To explore the role and possible mechanism of apoptosis and caspase-3 activity in the development of multicellular drug resistance of ovary cancer. Ovarian cancer cell A2780 multicellular spheroids （MCS） were obtain...To explore the role and possible mechanism of apoptosis and caspase-3 activity in the development of multicellular drug resistance of ovary cancer. Ovarian cancer cell A2780 multicellular spheroids （MCS） were obtained from three-dimensional culture. Drug sensitivity of monolayer cells （MC） and MCS were respectively tested by MTT staining and cytometry. The apoptosis of MC and MCS were determined by the flow cytometry （FCM）. The expression of bcl-2 and caspase-3 in A2780/MC and A2780/MCS were detected by using Western blot and caspase-3 assay kit, A2780/MC was compacted into mass after 2 days in three-dimensional cell culture model, and MCS had more than two layers of cells growing within 5 days. Compared with A2780/MC, A2780/MCS were more resistant to the anticancer drug, and the apoptosis rate was significantly lower than those of A2780/MC, The activity of caspase-3 in A2780/MCS was significantly lower than the A2780/MC. But the expression of bcl-2 in A2780/MCS was significantly higher than that in A2780/MC. It was suggested that the drug resistance of MCS might be associated with the overexpression of anti-apoptosis protein bcl-2 and the down-regulation of caspase-3 activity.展开更多
Objective: To investigate the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) and the relationship with cell cycle profiles in ovarian cancer SK-OV-3ip1 multicellular aggregates. ...Objective: To investigate the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) and the relationship with cell cycle profiles in ovarian cancer SK-OV-3ip1 multicellular aggregates. Methods: Liquid overlay system was employed to obtain multicellular aggregates. Expression of P-gp and MRP was detected with flow cytometry (FCM). Outer, intermediate and inner cells from multicellular aggregates were collected by layer-trypsinized method. Cell cycle profiles were also analyzed by FCM. Results: Compared with control cells, no expression of P-gp and MRP was detected in monolyer cells (P=0.128 and P=0.604), but expression of P-gp and MRP in aggregate cells was significantly elevated (P<0.01). P-gp expression in every layer cells was also obviously increased (P<0.01). Furthermore, P-gp expression in every layer cells was also obviously increased (P=0.071). Tendency to increased G0–G1 phase and reduced S phase cells existed from outer through intermediate to inner layers in multicellular aggregates but with no statistical difference. Cell percentages in G2-M phase also had no difference. However, compared with monolayer cells, cells in G0–G1 phase increased and cells in S and G2-M phases lowered significantly in every layer and in the whole multicellular aggregates. Expression elevation of P-gp and MRP was consistent with increased G0–G1 percentage in aggregate cells. Conclusion: Expression of P-gp and MRP increases in cells of SK-OV-3ip1 multicellular aggregates and is consistent with increased G0–G1 percentage, which implies the possible relationship between them and the possible role in multicellular-mediated drug resistance.展开更多
Objective: To detect mRNA levels and expression ofCD44, CD54, CD29 and E-cadherin (E-cad) and to discuss their relationship with formation and drug resistance ofovarian cancer SKOV3ip1 multicellular aggregates.Methods...Objective: To detect mRNA levels and expression ofCD44, CD54, CD29 and E-cadherin (E-cad) and to discuss their relationship with formation and drug resistance ofovarian cancer SKOV3ip1 multicellular aggregates.Methods: Liquid overlay system was employed to obtainmulticellular aggregates. mRNA levels and expression ofCD44, CD54, CD29 and E-cad were investigated with RTPCR and flow cytometry (FCM) respectively. Results:Compared with monolayer cells, RT-PCR results showed a decrease in CD44 mRNA level by 0.626-fold and a decrease in CD29 mRNA level by 0.792-fold in multicellularaggregates. However, an increase in CD54 mRNA level by 1.815-fold and an increase in E-cadherin mRNA level by1.344-fold were found in multicellular aggregates. Theresults revealed the downregulation of CD44 and CD29 and the