Vojnosanit Pregl 2013; 70(7): 675–678. VOJNOSANITETSKI PREGLED Strana 675
Correspondence to: Slavica Stojnev, Faculty of Medicine, University of Niš, Zorana đinĀiýa 81, 18 000 Niš, Serbia. Phone: +381 64 32 333 90. E-mail: slavicastojnev@gmail.com
G E N E R A L R E V I E W UDC: 576.36:>616-006.04-092:616-006-08
DOI: 10.2298/VSP1307675S
Reactive oxygen species, apoptosis and cancer
Reaktivne vrste kiseonika, apoptoza i kancer
Slavica Stojnev, Ana Ristiü-Petroviü, Ljubinka Jankoviü-Veliþkoviü
Faculty of Medicine, University of Niš, Niš, Serbia
Key words:
reactive oxygen species; apoptosis; neoplasms; oxidative stress.
Kljuÿne reÿi:
kiseonik, reaktivne vrste; apoptoza; neoplazme; stres, oksidativni.
Introduction
Reactive oxygen species (ROS) comprise oxygen free radicals, including superoxide anion (O2-), hydroxyl (HO•), peroxy (RO2•) and alkoxy (RO•) radicals, and oxygen-derived non-radical species like hydrogen peroxide (H2O2) and singlet oxygen (1O2) 1, 2. As a consequence of aerobic metabolism, ROS are continuously generated in biological systems and simultaneously detoxified by complex antioxi-dative mechanisms 3. Oxidative stress develops due to imbal-ance between the systems that generate and scavenge free radicals 4. Persisting oxidative stress leads to the accumula-tion of oxidative damage of the crucial biomolecules: geno-mic DNA, lipids and proteins.
Reactive oxygen species in carcinogenesis
Aside from DNA-damaging function, ROS can act as second messengers and control various signaling cascades which induce and maintain the oncogenic phenotype of can-cer cells 5. The effects of ROS include anticancer activities (cell cycle arrest, senescence, apoptosis or necrosis, inhibi-tion of angiogenesis) and pro-cancer activities (promoinhibi-tion of cell proliferation, invasiveness, angiogenesis, metastases and apoptosis suppression) 6.
The incidence of malignant neoplasms increases with age. Lifelong constant attacks of free radicals are considered one of the main culprits for this. The average rate of DNA oxidative products generation is about 1 in 105 DNA bases, but is this enough to cause the spontaneous malignancy re-lated to age? It was found that free radicals primarily damage specific genomic sequences that are important for carcino-genesis 6, 7. Per example, oxidative DNA damage in gastric mucosa in patients with Helicobacter pylori infection is un-evenly distributed among the genes, with a tropism for TP53 gene 8.
Significant number of enzymatic and non-enzymatic systems protects the cell from ROS toxicity. Their impair-ment could also lead to deteriorating effects of ROS. Gluta-thione (GSH), an important guardian against the oxidative damage, accumulates abundantly within the mitochondria of cancer cells 9. In vivo studies demonstrated that GSH deple-tion sensitizes tumor cells to oxidative cytolysis 3, 9. The study on knockout mice with the deficiency of copper-zinc superoxide dismutase (SOD), the enzyme which is main tracellular scavenger of superoxide radicals, revealed the in-creased liver cancer incidence in such animals 10. Deficiency of manganese SOD, which is main O2- cleaner in mitochon-drial matrix, leads to mice mortality quickly after birth 11. The animals that are heterozygotes for manganese SOD sur-vive, but carry increased risk to develop lymphoma, adeno-carcinoma and pituitary adenoma 12.
