INTRODUCTION:

Ovarian cancer (OC) is leading cause of high mortality among gynecologic malignancies in worldwide which is believed to start from a surface epithelial cell disturbed by ovulation (1,2). It contributes several factors to OC progress like hormonal, environmental, and viral-correlated stimuluses, these cases are initiated by oxidative stress (OS) (3,4). OS-mediated DNA damage which play a key role in initiation and progression of OC (5).

Oxidative DNA damage produced lesion base 8-OHG that has been identified to modify the enzyme-catalyzed methylation of close cytosine, altered methylation forms in DNA is an important for the regulating gene expression (6,7). Methylation of DNA provides an alternate pathway to gene mutation or deletion for losing tumor suppressor gene (TSG) role (8) that lead to prompts different mechanisms such as uncontrolled cell division, infiltrate adjacent tissues ability, metastasis and suppress the apoptosis (9,10). Cancer cells, involving OC augmented resistance to apoptosis, which can be as a reason of upregulation of antiapoptotic proteins (11), or downregulation of pro-apoptotic cytokine proteins (12). Cas-8 is an important initiator of cell death; this complicates deregulation of this gene in human malignancies. (13). Methylation of Cas-8 gene found in various human cancers (14). IL-8 is a pro-inflammatory chemokine that acts as activator of neutrophils. It has effects on cell growth, and angiogenic in different malignancies (15,16).

The aim of this study is to investigate the pro-apoptotic Cas-8 levels in patients with OCs, and its relationship with DNA methylation in Cas-8 gene and inflammation, as predictive biomarkers for OC.

MATERIAL AND METHODS:

A total of 90 subjects were involved in this study, healthy controls (n=30) (G1), and 30 premenopausal women with primary diagnosis of ovarian cancer (G2), (30) postmenopausal women with primary diagnosis of ovarian cancer (G3). The mean age of control group (45.12±11.9 years), patient groups age for G2 (30.33±14.7) years, G3 (54.33±8.7) years. Patient had not been received chemotherapy, were selected from January to Jun 2019. This study including blood samples and experiment protocols, was approved by ethical Committee of Al-Diwaniyah teaching hospital and college of Science, university of Al-Qadisiyah. In addition, informed consent was obtained from all study participants prior to sample collection.

5ml blood were collected in in Na-EDTA tubes, then plasma was separated by centrifuged (4000rpm, 0.894Xg) (Gottingen, Germany) at room temperature for 10 minutes. samples were divided in Eppendorf tubes and kept frozen at (-20°C) till used for the study assays.

Plasma SOD activity was determined spectrophotometry by (Misra and Fridovich.1972) method (17). Plasma levels of 8-OHG, IL-8 and Cas-8 were measured by ELISA kits (Elabscience, China).

The (cf DNA) were isolated from 2mL plasma using cfDNA Synthesis Kit (Quick-cfDNA TM Serum and plasma DNA Miniprep Kit, USA). The DNA was extracted and treated with bisulfite by using the EZ Methylation TM Kit (USA). Methylation specific primer was performed using 5μL of modified cf DNA, methylated of DNA by using gene-specific primers methylation Cas-8 MF(5'-TAGGGGATTTGGAGATTGTGA-3') (MR) (5'-CGTATATCTACATTCGAAACGA-(3') (Tm (60) Cº and unmethylated (59) Cº,(UMF)( 5-'TAGGGGATTTGGAGATTGTGA-3'),(UMR)(5'- CCATATATCTACATTCAAAACAA-3'), methylation (Cas-8) Tm (60)Cº with the cycling parameters: followed by 40 cycles the PCR products were examined by electrophoresis , PCR conditions were as follows: 95°C for 4 min and then 40 cycles of 95°C for 40 s, 60°C for20 s, and 72°C for 30 s, with a final extension for 5 min at 72°C.

