i ii !i I Mitochondria, Energy and Cancer: The ReTjationshipwith Ascorbic Acid 1 I I paper is dedicated to the memory of Dr. Brian Ixibovitz and Dr. Hugh D. Kiordan. Abstract Ascorbic Acid ( . has been used in thcprevention and treatment of [cancerwith reporlea' effictivenes.~.IbMucAondria may be ofre ofthe principal targets of ascorbatei celI21Zaractivity a d it may play an important role in the devehpnzent and progression ofcancer. Mitochondria, besides generati~rgadenosine triphosphate (ATP), ~ Q uS role in upuptosir regulation and in the prodaction ofregtilatory oxidative species that may be relevant in gene exprexriorz. At higher conce~~trations R4 may increase AT'. producpion dy increaring m itochondriaZ electronjgx, also may induce apoptotic cell c h t h in tumor cell li~zes,probab(y via il.cpro-oxidant action. In contrm at lower concent?-[ztions AA dispZt~ysaa~ioxidan~ p r o p i e s that may preumt the activation of oxidant-induced apuptoiis. Bese concentration dependent activities of ascorbate may zxplain inpart the seemingb co ztradictory results that have been re.rtedpreuio~s(y. Ascorbic Acid Ascorbic acid (idscorbate, vitamin C, AA) is a water-soluble vitamin needcd for the growth and repair of tissues in the body It is necessary for the formation of collagen, an important protein of skin, scar tissue, tendons, ligaments and blood vessels. AA is cssentid for the healing of wounds and for the repair and maintenance of cartilage, bones and teeth. AA is one of many molecules with antioxidant and pro-oxidant capacity. Proposed mechanisms of AA activity in the prevention and treatment of cancer include: Enhancement of the immune system by increased lymphocyte production and activity;' stimulation of: collagen formation, necessary for "walling off" tumors; inhibition of hyaluronidase by keeping the ground substance around the tumor intact and preventing metastasis;' inhibition of oncogenic viruses; correction of an ascorbate deficiency cornrnonly seen in cancer patients; expedition of wound healing after cancer s ~ r g e r y ; ~ enhancement of the anticarcinogenic effect of certain chemotherapy d r ~ g s ; reduction ~-~ of the toxicity of chemotherapeutic agent$ prevention of cellular free radical damage;? production of hydrogen p e r ~ x i d e ;and ~ * ~neu- an inhibitory action on malignant cell proliftralization of carcinogenic substances.I0." erationZ7by directly interfering with anaeroThe use of AA supplementation in large bic respiration (fermentation and lactic acid doses for the prevention and treatment of production), a common energy mechanism cancer has been reported by various groups. utilized by malignant cells. It would be worth Cameron and Pauling performed experiinvestigating the status of the mitochondria ments that utilized high doses of Ah for of malignant cells because Gonzkle.~and colthe treatment of cancer? l l e s e experiments leaguesZPbelieve this may be relevant in the proved that AA in high doses appears to be origin of malignancy. A problem in electron safe for the majority of individuals. Extentransfer in the malignant cells might well be sive epidemiological evidence points to the coupled to a defective mitochondria1 memcapacity of AA to prevent cancer at a numbrane and AA may help correct this electron ber of sites. Some of the few studies which transfer problem by balancing mitochondrial have been conducted on the use of high dose membrane potential of malignant tissue. The ascorbate in the treatment of cancer have inhibitory action on cancer cells by ascorbic yielded promising results.4" While AA alone acid has been described since 1952.29AAnot may not be enough of an intervention in the only has antioxidant properties but also protreatment of most active cancers, it appears oxidant activity capable of selective cytotoxic to improve quality of life and extend survival effects on malignant cells when provided in time in most cases and it should be considhigh ~oncentrations."~~~~ ered as part of the treatrnent protocol for all It has been suggested that ascorbate propatients-with cancer." motes oxidative metabolism by inhibiting the ?he main function of AA in small quanutilization of pyruvate for aerobic metabotities is as a hydrophilic antioxidant, b;t in 1isn-1.