upregulation of CD54 and E-cad genes activity. CD44 expression in monolayer cells and multicellular aggregates were 75.995?.046 and 50.700?.351 (%) respectively andthere was a significant decrease in multicellular aggregates (P=0.001). Compared with control cells, no expression of CD54 was detected in monolayer cells (P=0.563) but markedly elevated CD54 expression was detected in multicellular aggregates (15.780?.217) (%) (P<0.01). High expression of CD29 was seen in monolayer cells and also in multicellular aggregates with positive rates of 96.290+1.201 (%) and 92.494?.055 (%). However, the expression of CD29 in multicellular aggregates was significantly reduced (P=0.014). Also no expression of E-cadherin was found in monolayer cells compared with control cells (4.490?.283) (%) (P=0.65) while significantly increased expression in aggregates cells (17.258?5.572) (%) (P=0.003) was observed. Conclusion: Significant differences in mRNA levels and expression of CD44, CD54, CD29 and E-cadherin clearly exist between monolayer cells and multicellular aggregates, which may be associated with the formation of multicellular aggregates and its drug resistance.展开更多
Objective: To investigate the chemosensitivity of ovarian cancer SK-OV-3ip1 multicellular aggregates (MCA) to cisplatin and taxol and to explore the possible mechanisms. Methods: Liquid overlay system was employed to ...Objective: To investigate the chemosensitivity of ovarian cancer SK-OV-3ip1 multicellular aggregates (MCA) to cisplatin and taxol and to explore the possible mechanisms. Methods: Liquid overlay system was employed to obtain MCA. We detected the resistance using trypan blue exclusion testing, clonogenic assay, cell cycle profiles and apoptosis with flow cytometry (FCM). Results: After cisplatin exposure, MCA cells showed nearly equal cell viability with monolayer cells (P=0.05). After 40μM cisplatin exposure for 12 h, no clone (≥50 cells) was formed, but more viable cells attached to the bottom of 24-well plate in MCA group than monolayer. Furthermore, apoptosis rate and cell cycle profiles with FCM had no significant change between MCA and monolayer cells. After taxol exposure, however, trypan blue exclusion testing demonstrated higher cell viability in MCA cells (P=0.003) and higher clone formation rate in 100-cell group than monolayer cells (0.01<P<0.025). No significant difference was found in 50-cell or 200-cell group but more viable cells in MCA group were observed. Taxol exposure caused significantly decreased apoptosis rate in MCA cells than monolayer cells (P=0.012). Taxol induced significant cell arrest at G2-M phase in monolayer cells (P=0.001), but abrogation of G2-M arrest was observed in MCA cells (P=0.002). Conclusion: Compared with monolayer cells, MCA cells from the same SK-OV-3ip1 cell line appear to be more resistant to taxol but not to cisplatin. Cell cycle redistribution and multicellular-mediated inhibition of apoptosis can partially account for the resistance.展开更多
文摘To explore the role and possible mechanism of apoptosis and caspase-3 activity in the development of multicellular drug resistance of ovary cancer. Ovarian cancer cell A2780 multicellular spheroids （MCS） were obtained from three-dimensional culture. Drug sensitivity of monolayer cells （MC） and MCS were respectively tested by MTT staining and cytometry. The apoptosis of MC and MCS were determined by the flow cytometry （FCM）. The expression of bcl-2 and caspase-3 in A2780/MC and A2780/MCS were detected by using Western blot and caspase-3 assay kit, A2780/MC was compacted into mass after 2 days in three-dimensional cell culture model, and MCS had more than two layers of cells growing within 5 days. Compared with A2780/MC, A2780/MCS were more resistant to the anticancer drug, and the apoptosis rate was significantly lower than those of A2780/MC, The activity of caspase-3 in A2780/MCS was significantly lower than the A2780/MC. But the expression of bcl-2 in A2780/MCS was significantly higher than that in A2780/MC. It was suggested that the drug resistance of MCS might be associated with the overexpression of anti-apoptosis protein bcl-2 and the down-regulation of caspase-3 activity.
基金This work was supported by the National Natural Science Foundation of China (No. 30000177).