Statistical analysis:

Statistical data were analyzed using SPSS software 24. Data were expressed as mean± SEM. One-way ANOVA followed by the Tukey post hoc analysis or a non-parametric ranking (Kruskal-Wallis) were carried out as suitable to compare numerous groups for normal and non-normal distribution data respectively. A p value ˂ 0.05 was significant throughout.

RESULTS:

As shown in Fig.1 SOD activity were significantly decrease in G2 and G3 compared to G1, (P < 0.05). There were significant differences in 8-OHG concentration between three groups (P < 0.05), 8-OHG levels were significantly increase in G2 and G3 compared to G1 (Fig. 2), and there is significantly difference between G2 and G3 (Fig.2). As shown in Fig.3 IL-8 levels significantly increased G2 and G3 groups compared to control, (P < 0.05).

Plasma levels of Cas-8 were significantly decrease in the two patient groups G2 and G3 compared to G1, but there is no significantly difference between patient groups (P < 0.05).

Fig.1: Plasma SOD activity in the patients with primary diagnosis of ovarian cancer: control groups (G1), premenopausal women (G2), and postmenopausal women (G3). Data are expressed as means ± SEM, *indicates significant differences compared to the control, # significant differences between patient groups (P < 0.05).

Fig.2. Plasma 8-OHG levels in the patients with primary diagnosis of ovarian cancer: control groups (G1), premenopausal women (G2), and postmenopausal women (G3). Data are expressed as means ± SEM, *indicates significant differences compared to the control, # significant differences between patient groups (P < 0.05).

Fig.3. Plasma IL-8 levels in the patients with primary diagnosis of ovarian cancer: control groups (G1), premenopausal women (G2), and postmenopausal women (G3). Data are expressed as means ± SEM, *indicates significant differences compared to the control, # significant differences between patient groups (P < 0.05).

Fig.4. Plasma Cas-8 levels in the patients with primary diagnosis of ovarian cancer control groups (G1), premenopausal women (G2), and postmenopausal women (G3). Data are expressed as means ± SEM, *indicates significant differences compared to the control, # significant differences between patient groups (P < 0.05).

In the study found different methylation pattern of the Cas-8 gene in plasma samples between patients with ovarian cancer and control, ranging in all cases 20 (33.3%) and un-methylation in 40(66.6% than control group, methylation (0 0%)) and un-methylation 30 (100%). In this study association between the hyper methylation was no significantly with age (≥45y(10 (33.3%) and <45y (12. (40%)) and un methylation age (≥45y (20 (66.6%) and < 45y (18. (60%)) than normal cases also the association between the hypermethylation was shown in Menopause (Premenopause (13(43.3%), Postmenopause7 (23.3%)) and un methylation for Premenopause (17(56.6%), Postmenopause 23(76.6%)) than control group, methylation (0%), unmethylation30 (100%) as shown in Table 1. Fig.5 (A, B, C).

Table (1): Correlation analysis of Cas-8 gene methylation and un methylation ovarian cancer plasma

Casp-8 UM%	Casp-8 M%	No	Clinical information
40(66.6%)	20(33.3%)	60	Patients
20(66.6%) 18(60%)	10(33.3%) 12(40%)	30 30	age>45 ˂45
17(56.65) 23(76.6%)	13(43.3%) 7(23.3%)	30 30	Menopause Premenopause Postmenpause
30(100%)	0(0%)	30	Control
Casp-8 UM%	Casp-8 M%	No	Clinical information
40(66.6%)	20(33.3%)	60	Patients
20(66.6%) 18(60%)	10(33.3%) 12(40%)	30 30	age>45 ˂45
17(56.65) 23(76.6%)	13(43.3%) 7(23.3%)	30 30	Menopause Premenopause Postmenpause
30(100%)	0(0%)	30	Control