~~ Also, an inhibitory effect on growth high doses in malignant cells it may act as of several types of tumor cells has been propro-oxidant. Experiments in rodents suggest duced by ascorbate and/or its derivatives. that ascorbate administration increases host survival times and inhibit tunlor g r o ~ t h . ' ~ . ' ~T d s inhibitory action was not observed in normal fibroblast^.^^ This cytotoxic activity Clinical trials with M have yield mixed rcproduced by ascurhate in an array of maligsuits. In two Scottish studies, terminal cancer patients given intravenous AA (lQg/day) nant cell lines has been associated to its pro'-~~ can generate had longer - survival times than historical oxidant a ~ t i v i t y . ~Ascorbate 1 1,U2 (a reactive species) upon oxidation with controls."J6 A Japanese study yielded simioxygen in biological systems.3g Hz@ may iar results," but two double-blind studies further generate additional reactive species at the Mayo clinic using oral AA (lOg/day) such as the hydroxyl radical and aldehydes showed no benefit.l8.I9We should mention which can compromise ceU viability." Ihese that orill AA supplementation is unlikely to reactive species may induce strand breaks in produce plasma ascorbate levels sufficient to DNA, disrupt membrane function via lipid kill tumor cells directl~.~" Intravenous AA at .' peroxidation or deplete cellular ATP.39?he higher doses has bccn cffcctive in individual C'&ses.8,21-23 failure to maintain ilTP production may bc a consequence of oxidative inactivatiori of ?he energy producing process of makcy enzymes of the aerobic pathway on the lignant cells is mainly anaerobic and the ATP uses. The cytotoxicity induced by ascortransformation of norm21 cells to malignant bate seems to be primarily mediated by H202 may be due to defects in aerobic respiratory generated intracellularly by ascorbate's metp a t h ~ a y s . 2Oxygen, ~ ~ ~ the final electron acabolic oxidation to dehydr~ascorbate.~+l~fl-~~ ce~t0.r in the electron trans~ort svstem is of 1 .' In addition this antiproliferative action of great importance in the ascorbate-induced ascorbate in cultured malignant cells, animal inhibitory action, because of the production and human tumor has been increased by the of hydrogen peroxide (I--I,&) form a radical addition of the cupric ion, a catalyst for the out of the M molecule. Oxygen by itself has I *yv Mitochondriai ~ k e r gCancer ~ , and Ascorbic Acid ' oxidation of ascorhate."It has also been suggested that the selective toxicity of ascorbatc in malignant cells may be due to a reduced level of catalase in these cells, leading to cellular dainagc thmugh the accumulation of H ~ ~ ~ . I ) , 'There ? R , AisLa- 10 ~ . to ~ 100 fold greater content of catalase in normal cells than in tumor cells.2a For this reason the cornbination of rnegadoscs of ascorbate together with oxygen and copper seems logical as part of a non-toxic treatment protocol for cancer patient~.'~ Moreover, a lack of superoxide dismutase (SOD) has been detected in the mitochondria of cancer cells."' ?his deficiency will impair the function of the Krebs cycle fbrcin~anaerobic metabolism and the concornitant vroduction of lactic 3 c i ~ i . ~ ~ 'This ikformation suggests that the mitochondria may be one of the principal targets of ascorbate activity and play an important role in the development and progression of cancer. Since the Warburg research in the 1 9 3 0 ~ ~few " scientists have worked wit11 tlze mechanism of mitochondria1 respiratory alterations in cancer. Also, in relation to this it is relevant to understand the relationship between of AA, rnitochondrial al.terationi and the ztpoptosis process pathway. U Mitochondria and Apoptosis Mitochondria play a central role in oxidative metabolism in eukaryutcs." 'lhe primary purpose of mitochondria is to manufacture adenosine triphosphate (ATP), which is used as the primary source of energy by the cell. ~ i t c k h o n d i i ahave several important functions besides the production of ATP. htitochondria are also essential in, th,e processing of important metabolic in.termediates for various pathways involved in the metabolism of carbohydrates, amino acids, and fatty acids. In addition to oxidative phosphorylation, mitochondria are involved in other critical. ~rocesses.