consumption of the KD in unrestricted amounts can cause insulin insensitivity and glucose elevation despite the complete absence of carbohydrates in the diet (). Elevated consumption of the KD is not often seen, however, as humans usually restrict intake due to the high fat content of the diet.

Poff et al. also recently showed a synergistic interaction between the KD and hyperbaric oxygen therapy (HBO2T) (Figure 6). The KD reduces glucose for glycolytic energy while also reducing NADPH levels for anti-oxidant potential through the pentose-phosphate pathway. HBO2T will increase ROS in the tumor cells, whereas the ketones will protect normal cells against ROS damage and from the potential for central nervous system oxygen toxicity (,). Glucose deprivation will enhance oxidative stress in tumor cells, whereas increased oxygen can reduce tumor cell proliferation (,). A dependency on glucose and an inability to use ketones for energy makes tumor cells selectively vulnerable to this therapy. Although this metabolic therapy is effective against those tumor cells that contain mitochondria, it remains to be determined if this therapy would be equally effective against those tumor cells containing few or no mitochondria (). In contrast to radiation therapy, which also kills tumor cells through ROS production (), the KD + HBO2T will kill tumor cells without causing toxic collateral damage to normal cells. Cancer patients and their oncologists should know about this. Some KDs might also enhance the therapeutic action of radiation therapy against brain and lung tumors (,). It will be important to compare and contrast the therapeutic efficacy of conventional radiation therapy with HBO2T when used with the KD-R. Although radiation is widely used as a cancer therapy, it should be recognized that radiation damages respiration in normal cells and can itself cause cancer (). Radiation therapy for malignant brain cancer creates a necrotic microenvironment that can facilitate recurrence and progression through enhanced glucose and glutamine metabolism (,).

Besides drugs that target glucose, drugs that target glutamine can also be effective in killing systemic metastatic cancer cells (,,). Many metastatic cancers express multiple characteristics of macrophages (,). Glutamine is a major fuel of macrophages and other cells of the immune system (,). As glutamine is the most abundant amino acid in the body and is used in multiple metabolic reactions, targeting glutamine without toxicity might be more difficult than targeting glucose (,). Although glutamine interacts synergistically with glucose to drive energy metabolism in cultured tumor cells, there are reports suggesting that glutamine can have chemo-preventive effects (). Further studies are needed to evaluate the role of glutamine as a facilitator of tumor energy metabolism in vivo.

The novelty of the metabolic approach to cancer management involves the implementation of a synergistic combination of nutritional ketosis, cancer metabolic drugs and HBO2T. This therapeutic approach would be similar to the ‘Press-Pulse’ scenario for the mass extinction of organisms in ecological communities (,). The KD-R would act as a sustained ‘Press’, whereas HBO2T and metabolic drugs would act as a ‘Pulse’ for the mass elimination of tumor cells in the body. Some of the cancer metabolic drugs could include 2-deoxyglucose, 3-bromopyruvate and dichloroacetate (,,). This therapeutic strategy produces a shift in metabolic physiology that will not only kill tumor cells but also enhance the general health and metabolic efficiency of normal cells, and consequently the whole body (,). We view this therapeutic approach as a type of ‘mitochondrial enhancement therapy’ (). As we consider OxPhos insufficiency with compensatory fermentation as the origin of cancer, enhanced OxPhos efficiency would be anti-carcinogenic.

Many cancers are infected with human cytomegalovirus, which acts as an oncomodulator of tumor progression (). Products of the virus can damage mitochondria in the infected tumor cells, thus contributing to a further dependence on glucose and glutamine for energy metabolism (,). The virus often infects cells of monocyte/macrophage origin, which are considered the origin of many metastatic cancers (,,,). We predict that the KD-R used together with anti-viral therapy will also be an effective Press-Pulse strategy for reducing progression of those cancers infected with human cytomegalovirus ().

Advanced metastatic cancers can become manageable when their access to fermentable fuels becomes restricted. The metabolic shift associated with the KD-R involves ‘keto-adaptation’. However, the adaptation to this new metabolic state can be challenging for some people. The administration of ketone esters could conceivably enable patients to circumvent the dietary restriction generally required for sustained nutritional ketosis. Ketone ester-induced ketosis would make sustained hypoglycemia more tolerable and thus assist in metabolic management of cancer (,). As each person is a unique metabolic entity, personalization of metabolic therapy as a broad-based cancer treatment strategy will require fine-tuning based on an understanding of individual human physiology. Also, personalized molecular therapies developed through the genome projects could be useful in targeting and killing those tumor cells that might survive the non-toxic whole body metabolic therapy. The number of molecular targets should be less in a few survivor cells of a small tumor than in a heterogeneous cell population of a large tumor. We would therefore consider personalized molecular therapy as a final strategy rather than as an initial strategy for cancer management. Non-toxic metabolic therapy should become the future of cancer treatment if the goal is to manage the disease without harming the patient. Although it will be important for researchers to elucidate the mechanistic minutia responsible for the therapeutic benefits, this should not impede an immediate application of this therapeutic strategy for cancer management or prevention.

Glossary

Abbreviations:

ATP adenosine triphosphate
HBO2T hyperbaric oxygen therapy
KD ketogenic diet
OxPhos oxidative phosphorylation
ROS reactive oxygen species
SLP substrate level phosphorylation
TCA tricarboxylic acid.
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Do gut bacteria inhibit weight loss?

Content Source ~ Harvard Health Publishing-Harvard Medical School

Q. I just can’t lose weight. A friend says that my problem might be due to the types of bacteria that live in my gut. That sounds crazy to me, but is it true, and can I do something about it?

A. Ten years ago, I also would have thought your friend was crazy. Today, I’d say she could well be right. Here’s why. We’ve known for a century that bacteria live in our intestines, but we’ve assumed that they did little to affect our health. We thought that they were just mooching off of us — taking advantage of the warmth and nutrients in our gut.

In the past decade, however, remarkable breakthroughs have allowed scientists to count and characterize the genes in our gut bacteria. The results have been astonishing. Our gut bacteria have 250 to 800 times more genes than we have human genes. Even more remarkable, these bacterial genes make substances that get into the human bloodstream, affecting our body chemistry. That means it is entirely plausible that the bacteria in our gut could be affecting our health.

How could they affect our weight? When we eat food, our gut breaks it down into small pieces. Only the smallest pieces get absorbed into our blood. The rest is eliminated as waste material. In other words, not all of the calories in the food we eat get into our body and increase our weight. The gut bacteria help break down food. Some bacteria are better able to chop food into those smallest pieces that get digested, add calories to our body and thereby tend to increase our weight. Theoretically, if our guts have more of those kinds of bacteria, it should be harder to lose weight.

But is there evidence that it really is true? Several studies in animals, and some in humans, say that it is. For example, scientists transferred bacteria from the guts of two strains of mice — one that naturally becomes obese and one that naturally stays lean — into a third lean strain raised from birth to have no gut bacteria. Gut bacteria transferred from the naturally obese mice made the germ-free mice become fat, but gut bacteria transferred from the naturally lean mice kept them lean.

Then scientists took bacteria from the guts of human identical twins, one of whom was obese and one of whom was lean, and transferred those bacteria into the guts of lean, germ-free mice. Bacteria from the obese twin made the mice become fat, but bacteria from the lean twin did not.

We are just beginning to understand the role of gut bacteria in obesity, and the science hasn’t led yet to treatments that will make it easier to lose weight. However, I believe that day is coming.

— by Anthony L. Komaroff, M.D.
Editor in Chief, Harvard Health Letter