Fig.5: The product Photograph of PCR products was showing methylation and Un methylation for. Cas-8 gene in A. Healthy control group (G1), B. premenopausal women (G2) and postmenopausal women (G3). The product was electrophoresis on 2% agarose gel at 50 volts and ,0.5 TBE buffer about 45 min M: The DNA ladder (25 -1000), under U.V light after staining with Ethidium Bromide (U: un methylation, M: methylation)

DISCUSSION:

The mechanisms that are involved in pathogenesis of OC is not yet clear and there is defect of exact and early diagnosis. Therefore, mortality rate of ovarian cancer was increased among women. A decreased levels of plasma SOD activity and an increased levels of circulating 8-OHG were observed in ovarian cancer patient groups as a marker of augmented oxidative stress that lead to depletion of antioxidants enzyme activities, which is linked with the initiation and development of cancer (3). Increase of superoxide anion is triggering harmful effects at sites far from the tumor, because it has the ability of crossing membranes (18,19). studies showed the loss of SOD activity, due to increase consumption and sequestration the antioxidant by tumor cells (19).

ROS can induce, promote and modulate carcinogenesis (20,21,22), 8-oxoguanine DNA glycosylase 1, is the key repair enzyme linked with 8-OHdG, which is cleaves the damaged guanosine from DNA and afterward it is secreted to extracellular fluids (23,24). Numerous studies were considered high level of plasma or serum 8-OHdG as a predictor of OC patients (25,26), and found the tissue expression of 8-OHdG in OC is increased significantly than in benign state, supposing a role of 8-OHdG in ovarian epithelial carcinogenesis (27,28).

A pro-inflammatory chemokines IL-8 levels were increased significantly in OC groups compared to control groups. IL-8 performed key roles in the progress of OC that causing declined survival (29,30), several reports was shown that IL-8 levels are raised in serum, plasma, and tumor tissue in patients with OC (31,32). IL-8 is stimulating the angiogenesis, and cancer metastasis via binding to CXCR1 and CXCR2 receptors (33), IL-8 expression is primarily mediated by NF-κB pathway activation (34), suggesting that IL-8 may act as key regulatory factor within the tumor microenvironment, therefore IL-8 could play a central role in this process.

Low levels of Cas-8 in patients’ groups especially in post-menopausal group reflects a decreased in apoptosis. When the death-inducing signaling complex is formed, Cas-8 is activated and cleaved diverse apoptotic proteins, result in execution of apoptosis (35,36). Cas-8 is complicated in extrinsic and intrinsic pathways of apoptosis. cas-8 is inactivated in numerous human cancers, because of deficiency of apoptosis, which may lead to tumor development and resistance to chemotherapeutic agents (37). Several studies were shown that Cas-8 plays a role in the suppression of oncogenic transformation, irrespective of its function in apoptosis. The deficiency of Cas-8 can promote further oncogenic mutations (38,39).

This study was used the methylation specific primer (MSP) method for determination of Cas-8 gene levels in plasma of OC patients, the results showed different methylation and variation significantly pattern of the Cas-8 gene in plasma samples between the patients with OC and control as well as between methylation and un methylation, the results were shown that in some patients the methylation were appeared, while the bands were not appeared in others, which suggest that patients are not necessary suffered from the mutation in cas-8 gene.

Low levels of Cas-8 in OC samples may explain the possibility of preventing intrinsic and extrinsic apoptosis signaling pathways, consequently decline or resistance to apoptosis in epithelial ovarian cells, an important step in ovarian carcinogenesis. The abnormal methylation as a suppressor mechanism of gene transcription was frequently reported in promoter regions of tumor suppressors in OC (40). The silencing of Cas-8 expression through DNA methylation has been defined in OC, and has been found promising molecular markers for use in early cancer diagnosis of disease development and treatment (41).

A negative correlation between the level of IL-8 and Cas-8 in both patients’ groups were found (Fig.5), These result were in agreement with other studies which showed that IL-8 inhibits TRAIL-induced apoptosis which is a part of the TNF family and has been displayed to stimulate apoptosis (42,43,44), especially in tumor cells that decrease caspase splitting in EOC cell lines through declining the death receptor 4 expression (45).

