Study Of The Effects And Underlying Mechanisms Of IL-17A On The Cisplatin-based Resistance Of Ovarian Cancer

Objective: Human OVCA cell lines, IL-17A-/- mice, a mouse OVCA cell line and clinical OVCA specimens were utilized to study the effects and underlying mechanisms of IL-17 A on DDP-based resistance of OVCA, which may provide a novel strategy to improve the chemoresistance of OVCA in the clinic.Methods: 1. Western blotting assay was used to detect the protein level of IL-17 RA in human OVCA cell lines, including DDP-sensitive A2780 and DDP-resistant OVCAR3 and a mouse OVCA cell line ID8.2. MTT assay was used to detect the effect of rhIL-17 A on proliferation of A2780 and OVCAR3 cells.3. MTT assay was used to detect the IC50 of A2780 and OVCAR3 cells to DDP.4. Western blotting assay was used to detect the dose-dependent and time-dependent effects of rhIL-17 A on protein levels of ABCG2 and MDR1 in A2780 and OVCAR3 cells.5. MTT assay was used to detect the effect of viability of rhIL-17A(1ng/ml, 24h) on A2780 and OVCAR3 cells treated with DDP(A2780, 10μM; OVCAR3, 100μM), and then neutralizing IL-17 RA mAb(using m Ig as an isotype control) and Gant61 were used to do the blocking test respectively.6. Three pairs of siRNA sequence targeting IL-17RA(designated as #1、#2、#3 siIL-17RA) were synthesized by company and were respectively transfected into A2780 and OVCAR3 cells with Lipofectamine 2000. Western blotting assay was used to detect the efficiency of knockdown after transfection.7. MTT assay was used to detect the effect of viability of rhIL-17A(1ng/ml, 24h) on DDP-based resistance of #3siIL-17 RA transfected A2780 and OVCAR3 cells.8. Flow cytometry was used to detect the cell apoptotic effect of rhIL-17A(1ng/ml, 24h) on DDP-induced resistance of A2780 and OVCAR3 cells(A2780, 10μM;OVCAR3, 100μM), and then neutralizing IL-17 RA mAb(using m Ig as an isotype control) was used to do the blocking test.9. Western blotting assay was used to detect the protein levels of ABCG2, MDR1 and Gli1 on rhIL-17 A treated(1ng/ml, 24h) A2780 and OVCAR3 cells, and then neutralizing IL-17 RA mAb(using m Ig as an isotype control) and Gant61 were used to do the blocking test respectively.10. Western blotting assay was used to detect the effects of rhIL-17A(1ng/ml, 24h) on ABCG2, MDR1 and Gli1 levels in #3siIL-17 RA transfected A2780 and OVCAR3 cells.11. The ID8-burden mouse model was first established with C57BL/6 background WT and IL-17A-/- mice and then the tumor-bearing mice were intraperitoneally(i.p.) injected with DDP. IHC staining assay was used to detect the expression of IL-17 A, ABCG2, MDR1 and Gli1 in tumor samples from ‘WT+DDP’ and ‘deficient +DDP’ group mice. Western blotting assay was used to detect the protein levels of IL-17 A, ABCG2, MDR1 and Gli1 in tumor samples from WT, IL-17A-/-, ‘WT+DDP’ and ‘deficient +DDP’ group mice.12. About 55 EOC and 9 normal ovary specimens were collected to study. IHC staining assay was used to analyze the relationship between the expression of IL-17 A and FIGO stage, and ABCG2, MDR1, Gli1 levels in clinical settings.Results:1. IL-17 RA was present in DDP-sensitive A2780, DDP-resistant OVCAR3 human OVCA cell lines and a mouse OVCA cell line ID8(Fig 1A).2. rhIL-17 A had no obvious effect on the proliferation of A2780 and OVCAR3 cells(Fig 1B).3. The IC50 values of DDP from MTT assay were determined to be 25.78 and 322.00μM