Radiation induced carcinogenesis is largely based on generation of very reactive HO· radical, which interacts with DNA causing the formation of mutagenic purines, pyri-midines and oxidative deoxyribose products, such as 8-hydroxy-2-deoxyguanosine (8OHdG) 6, 13. The defects in en-zymes responsible for reparation of oxidative DNA damage increase the level of 8OHdG and other mutagenic bases, leading to higher incidence of age-related cancer in animals 14. The DNA itself or its precursors can be altered by ROS. In mice with MTH1 enzyme deficiency, which hydrolyses DNA precursors damaged by oxidative mechanisms, the in-corporation of these defective nucleotides in DNA is en-abled. This increases the rate of spontaneous tumorigenesis primarily in lungs, stomach and liver 14, 15.
The role of ROS in apoptosis
Strana 676 VOJNOSANITETSKI PREGLED Volumen 70, Broj 7
Stojnev S, et al. Vojnosanit Pregl 2013; 70(7): 675–678. Apoptosis can be induced by extracellular or intracellular
signals and is conducted through two major pathways: mito-chondrial (intrinsic) or death receptor pathway (extrinsic). Ex-trinsic pathway of apoptosis is triggered by interaction of death receptor and its ligand in cellular plasma membrane 16, which activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and generation of ROS. This further leads to activation of acid sphingomielinase, generation of ceramide and clustering of the receptors. The described processes form signal platform that induces apoptotic cascade 17. Activation of NF-țB survival path increases the transcription of anti-apoptotic proteins (FLIP, MnSOD, Bcl-X, IAP) and blocks the apoptosis. In the presence of high ROS concentrations, the im-possibility of NF-țB induction triggers the activation of ASK1/JNK kinases, which initiates apoptosis 18.
Intrinsic apoptotic cascade is associated with the changes in permeability of outer mitochondrial membrane, which allows the release of proapototic proteins. ROS are well known triggers of intrinsic apoptotic pathway through interaction with outer mitochondrial membrane proteins 19. Proteins of the Bcl-2 family determine the susceptibility to apoptosis, shifting the balance between pro-apoptotic (Bax, Bak, Bad, Bim and Bid) and anti-apoptotic (Bcl-2, Bcl-XL and Bcl-w) members of the family in favour of apoptosis or against it. In the presence of apoptotic stimuli like ROS, truncated form of Bid protein causes Bax/Bak oligomeriza-tion which leads to creaoligomeriza-tion of megapores in mitochondrial membrane 20. Subsequently, apoptosome complex is formed in the cytosol, activating initiator Caspase 9 and than Caspase 3, which executes the final steps of apoptosis. Caspase activation is further enhanced due to neutralization of caspase inhibitors by proteins released from mitochondria, like Smac/Diablo and Omi/HtrA2 19. In addition, mitochon-drial proteins like apoptesis-inducing factor and Endo G stimulate caspase-independent apoptosis via translocation into nucleus, where they mediate genomic DNA fragmenta-tion 21.
Programmed cell death is often characterized by gen-eration of large quantities of ROS or transient oxidative burst 22. Catalase prevents H2O2 mediated apoptosis, indi-cating the important role of H2O2 in apoptosis 2. Recently it has been found that H2O2 initiates caspase activation which is dependant on mitochondrial cytochrome c release 23. How-ever, very high doses of H2O2 or other ROS induce cell ne-crosis 13, 23. This implies that the modus of cell death induced by oxidative stress is dose dependant. Since apoptosis repre-sents active, energy dependant process, it seems that the in-tracellular ATP level is crucial determinant weather the apoptotic cascade would eventuate.
The interplay of ROS and apoptosis in cancer
Although apoptosis and cancer represent opposite enti-ties, ROS play an important role in both of the processes. Moderate levels of ROS, where “moderate” depends on the cell type, promote apoptosis in tumor cells 24. Nevertheless, in some malignant cells ROS can exert the opposite effect. In melanoma cell line M14, overexpression of zinc-copper SOD
causes the decrease in O2- concentration and thus promotes apoptosis 25. Generation of ROS by NADPH oxidase enzy-matic system acts as an anti-apoptotic, pro-proliferative stimulus in pancreatic cancer cell lines 26. The ability of mi-tochondrial apoptosis-inducing factor to oxidase NADH and generate superoxide anion contributes to viability of some cancer cell lines 27.
