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

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 broblasts: redox state and programmed cell death (x)
Askorbik asit olarakta blnen genel olarak hcre metabolizmasinda oluan oksjen radikallerini indirgeyici roluyle taninmaktadir. Bunun yannda mtokondrial membrane potansiyelini s tabilize 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 M kadar 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

(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 disulde 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 broblasts stimulated with

PDGF[7,8]. The oxidation of PTEN by nitrosothiols or oxidized GSH led to modications 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 benets 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-

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 receptormediated 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 disulde 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 broblasts stimulated with PDGF[7,8]. The oxidation of PTEN by nitrosothiols or oxidized GSH led to modications 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

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 Bcl2. 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 NFkappaB 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, 73 91. 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, 1 11.