Mechanism Of Norcantharidin-induced Apoptosis In Human Ovarian Cancer SKOV3 Cells

Purpose:In this study,we investigated the effect of norcantharidin treatment on SKOV3 cells proliferation and apoptosis.It was detected whether norcantharidin can enhance cisplatin sensitivity in SKOV3 cells.The mechanism of norcantharidin and cisplatin combination was explored.Our study showed scientific basis on auxiliary treatment of norcantharidin in ovarian cancer and provided the reference to combine traditional Chinese and western medicine in clinical application.Material and method:Part 1:1.MTT assay was used to detect the growth inhibition effect of norcantharidin on SKOV3cells.The cytotoxicity of norcantharidin at 12h,24h and 48h was detected by adding different concentrations(0-100?mol/L)of norcantharidin.2.The morphological changes of ovarian cancer cells treated with norcantharidin(52?mol/L)and active oxygen scavenging agent NAC plus norcantharidin(52?mol/L)were observed under inverted microscope.3.Flow cytometry was used to detect the cell cycle DNA content and cell apoptosis rate after norcantharidin treatment.Fluorescent probe DCF-DA was used to stain the cells and then flow cytometry was used to detect the production of intracellular reactive oxygen species after treatment,so as to explore the mechanism of apoptosis.4.The protein expressions of Bax,Bcl-2,procaspase-3,cyclin B1 and p21 were detected by western blot to explore the mechanism of norcantharidin acting on SKOV3 cells.Part 2:1.MTT assay was used to detect the growth inhibitory effect of norcantharidin combined with cisplatin on SKOV3 cells.2.The morphological changes of SKOV3 cells treated with norcantharidin(30?mol/L),cisplatin(10?g/m L)and norcantharidin+cisplatin(30?mol/L+10?g/m L)were observed under inverted microscope.3.The apoptosis rate of ovarian cancer cells after treatment with norcantharidin(30?mol/L),cisplatin(10?g/m L)and norcantharidin+cisplatin(30?mol/L+10?g/m L)was analyzed by flow cytometry.4.The morphology and distribution of mitochondria of SKOV3 cells after treated with norcantharidin(30?mol/L),cisplatin(10?g/m L)and norcantharidin+cisplatin(30?mol/L+10?g/m L)were observed under fluorescence microscope.5.Western blotting was used to detect the protein expression of procaspase-3 after treatment with norcantharidin(30?mol/L),cisplatin(10?g/m L)and norcantharidin+cisplatin(30?mol/L+10?g/m L).Results:Part 1:1.Norcantharidin inhibits SKOV3 cell growthMTT results showed that the inhibition of norcantharidin on cell growth was time and concentration dependent compared with blank control group.2.Norcantharidin induces apoptosis and G₂/M phase cell cycle arrest in SKOV3 cellsUnder the inverted microscope,SKOV3 cells had obvious morphological changes after drug treatment,and the cell morphology became wrinkled and rounded,and bud formed obvious apoptotic corpuscle.Flow cytometry with PI staining showed that compared with the control group,the proportion of sub G1 peak and the proportion of double amount of DNA were significantly increased after norcantharidin treated cells for 12h and 24h,indicating apoptosis and G₂/M phase arrest in the cells.3.Norcantharidin induces ROS production in SKOV3 cellsFlow cytometry showed that the ROS level in SKOV3 cells increased in a time-dependent manner after treated with 52?mol/L norcantharidin for 0-24h.4.Effects of reactive oxygen species on norcantharidin apoptosis and G₂/M phase cycle arrestFlow cytometry was used to detect the production of intracellular ROS,and it was found that 5mmol/L NAC pretreatment significantly reduced the production of intracellular ROS induced by norcantharidin.DCF staining was observed under fluorescence microscope,and it was found that NAC pretreatment significantly reduced the green fluorescence intensity of norcantharidin-induced DCF.5.Effect of norcantharidin on protein expression in SKOV3 cellsWestern blot results showed that the expression of Bcl-2 decreased significantly,Bax increased and procaspase-3 decreased after treatment with norcantharidin for 24h.The expression level of p21,an important regulatory factor in G₂/M phase,was up-regulated,and the expression level of cyclin B1 was down-regulated.The addition of NAC could significantly reverse the expression changes of Bax,Bcl-2,procaspase-3,p21 and cyclin B1caused by norcantharidin alone.Part 2:1.Growth inhibitory effect of norcantharidin combined with cisplatin on ovarian cancer cellsNorcantharidin(30?mol/L),cisplatin(10?g/m L)and norcantharidin(30?mol/L)+cisplatin(10?g/m L)were added into ovarian cancer cells,respectively.The drug-induced cytotoxicity was detected at 24h,48h and 72h,respectively.After the combination of norcantharidin and cisplatin,norcantharidin could effectively improve the sensitivity of ovarian cancer SKOV3 cells to cisplatin in a time-dependent manner.2.Effect of norcantharidin combined with cisplatin on SKOV3 cell morphologyUnder the inverted microscope,compared with the normal control group,SKOV3 cells in the cisplatin group and the combined treatment group were smaller and rounder.With the change of cell morphology,the intercellular connections were loosened,the adhesion ability was weakened,and the growth ability was obviously inhibited.3.Norcantharidin combined with cisplatin induced apoptosis of ovarian cancer cellsFlow cytometry results showed that the apoptosis rate of norcantharidin and cisplatin group was significantly increased,especially in the early apoptosis,norcantharidin could significantly promote the apoptosis of ovarian cancer cells induced by cisplatin.4.The morphological changes and distribution of mitochondria were observed under fluorescence microscopeThe mitochondria of SKOV3 cells were specifically labeled with Mito-tracker Green.The morphological changes of mitochondria were observed under fluorescence microscope.Compared with the normal control group,the mitochondrial structure of SKOV3 cells in the combination of norcantharidin and cisplatin was ambiguous,and the number of mitochondria gathered around the nucleus was significantly increased.5.Effect of norcantharidin combined with cisplatin on protein expression of procaspase-3 in ovarian cancer cellsWestern blots were used to detect the protein expression of procaspase-3 in ovarian cancer cells after treatment,and it was found that the protein expression of procaspase-3decreased to varying degrees in both the norcantharidin and cisplatin groups,while the protein expression of procaspase-3 obviously decreased in the norcantharidin and cisplatin combination group.Conclusion:1.Norcantharidin could effectively inhibit the growth and change the cell morphology in SKOV3 cells.2.Norcantharidin could induce apoptosis and G₂/M cell cycle arrest by increasing intracellular ROS content in SKOV3 cell.3.Norcantharidin could obviously promote the apoptosis of ovarian cancer cells induced by cisplatin.4.The combination of norcantharidin and cisplatin could significantly change the cell morphology and mitochondrial morphology of SKOV3.5.The combination of norcantharidin and cisplatin could decrease the expression of procaspase-3 protein.