| Objective:Melanoma is the most invasive skin tumor,derived from melanocytes,is the most deadly type of skin tumor.Skin melanoma accounts for 1.6%of new malignant tumors worldwide,and the incidence is increasing at the rate of 2-7%per year.The incidence of melanoma has been increasing over the past few decades.Although traditional therapies such as surgery,chemotherapy,and radiotherapy can improve the prognosis of patients,but the effect is still limited.The researchers are committed to finding drugs that have the greatest impact on cancer cells and the least toxic to normal cells.In recent years,the role of traditional Chinese medicine has been recognized by researcheres world wide.Studies on the effect of active ingredients of Chinese herbal medicine on tumor are new directions in the field of cancer research.Cardamonin is an alkaloid monomer component extracted from the seeds of Alpinia katsumadai.Studies have confirmed the inhibitory effect of cardamonin on various tumor cells,but there is still no research on the effect of cardamonin on melanoma.In this study,melanoma M14 cells was treated with cardamonin,and the effects of M14 cell proliferation,apoptosis and apoptotic related proteins in M14 cell line were observed,and the potential mechanism was explored.Methods:1.Melanoma M14 cells were cultured in 1640 media containing 10%fetal bovine serum and cultured in a 37°C,5%CO 2 incubator.2.The effect of cardamonin on proliferation of melanoma M14 cells was examined by MTS assay.In the experiment,the blank control group was only added with media without cardamonin,and the negative control group.The negative control group was cells cultured in media containing different concentrations of cardamonin（30,60,90,120μmol/L）.Each group and concentration has 6 wells.After 24 h,48 h,72 h,MTS was added to detect the effect of cardamonin on the proliferation of melanoma M14 cells,and the survival rate of cells at different concentrations of drug was calculated.3.The apoptosis of melanoma M14 cells induced by cardamonin was detected by flow cytometry.Two groups were set up in the experiment.The negative control group was cells cultured in media without cardamonin,and the treated group was cells cultured in media with following concentrations（30,60,90 micromol/L）of cardamonin.After 24 hours of incubation,the assay was performed.4.Real-time quantitative PCR（RT-qPCR）was used to detect apoptosis-related genes in M14 cells treating with cardamonin.Two groups were set up in the experiment.The negative control group was cells cultured by media without cardamonin,and the treated group was cells cultured by media with following concentrations（30,60,90 micromol/L）of cardamonin.After 24 hours of incubation,the assay was performed.5.Western blot was used to detect the expression of apoptosis-related proteins（BAX,BCL2,Cleaved Caspase8,Cleaved Caspase9 and Cleaved PARP）in M14 cells treating with Cardamonin.The negative control group was cells cultured by media without cardamonin,and the treated group was cells cultured by media with following concentrations（30,60,90 micromol/L）of cardamonin.Results:1.Results of MTS assay showed that the viability of melanoma M14cells after treatment with different concentrations of cardamonin for 24h were（87.9±4.2）%,（78.2±2.4）%,（70.1±2.2）%,（44.0±3.2）%,respectively.By comparison,the difference between the experimental group and the control group was statistically significant（P<0.05）;the viability of the cardamonin treatment for 48 hours was（76.0±1.6）%,（35.5±2.2）%,（32.0±1.3）%,（23.5±1.7）%,the difference between the experimental group and the control group was statistically significant（P<0.05）;the viability of the adzuki bean treatment for 72 hours was（40.1±1.3）%,（34.6±2.3）%,（23±2.2）%,（18.3±2.0）%,the difference between the experimental group and the control group was statistically significant（P<0.05）.2.Flow cytometry showed that,the proportion of Annexin V positive cells（the sum of early apoptotic cells and late apoptotic cells）increased gradually with the increasing of cardamonin dosage.3.The results of RT-qPCR showed that after treated with different concentration of cardamonin for 24h,the expression of Bax at the mRNA level was 1.595±0.141,2.201±0.388,2.411±0.442,and the negative control group was 1.002±0.087.The difference between the control group and the experimental groups was statistically significant（P<0.05）.There was no significant difference between the other groups（P>0.05）.At the mRNA level,the expression of BCL2 gene were 0.755±0.044,0.617±0.004,0.536±0.074,and the negative control group was 1.008±0.180.The difference was statistically significant（P<0.05）between the control group and the experimental groups,and the difference between the other groups was not statistically significant（P>0.05）.4.The results of Western blot showed that the expression of Bax in M14cells was 0.326±0.017,0.585±0.013,0.307±0.008,and 0.643±0.005 in the negative control group,and after comparison,the difference between the experimental group and the negative control group were statistically significant（P<0.05）;BCL2 protein expression was 0.501±0.007,0.465±0.011,0.409±0.01,the negative control group was 0.537±0.007.After compari-son,the difference between the experimental group and the negative control group was statistically significant（P<0.05）.The expression of Cleaved Caspase 8 protein was 0.592±0.023,0.872±0.022,0.949±0.0259,respectively.The negative control group was 0.437±0.003,and after comparison,the difference between the experimental group and the negative control group was statistically significant（P<0.05）.The expression of Cleaved Caspase 9protein was 0.666±0.013,0.915±0.013,0.979±0.007,and the negative control group was 0.507±0.014.After comparison,the difference between the experimental group and the negative control group was statistically significant（P<0.05）;the expression of Cleaved PARP protein was 0.320±0.006,0.900±0.025,0.979±0.009,and the negative control group was 0.175±0.003.After comparison,the difference between the experimental group and the negative control group was statistically significant（P<0.05）;the expression of P65 protein was 0.720±0.006,0.630±0.008,0.439±0.003,and the negative control group was 0.811±0.011.After comparison,the difference between the experimental group and the negative control group was statistically significant（P<0.05）.Conclusion:1.In a certain concentration and time range,cardamonin has a significant inhibitory effect on the proliferation of melanoma M14 cells in a time-dose dependent manner.2 Cardamonin can induce apoptosis of M14 melanoma cells in a certain concentration and time range.As the concentration increases,the induction of apoptosis is growing.4.The apoptosis of melanoma M14 cells induced by cardamonin may be related to endogenous and exogenous apoptotic pathways. |
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