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High-dose Vitamin C (Ascorbic Acid) Therapy in the Treatment

of Patients with Advanced Cancer (Y)

Laboratory data show that ascorbate is toxic to a variety of cancer cell lines (25-27). Extracellular

concentrations as low 100-200 M are toxic to some cell lines, but many types of malignant cells are

killed only at concentrations approaching the mM range (19)

As established by seminal studies by Chen et al.(28, 29), in concentrations higher than 1 mM, ascorbate

can cause a build-up of hydrogen peroxide (H2O2 ), which is preferentially toxic toward tumor cells.

The

killing of cancer cells is dependent on extracellular H2O2

formation with the ascorbate radical as an intermediate.

Ascorbate generates detectable levels of H2O2

in the

extracellular medium only in the presence of 0.5-10%

serum. Moreover, H2O2

generation is dependent on time and

ascorbate concentration. Human whole blood inhibits H2O2

and ascorbate radical generation from ascorbate The mechanism of cytotoxicity to cancer cells remains

unsolved. Possibilities include stimulatory effects on

apoptotic pathways, accelerated pro-oxidant damage that

cannot be repaired by tumor cells and increased oxidation of

ascorbate at high concentrations in the plasma to the unstable

metabolite dehydroascorbic acid, which in turn can be toxic.

It remains possible that toxicity is an artifact of cell culture

(30), perhaps due to contamination of media by iron (31) or


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other cations resulting in excessive oxidation

Resent researches suggest that H2O2

plays a vital role as a

cytotoxic mediator in high-dose vitamin C therapy. Chen et

al.(28, 29)

Vitamin C has been reported to have perhaps the lowest

toxicity of all vitamins (48)

Apoptotic effects of hydrogen peroxide and vitamin C

on chicken embryonic fibroblasts: redox state

and programmed cell death (x)

Askorbik asit olarakta blnen genel olarak hcre metabolizmasinda oluan oksjen radikalleriniindirgeyici roluyle taninmaktadir. Bunun yannda mtokondrial membrane potansiyelini stabilize

edilmekte ve kaspaz 3 ve kaspaz 9 aktivasyonunu azaltmaktadir( X Mandl et al.2009). Gnlk

2000 mg dozlarda ble organzmada cdd yan etkler olusturmamasna ragmen son

calsmalarda degsk kanser hucre dzler uzernde toksik etkileri olduu gsterilmitir( Y 25-27) .

Bazi hcre dizilerinde 100-200 Mkadar dk ekstraseller konstrasyonlarda toksik etki grlrken,

baz hcre dizilerinde ancak mM duzeylernde toksik etki gzlenmektedir (Y 19). Chen ve arkadaslar

1mM`un uzerindeki ekstraseluler askorbikasit konsantrasyonlarinin H2O2 uretimine neden oldugunu

bununda tumor hucreleri uzerinde toksik etki gsterdiini belirlediler.Bununla birlikte H2O2 uretiminin

zamana ve askorbik asit konsantrasyonuna bagimli oldugu gosterilmistir. Yneden C vtamnn bu

sitotoksik etkisinin mekanizmas tam olarak anlaslamamstr. Hucrelerin uzun sure H2O2` maruz

kalmalarinin PTEN sitein rezudulerinde oksidasyona yolactigi bu oraninda % 5-16 arasinda degisebilecegi

gosterilmistir (Z 6). PTEN oksidasyonu insulin ile situmule edilen roblastoma hucreleri, epidermal

growth faktor ile situmule edilen HeLa hucrelerinde ve Epidemal growth faktor ile situmule edilen

fibroblast hucrelerinde PTEN`nin oksidasyonu gosterilmistir (Z 9,10). Ayrica okside PTEN bi antioksidan

olan glutathione yoluyla deokside edilebilmistir (Z 5). PTEN PI3/Akt yolagnn negatf regulatoru olarak

Aslev gormektedr. Down regulasyonu durumunda aktive olmus Akt pro-apoptorik Bcl-2 ailei uyelerinin


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(Bad, Bax, caspase-9, GSK-3 and FoxO1) aktive olmasina neden olmaktadir. Over regulasyonu

durumlarininda apotozisi tetikledigi bilinmektedir. PDGF gibi ligand ve EGFR gibi reseptorler araciligiyla

tetiklenen H2O2 artisina bagli oksidasyon artislarina yuksek doz C vitamini alumina bagli H2O2

artislarinin PTEN oksidayonunu artiracagi ve bunununda aktivasyon artisina bagli apoptosisi

tetikleyecegini duzunmekteyiz. Bu amala kanser hucre dzlernde yuksek doz C vtamnn apoptotk ve

antapoptotk etklern ve bunun PTEN, PDGF, EGFR ekspresyon duzeylerine etkisini arastirmayi

amaladk.

METHOD: ATCC den temin edilen hucre dizilerine IC50 belirlemek amaciyla artan dozlarda askorbik asit

uygulandi ve MTT assay araciligyla doz ve sure belirlendi. Apotozis MTT ve Tunnel assay yoluyla

belirlendi. PTEN, PDGF ve EGFR gen ekspresyonlari Real Time PCR (Roche light cycle) araciligiyla

belirlendi.

PTEN is one of the most mutated and deleted tumor

suppressors in human cancer but, importantly, it is also found

partially downregulated in cancer in the absence of genetic

loss or mutation (10)

PTEN negatively regulates the PI3K/Akt pathway. Activated Akt inhibits the pro-apoptotic Bcl-2 family

member Bad, Bax, caspase-9, GSK-3 and FoxO1 by phosphorylation.