CONCLUSION:

This study described that several factors may lead to enhance tumorigenesis. Increased oxidative stress is associated with defects in DNA repair mechanisms by an increase in 8-OHdG levels and consequently decrease antioxidant enzyme defense. Oxidative stress enhanced the inflammatory chemokines that probable can effect on Cas-8 regulation, besides to promoter hyper methylation of Cas-8 that occur through interaction these mechanisms. This suggest that apoptosis is related with an inflammatory response and oxidative stress. Increased resistance to apoptosis has been known as one of the major mechanisms leading to OC progression. The results identify a role of Cas-8 in OC

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of A.N. Zainb. College of science, University of Al-Qadisiyah for the facilities.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

REFERENCES:

1. Momenimovahed Z., Tiznobiak A., Taheri S., and Salehiniya, H. Ovarian cancer in the world: epidemiology and risk factors. International Journal of Women's World Health.2019; 11: 287-299.

2. A Case Study on Ovarian Cancer – Palliative Perspective. Asian J. Nur. Edu. and Research 5(3): July- Sept.2015; Page 446-448.

3. Gloria M. C., Urzua U. Lara Termini L., and Aguayo F. Oxidative stress in female cancers. Oncotarget, 2018; 9(34): 23824-23842.

4. Dibyajyoti Saha, Ankit Tamrakar. Xenobiotics, Oxidative Stress, Free Radicals Vs. Antioxidants: Dance of Death to Heaven’s Life. Asian J. Res. Pharm. Sci. 1(2): April-June 2011; Page 36-38.

5. William J. M., and James F. M. Oxidative Damage to DNA of Ovarian Surface Epithelial Cells Affected by Ovulation: Carcinogenic Implication and Chemoprevention. Experimental Biology and Medicine, 2004; 229: 546-552

6. Helen Wiseman and Barry Halliwell. Damage to DNA by Reactive Oxygen and Nitrogen Species: Role in Inflammatory Disease and Progression to Cancer. Biochem. J. 1996; 313: 17-29.

7. Chandrasekar. R, Sivagami. B, M. Niranjan Babu. A Pharmacoeconomic Focus on Medicinal Plants with Anticancer Activity. Res. J. Pharmacognosy and Phytochem. 2018; 10(1): 91-100.

8. Garcia-Manero G, Daniel J, Smith TL, et al. DNA methylation of multiple promoter-associated CpG islands in adult acute lymphocytic leukemia. Clin Cancer Res. 2002; 8: 2217-2224.

9. Lam Kah Yuen, Puteri Jamilatul Noor Megat Baharuddin, Zubaidah Zakaria: The Pattern of Aberrant DNA Methylation in Adult Acute Lymphoblastic Leukemia. International Journal of Health Sciences & Research. 2015; 5 (5): 134-143.

10. Milena K, Maja PJ, Biljana M, et al. Hyper methylation of p15 Gene in Diffuse – Large B-Cell Lymphoma: Association with Less Aggressiveness of the Disease. Clinical and Translational Science. 2014; 7(5): 384-390.

11. Schimmer, A. D. Inhibitor of apoptosis proteins: translating basic knowledge into clinical practice. Cancer Res 2004; 64:7183-90.

12. Banelli, B., Casciano, I., Croce, M., et al. Expression and methylation of CASP8 in neuroblastoma: identification of a promoter region. Nat Med. 2002; 8: 1333-5.

13. Gudmundsdottir K., Ashworth A. The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability. Oncogene.2006; 25: 5864-5874

14. Nikbakht M., Shabanizadeh A., Salehi M., Talebi A. BRCA1 Promoter Methylation Status in Ovarian Cancer. Laboratory Medicine. 2012; 43(4): 18–21.

