Antitumor Effect And Mechanisms Of DMDD Isolated From The Root Of Averrhoa Carambola L.on Breast And Liver Cancer In Vitro

Objective: To investigate the antitumor effect of 2-dodecyl-6-methoxy-cyclohexa-2,5-diene-1,4-dione(DMDD)isolated from the root of Averrhoa carambola L.on 4T1 breast cancer cells and BEL-7404 liver cancer cells and the regulation of DMDD on MAPK signaling pathway.Methods:1.The part of anti-breast cancer:4T1 breast cancer cells were cultured in vitro and treated with different concentrations of DMDD.The effect of DMDD on the proliferation of 4T1 cells was detected by MTT method and clone formation experiment.Effect of DMDD on apoptosis of 4T1 cells was observed with AO/EB double staining method.Effect of DMDD on 4T1 cell cycle was detected by flow cytometry.The effects of DMDD on the migration and invasion of 4T1 cells were detected by scratch test and Transwell chamber method.The expression levels of related genes and related proteins were detected by PCR and WB.2.The part of anti-liver cancer:BEL-7404 liver cancer cells were cultured in vitro and treated with different concentrations of DMDD.The effect of DMDD on the proliferation of BEL-7404 cells was detected by MTT method and clone formation experiment.Effect of DMDD on apoptosis of BEL-7404 cells was observed with AO/EB double staining method.Effect of DMDD on BEL-7404 cell cycle was detected by flow cytometry.The effects of DMDD on the migration and invasion of BEL-7404 cells were detected by scratch test and Transwell chamber method.The expression levels of related genes and related proteins were detected by PCR and WB.Results:1.The part of anti-breast cancer: The result of MTT showed that DMDD could inhibit the proliferation of 4T1 cells in a dose-and time-dependent manner(P<0.01).Clone formation experiments showed that DMDD can inhibit the formation of 4T1 cell colonies in a dose-dependent manner(P<0.01).AO/ EB double staining method showed that DMDD can significantly induce apoptosis in 4T1 cells.The results of flow cytometry showed that compared with the control group,with the increase of DMDD concentration,the number of 4T1 cells in the G1 phase gradually increased,and the number of cells in the S/G2 phase gradually decreased(P<0.05 or P<0.01).The scratch test results showed that compared with the control groups,the cell migration rates of the three concentration experimental groups decreased significantly(P<0.05 or P<0.01).Transwell invasion experiment results showed compared with the control group,the number of invasive cells of the three concentration experimental groups decreased significantly(P<0.01).The results of RT-PCR showed that compared with the control group,the raf1,mek1,mek2,erk1,erk2,and bcl2 gene expressions in the DMDD dose groups were significantly down-regulated(P<0.05 or P<0.01),and the bax gene expression in the DMDD dose groups was significantly up-regulated(P<0.01).WB experiment results show that compared with the control group,p-RAF1,p-MEK,p-ERK,p-p38,Bcl2,MMP2 and MMP9 protein expression in the DMDD dose groups were down-regulated,and p-JNK and Bax protein expression in the DMDD dose groups were up-regulated(P<0.05 or P<0.01).2.The part of anti-liver cancer:The result of MTT showed that DMDD could inhibit the proliferation of BEL-7404 cells in a dose-and time-dependent manner(P<0.01).Clone formation experiments showed that DMDD can inhibit the formation of BEL-7404 cells colonies in a dose-dependent manner(P<0.01).AO/EB double staining method showed that DMDD can significantly induce apoptosis in BEL-7404 cells.The results of flow cytometry showed that compared with the control group,with the increase of DMDD concentration,the number of BEL-7404 cells in the G1 phase gradually increased,and the number of cells in the S/G2 phase gradually decreased(P <0.05 or P <0.01).The scratch test results showed that compared with the control groups,the cell migration rates of the three concentration experimental groups decreased significantly(P<0.01).Transwell invasion experiment results showed compared with the control group,the number of invasive cells of the three concentration experimental groups decreased significantly(P<0.01).The results of RT-PCR showed that compared with the control group,the raf1,mek1,mek2,erk1,erk2,and bcl2 gene expressions in the DMDD dose groups were significantly down-regulated(P<0.05 or P<0.01),and the bax gene expression in the DMDD dose groups was significantly up-regulated(P<0.01).WB experiment results show that compared with the control group,p-RAF1,p-MEK,p-ERK,p-p38,Bcl2,MMP2 protein expression in the DMDD dose groups were down-regulated,and p-JNK and Bax protein expression in the DMDD dose groups were up-regulated(P<0.05 or P<0.01).Conclusions: DMDD can inhibit the proliferation,migration,and invasion of4T1 breast cancer cells and BEL-7404 liver cancer cells,promote the apoptosis of 4T1 breast cancer cells and BEL-7404 liver cancer cells,andinduce G1 cell cycle arrest.The mechanism may be related to MAPK signal pathways.