1. REACTIVE OXYGEN SPECIES MANAGEMENT; A STEP TO DEVELOP ANTI-CANCER THERAPY Ms. Bushra Allah Rakha Lecturer Department of Wildlife Management PMAS-AAUR

2. Reactive oxygen species (ROS) act as a second messenger in cell signaling and are essential for various biological processes in normal cells. Any aberrance in redox balance may relate to human pathogenesis including cancers. Since ROS are usually increased in cancer cells due to oncogene activation, relative lack of blood supply or other variances and ROS do involve in initiation, progression and metastasis of cancers, ROS are considered oncogenic. The mechanism of cancer initiation is shown in figure 1.

3. Endogenous source: Mitochondria Peroxisomes Cytochromes P450 Exogenous source: UV light Ionizing Radiation Inflammatory cytokines Pathogens ROS Oxidative stress Damage to Nucleic acid, proteins and lipids Chromosomal Instability Mutations Loss of organelle function Membrane damage Cancer Figure No: 1

4. ROS, mainly consisting of superoxide anion radical, singlet oxygen, hydrogen peroxide (H2O2) and the highly reactive hydroxyl radical, as a group of molecules harmful to cells, tissues and organisms. However, ROS serve as a second messenger in cell signaling and are essential for various biological processes in normal cells. Physiologically generated ROS are normally reduced by non-enzymatic and enzymatic anti-oxidizing agents, such as glutathione(GSH), thioredoxin (Trx), superoxide dismutase (SOD), catalase and peroxidases. Cellular oxidative stress, an imbalance of redox state, results from exposure to higher levels of ROS, which are not detoxified by cellular antioxidative agents.

5. Therefore, as well known, ROS, when present in a very high concentration, can damage cellular proteins, lipids and DNA, giving rise to senescent, degenerative or fatal lesions in cells, which are related to many human diseases including cancers, cardiovascular and neuro- degenerative diseases. This DNA damage and division of cells with unpaired or mispaired damage leads to mutations. Mutations involve modification of guanine causing G T tranversions and p53 gene mutations.

6. Mutations of the p53 gene are found in more than 50% of malignancies and are the single most common molecular abnormality in human cancer. Loss of p53 function is associated with loss of cell- cycle control, decreased apoptosis, and genomic instability. The p53 protein can be regulated by different post- translational modifications such as phosphorylation of serine and/or threonine residues, acetylation, ubiquitylation, or sumoylation of lysines residues.

7. ROS can function upstream of p53 and regulate p53 activity and that ROS production can also be a downstream effect of p53 activation. The redox status and consequently the function of p53 can be affected by redox molecules such as glutathione and thioredoxin/thioredoxin reductase. For example, S-glutathionylation or oxidation of p53 cysteine residues under oxidative stress was associated with a loss of p53 protein function.

8. THE ROLE OF REACTIVE OXYGEN SPECIES (ROS) IN CANCER CELL GROWTH AND METASTASIS.

9. P ARADOXICAL ROS-M ANIPULATION S TRATEGIES IN C ANCER T REATMENT ROS molecules due to their chemical composition are considered as harmful to cells, tissues and organisms. ROS serve as a second messenger in cell signaling and are essential for various biological processes in normal cells. Physiologically generated ROS are normally reduced by non-enzymatic and enzymatic anti- oxidizing agents, such as glutathione(GSH), thioredoxin (Trx), superoxide dismutase (SOD), catalase and peroxidases.

10. R EASONS FOR A NTI - CANCER THERAPY ROS contribute to cancer initiation, promotion and progression as well as maintenance of tumor cell phenotypes. Cancerous cells have increased ROS generation Increased ROS is usually accompanied with oncogene activation that is the initial steps of malignant transformation Although the causative relationship of ROS increase and oncogene activation remains unclear, oxidative DNA damage has long been thought to play a role in carcinogenesis and malignant transformation. However, oxidative DNA damage may be necessary, but not sufficient, for cancer development.

11. ROS VS GENOMIC INSTABILITY ROS act of pyrimidines, purines, chromatin proteins Cause base modification, DNA adduction, gene mutation Point mutations become carcinogenic

12. ROS VS PROLIFERATION Ligand mediated activation Growth factor receptor supression by PTP ROS inhibit PTP and growth related ligase C-Cb1 Accumulation of ROS cause degradation of MAPK phosphatase 3 that cause tumorigenicity

13. ROS VS ANGIOGENESIS ROS Cause neo-vascularization Neo-vascularization is due to presence to H2O2 in ROS, which increase expression of Vascular Endothelial Growth Factor (VEGF), receptor and MMP activity Increased vasularization leads to rapid tumor expansion

14. ROS VS METASTASIS Exogenously administration of ROS would enhance certain stages of metastatisis, while anti-oxidant treatment could attenuate metastatic progress. Even surgical procedures, a primary option for treating tumors, can lead to the increased growth of metastatic tumors by ROS generation. Possible mechanisms involve aberrant expression of integrins and MMPs and suppression of anoikis. Intriguingly, causative relationship between ROS and tumor metastasis is due to replacement with mitochondria DNA (mtDNA) derived from a highly metastatic mouse tumor cell line, an originally poorly metastatic cell line acquires the metastatic potential. The transferred mtDNA contain mutations producing a deficiency in respiratory complex I activity and are associated with overproduction of ROS.