~' 'These variety of functions corresponds to the variety of rnitochoodrial diseases including diabetes, cardiornyopathy, infertility, migraine, blinctness, deafness, kidney and liver diseases, stroke, age-related neurodegenerative disorders such as Parkinson's, AlzheirnerS A 1 and Huntington's disease, and cancer.?? Mitochondria also play a role in the regulation of apoptosis.1his organelle can trigger cell death in a nuinber of ways: by disrupting electron transport and energy metabolism, by releasing and/or activating proteins that mediate apoptosis and by a1.tering cellular redox potential. Apoptosis, a physiological process for killing cells, is critical for the normal development and fu~lctionof rnulticellular organisms, Abnormalities in ccll death control contribute to a variety of diseases, including cancer, autoimmunity and degenerative disorder~."~ Electron microscopic analysis has identified the morphological changes that occur during apoptosis, which 'i-ncludechromatin condensation, cytoplasmic shrinkage, and plasma mcmbrane b1~bbing.l.~~ 'Ihese studies have also noted that during the early stages of apoptosis, no visible changes occur in mitochondria, the endoplasmic reticulum, or the Colgi apparatus. However, others have more recently rcported swelling of the outer mitochondria1 and release of cytochrome ~ 5 and % ~ ayoptosis ~ inducing factor, an oxidoreductase-relatcd flavoprot e i ~ from ~ , ~ the ~ mitochondria1 interm&brane space." Molecular changes induced during apoptosis include internucleosomal DNA cleavage" and randomization of the distribution of phosphatidyl scrine between the inner and outer leaflets of the plasma &sc morphological and molecular changes are elicited by a broad range of physiological or experimentally applied death. stimuli and are observed in cells 'from diverse tissue tvrjes and s~ecies.~' .' Apoptosis research has reccntly experienced a paradigm change in which the nucleus of the cell no longer determines the apoptotic process. ?he new paradigm states that caspases and more recently, rnitochondria conbtiture the center of death control..58 Severaf observations are coxnuatible .with the 1. hmothesis that mitocholldria controls cell death: (i) mitochondria1 membrane perrneabilization generally precedes the signs of advanced apoptosis or necrosis, irrespective of the cell type or the death i~lducingstimulus; I il 1 31 lines exhibit differences in the number, size and shane of their mitochondria relative to normal controls. The mitochondria of ra~idlv growing tumors tend to be fewer in number, smaller in size and have fewer cristae than mitochondria from slowly growing tumors; the latter are larger and have characteristics more closely resembling those of normal cells.49 Chemotherapeutic anti-cancer drugs and ganma-irradiation induce apoptosis in tumor ~ells."~'~ Oncogenes and tumor suppressor genes that regulate cell death influence the sensitivity of tumor cells to anti-cancer therapy. Overexpression of Rcl-2 and its pro-survival hornologs or the inactivation of Bax not only provides short term protection against apoptosis, but can significantly increase long-term survivd with retention ofclonoger~icityin certain tumor cells that have been treated with anticancer drugs or garnrna-irradi ation?0373 Thus, the response of cancer cells to therapy is determined bv at least two Drocesses: the propensity to undergo mitotic death and the sensitivity to apoptotic stimuli. Many antitumor therapies rely on inducing apoptosis in their target cells. I h e role of caspases in the response or resistance to such drugs has therefore been under intense scrutiny Because the various caspases can process each other, most of them eventually become activated in cells undergoing apopGsis and this appears to be the case in drug-treated tumor ~ e l l s . ~ ~ - ~ ~ Zhe observation th.at in some cancer DaL tients "spontaneous" tumor regression was dependent on circulating levels of TNF80 was the first indication that cell surfice receptors might he viable anti-cancer targets. Mitochondria and Cancer It is now well established that some memMitochondria are involved either directly bers of the TNF receptor supcrfamily induce or indirectly in many aspects of the altered apoptosis in cells.6o Receptor-mediated inmetabolism in cancer Cancer ceffs duction of apoptosis is preceded by ligandhave altered metabolic characteristics