Mechanisms Of The Anticancer Effects Of Norcantharidin On Human Choriocarcinoma And Skin Cancer

Norcantharidin(NCTD)is a compound derived by removing 2,3-methyl of cantharidin molecule.It is the first synthetic and new anticancer drugs with independent intellectual property rights in China.Compared with cantharidin,norcantharidin has a lower toxicity and a unique effect of increasing white blood cells.Previous studies have shown that norcantharidin protected normal hepatocytes from damage caused by lipopolysaccharide(LPS).It could reduce tubulointerstitial fibrosis and protect the kidneys.It played some role in the regulation of human immune system.In addition,norcantharidin also had a strong anti-cancer effect on a variety of tumors,which included ovarian cancer,prostate cancer,cervical cancer and liver cancer etc.Using anticancer drugs to increase the level of reactive oxygen species in tumor cells is considered to be a potential strategy of anticancer therapy.Elevated reactive oxygen species can induce the apoptosis and autophagy of cancer cells,which result in cell death.However,little is known about the anticaner effect of NCTD on human choriocarcinoma and skin cancer.In the preliminary study,we found NCTD could increase the level of reactive oxygen species in human choriocarcinoma JEG-3 cells and skin squamous cell carcinoma A431 cells.Therefore,it is necessary to study whether NCTD can induce the two kinds of cancer cell apoptosis and autophagy,etc.Part 1: Effects of NCTD on proliferation,apoptosis and autophagy of JEG-3 cells,and its chemosensitivityObjective:To investigate the effects of NCTD on the proliferation,apoptosis and autophagy of human choriocarcinoma JEG-3 cells and its molecular mechanism.In addition,to study the effects of NCTD on the chemosensitivity of 5-FU in JEG-3 cells.Methods:In this study,MTT assay was used to detect cell proliferation.Cell cycle was detected by flow cytometry.Apoptosis was investigated by PI-FITC fluorescent double staining.DAPI staining and Hochest33258 staining were also used to detect apoptosis.The changes of cell morphology of apoptosis and autophagy were observed by electron microscopy.We also used kinds of kits to detect the changes in apoptosis,reactive oxygen species,membrane potential,autophagic positive cells,MDA and Caspase-3,-9 activities.The expression of P21,Cyclin B1,Fas,Fas L,Bcl-2,Bax,m TOR,P62,Beclin1 and LC3-Ⅱ protein was detected by Western blot.Results:1.NCTD significantly inhibited the growth and proliferation of human choriocarcinoma JEG-3 cell line.NCTD could cause G2 arrest of JEG-3 cells,which might be caused by up-regulating the expression of P21 protein and down-regulating Cyclin B1 protein.2.It was found that NCTD could increase the level of ROS and induced JEG-3 cell apoptosis.And the expression of Bcl-2 protein was decreased after NCTD treatment,while Bax,Fas and Fas L protein expression were increased.3.To investigate whether apoptosis was induced by elevated levels of reactive oxygen species,active oxygen scavenger N-acetylcysteine(NAC)was used to treat JEG-3 cells with NCTD.The results showed that NAC could reduce NCTD-induced apoptosis and the activity of Caspase-3 and 9.4.We also found that NCTD could depress the expression of antioxidant enzymes SOD1,PRDX1,PRDX2,PRDX3 as well as cytokine IL-6 in JEG-3 cells,and the MDA content was increased,while the total SOD activity in the cells was decreased.These results suggested that NCTD induces apoptosis of human choriocarcinoma JEG-3 cells through ROS pathway.5.It was also found that NCTD could enhance the chemosensitivity of 5-FU in JEG-3 cells,which might be caused by the increased Caspase-3,-9 enzyme activity in JEG-3 cells.6.In addtion,we found that NCTD induced autophagy of JEG-3 cells,which might be caused by the changes of expression of m TOR,Akt,P62,LC3-Ⅱ and Beclin1 proteins.In order to explore the relationship between autophagy and reactive oxygen species,we treated the cells with the combation NAC and NCTD,and found that NAC could significantly attenuate NCTD-induced autophagy and change the expression of autophagy-related proteins Beclin1 and P62.These results suggested that NCTD mainly induced autophagy of JEG-3 cells by increasing intracellular ROS levels.Conclusions:NCTD can increase the level of reactive oxygen species in JEG-3 cells and induce the apoptosis and autophagy.The mechanism may be related to the changes of P21,Cyclin B1,Fas,Fas L,Bcl-2,Bax,m TOR,P62,Beclin1,LC3-Ⅱ protein expression.In addition,NCTD can inhibit cell proliferation,induce cell cycle arrest in G2 phase.NCTD also promotes the chemosensitivity of 5-FU on choriocarcinoma.This experiment provides an important experimental basis for the treatment of human choriocarcinoma by NCTD,and expanding the application of NCTD in the treatment chemotherapy of cancers.Part II: Effects of NCTD on the proliferation,apoptosis and metastasis of human skin squamous cell carcinoma A431 cellsObjective:To investigate the effects of NCTD on the proliferation,apoptosis and metastatic potentials of human skin squamous cell carcinoma A431 cells and its molecular mechanism.Methods:The cell proliferation was examined by MTT assay,cell cycle changes were detected by flow cytometry,apoptosis was examined with PI-FITC fluorescent double staining,and cell morphology changes were observed using DAPI staining.We also used various kits to assay the changes of reactive oxygen species,membrane potential,MDA and Caspase-3,-9 activities.In addition,transwell chamber was used to investigate cell invasion and metastasis ability,and the ability of cell migration was detected by scratch repair method.The expression levels of P21,Cyclin B1,Fas,Fas L,Bcl-2,Bax,VEGF,MMP-2 and MMP-9 were detected by Western blot.Results:1.It was found that NCTD significantly inhibited the growth and proliferation of A431 cells.NCTD could also cause the arrest of G2 phase in A431 cells,which was induced by the increased P21 protein expression and decreased Cyclin B1 protein expression.2.NCTD could increase the level of ROS and the apoptosis rate of A431 cells.It was also found that the expression of Bcl-2 protein was decreased and the expression of Bax,Fas and Fas L were increased.3.In order to explore the induction of A431 cell apoptosis was due to the elevated levels of reactive oxygen species,the active oxygen scavenger,N-acetylcysteine(NAC)was used to treat A431 cells with NCTD.It was showed that NAC could reduce NCTD-induced apoptosis and decrease the activity of Caspase-3 and 9.These findings indicated that NCTD induced A431 cell apoptosis through ROS pathway.4.In addition,we found that NCTD could inhibit the migration and adhesion of A431 cells,which might be related with the decreased expression of VEGF,MMP-2 and-9 proteins after NCTD treatment.Conclusions:NCTD could significantly inhibit the proliferation and metastasis of A431 cells,and induce apoptosis by increasing reactive oxygen species pathway.These findings provide an important experimental basis for the treatment of skin cancer by NCTD,and further expanding the application of NCTD as an anticancer drug.