The Inhibitory Effects And Mechanism Of Oridonin Derivative Compound CYD0618 On Chemoresistant Breast And Ovarian Cancer Cells

Objective:Malignant tumor is a serious threat to human health.Chemotherapy remains the main treatment option for patients with malignant tumors.However,the development of multidrug resistance(MDR)to chemotherapy is the major obstacle that compromises therapeutic efficacy to achieve a favorable outcome and prognosis.Therefore,exploring effective anti-multidrug resistance drugs with novel mechanisms and targets is an important way to improve outcomes of cancer treatment,and has important clinical significance.We previously synthesized a series of oridonin derivative compounds based on the anti-tumor activity of oridonin and its structure-activity relationship,which exhibit high anti-tumor activity.However,the role of these compounds in the chemoresistance is unclear.In this study,we further invesitaged the inhibitory effect and mechanism of oridonin derivative CYD0618 on adriamycin-resistant breast cancer cells(MCF-7/ADR)and cisplatin-resistant ovarian cancer cells(A2780/CP).Methods:1.The effects of oridonin-derived compounds including YD0514,CYD0618 and CYD0686 on MCF-7/ADR and A2780/CP cell proliferation were measured by MTT and colony formation assay.2.The effects of CYD0618 on MCF-7/ADR and A2780/CP cell migration and invasion were measured by scratch assays and transwell assay.3.The effects of CYD0618 on MCF-7/ADR and A2780/CP cell apoptosis were measured by Flow cytometry.4.Nude mice xenograft model was established to assess the effects of CYD0618 on xenograft tumor growth.5.MCF-7/ADR and A2780/CP cells were transfected with SATA3 siRNA,the effect of STAT3 in chemoresistance was analyzed.6.MCF-7 and A2780 cells were transfected with pcDNA-STAT3 or treated with IL-6,the effect of STAT3 in chemoresistance was analyzed.7.Western blot was used to measure the effects of CYD0618 on the expression of STAT3 and its targets in MCF-7/ADR and A2780/CP cells.8.Immunohistochemistry was used to measure the effects of CYD0618 on the expression of STAT3 and its targets in MCF-7/ADR xenograft tissues.9.Immunofluorescence was used to analyze the effects of CYD0618 on the expression and cellular location of STAT3.10.Molecular docking analysis was used to investigate the interaction domain of CYD0618 and STAT3.11.Co-immunoprecipitation method and cellular thermal shift assay(CETSA)were used to determine the binding of CYD0618 and STAT3.Results:1.MTT assay showed that MCF-7/ADR and A2780/CP cells were resistant to adriamycin and cisplatin,respectively.Morphologically,both resistant cell lines exhibited high invasive ability.2.Oridonin-derived compounds YD0514,CYD0618 and CYD0686 significantly suppressed MCF-7/ADR and A2780/CP cells proliferation in time-and dose-depended manners.In which,CYD0618 exhibited the best anti-tumor activity against MCF-7/ADR and A2780/CP cells.3.Scratch assays and Transwell assays results showed that CYD0618 significantly inhibited migration and invasion of MCF-7/ADR and A2780/CP cells.4.Flow cytometry analysis showed that CYD0618 markedly induced MCF-7/ADR and A2780/CP cell apoptosis.5.In vivo assay showed that CYD0618 significantly suppressed xenograft tumor growth.6.STAT3 is significantly up-regulated in MCF-7/ADR cells and A2780/CP cells.Knockdown of STAT3 increased adriamycin and cisplatin sensitivity of MCF-7/ADR cells and A2780/CP cells respetively,whereas overexpression of STAT3 increased adriamycin and cisplatin resistance of MCF-7 and A2780 cells.7.Western blot results showed that CYD0618 significantly inhibit the expression of STAT3 and its downstream signaling molecules related to proliferation,apoptosis,margration and invasion in MCF-7/ADR cells and A2780/CP cells.Immunohistochemistry resulted comfirmed that CYD0618 significantly inhibited the expression of STAT3,p-STAT3 and Cyclin D1,but increased the expression of cleaved caspase-3 in MCF-7/ADR xenograft tissues.Immunofluorescence results showed that CYD0618 could inhibit nuclear translocation of STAT3 in MCF-7/ADR cells.8.Molecular docking indicated that CYD0618 selectively binds to STAT3-SH2 domain,thereby interrupting the phosphorylation of STAT3 at the Tyr705 residue.Co-immunoprecipitation and CETSA further comfirmed that CYD0618 directly bound to STAT3,thereby inhibiting its activation.Conclusion:STAT3 up-regulation is associated with chemoresistance in breast and ovarian cancer cells.It suggests that targeting and regulating STAT3 may be a rational strategy to overcome multiple drug resistance.The oridonin derivative CYD0618 exhibits a significant inhibitory effect on chemoresistant breast and ovarian cancer cells both in vivo and in vitro.CYD0618 directly binds to and inhibits STAT3,thereby inhibiting cell proliferation,migration and invasion of MCF-7/ADR and A2780/CP cells,and promotes apoptosis of MCF-7/ADR and A2780/CP cells.The results suggest that the oridonin derivative compound CYD0618,is a potential candidate of STAT3 inhibitor.