that induced trimerization; recruitment of the includes: a higher rate of glycolysis," an indeath-inducing signaling complex (DISC) increased creased rate of glucose tran~port,~' and activation of p r ~ c a s p a s e - 8 . ~ Activat*~~ gluconeogenesis,b6 reduced pyruvate oxidation and increased lactic acid p r o d ~ c t i o n , ~ ~ed caspase-8 initiates a proteolytic cascade, resulting in apoptotic cell death. However, increased g1u:lutaminolytic activi&?Qeduced dle use of TNF and other seauence-related fatty acid 0xidation,6~increased glycerol and ligdnds as pro-apoptotic agonists has been fatty acid turn0ver,7~modified amino acid limited because of the zssociated systemic metabolism7l and increased pentose ph.osphate pathway activityT2Various tumor cell (ii) this perrneabilization event has a better predictivi value for cell dcath than other pa&meters including caspase activation; (iii) an increasing number of pro-apoptotic effectors ' d ~on t mitochondrial membranes to induce their permeabil?tation; (iv) anti-apoptotic members of the Bcl-2 family physically interact with mitochondria1 membrane proteins and inhibit cell death by virtue of their capacity to prevent rnitochondrial membrane aerrncabilizition; (v) inhibition of rnitochondrial membrane perrneabilization by specific pharmacological intenrentions prevents or retards cell death; (vi) cell-free systems have identified several mitochondrial proteins, which are rate-limiting for the activation of catabolic hydrolases (caspses and nucleases). 'These observations suggest a three-step model of apoptosis: a pre-mitochondrial phase during which signals transduction cascades or pathways is activated (initiation phase); a mitochondria1 phase, during which rnitochondrja1 membranes are I~ermeabilized (decisiodeffector phase); and a post-mitochondrial phase during which proteins are released from mitochondria and cause the activation of proteases and nucleases (degradation phase).58 Inducers of apoptosis are relatively diverse and include death factors such as Fas ligand (FasL), Tumor Necrosis Fact0.r (TNF) and TNF-related apoptosis-inducing ligand (?'RAIL), genotoxic agents such as anti-cancer drugs, gamma-irradiation and oxidative s t r e s ~ - ~ ~ - ~ ~ i I J L I d Mitochondria, Oxidative Species and differentiation Cardiolipin is essential for the functionality of several mitochondrial proteins. Its distribution between the inner and outer leaflet of the mitochondrial internal membrane is crucial for ATP synthesis. GarciaFernandez and colleagueQ4 investigated alterations in cardioli~in distribution durJ. ing the early phases of apoptosis. Using two classical models (staurosporine-treated HL60 cells and TNF-alpha-treated U937 cells); they found that in aioptotic cells cardiolipi'n moves to the outer leaflet of the mitochondrial inner membrane in a time-dependent rnanner.?his occurs before the appearance of apoptosis markers such as plasma-memblrane exposure of phosphatidylserine, changes in mitochondrial membrane potential, DNA fragmentation, but after thk production of reactive oxygen species (ROS). The exposure of phospholipids on the outer surface doring apoptosis, thus occurs not only at the plasma membrane level but also in the mitochondria, reinforcing the hypothesis of "mitoptosis" (cell death induce by mitochondria) as a crucial regulating system from programmed cell death, also occurring in cancer cells after treatment with antineo~lastica ~ e n t s84. Several potentially damaging ROS species may arise as by-products of normal metabolism and from chemical accident^.^' Superoxide is a one-electron reduction product of molecular oxygen that is formed during normal respiration in mitochondria and by autoxidation reactions.'The electron transport chain is not completely secure and electrons may leak onto molecular oxygen prior to their transfer to cytochrome oxidase forming superoxide. H202 is another ROS that can be fo;med during normal r n e t a b ~ l i s m . ~ ~ Not all ROS production is accidental, however, since thebody can use these substances for its own benefit. There is some indications that these oxidative products may control cell growth and differentiationag7Nitric oxide finds a multiplicity of uses, for example as a regulator of vascular tone and as a messenger of the central