| ObjectiveTo investigate C21 steroids from Tong-Guang-San (Marsdenia tenacissima), a Chinese medicine, for their activities and possible mechanisms in enhancing growth inhibitory effect of paclitaxel in tumor cells and tumor xenografts.MethodsEvaluation of growth inhibitory effect of paclitaxel in tumor cells or tumor xenografts:MTT assay was used for measurement of IC5o value (concentration to inhibit 50% of the cell growth) of paclitaxel in tumor cells including human cervical carcinoma cells (Hela), human colorectal adenocarcinoma cells (HCT-15) and human breast cancer cells (MCF-7), in the presence or absence of a test C21 steroid; tumor bearing nude mouse model was used to evaluate the inhibitory effect of paclitaxel alone or paclitaxel in combination with a test compound on the growth of HeLa xenograft.Mechanistic investigation:In HeLa cells, cell cycle was analyzed by flow cytometry combined with PI staining, while apoptosis was detected by flow cytometry combined with Annexin V-FITC/PI double staining; expression of cellular bcl-2, bax, p53 and caspase-9 was detected by Western-blot analysis, for investigating the mechanism of Marsdenia tenacissima monomer component W-9 in enhancing antitumor effect of paclitaxel.Results(1) Two C21 steroidal aglycones and their glycosides were investigated, they are 11α-O-2-methylbutanoyl-12β-O-tigloyl-tenacigenin B (W-7),11a-O-2-methylbutanoyl-12β-O-benzoyl-tenacigenin B (W-9), marsdenoside A (W-13) and marsdenoside C (W-17).(2) All four compounds, at their non-toxic concentrations, dose-dependently decreased the IC50 values of paclitaxel in human cervical carcinoma cell line HeLa and human colorectal cancer cell line HCT-15, but not in human breast cancer cell line MCF-7.(3) In nude mice bearing HeLa tumors, W-9 or W-13 alone (30 mg/kg/d,10 d, orally) did not affect tumor growth. Paclitaxel alone (10 mg/kg every two days,10 d, intraperitoneally) showed certain inhibition on tumor growth. But the combination of paclitaxel and W-9 or the combination of paclitaxel and W-13 achieved significant inhibition to tumor growth. Tumors in these two groups were obviously smaller and lighter than those in mice treated with paclitaxel alone (P<0.001).(4) In HeLa cell, paclitaxel could arrest cell cycle in S phase and induce apoptosis,10μM of W-9 alone could not affect the cell cycle and apoptosis, while the combination of 2.5 nM paclitaxel and non-toxic concentration of W-9 achieved stronger cell cycle arrest and higher apoptosis rate than paclitaxel alone (P<0.001).(5) 10μM W-9 and 2.5 nM paclitaxel alone or in combination with each other were applied to HeLa cells for 48h. Cells were collected by centrifugation, cell apoptosis was detected by flow cytometry. It was found that apoptosis rate in HeLa cells was 34.3±1.1% for treatment with paclitaxel in combination with W-9, which was significantly greater than 14.6±2.7% or 7.3±0.2% in treatment with paclitaxel or W-9 alone (P<0.001).(6) In Western blot analysis, lower level of bcl-2, higher level of Bax and p53, and activated caspase-9 were observed in HeLa cells treated with 2.5 nM paclitaxel, or the combination of W-9 and paclitaxel. The most significant effect was achieved by the combination of W-9 and paclitaxel, Bax/Bcl-2 ratio (P<0.05) and p53 expression level (P<0.01) in these cells were obviously higher than those in cells treated with paclitaxel alone.ConclusionTenacigenin B ester derivatives from Marsdenia tenacissima enhanced growth inhibitory effect of paclitaxel in HeLa cells and HCT-15 cells but not in MCF-7 cells. Activity of W-9 and W-13 in enhancing paclitaxel antitumor effect had been confirmed using HeLa tumor-bearing nude mouse model. These compounds may play a role in common mitochondrial pathway by altering the permeability of the outer membrane of mitochondria to induce apoptosis in HeLa cell. |
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