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Mitochondria, Energy and Cancer:
The ReTjationshipwith Ascorbic Acid
1
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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
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Mitochondriai ~ k e r gCancer
~ , and Ascorbic Acid
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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;
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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 ~ - ~ ~ - ~ ~
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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
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A
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U
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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.
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