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    • The SKOV and SKOV TR cells were exposed to stepwise

    2024-03-07

    The SKOV3 and SKOV3TR 3,4-DAA were exposed to stepwise increased concentration of paclitaxel with or without a different concentration of autophagy inhibitor (3-MA or HCQ) to further evaluate whether suppression of autophagy affected the growth and drug sensitivity of ovarian cancer cells in vitro. As observed in the MTT assay, SKOV3TR cells exhibited stable growth in culture medium containing 0.3 μM of paclitaxel, while SKOV3 cells were unable to survive in a culture medium supplemented with >0.03 μM of paclitaxel (Fig. 3A). This result indicated that SKOV3TR cells were resistant to paclitaxel whereas SKOV3 cells were sensitive to paclitaxel. Nevertheless, when HCQ or 3-MA was added alone, no significant difference was observed in the sensitivity to autophagy inhibitors between the paired sensitive and resistant cell lines (P > 0.05) (Fig. 3B and C). Both paclitaxel and autophagy inhibitors suppressed cell proliferation in a dose-dependent manner in SKOV3TR cells. Next, we set out to assess the combination effect of paclitaxel and autophagy inhibitors. As expected, the combination of paclitaxel and autophagy inhibitors had an enhanced antiproliferative effect when compared with either agent alone (Fig. 3D and E). This inhibition effect was does-dependent. The peak synergistic effect was associated with either 0.01 μM of paclitaxel with 20 μM of HCQ (CI = 0.02, Table 1) or 0.01 μM of paclitaxel with 5 mM 3-MA (CI = 0.01, Table 1). When these SKOV3TR cells were treated with a combination of 1 μM of paclitaxel and 20 μM of HCQ or 5 mM of 3-MA, the SKOV3TR survival rate decreased to 40% of the control group (Fig. 3F and Table 1). Additionally, effects of combination treatment with paclitaxel and autophagy inhibitors on the viability of multidrug resistant osteosarcoma cell line U2OSR2 were also evaluated (Supplementary Fig. S3). Taken together, these results suggest that when combined with autophagy inhibitors, MDR ovarian cancer cells have an enhanced response to paclitaxel. Autophagy inhibitors reduce MDR ovarian cancer cell migration in vitro. Migration of ovarian cancer cells causes metastasis and recurrence. Considering that the TMA results of ovarian cancer patients showed that autophagy expression was significantly correlated with metastasis and recurrence, we investigated the role of autophagy in the modulation of cell mobility in vitro. The wound healing assay was performed in a MDR ovarian cancer cell line post autophagy inhibitor treatment. As illustrated in Fig. 4A, exposure to 20 μM of HCQ and 5 mM of 3-MA for 24, 48, and 72 h, the cell migration activities were time-dependently repressed in the SKOV3TR cell line, as compared with the control groups (P < 0.05). During the 72-hour incubation, the relative migratory distances of SKOV3TR cells treated with 20 μM of HCQ and 5 mM of 3-MA were 70 and 45 μm, while relative migratory distance of untreated cells was 170 μm (Fig. 4B). The results demonstrate that the inhibition of autophagy suppresses the migratory capabilities of drug resistant ovarian cancer cells. p62 accumulation enhances paclitaxel sensitivity in MDR ovarian cancer cells in vitro. To further evaluate whether inhibition of autophagy affects drug sensitivity, we set out to enhance p62 expression by transfection of mCherry-Sequestosome1-N-18 plasmid in SKOV3TR cells. Considering that p62 is inversely correlated with autophagy activity, p62 accumulation may induce autophagy reduction. Total proteins were extracted to detect the efficacy of p62 plasmid transfection in vitro. It was found that p62 expression was three times greater than the p62 level of untreated cells, which was determined after cell sorting by flow cytometry to separate the cells with successful mCherry-Sequestosome1-N-18 plasmid transfection (Fig. 5A and B). Conversely, the expression of autophagy marker proteins, LC3-II, Atg5 and Beclin-1, were significantly downregulated in transfection cells (P < 0.05). Additionally, we found that, compared to untreated cells, sensitivity to paclitaxel was promoted when p62 was overexpressed (Fig. 5C). Taken together, we conclude that autophagy may play a critical role in maintaining drug sensitivity in ovarian cancer cells.