ABSTRACT
Objective: To explore the effect of gomisin A (GA) in combination with carboplatin (CBP) on the apoptosis of ovarian cancer Skov3 cells, and to illustrate the synergistic antitumor effect of GA. Methods: The ovarian cancer Skov3 cells were treated with GA combined with CBP. MTT assay was used to detect the inhibitory rates of proliferation and the appropriate concentrations of GA and CBP were selected according to the results. The Skov3 cells were divided into control group, GA (0. 04 μmol · L-1) group, CBP (16 mg · L-1) group and GA (0.04 μmol · L-1) + CBP (16 mg · L-1) group. After 48 h treatment, the cell morphology was observed by microscope, the apoptotic rate was measured by flow cytometry, the apoptotic index (AI) was observed by TUNEL staining, the mitochondrial membrane potential was detected by JC-1 staining, and the mRNA and protein expression levels of apoptosis-related genes (Bax, caspase-3, Stat3) were determined by RT-PCR and Western blotting method. Results: The inhibitory rate of proliferation of Skov3 cells in GA + CBP group (52. 1%) was significantly higher than those in GA and CBP groups (P<0. 01). The concentrations of GA and CBP for the best dose ratio were 0. 04 μmol · L-1 and 16 mg · L-1, respectively. Compared with control group, the cell refractivities were decreased, the cells retracted, and part of cells were suspended in GA group and CBP groups; there were more suspended cells and cell retraction phenomenon was more prominent in GA + CBP group. The results of TUNEL staining and flow cytometry showed that the AI and apoptotic rate of cells in GA + CBP group were significantly increased compared with control group, GA and CBP group (P<0. 05 or P<0. 01); the JC-1 staining results suggested that the mitochondrial membrane potential of Skov3 cells in GA + CBP group was decreased (P<0. 05 or P<0. 01). The RT-PCR and Western blotting results showed that the expression levels of Bax and caspase-3 mRNA and protein were up-regulated (P<0. 05) and the expression levels of Bcl-2 and Stat3 mRNA and protein in GA + CBP group were down-regulated compared with control, GA and CBP groups (P< 0. 05). Conclusion: GA can enhance the ability of CBP to induce the apoptosis of human ovarian cancer Skov3 cells, and its mechanisms may be related to the up-regulation of the Bax and Caspase-3 expressions and the down-reguation of the Bcl-2 expression; GA can synergize the induction of CBP on apoptosis.
ABSTRACT
To explore the inhibitory effects of gomisin A (GA) in combination with carboplatin (CBP) on the proliferation, invasion and metastasis of human ovarian cancer Skov3 cells and their mechanisms, and to illustrate the synergistic antitumor effect of GA. Methods: The ovarian cancer Skov3 cells were treated with different concentrations of GA combined with different concentrations of CBP. MTT assay was used to detect the inhibitory rates of proliferation. According to the results, the appropriate concentrations of GA and CBP were conformed. The Skov3 cells were treated with PBS, GA 0.04 μmol · L-1), CBP 16 mg · L-1) and GA 0.04 jumol · L-1) in combination with CBP 16 mg · L-1), respectively, and used as control group, GA group, CBP group and GA+CBP group. After 48 h of treatment, the cell morphology was observed by optical microscope, the cell reproductive ability was determined by clonogenic cell survival assay, and the cell migration ability was measured by cell wound healing test; Transwell experiment was used to detect the cell invasion ability. The expression levels of MMP-2 and MMP-9 mRNA were determined by RT-PCR and the protein expression levels of MMP-2, MMP-9, AKT1 and pAKT1 were determined by Western blotting method. Results: Compared with GA and CBP groups, the inhibitory rate of proliferation of Skov3 cells in GA + CBP group was significantly increased (P<0.01). The concentrations of GA and CBP for the best dose ratio were 0.04 μmol · L-1 and 16 mg · L-1, respectively. The results of clone formation assay showed that the colony formation rates in CBP and GA+CBP groups were decreased compared with control group (P<0.05 or P<0.01); the colony formation rate of cells in GA + CBP group was significantly decreased compared with GA and CBP groups (P<0.01); the wound scratch assay results suggested that the number of metastic Skov3 cells in GA and CBP groups were decreased compared with control group; and the number of metastic Skov3 cells in GA + CBP group was decreased compared with GA and CBP groups. The results of Transwell expriment showed that the capacities of cells passing the ECM in GA and CBP groups were decreased compared with control group; the capacity of cells passing the ECM was decreased compared with GA and CBP groups. The RT-PCR and Western blotting results showed that the expression levels of MMP-2 and MMP-9 mRNA in GA+CBP group were down-regulated compared with GA and CBP groups (P< 0.01), and the expression levels of MMP-2, MMP-9, AKT1 and pAKTl protein were down-regulated (P<0.01). Conclusion: GA can enhance the ability of CBP to inhibit the proliferation, invasion and metastasis of human ovarian cancer Skov3 cells, and thus play a role in decreasing the toxicity and increasing the efficacy of chemotherapy.