in A2780 and OVCAR3 cells, respectively.4. The protein levels of ABCG2 and MDR1 were significantly enhanced by the treatment of 1ng/ml rhIL-17 A for 24h(Fig 3).5. IL-17 RA protein level was significantly knockdown in #3 siRNA transfected A2780 and OVCAR3 cells(Fig 5A).6. rhIL-17 A alone had no effect on the viability of A2780 and OVCAR3 cells, but it could increase the viability of A2780 and OVCAR3 cells with DDP treatment(A2780:0.72±0.01 vs 0.49±0.02;OVCAR3:0.75±0.01 vs 0.55±0.04,P<0.05). Furthermore, neutralizing IL-17 RA mAb, knockdown of IL-17 RA and Gant 61 could respectively remarkably block the above effect of rhIL-17 A on DDP-sensitivity(neutralizing IL-17 RA mAb, A2780: 0.44±0.01 vs 0.72±0.01, P<0.01; OVCAR3:0.63±0.01 vs 0.75±0.01, P<0.05, Fig 4; knockdown of IL-17RA: A2780: 0.82±0.01 vs 0.51±0.03, P<0.01, OVCAR3: 0.85±0.01 vs 0.77±0.04, P<0.05, Fig 5B; Gant 61, A2780 : 0.48±0.02 vs 0.72±0.01; OVCAR3:0.41±0.01 vs 0.75±0.01, P<0.01, Fig 7C).7. rhIL-17 A alone had no effect on apoptosis of A2780 and OVCAR3 cells, but it could decrease the percentage of the apoptosis of A2780 and OVCAR3 cells with DDP treatment(A2780:14.97±0.80% vs 25.77±3.31%,P<0.05;OVCAR3:11.67±1.90% vs 19.20±0.98%,P<0.01). Furthermore, neutralizing IL-17 RA mAb could partially reverse the apoptotic-inhibition effect by rhIL-17A(A2780:14.97±0.80 vs 24.77±2.51%;OVCAR3:11.67±1.90 vs 17.97±0.72%,P<0.05)(Fig6).8. rhIL-17 A could increase the expression of ABCG2, MDR1 and Gli1 in A2780 and OVCAR3 cells. Furthermore, neutralizing IL-17 RA mAb, knockdown of IL-17 RA and Gant 61 could respectively block the enhanced effects of rhIL-17 A on ABCG2, MDR1 and Gli1 levels in A2780 and OVCAR3 cells(Fig 7).9. After DDP treatment to the WT and IL-17A-/- mice burden with ID8 tumor, the result illustrated that:(1) ‘deficient +DDP’ group mice exhibited extremely reduced tumor load compared to the ‘WT +DDP’ group mice(Fig 8).(2) IHC staining assay showed that IL-17 A expression was only detected in the samples from ‘WT+DDP’ group mice not in ‘deficient +DDP’ group mice and the ABCG2, MDR1 and Gli1 levels were significantly higher in ‘WT+DDP’ group mice than those in ‘deficient +DDP’ group mice(Fig 9A).(3) Western blotting analysis of protein extraction of tumor samples from the four groups mice respectively showed that the expression of ABCG2, MDR1 and Gli1 in WT group mice significantly increased compared to those in deficient group mice,and compared with WT group, those were upregulated in ‘WT +DDP’ group mice, and that compared with deficient group, those in ‘deficient +DDP’ group mice had no change(Fig 9B).10. Result of IHC staining in the clinical EOC settings displayed that: IL-17 A level in OVCA markedly increased as the disease progresses based on FIGO staging(Fig 10); ABCG2, MDR1 and Gli1 levels were basically absent or at a the low level in the normal ovary tissues, as the disease progressed, the proportion of OVCA cells positive for ABCG2, MDR1 or Gli1 increased(Fig 11A); IL-17 A level significantly positively related with the levels of ABCG2, MDR1 and Gli1 in the clinical specimens(Fig 11B).Conclusion:Our present study found that IL-17 A could exacerbate DDP-induced resistance of OVCA via upregulating the expression levels of ABCG2 and MDR1 through Gli1-mediated Hh signal pathway. These findings may provide a novel strategy to improve the chemoresistance of OVCA in the clinic.