Beta-galactoside α2,3-sialyltransferase Ⅲ Modulates The Paclitaxel Sensitivity Of Human Ovarian Cancer Cells In Vitro

Objective: The study is to investigate the effect of α2,3-sialyltransferase Ⅲ(ST3Gal-Ⅲ) on the sensitivity of ovarian cancer cells to paclitaxel(PTX) in vitro, and explore the synergistic effects of ST3Gal-Ⅲ silencing and anti-cancer drugs on the apoptosis of human ovarian cancer, which may provide innovative strategies for clinical treatment of recurrent and resistant ovarian cancer.Methods: 1. Identification of the differential expression of ST3Gal-Ⅲ in human ovarian cancer cell lines HO8910 PM and SKOV3. The ST3Gal-Ⅲ m RNA expression was detected by real-time fluorescence quantitative PCR(q RT-PCR). The mean fluorescence intensity of biotinylated Maackia amurensis lectin II(MALII) on the cell surface was determined by flow cytometry. 2. Detection of the PTX sensitivity of ovarian cancer cell lines with high and low expression of ST3Gal-Ⅲ. The change of cell proliferation was observed by Cell Counting Kit-8(CCK-8) assay after treatment with different doses of PTX for 24 h, and the 50% inhibitory concentration(IC50) of PTX was calculated to analysis the correlation between the PTX sensitivity of ovarian cancer cell lines and the expression of ST3Gal-Ⅲ. 3. Determination of the silencing effect of ST3Gal-Ⅲ si RNA. The m RNA expression of ST3Gal-Ⅲ in HO8910-PM cells with high expression of ST3Gal-Ⅲ after transfection with ST3Gal-Ⅲ si RNA was detected by q RT-PCR. 4. Detection of the apoptosis of HO8910-PM cells treated with ST3Gal-Ⅲ si RNA and PTX. Western blot analysis was used to detect the protein expression of caspase-3 and caspase-8 in HO8910-PM cells which were successively treated with ST3Gal-Ⅲ si RNA and PTX. Meanwhile, the apoptotic rate was measured by flow cytometry and TUNEL staining. 5. Measurement of the effect of PTX on ST3Gal-Ⅲ m RNA expression in SKOV3 cells with low expression of ST3Gal-Ⅲ. The m RNA expression of ST3Gal-Ⅲ in SKOV3 cells treated with different concentrations of PTX for different time was detected by q RT-PCR.By comparing the difference expression of ST3GalⅢ genes in SKOV3 cells which were treated with different concentrations of paclitaxel in different time,the effecting law and action mechanism of paclitaxel have been discussed. 6. q RT-PCR was used to detect ST3Gal-Ⅲ m RNA expression in SKOV3 cells which were successively treated with PTX and ST3Gal-Ⅲ si RNA. 7. Detection of the apoptosis of SKOV3 cells treated with ST3Gal-Ⅲ si RNA and PTX. Western blot analysis was used to detect the protein expression of caspase-3 and caspase-8in SKOV3 cells which were successively treated with PTX and ST3Gal-Ⅲ si RNA. Meanwhile, the apoptotic rate was measured by flow cytometry and TUNEL staining.Results: 1. SKOV3 and HO8910 PM cells showed significantly different expression of ST3Gal-Ⅲ on the cell surface. q RT-PCR results elucidated that ST3Gal-Ⅲ m RNA expression in HO8910 PM cells was higher than that in SKOV3 cells(P<0.05). Flow cytometric analysis showed that the mean fluorescence intensity of biotinylated MALII(indicating α2,3-sialic acid on the cell surface) in HO8910 PM cells was higher than that in SKOV3 cells(P<0.05). Therefore, HO8910 PM and SKOV3 were identified as a high ST3Gal-Ⅲ expression cell line and the low expression one 2. SKOV3 and HO8910 PM cells with different expression levels of ST3Gal-Ⅲ had different sensitivities to PTX. The CCK-8 results indicated that the inhibitory rate of HO8910 PM cells treated with different doses of PTX for 24 h was obviously lower than that of SKOV3 cells, and the IC50 of PTX for HO8910 PM cells(395.078 nmol/L) was almost 3 times as high as that for SKOV3 cells(130.645 nmol/L). 3. ST3Gal-Ⅲ si RNA effectively down-regulated the expression of ST3Gal- Ⅲ m RNA in HO8910 PM cells. q RT-PCR analysis showed that the relative expression of ST3Gal-Ⅲ m RNA in si RNA group was 0.239±0.859 times as much as that in control group(P<0.01), while that in PTX+si RNA group was 0.184±0.037 times as much as that in PTX+non-si RNA group(P<0.01). 4. Apoptosis and increase in apoptosis-related protein expression were found in HO8910 PM cells which were successively treated with ST3Gal-Ⅲ si RNA and PTX. Western blot assay indicated that ST3Gal-Ⅲ protein expression in PTX+si RNA group and si RNA group was lower than that in other groups, and the apoptosis-related protein expression in si RNA group was significantly higher than that in other groups. The results of apoptotic rates measured by flow cytometry and TUNEL staining also validated the above-mentioned result. 5. The ST3Gal-Ⅲ m RNA expression in SKOV3 cells with low expression of ST3GalⅢ was up-regulated after treatment with PTX. q RT-PCR analysis showed that PTX, as a cell cycle-specific drug, up-regulated ST3Gal-Ⅲ m RNA expression in SKOV3 cells in a dose- and time-dependent manner. Treatment with 50 nmol/L PTX for 48 h could exert the best up-regulation effect. 6. The ST3Gal-Ⅲ m RNA expression in SKOV3 cells which were successively treated with PTX and ST3Gal-Ⅲ si RNA. q RT-PCR results showed that ST3Gal-Ⅲ m RNA expression in PTX+si RNA group was lower than that in other groups, and that in PTX96 h group, PTX+non-si RNA group and PTX48 h group was higher than that in control group. 7. Apoptosis and increase in apoptosis-related protein expression were found in SKOV3 cells which were successively treated with PTX and ST3Gal-Ⅲ si RNA. Western blot analysis showed that the apoptosis-related protein expression in si RNA group was significantly higher than that in other groups. The results of apoptotic rates measured by flow cytometry and TUNEL staining also validate the above-mentioned result.Conclusions: 1. The ST3Gal-Ⅲ expression in HO8910 PM cells are higher than those in SKOV3 cells. 2. SKOV3 cells with low expression of ST3Gal- Ⅲ are more sensitive to PTX, whileHO8910PM cells with high expression of ST3Gal-Ⅲ show stronger drug resistance. 3. Treatment with ST3Gal-Ⅲ si RNA plus PTX enhances chemotherapeutic response and reverses anti-cancer drug resistance in HO8910 PM cells with high expression of ST3Gal-Ⅲ. 4. PTX up-regulates the ST3Gal-Ⅲ m RNA expression in SKOV3 cells and enhances their resistance to PTX. Down-regulation of PTX-induced high expression of ST3Gal-Ⅲ in SKOV3 cells can reduce their resistance to PTX. 5. ST3Gal-Ⅲ can enhance the resistance of ovarian cancer cells to PTX, and might protect ovarian cancer cells against apoptosis through caspase signaling pathway.