ABSTRACT
Objective:To explore the inhibitory effects of gomisin A(GA)in combination with carboplatin (CBP)on the proliferation,invasion and metastasis of human ovarian cancer Skov3 cells and their mechanisms,and to illustrate the synergistic antitumor effect of GA.Methods:The ovarian cancer Skov3 cells were treated with different concentrations of GA combined with different concentrations of CBP.MTT assay was used to detect the inhibitory rates of proliferation.According to the results,the appropriate concentrations of GA and CBP were conformed.The Skov3 cells were treated with PBS,GA(0.04 μmol·L-1),CBP(16 mg·L-1)and GA (0.04 μmol·L-1)in combination with CBP(16 mg·L-1),respectively,and used as control group,GA group, CBP group and GA+CBP group.After 48 h of treatment,the cell morphology was observed by optical microscope, the cell reproductive ability was determined by clonogenic cell survival assay,and the cell migration ability was measured by cell wound healing test;Transwell experiment was used to detect the cell invasion ability.The expression levels of MMP-2 and MMP-9 mRNA were determined by RT-PCR and the protein expression levels of MMP-2,MMP-9,AKT1 and pAKT1 were determined by Western blotting method.Results:Compared with GA and CBP groups,the inhibitory rate of proliferation of Skov3 cells in GA + CBP group was significantly increased (P<0.01).The concentrations of GA and CBP for the best dose ratio were 0.04 μmol·L-1and 16 mg·L-1, respectively.The results of clone formation assay showed that the colony formation rates in CBP and GA+CBP groups were decreased compared with control group(P<0.05 or P<0.01);the colony formation rate of cells in GA + CBP group was significantly decreased compared with GA and CBP groups(P<0.01);the wound scratch assay results suggested that the number of metastic Skov3 cells in GA and CBP groups were decreased compared with control group;and the number of metastic Skov3 cells in GA+ CBP group was decreased compared with GA and CBP groups.The results of Transwell expriment showed that the capacities of cells passing the ECM in GA and CBP groups were decreased compared with control group;the capacity of cells passing the ECM was decreased compared with GA and CBP groups.The RT-PCR and Western blotting results showed that the expression levels of MMP-2 and MMP-9 mRNA in GA+CBP group were down-regulated compared with GA and CBP groups(P<0.01),and the expression levels of MMP-2,MMP-9,AKT1 and pAKT1 protein were down-regulated(P<0.01).Conclusion:GA can enhance the ability of CBP to inhibit the proliferation,invasion and metastasis of human ovarian cancer Skov3 cells,and thus play a role in decreasing the toxicity and increasing the efficacy of chemotherapy.
ABSTRACT
Objective:To explore the effect of gomisin A (GA) in combination with carboplatin (CBP) on the apoptosis of ovarian cancer Skov3 cells,and to illustrate the synergistic antitumor effect of GA.Methods:The ovarian cancer Skov3 cells were treated with GA combined with CBP.MTT assay was used to detect the inhibitory rates of proliferation and the appropriate concentrations of GA and CBP were selected according to the results.The Skov3 cells were divided into control group,GA (0.04 μmol · L-1) group,CBP (16 mg · L 1) group and GA (0.04 μmol· L-1)+ CBP (16 mg · L-1) group.After 48 h treatment,the cell morphology was observed by microscope,the apoptotic rate was measured by flow eytometry,the apoptotic index (AI) was observed by TUNEL staining,the mitochondrial membrane potential was detected by JC-1 staining,and the mRNA and protein expression levels of apoptosis-related genes (Bax,caspase-3,Stat3) were determined by RT PCR and Western blotting method.Results:The inhibitory rate of proliferation of Skov3 cells in GA + CBP group (52.1％) was significantly higher than those in GA and CBP groups (P＜0.01).The concentrations of GA and CBP for the best dose ratio were 0.04 μmol· L-1 and 16 mg · L-1,respectively.Compared with control group,the cell refractivities were decreased,the cells retracted,and part of cells were suspended in GA group and CBP groups;there were more suspended cells and cell retraction phenomenon was more prominent in GA + CBP group.The results of TUNEL staining and flow cytometry showed that the AI and apoptotic rate of cells in GA + CBP group were significantly increased compared with control group,GA and CBP group (P＜0.05 or P＜0.01);the JC-1staining results suggested that the mitochondrial membrane potential of Skov3 cells in GA + CBP group was decreased (P＜0.05 or P＜0.01).The RT-PCR and Western blotting results showed that the expression levels of Bax and caspase-3 mRNA and protein were up-regulated (P＜0.05) and the expression levels of Bcl-2 and Stat3mRNA and protein in GA + CBP group were down-regulated compared with control,GA and CBP groups (P＜0.05).Conclusion:GA can enhance the ability of CBP to induce the apoptosis of human ovarian cancer Skov3 cells,and its mechanisms may be related to the up-regulation of the Bax and Caspase-3 expressions and the down-reguation of the Bcl-2 expression;GA can synergize the induction of CBP on apoptosis.