15. Araki S., Omori Y., Lyn D., Singh R.K., Meinbach D.M., Sandman Y., Lokeshwar V.B., Lokeshwar B.L. Interleukin-8 is a molecular determinant of androgen independence and progression in prostate cancer. Cancer Research. 2007; 67: 6854–6862.

16. Gabellini C, Trisciuoglio D, Desideri M, Candiloro A, Ragazzoni Y, Orlandi A, Zupi G, Del Bufalo D. Functional activity of CXCL8 receptors, CXCR1 and CXCR2, on human malignant melanoma progression. European journal of cancer. 2009; 45: 2618–2627.

17. Misra, H.P., and Fridovich, I. The role of Superoxide anion in theauto-oxidation of epinephrine and a simple assay for Superoxide Dismutase. J. Biol. Chem. 1972; 247: 3170-3175.

18. Oberley, L.W. and Buettner G.R. Role of superoxide dismutase in cancer. Cancer Res., 39: 1141-1149.

19. V Jaydeokar, DD Bandawane, SS Nipate, PD. Chaudhari. Natural Antioxidants: A Review on Therapeutic Applications. Research J. Pharmacology and Pharmacodynamics. 2012; 4(1): 55-61.

20. Manimaran A. and Rajneesh C. P. Activities of Antioxidant Enzyme and Lipid Peroxidation in Ovarian Cancer Patients. Academic J. Cancer Res. 2009; 2(2): 68-72.

21. Karnakumar V Biradar, Chandrashekhar B Patil, Basavaraj V Chivde, MH Malipatil, Sanjivkumar D Biradar. Effect of Superoxide Dismutase Mimetic Tempol on Dexamethasone Induced Insulin Resistance-Role of Oxidative Stress. Research J. Pharmacology and Pharmacodynamics. 2011; 3(3): 134-137.

22. Vadivelan R, Dhanabal SP, Raja Rajeswari, Shanish A, Elango K, Suresh B. Oxidative Stress in Diabetes- A Key Therapeutic Agent. Research J. Pharmacology and Pharmacodynamics. 2010; 2(3): 221-227.

23. Hirano T. Repair system of 7,8-dihydro-8-oxoguanine as a defense line against carcinogenesis. J Radiat Res. 2008;49: 329–40.

24. Karnakumar V Biradar, Chandrashekhar B Patil, Basavaraj V Chivde, MH Malipatil, Sanjivkumar D Biradar. Effect of Superoxide Dismutase Mimetic Tempol on Dexamethasone Induced Insulin Resistance-Role of Oxidative Stress. Research J. Pharmacology and Pharmacodynamics. 2011; 3(3): 134-137.

25. Pylväs M., Puistola U., Laatio L., Kauppila S., Karihtala P. Elevated serum 8-OHdG is associated with poor prognosis in epithelial ovarian cancer. Anticancer Res. 2011; 21: 1411–5.

26. Xu X, Wang Y, Guo W, Zhou Y, Lv C, Chen X, et al. The significance of the alteration of 8-OHdG in serous ovarian carcinoma. J Ovarian Res. 2013; 6-74: 1757-2215.

27. Pylväs M, Puistola U, Kauppila S, Karihtala P. Oxidative stress-induced antioxidant enzyme expression is an early phenomenon in ovarian carcinogenesis. Eur J Cancer. 2010; 46: 1661–7.

28. Nirjala Laxmi Madhikarmi, Kora Rudraiah Siddalinga Murthy. Study of oxidative stress and antioxidants status in iron deficient anemic patients. Research J. Science and Tech. 2012; 4(4): 162-167.

29. Masoumi-Moghaddam S., Amini A., Wei, A.Q., Robertson, G., Morris, D.L. Intratumoral interleukin-6 predicts ascites formation in patients with epithelial ovarian cancer: A potential tool for close monitoring. J. Ovarian Res. 2015; 8: 58.