It was shown that exposure of cells to H2O2resulted in the oxidation of PTEN through the

formation of a disulfide bond between cysteine 124 (Cys 124 ) residue at the active site and a

nearby Cys 71 residue, suggesting that this might function to regulate PTEN activity[5].

Furthermore, stimulation of macrophages with lipopolysaccharides and phorbol ester caused

the fraction of oxidized PTEN to increase from 5% to 16%[6]. More recently, the oxidation of

PTEN has been observed in neuroblastoma cells or HEK293 cells stimulated with insulin, HeLa

cells stimulated with epidermal growth factor (EGF), and fibroblasts stimulated with


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PDGF[7,8]. The oxidation of PTEN by nitrosothiols or oxidized GSH led to modifications of the

PTEN thiol such asS-nitrosylation orS-glutathionylation[9,10].

It was previously shown biochemically that oxidized PTEN was

more effectively converted back to the reduced form by thioredoxin than by glutathione[5]

Vitamin C, whose chemical name is ascorbic acid,

is generally considered as a potent reductant. Vitamin

C in cells undergoing hypoxia-reperfusion are linked

with a reduction of ROS level, prevention of cytochrome c release and a stabilized mitochondrial

membrane potential and a decreased activation of

caspase-3 and caspase-9 (Mandl et al.2009). While

high intake of Vitamin C (2,000 mg/d) has not been

consistently reported to cause any side effects, its

benefits to normal people have never been established, and what have been discovered is only that

Vitamin C exerts some inhibitory effects on gastric

metaplasia, chronic gastritis and lung and colorectal

cancer in vulnerable population (Valko et al.2

Reversible Inactivation of the Tumor Suppressor PTEN by

H2O2

The generation of H2O2 also appears to be required, however, for many normal cellular

functions, including propagation of receptor signaling (1, 2). Ligands that induce an increase in

the intracellular concentration of H2O2 include peptide growth factors such as platelet-derived

growth factor (PDGF) 1 and epidermal growth factor, cytokines such as transforming growth

factor1 and tumor necrosis factor , and agonists of heterotrimeric

GTP-binding protein (G protein)-coupled receptors such asN-formyl-methionyl-leucyl-


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phenylalanine (fMLP) and angiotensin II (1, 2). The essential role of H2O2 production in

intracellular signaling triggered by PDGF (3, 4), epidermal growth factor (5), angiotensin II (6),

and cell-cell contact (7) has been demonstrated by theobservation that corresponding receptor-

mediated events are abrogated by blocking the accumulation of H2O2with enzymes such as

catalase or small molecules such asN-acetylcysteine.

Redox regulation of the tumor suppressor PTEN by

glutathione (Z)

It was shown that exposure of cells to H2O2resulted in the oxidation of PTEN through the

formation of a disulfide bond between cysteine 124 (Cys 124 ) residue at the active site and a

nearby Cys 71 residue, suggesting that this might function to regulate PTEN activity[5].

Furthermore, stimulation of macrophages with lipopolysaccharides and phorbol ester caused

the fraction of oxidized PTEN to increase from 5% to 16%[6]. More recently, the oxidation of

PTEN has been observed in neuroblastoma cells or HEK293 cells stimulated with insulin, HeLa

cells stimulated with epidermal growth factor (EGF), and fibroblasts stimulated with

PDGF[7,8]. The oxidation of PTEN by nitrosothiols or oxidized GSH led to modifications of the

PTEN thiol such asS-nitrosylation orS-glutathionylation[9,10].

PTEN Level in Tumor Suppression: How Much Is Too Little?

PTEN is one of the most mutated and deleted tumor

suppressors in human cancer but, importantly, it is also found

partially downregulated in cancer in the absence of genetic


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loss or mutation (10)

PTEN negatively regulates the PI3K/Akt pathway. Activated Akt inhibits the pro-apoptotic Bcl-2

family member Bad, Bax, caspase-9, GSK-3 and FoxO1 by phosphorylation. Many growth factors

and cytokines induce anti-apoptotic Bcl-2 family members. The Jaks and Src phosphorylate and

activate Stat3, which in turn induces the expression of Bcl-xL and Bcl-2. Erk1/2 and PKC activate

p90RSK, which activates CREB and induces the expression of Bcl-xL and Bcl-2. These Bcl-2 family

members protect the integrity of mitochondria, preventing cytochrome c release and the

subsequent activation of caspase-9. TNF- may activate both pro-apoptotic and anti-apoptotic

pathways; TNF- can induce apoptosis by activating caspase-8 and -10, but can also inhibit

apoptosis signaling via NF-B, which induces the expression of anti-apoptotic genes such as Bcl-

2. cIAP1/2 inhibit TNF- signaling by binding to TRAF2. FLIP inhibits the activation of caspase-8.

Selected Reviews:

Arya R, Mallik M, Lakhotia SC (2007) Heat shock genes - integrating cell survival and death. J.

Biosci. 32(3), 595610.

Brumatti G, Salmanidis M, Ekert PG (2010) Crossing paths: interactions between the cell death

machinery and growth factor survival signals. Cell. Mol. Life Sci. 67(10), 161930.

Fan Y, Dutta J, Gupta N, Fan G, Glinas C (2008) Regulation of programmed cell death by NF-

kappaB and its role in tumorigenesis and therapy. Adv. Exp. Med. Biol. 615, 22350.

Rong Y, Distelhorst CW (2008) Bcl-2 protein family members: versatile regulators of calcium

signaling in cell survival and apoptosis. Annu. Rev. Physiol. 70, 7391.

Srinivasula SM, Ashwell JD (2008) IAPs: what's in a name? Mol. Cell 30(2), 12335.

Zakeri Z, Lockshin RA (2008) Cell death: history and future. Adv. Exp. Med. Biol. 615, 111.


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