The Mechanism Of Garcinol Inhibition On Endometrial Cancer Growth

Objective:Endometrial cancer(EC)is one of the most common gynecological cancers and one of the most important causes of cancer-related death in women worldwide.The long-term survival rate of advanced and recurrent EC is low.Therefore,it is important to further explore the pathogenesis of EC and find new anti-cancer drugs.Garcinol,a polyisoprenylated benzophenone,is a promising anti-cancer drug for various cancer types but its effects on EC remain unclear.The purpose of this project was to study the effects of garcinol on the proliferation and apoptosis of the endometrial cancer cell lines Ishikawa(ISH)and HEC-1B,and to explore the mechanism of garcinol’s anti-endometrial cancer cell growth effects,so that it can be used in clinical EC treatment in the future.Methods:The two EC cell lines were treated with different concentrations of garcinol(0,1,5,10 and 20 μM)and cell proliferation was assessed by real-time cell analysis(RTCA)and cell counting methods;colony formation assay was used to detect the effect of garcinol on EC cell clone formation;flow cytometry was used to detect cell cycle and apoptosis;the incorporation assay of thymidine analogues EdU(5-ethynyl-2’-deoxyuridine)was used to detect the effect of garcinol on DNA synthesis in S phase of EC cells;Western blotting was used to evaluate the expression levels of cell cycle-related proteins p53,p21,CDK1,CDK2,CDK4,Cyclin D1 and Cyclin B1,and the expression of apoptotic proteins PARP,Cleaved PARP Bcl-2 and Bax,and the expression levels of the phosphorylated c-JUN N-terminal kinase(JNK)and c-JUN.Results:Garcinol inhibited ISH and HEC-1B cell proliferation in a dose-dependent manner,induced ISH and HEC-1B cell cycle arrest in G1 and G2/M phases,respectively,and reduced S phase and DNA synthesis.After garcinol treatment of ISH and HEC-1B cells,the expression levels of p53 and p21 increased,and the expression levels of CDK2,CDK4,cyclin D1 and cyclin B1 gradually decreased in a dose-dependent manner.There was no significant difference in the expression of CDK1 in ISH cells;however,the expression of CDK1 in HEC-1B cells decreased.Garcinol can promote cell apoptosis in EC cell lines.At higher concentrations(20 and 30 μM)of garcinol induced apoptosis in ISH cells,but at least 30 μM garcinol was required to induce apoptosis in HEC-1B cells.Compared with the control group,the cleaved PARP/PARP increased(p=0.02)in the 20 μM garcinol treatment group and the expression of the anti-apoptotic protein Bcl-2 decreased(p=0.03)in the ISH cells.In HEC-1B cells,compared with the control group,there was no significant change in cleaved PARP/PARP after treatment with 20 μM garcinol,the expression of Bax protein increased(p=0.045),and the expression of Bcl-2 protein decreased.In addition,the expression levels of the phosphorylated JNK and c-JUN of the two EC cells were significantly increased after the transient stimulation with garcinol.Conclusions:1.Garcinol can significantly inhibit the growth of endometrial cancer ISH and HEC-1B cell lines.2.Garcinol induces cell cycle arrest by up-regulating cell cycle inhibitors p53 and p21,inhibiting the expression of cyclin kinases CDK1,CDK2,CDK4,and cyclin D1 and cyclin B1.3.Garcinol can promote EC cell apoptosis,and its sensitivity to different EC cells is different.4.Garcinol can activate the JNK/c-JUN signaling pathway in EC cells.5.Garcinol is a promising EC treatment drug,but further research is still needed.