The mechanisms by which ROS exert their anti-apoptotic effect have not been completely elucidated so far. It has been hypothesized that caspase inactivation could be responsible. In some cells elevated O2- causes the increase in cytosolic pH value, which halts the caspase activation 28. In opposite, H2O2 lowers pH in cytosol and thus acts in favor of apoptosis initiation. In addition, H2O2 promotes apoptosis through conversion in HO· radical that directly attacks DNA or damages mitochondria 28. An alternative mechanism in-cludes inactivation of PTEN protein (product of tumor sup-pressor gene PTEN), which increases the activation of Akt signaling pathway, thus promoting cell survival. However, Akt signaling is often attributed ambiguous roles, since some studies have indicated that the activation of this path sup-ports cancer cell survival, but suppresses invasiveness and metastases 29.
The phenomenon of apoptosis has been investigated in many types of cancer and its significance has been well-established for urothelial carcinoma 30–32. Recent findings have suggested that oxidative stress sensitizes tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by downregulation of anti-apoptotic proteins 33. Moreover, TRAIL protein has gained a lot of attention as a new therapeutic target because of its property to induce apoptosis only in cancer cells, but not in normal cells 34. However, some urothelial carcinomas de-velop resistance to TRAIL, therefore the reestablishment of this sensitivity is of great importance. Induction of oxidative stress with low concentration of H2O2 can reverse the resis-tance to TRAIL by significantly decreasing the expression of short living anti-apoptotic proteins such as FLIP, XIAP and Survivin 33. Further studies are to determine the relevancy of H2O2 application as adjuvant intravesical agent in urothelial cancer treatment, in order to lower the threshold for initiating apoptosis in malignant cells.
Apoptosis induced by ROS in anticancer treatment
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ROS generating anticancer drugs lead to depletion of intracellular antioxidative capacity and when the ROS concentration reaches a certain threshold, apoptosis is initiated 13. In the absence of adequate antioxidative de-fense, damage caused by ROS induces activation of apopto-sis-related genes. In addition, intracellular ROS increase can also activate redox sensitive JNK/SAPK signaling pathway, often implicated in genes transactivation and posttransla-tional modifications of proteins required for apoptosis 23.
Future implications
Pharmacological manipulations are able to shift the in-tracellular redox equilibrium toward the increase in ROS and/or the depletion of protective agents, inducing the apop-totic cascade in cancer cell. The studies investigating appli-cation of antioxidant inhibitors and/or ROS generating agents in order to induce apoptosis or to overcome the resis-tance to chemotherapy 39, 40 have provided significant ad-vances, but further validation of the results is necessary.
Genomics and proteomics represent powerful and promising tools in unraveling complex molecular net-works 41, 42. Investigators have attempted to understand the subset of proteins whose expression levels are directly al-tered by oxidants, or those proteins that are
posttranslation-ally modified in a redox-dependent fashion 43. Recent pro-teomics based study identified gold (III) porphyrin 1a as a new, potent anticancer drug that induces apoptosis through both caspase-dependent and caspase-independent mito-chondrial pathways, and demonstrated that intracellular oxidation affected gold (III) porphyrin 1a-induced apopto-sis 44. The difficulty with advanced “omics” methodologies is how to translate the provided data into a meaningful clinical context, ie into clinical trial design and subse-quently into routine clinical use 41.
One of the most important goals in cancer studies is di-rected to the possibility of treating cancer by inducing apop-tosis via caspase activation. ROS could play a major role in this process, acting as potent mediators. The greatest chal-lenge remains the issue of inducing apoptosis selectively within the neoplastic cells, with maximal spearing of normal tissue. The therapeutic strategy based on triggering apoptosis by modulation of ROS levels, selectively within the neoplas-tic cells, carries great expectations and holds promise of a significant advance in clinical oncology.
Acknowledgements
This work was supported by the Ministry of Education Science and Technological Development of the Republic of Serbia (Grant No. 175092).
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