Effects of L-OHP and DAPT, alone or in combination, on the proliferation of human ovarian cancer stem cells
Different concentrations of L-OHP inhibited the proliferation of ovarian cancer stem cells to varying degrees. This effect was significant when compared with the control group (P < 0.05). Pearson correlation analysis revealed clear dose- (r = 0.773) and time-dependent effects (r = 0.591; Table 1). Similarly, different concentrations of DAPT also inhibited the proliferation of ovarian cancer stem cells to varying degrees. Compared with control, these effects were statistically significant (P <0.05). Pearson correlation analysis revealed obvious dose- (r = 0.819) and time-dependent effects (r = 0.520; Table 2).
Next, the effect of co-treatment with L-OHP (2.5, 5, and 7.5 µg/ml) and DAPT (20, 40, 80 and µmol/L) was assessed in ovarian cancer stem cells. Increasing drug concentrations inhibited cell proliferation significantly (P <0.05) compared with the control group and the corresponding monotherapy groups. According to the Q method reported by Zhang SQ [6], co-treatment with 2.5 µg/ml L-OHP and 7.5 µg/ml DAPT had a clear additive effect (q = 0.85–1.15). When 5µg/ml L-OHP was used, synergistic effects occurred after co-treatment with 20, 40, or 80 µmol/L DAPT (q >1.15). Treatment with 5µg/ml L-OHP alone inhibited proliferation by 38.24%; co-treatment with 20, 40, and 80 µmol/L DAPT inhibited the 24 h proliferation rate of ovarian cancer stem cells by 60.13%, 68.17%, and 71.33%, respectively. Therefore, the inhibition rate increased with increasing concentrations of DAPT. This result demonstrates that DAPT synergizes with L-OHP to inhibit the proliferation of ovarian cancer stem cells (Table 3).
Effects of L-OHP and DAPT, alone or in combination, on the morphology of human ovarian cancer stem cells
Under an inverted microscope, control cells exhibited an oval or polygonal morphology, with a round shape and a large nucleus. However, after 24 h treatment with 5 µg/ml L-OHP, blurred cell membranes were observed, with an enlarged cell gap, and increased apoptosis. Similarly, 24 h treatment with 40 µg/ml DAPT resulted in fusiform, shrunk, and cracked cells with a reduced cell density, enlarged cell gaps, an increased number of nuclear particles, and enhanced apoptosis. After 24 h of treatment with the combination of 40 µg/ml DAPT and 5 µg/ml L-OHP, the cell number was reduced further; in addition, more cells had disintegrated and were undergoing apoptosis (Fig.1).
Increased apoptosis in human ovarian cancer stem cells treated with L-OHT and DAPT alone and in combination
Flow cytometry revealed that the apoptosis rate of cells in the L-OHP (17.7 ± 0.6%) and DAPT groups (15.3 ± 0.2%) was increased significantly compared with the control group (8.1 ± 0.6%; P < 0.05). As expected, the apoptosis rate in the DAPT + L-OHP group (36.5 ± 0.3%) was significantly higher than in the L-OHP or DAPT groups (P <0.05; Fig.2).
Synergistic effects of DAPT and L-OHP on inhibiting the invasion of human ovarian cancer stem cells
The rates of invasion inhibition in the DAPT and L-OHP alone groups were 40.3 ± 4.2% and 50.2 ± 7.8%, respectively. However, co-treatment inhibited invasion by 75.1 ± 2.9%. This suggests that L-OHP could enhance the DAPT-mediated inhibition of cell invasion in ovarian cancer stem cells (Fig.3).
Effects of L-OHP and DAPT, alone or in combination, or Notch-1 and LRP expression in human ovarian cancer stem cells
There were no significant differences in Notch-1 protein expression between the L-OHP (0.849 ± 0.02) and control groups (0.861 ± 0.01; P > 0.05). However, Notch-1 expression was reduced significantly in the DAPT (0.644 ± 0.02) and DAPT + L-OHP groups (0.517 ± 0.05; both P < 0.05). In addition, the DAPT + L-OHP group had significantly lower Notch-1 expression than the DAPT group (P < 0.05).
LRP protein levels in the L-OHP group (0.811 ± 0.03) were significantly higher than in the control group (0.686 ± 0.03; P < 0.05). In contrast, levels in the DAPT (0.487 ± 0.01) and DAPT + L-OHP groups (0.318 ± 0.04) were significantly lower than in the control and L-OHP groups (both P < 0.05). Finally, LRP expression was significantly lower in the DAPT + L-OHP group than in the DAPT group (P <0.05; Fig.4).