15. ROS VS. ESCAPING FROM IMMUNO - ATTACK The intratumoral lymphocytes in many human malignant tumors are responsible for attacking tumor cells. However, they could be inhibited by ROS derived from NADPH oxidase in adjacent monocytes/macrophages (MO). In vitro data suggest that immunotherapeutic cytokines such as interleukin-2 (IL-2) or interferon-α (IFNα) only weakly activate T cells or natural killer (NK) cells in a reconstituted environment of oxidative stress.

16. ANTI-OXIDANT CANCER THERAPIES To intake dietary or supplementary antioxidants To enhance ROS scavenging enzymes To target NADPH oxidase To manipulate nitroxide compounds

18. P RO - OXIDANT CANCER THERAPY ROS are responsible for triggering cell death and reversing chemo-resistance in tumors ROS-inducing tumors with antioxidants is reasonable, ironically, the mechanism underlying that many chemotherapeutic agents and ionizing radiation exert on tumor cell kill is not associated with the increase of antioxidants, but rather the production of more ROS leading to irreversible oxidative stress.

19. ROS VS. APOPTOSIS Both death receptor- and mitochondriamediated apoptosis depend a lot on ROS. Fas ligand NADPH oxidase Triggers ROS formation Involve a siphingomylinase and Pkczeta-dependant phosphorylation pf p47 phox Required for yes/EGFR/FAS interactions of Fas- tyrosine phosphorylation Cause Fas-associated death domain, caspase8 and apoptosis

20. Mitochondria-mediated apoptosis is haracterized by an opening of permeability transition (PT) pore complex which results in cytochrome c release, apoptosome formation and culminate caspases activation. ROS are known to impact the stability of PT pore complex both through cell signaling cascade and through oxidative modification of components of PT pore complex. This occurs through the following mechanism

21. To induce the dimerization and activation of ASK1 To release MEKKI from binding with inhibitory molecules such TRX, GST To inhibit activity of protein tyrosine phosphotase to relieve the activity of SRC to initiate downstream cascade This activate the ROS, JNK would translocate to close mitochonrial membrane to activate destabilizing proteins leading to opening of PT pore complex. This complex affcets both the inner and outer mitochonrial membranes.

22. ROS VS NECROSIS Necrotic cell death invole ROS accumulations This accumulation is due to RIP, TRAF2 and FADD

23. ROS VS AUTOPHAGIC CELL DEATH Autophagy play important role in cellular response to oxidative stress. The outcome of autophagy results in removal of pathogens, damaged organelles and proteins to programmed cell death. ROS is used recently in killing cancer cells through autophagy. The effectiveness and selectivenss are indicated for a few type of cancer cells that are resistant to proapoptotic therapies, such as radiotherapy and chemotherapy

24. ROS VS CHEMO-SENSITVITY

25. P RO - OXIDANT CANCER THERAPIES 1. Generation of ROS directly in tumour cells

26. I NHIBTING THE ANTIOXIDATIVE ENZYME SYSTEM OF TUMOUR CELLS.

28. C AUTION IN DECETION OF ROS : OXIDATIVE OR REDUCTIVE To establish the role of ROS in cancer treatment involves to measure them correctly. Due to difference in methodology, some agents called as oxidants and some anti-oxidants. Delayed measurement also results in opposite conclusion about the redox role of the agent.

29. CONLUSIONS Targeting cancer cells is currently not only an idea but also start to go to patient beds. Both oxidant and anti-oxidant elevated levels are shown to be effective in cancer treatment. Selectivity between tumor and nontumor cells may depend on difference of their redox status. However, a combinational set of parameters including redox status, antioxidant enzymes expression, cell signaling and transcription factor activation profiles, namely “redox signaling signature” in a given type of cancer cells, is waiting for being developed. And then it can be used as an indicative for choosing ROS-elevating or ROS-depleting therapy specific to certain type of cancer cells. Further research also needs to explain in respect of molecular mechanism why each strategy exerts different effects on cancer and normal cells. In clinical setting individualized choice of an optimal ROS-manipulation therapy may require more accurate and convenient measurements for ROS as well as an integrative “redox signaling signature” for prediction of efficacy and systemic toxicity.

30. Thanks