Costunolide Enhances Doxorubicin-induced Apoptosis In Prostate Cancer Cells Via Activated Mitogen-activated Protein Kinases And Generation Of Reactive Oxygen Species

Prostate cancer is commonly diagnosed in men living in western countries,and the incidence of prostate cancer in developing countries has been rapidly increasing in past decades.Although surgery and androgen ablation therapy are frst-line treatments for prostate cancer,they often fail to be effective for advanced stage and castration-resistant prostate cancers(CRPCs).Thus,chemotherapy remains a very important option for advanced-stage prostate cancer patients.Doxorubicin has been extensively used in many different tumors and is often combined with other drugs to enhance effects and reduce toxicity.Costunolide is a natural sesquiterpene lactone with anti-cancer properties.In this research,we study the effect of CTN and DOX on the proliferation and apoptosis of PC-3 and DU-145 cells and explore the potential molecular mechanisms;and then study the anti-cancer effects in the vivo study.From the study,the main results are as follows:(1)Cell Counting Kit-8(CCK-8)assay showed both costunolide and Dox inhibited the growth of these cells in a concentration-dependent manner.To analyze the combinational effects,we used CompuSyn software and the Chou-Talalay method to calculate the combination index(CI),which offers a quantitative defnition for additive(CI =1),synergistic(CI<1),and antagonistic(CI>1)effects of drug combinations.The results showed the CTN combined with the lower dose DOX had synergistic effects in the PC-3 and DU-145 cells.(2)Annexin V/PI kit was used to analyze the induction of apoptosis in PC-3 and DU-145 cells treated with costunolide,Dox,or their combination for 24 h.The results showed costunolide synergistically enhanced Dox-induced apoptosis over that of either drug alone.To analyze costunolide-and Dox induced apoptosis,we detected caspase and poly(ADPribose)polymerase(PARP)activation via western blotting.Exposure to the combined agents led to higher levels of cleaved caspase-9,cleaved caspase-3,and cleaved PARP than that of costunolide or Dox alone in PC-3 and DU-145 cells.Taken together,these results demonstrated that costunolide greatly enhanced Dox-induced apoptosis in PC-3 and DU-145 cells.(3)JC-1 staining was used to detect the mitochondrial membrane potential in PC-3 and DU-145 cells treated with costunolide,Dox,or their combination for 24 h.The results showed costunolide synergistically enhanced Dox-decreased △ψm over that of either drug alone.Furthermore,to analyze costunolide-and Dox induced mitochondrial dysfunction,we detected changes in the expression of proapoptotic Bcl-2 members(Bax and Bak),antiapoptotic Bcl-2 members(Bcl-2 and Bcl-xL),mitochondrial and cytosolic cytochrome C via western blotting.the expressions of Bcl-2 and Bcl-xL were decreased,whereas the levels of Bax and Bak were increased in cells treated with costunolide combined with Dox.Mitochondrial cytochrome C expressions were decreased,whereas the levels of cytosolic cytochrome C were increased in PC-3 and DU-145 cells treated with costunolide combined with Dox,compared to that of costunolide or Dox alone.(4)Using DCFH-DA staining and western blotting,we found that this drug combination signifcantly increased the production of reactive oxygen species(ROS),as well as phosphorylation of c-jun N-terminal kinase(JNK)and p38 mitogen-activated protein kinases compared to that of costunolide or Dox alone,which play upstream roles in mitochondria-mediated apoptosis.Further studies showed that N-acetyl cysteine blocked JNK and p38 phosphorylation,suggesting that ROS were upstream activators of JNK and p38.However,a JNK inhibitor,but not a p38 inhibitor,blocked the increase in ROS observed in cells treated with a combination of costunolide and doxorubicin,suggesting that ROS and JNK could activate each other.(5)In vivo,inhibition of tumor growth and induction of apoptosis were greater in mice treated with the costunolide and doxorubicin combination than in mice treated with either drug alone,without an increase in toxicity.Therefore,our results firstly showed that costunolide enhanced Dox-induced apoptosis in the prostate cancer cells through a mitochondria-mediated apoptotic pathway by facilitating crosstalk of p38/JNK pathways and ROS generation.We suggested that costunolide in combination with doxorubicin was a new potential chemotherapeutic strategy for treating prostate cancer.