nervous svtern? Production of reactive species by activa:ted L L I U A - U - J neutrophils, macrophages and several other cell types is used in bacterial killing. Indeed ROS may have a variety of functions including regulation of gene expressiong7and induction of a p o p t o s i ~ ;programmed ~~~~~ cell death involved in fetal development and tissue remodeling where changes in the type and distribution of tissue occur. A n involvement of ROS in the process of cell differentiation has been suggested on the basis of studies with slime mold and Neurospora c r a ~ s a . I~n' both cases differentiation appears to be accompanied by increased production of ROS. Indeed, addition of antioxidants can modulate the process.g6 W e should remember that cells, in order to be able to divide, need to reduce cohesiveness and dismount part of their structure: in other words dedifferentiate. ?his unstable state of cellular organization facilitates oxidative damage in dividing cells. W i t h this taken into account, AA in-high doses in the presence of divalent cations and oxygen may act as a chemotherapeutic pro-oxidant. I t should also be pointed out that iron is imbedded in the center of the DNA double helix at certain loci where it seems to function as an oxygen sensor to activate DNA in response to oxidative stress. Because iron is a big molecule, it distorts the outer shape of the DNA helix this distortion depends on the oxidation state of iron. Under nonoxidized (reduced) conditions, iron is present in the ferrous state and the DNA helix is fairly tight around the iron atom; but when the ion is oxidized to the ferric state it opens up the DNA so that transcription into RNA becomes more easyg7This mechanism might as well be a way in which oxidative molecules influence and/or determine differentiation. AA enters the picture since AA may assure a continuing electron exchange among body tissues and cell m i t ~ c h o n d r i a .A ~~ greater amount of AA in the body enhances the flow of electricity optimizing the ability of the cells to maintain aerobic energy production and production of metabolic intermediaries that arise from this pathway facilitates cell to cell communication and probably enhances cell differentiation. In support of I this theory, it has been documented that osteoblast cells treated with ascorbic acid had a four fold increase in respiration and there-fold increase in ATP production that provided the necessary environment for cell differentiati~n.~~The antioxidant defenses consist of redox molecules such as AA and vitamin E and enzymes (e.g. superoxide dismutase). Their function,is to act as a coordinated and balanced system to protect tissues and body fluids from damage by ROS whether produced physiologically or as a response to inflammation, infection and/or disease.86 Many antioxidant defenses depend on micronutrients or are micronutrients themselves. Examples of the latter are vitamin E (protects against lipid peroxidation) and AA (scavenges some aqueous ROS directly and recycles lipophylic vitamin E). Superoxide dismutase removes superoxide by converting it to H202which is then removed by glutathione peroxidase or c a t a l a ~ e . ~ ~ Antioxidants most likely regulate the activation phase of oxidant-induced apoptosis. Oxidants can induce mitochondrial permeability transition and release rnitochbndrial intermembrane proteins, such as cytochrome c, apoptosis inducing factor (AIF) and mitochondrial ~ a s p a s e . ~ ~ In health, the balance between ROS and the antioxidant defenses lies slightly in favor of the reactive species so that they are able to fulfiU their biological roles and maintain the electrical flow of life. Repair systems take care of damage which occurs at a low level even in healthy individ~als.9~ Oxidative stress occurs when there is a change in this ratio by increasing ROS and this may occur in several circmstances, for instance in disease or malnutrition where there are insufficient redox micronutrients to meet the needs of the antioxidant defenses? Oxidative stress can be significant especially if the individual is exposed to intra or inter environmental challenges which increase the production of reactive species above "normd7'levels,for instance, infection. Oxidative stress may be an important factor in infection if redox minonutrients (e.g. vitamins C and Ef are deficientE6 I. Mitochondria and Ascorbic Acid Different groups are studying the role of AA in mitochondria and apoptosis. PerezCruz and colleagues,97proposed that the AA inhibits FAS-induced apoptosis in monocytes and U937 cells.?he FAS receptor-FAS ligand system is a key apoptotic pathway for cells of the immune system. Ligation of the FAS-receptor induces apoptosis by activation of pro-caspase-8 followed by downstream events, including and increase in ROS and the release of pro-apoptotic factors from the mitochondria, leading to caspase-3 activation. They found that AA inhibition of FAS-mediated apoptosis was associated with reduced activity of caspase-3, -8, and -10,as well as diminished levels of ROS and preservation of mitochondria1 membrane integrity. Mechanistic studies indicated that the major effect of AA was inhibition of the activation of caspase-8 with no effect on it enzymatic activity. This group suggests that AA can modulate the immune system by inhibiting FAS-induced monocyte death.96 AA has been reported to play a role in the treatment and prevention of cancer by Kang and others.97They reported that AA induces the apoptosis of B16 murine melanqma cells via a caspase-8-independent pathway AAtreated B16F10 melanoma cells showed increased intracellular ROS levels. Also, their results indicated that AA induced apoptosis in these cells by acting as a prooxidant. However, AA-induced apoptosis is not mediated by caspase-8. Taken altogether, it appears that the induction of a pro-oxidant state by A A and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway on B16F10 murine melanoma cells and that t h i s occurs in a caspase-8-independent manner.97 Although a high intake of antioxidant vitamins such as AA and vitamin E may play an important role in cancer prevention, a high level of antioxidants may have quite different effects at different stages of the transformation process. In cancer development, the resistance of cells to apoptosis is one of the most crucial steps. Recently, Wenzel and colleagues,9*tested the effects of AA on. apoptosis in HT-29 human colon carcinoma cells when induced by two potent apoptosis inducers, the classical antitumor drug camptothecin or the flamne flavonoid. AA dose-dependently inhibited the apoptotic response of cells to camptothecin and flavone. Also, AA specifically blocked the decrease of bcl-XL by these treatments. An increased generation df mitochondria1 O2precedes the down regulation of bcl-XL by camptothecin and flavone and AA at a concentration of I& prevented the generation of this ROS. In conc2usion ascorbic acid at concentrations of I d functions as an antioxidant in mitochondria of human colon cancex and thereby blocks drug-mediated apoptosis induction allowing cancer cells to become insensitive to chemotherapeutics. Studies utilizing higher concentrations are necessary to completely understand the mechanism involved. It seems that the different actions of AA in the mitochondria may be dependent on different. concentration levels., Conclusion AA shows both reducing and oxidizing activities, depending on the environment in which this vitamin is present and its concentration. Higher concentrations of AA induce apoptotic cell death in various tumor cell lines, probably via its pro-oxidant action.' On the other hand, at lower concentrations, ascorbic acid displays antioxidant properties, preventing the activation of oxidant-induced a p ~ p t o s i sHowever, .~~ the AA specific mechanistic pathways in relation with mitochondria stiU remain unclear. Acknowledgments We would like to thank the Puerto Kco Cancer Center and The Center for the Improvement of Human Functioning for their financial support in our research. References 1. Cameson E, Pauling L, Leibovitz B: Ascorbic acid and Cancer: a review. Cancer Res, 1979; 39: 663-681. 2. Sukolinskii VNI, MorozkinaTS: Prevention of postoperative complicaton in patients with stomach cancer using an antioxidant complex. Y p r Onkol, 1989; 35: 1242-1245. 3. Kurbacher CM, Wagnber U, Kolster B, et al: Ascorbic acid (vitzrnin C) improves the antineoplastic activity of doxorubidd cisplatin, and paclitaxef in human breast carcinoma cells in vitxo. Cancer Lett, 1996;203:183-189. 4. 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