文摘Objective: To investigate the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) and the relationship with cell cycle profiles in ovarian cancer SK-OV-3ip1 multicellular aggregates. Methods: Liquid overlay system was employed to obtain multicellular aggregates. Expression of P-gp and MRP was detected with flow cytometry (FCM). Outer, intermediate and inner cells from multicellular aggregates were collected by layer-trypsinized method. Cell cycle profiles were also analyzed by FCM. Results: Compared with control cells, no expression of P-gp and MRP was detected in monolyer cells (P=0.128 and P=0.604), but expression of P-gp and MRP in aggregate cells was significantly elevated (P<0.01). P-gp expression in every layer cells was also obviously increased (P<0.01). Furthermore, P-gp expression in every layer cells was also obviously increased (P=0.071). Tendency to increased G0–G1 phase and reduced S phase cells existed from outer through intermediate to inner layers in multicellular aggregates but with no statistical difference. Cell percentages in G2-M phase also had no difference. However, compared with monolayer cells, cells in G0–G1 phase increased and cells in S and G2-M phases lowered significantly in every layer and in the whole multicellular aggregates. Expression elevation of P-gp and MRP was consistent with increased G0–G1 percentage in aggregate cells. Conclusion: Expression of P-gp and MRP increases in cells of SK-OV-3ip1 multicellular aggregates and is consistent with increased G0–G1 percentage, which implies the possible relationship between them and the possible role in multicellular-mediated drug resistance.
基金This work was supported by the National Natural Science Foundation of China (No. 30000177).
文摘Objective: To detect mRNA levels and expression ofCD44, CD54, CD29 and E-cadherin (E-cad) and to discuss their relationship with formation and drug resistance ofovarian cancer SKOV3ip1 multicellular aggregates.Methods: Liquid overlay system was employed to obtainmulticellular aggregates. mRNA levels and expression ofCD44, CD54, CD29 and E-cad were investigated with RTPCR and flow cytometry (FCM) respectively. Results:Compared with monolayer cells, RT-PCR results showed a decrease in CD44 mRNA level by 0.626-fold and a decrease in CD29 mRNA level by 0.792-fold in multicellularaggregates. However, an increase in CD54 mRNA level by 1.815-fold and an increase in E-cadherin mRNA level by1.344-fold were found in multicellular aggregates. Theresults revealed the downregulation of CD44 and CD29 and the upregulation of CD54 and E-cad genes activity. CD44 expression in monolayer cells and multicellular aggregates were 75.995?.046 and 50.700?.351 (%) respectively andthere was a significant decrease in multicellular aggregates (P=0.001). Compared with control cells, no expression of CD54 was detected in monolayer cells (P=0.563) but markedly elevated CD54 expression was detected in multicellular aggregates (15.780?.217) (%) (P<0.01). High expression of CD29 was seen in monolayer cells and also in multicellular aggregates with positive rates of 96.290+1.201 (%) and 92.494?.055 (%). However, the expression of CD29 in multicellular aggregates was significantly reduced (P=0.014). Also no expression of E-cadherin was found in monolayer cells compared with control cells (4.490?.283) (%) (P=0.65) while significantly increased expression in aggregates cells (17.258?5.572) (%) (P=0.003) was observed. Conclusion: Significant differences in mRNA levels and expression of CD44, CD54, CD29 and E-cadherin clearly exist between monolayer cells and multicellular aggregates, which may be associated with the formation of multicellular aggregates and its drug resistance.
基金This work was supported by the National Natural Science Foundation of China (No. 30000177).
文摘Objective: To investigate the chemosensitivity of ovarian cancer SK-OV-3ip1 multicellular aggregates (MCA) to cisplatin and taxol and to explore the possible mechanisms. Methods: Liquid overlay system was employed to obtain MCA. We detected the resistance using trypan blue exclusion testing, clonogenic assay, cell cycle profiles and apoptosis with flow cytometry (FCM). Results: After cisplatin exposure, MCA cells showed nearly equal cell viability with monolayer cells (P=0.05). After 40μM cisplatin exposure for 12 h, no clone (≥50 cells) was formed, but more viable cells attached to the bottom of 24-well plate in MCA group than monolayer. Furthermore, apoptosis rate and cell cycle profiles with FCM had no significant change between MCA and monolayer cells. After taxol exposure, however, trypan blue exclusion testing demonstrated higher cell viability in MCA cells (P=0.003) and higher clone formation rate in 100-cell group than monolayer cells (0.01<P<0.025). No significant difference was found in 50-cell or 200-cell group but more viable cells in MCA group were observed. Taxol exposure caused significantly decreased apoptosis rate in MCA cells than monolayer cells (P=0.012). Taxol induced significant cell arrest at G2-M phase in monolayer cells (P=0.001), but abrogation of G2-M arrest was observed in MCA cells (P=0.002). Conclusion: Compared with monolayer cells, MCA cells from the same SK-OV-3ip1 cell line appear to be more resistant to taxol but not to cisplatin. Cell cycle redistribution and multicellular-mediated inhibition of apoptosis can partially account for the resistance.
