Study On IL-17(A) In Regulating Omentum Metastasis And Drug Resistance Of Ovarian Cancer

Objective: To investigate the effects of interleukin-17 A on proliferation, omentum metastasis and cisplatin(DDP) sensitivity of ovarian cancer, and explore its possible mechanisms.Methods: 1. To provide the basis for following tests, western blot assay was used to detect the protein expression of IL-17 RA in human ovarian cancer cell lines A2780, OVCAR3, SKOV3, HO8910 and HO8910/PM. 2. MTT test was used to detect the effect of exogenous rh IL-17 A on proliferation of human ovarian cancer cells. Real-time PCR and western blot assay were used to analyze the expression of m RNA and protein of CSCs’ biomarkers.3. Wound healing assay was used to detect the effect of exogenous rm IL-17 A on migration ability of ID8. Transwell chamber invasion test(8.0μm) was used to detect the effect of exogenous rm IL-17 A and/or mice primary omentum adipocytes on invision ability of ID8. After co-culturing with exogenous rm IL-17 A and/or mice primary omentum adipocytes in 3.0μm transwell chamber, protein in ID8 cells was extracted and western blot assay was used to analyze the protein expression of FABP4 in ID8 cells. Real-time PCR and western blot assay were used to analyze the effect of exogenous IL-17 on the expression and activation of STAT3 and NF-κB. 4. To establish murine ovary carcinoma models, C57 BL/6 wide type and IL-17-/-mice were used for intraperitoneal(i.p.) injection or orthotopic(intrabursal) injection with ID8 cells. The effect of endogenous IL-17 A on tumor metastasis was evaluated. Immunohistochemical staining assay was used to detect the expression of IL-17 A, MMP-2 and FABP4 in tumors.5. Clinical samples of human ovarian epithelial tumors were collected. Immunohistochemical staining assay was used to analyze the role of IL-17 A, MMP-2 and FABP4 on development, metastasis and prognosis of OVCA. 6. MTT test was used to detect the DDP sensitivity and analyze the effect of exogenous rh IL-17 A on DDP sensitivity of human ovarian cancer cells. DDP sensitive OVCA cell line A2780 and DDP resistant OVCA cell line OVCAR3 were used in the following tests. Flow cytometry assay was used to detect the effect of exogenous rh IL-17 A on cell apoptosis and cell cycle distribution. 7. Real-time PCR assay was used to detect the m RNA expression of ABCG2 and TUBB3. Western blot assay was used to detect the protein expression of ABCG2. 8. To establish murine ovary carcinoma models, C57 BL/6 wide type and IL-17-/-mice were used for intraperitoneal(i.p.) injection with ID8 cells. The effect of endogenous IL-17 on DDP sensivity was evaluated. 9. Clinical samples of primary and recurrent human ovarian epithelial tumors were collected. Immunohistochemical staining assay was used to analyze the role of IL-17 A and ABCG2 on development and prognosis of OVCA.Results: 1. IL-17 RA expression was observed in human ovarian cancer cell lines. 2. Comparing with control group, exogenous rh IL-17 A has no effect on proliferation of OVCA cells. The m RNA expression of CSCs’ biomarkers, Oct3/4, in OVCA cells were promoted by 0.1ng/m L and 100ng/m L exogenous rh IL-17 A, whereas there was no change detected on the protein level of Oct3/4.3. IL-17 RA expression was observed in mice ovarian cancer cell line ID8 and mice primary omentum adipocytes. Comparing with control group, the migration ability of ID8 was promoted by exogenous rm IL-17 A in a dose dependent manner. The invision ability of ID8 could be increased by exogenous rm IL-17 A, or co-culturing with primary omentum adipocytes from IL-17-/- mice or wide type mice, or co-culturing with primary omentum adipocytes from wide type mice after treating with rm IL-17 A. The invision of ID8 could be up-regulated by exogenous IL-17, or co-culturing with primary adipocytes from IL-17-/- mice or WT mice. The protein expression of MMP-2 and FABP4 in OVCA cells were promoted after treated with rh IL-17 A. After treating with BMS 309403, inhibitor of FABP4, the invision of ID8 could be inhibited apparently. The protein level of STAT3 in HO8910, HO8910/PM and ID8 cells were increased by exogenous IL-17 A. After treating with HO-3867, inhibitor of STAT3, the promotion of p STAT3 by exogenous IL-17 A was blocked.4. The murine ovary carcinoma models were established. Comparing with IL-17-/-mice, the survival time of wide type mice lessened and the tumor counts increased apparently. The expression of IL-17 A, MMP-2 amd FABP4 in tumors from wide type mice were increased than those from IL-17-/- mice. There was no IL-17A+ cells detected in IL-17-/- mice.5. Results of IHC staining showed the positive correlation between expression of IL-17 A, MMP2 and FABP4 and malignant degree of ovarian epithelial tumors. 6. MTT results showed that A2780 was a DDP sensitive OVCA cell line and OVCAR3 and SKOV3 were DDP resistant OVCA cell lines. Comparing with the control group, the cytotoxicity of DDP to A2780 and OVCAR3 was inhibited after treating with low or high concentration of rh IL-17 A, whereas only high concentration of rh IL-17 A might decrease the DDP cytotoxicity to SKOV3. Comparing with the control group, cell apoptosis of A2780 and OVCAR3 were not affected by 0.1ng/m L rh IL-17 A. Pre-treating with 0.1ng/m L rh IL-17 A, the cytotoxicity of DDP to A2780 was inhibited apparently. There was no effect of rh IL-17 A on cell cycle distribution of OVCA cells. After treating with DDP, A2780 and OVCAR3 cells had a decreasement in the G0/G1 phase and a significant increasement in the G2+S phase, suggesting that DDP might inhibit proliferation of OVCA cells by altering cell cycle distribution. After pre-treating with 0.1ng/m L rh IL-17 A, the DDP effect on cell cycle might be reverse partly by increasing the G0/G1 phase and decreasing the G2+S phase.7. The expression of drug-resistant related protein, ABCG2, was different in OVCA cells, which is lower in A2780 but higher in OVCAR3 and SKOV3 cells. Comparing with control group, exogenous rh IL-17 A increased mainly the ABCG2 expression in A2780, but not TUBB3. 8. Comparing with IL-17-/- mice, DDP sensitivity of OVCA was inhibited markedly by endogenous IL-17. 9. Results of IHC staining showed the positive correlation between expression of IL-17 A and ABCG2 and malignant degree and prognosis of ovarian epithelial tumors.Conclusion: From the results of experiments in vitro, animal experiments and immunohistochemical analysis of clinical specimens, this study reveals for the first time IL-17 A can increase the expression of FABP4 in ovarian cancer cells, make tumor cells migrate to the omentum adipose tissue, promote the omentum metastasis occur directly and/or indirectly via omentum adipocytes, by combining with IL-17 RA on ovarian cancer cells and omentum adipocytes. IL-17 A and FABP4 maybe the new targets for inhibition of ovarian cancer omentum metastasis. This study also reveals for the first time IL-17 A can enhance the cisplatin resistance by up-regulating ABCG2 in ovarian cancer cells, which provide the new targets for improving the resistance of ovarian cancer.