30. Dalal, V.; Kumar, R.; Kumar, S.; Sharma, A.; Kumar, L.; Sharma, J.B.; Roy, K.K.; Singh, N.; Vanamail, P. Biomarker potential of IL-6 and VEGF-A in ascitic fluid of epithelial ovarian cancer patients. Clin. Chim. Acta. 2018; 482: 27–32.

31. Darai E., Detchev R., Hugol D., Quang NT. Serum and cyst fluid levels of interleukin (IL) -6, IL-8 and tumour necrosis factor-alpha in women with endometriomas and benign and malignant cystic ovarian tumours. Hum Reprod. 2003.

32. Kassim S.K., El-Salahy E.M., Fayed S.T., Helal S.A.; Helal T.; Azzam Eel, D., Khalifa, A. Vascular endothelial growth factor and interleukin-8 are associated with poor prognosis in epithelial ovarian cancer patients. Clin. Biochem. 2004; 37: 363–369.

33. Lokshin A.E., Winans M., Landsittel D., Marrangoni A.M., Velikokhatnaya L., Modugno F. Circulating IL-8 and anti-IL-8 autoantibody in patients with ovarian cancer. Gynecologic Oncology. 2006; 102: 244–251.

34. Merritt W.M., Lin Y.G., Spannuth W.A., Fletcher M.S., Kamat A.A., Han L.Y. Effect of interleukin-8 gene silencing with liposome-encapsulated small interfering RNA on ovarian cancer cell growth. Journal of the National Cancer Institute. 2008;100: 359–372.

35. Duiker E.W., Van der Zee A.G, De Graeff P., et al. The extrinsic apoptosis pathway and its prognostic impact in ovarian cancer. Gynecol Oncol. 2010; 25(116): 549-55.

36. Merlin NJ Parthasarathy V, Manavalan R, Devi P, Meera R. Apoptosis Significance and Molecular Mechanisms- A Review. Asian J. Research Chem. 2(4):Oct.-Dec. 2009: 369-375.

37. Fulda S. Caspase-8 in cancer biology and therapy. Cancer Lett 2009; 281: 128-33.

38. Olsson M., Zhivotovsky B. Caspases and cancer. Cell Death Differ 2011;18 (9): 1441–9.\

39. Krelin Y., Zhang L., Kang T., Appel E., Kovalenko A., Wallach D. Caspase-8 deficiency facilitates cellular transformation in vitro. Cell Death Differ 2008;15(9): 1350–5.

40. Esteller M., Sanchez-Cespedes M., Rosell R., Sidransky D., Baylin S.B., and Herman J.G. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res. 1999;59: 67–70.

41. Hopkins-Donaldson S., Ziegler A., Kurtz S., Bigosch C., Kandioler D., Ludwig C., Zangemeister-Wittke U., and Stahel R. Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation. 2003; 10: 356–364

42. Wiley S.R., Schooley K., Smolak P.J., Din W.S., Huang, C.P., Nicholl J.K., Sutherland G.R., Smith, T.D., Rauch C., Smith C.A., et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 1995; 3: 673–682.

43. Walczak H., Miller R.E., Ariail K., Gliniak B., Griffith T.S., Kubin M., ChinW., Jones J., Woodward A., Le T., et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat. Med. 1999; 5: 157–163.

44. Namrata Dwivedi, Bhavna Dwivedi, Skand Mishra, Yogeshwer Shukla. Lupeol Induced Apoptosis in Human Lung Cancer Cell Line: A Flow Cytometry Study. Research Journal of Pharmacology and Pharmacodynamics. 2014; 6(4): 197-203.

45. Abdollahi T., Robertson N.M., Abdollahi A., Litwack G. Identification of interleukin 8 as an inhibitor of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in the ovarian carcinoma cell line OVCAR3. Cancer Res. 2003; 63: 4521–4526.

Received on 17.03.2020 Modified on 19.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2676-2680.

DOI: 10.52711/0974-360X.2021.00472