目的 评价通关藤中4种C21甾体化合物(通关藤苷I、通关藤苷G、通关藤苷H和17β-通关藤苷元B)对人卵巢癌紫杉醇耐药细胞A2780/Taxol(A2780/T)耐药的逆转作用及相关机制。方法 将人卵巢癌A2780/T细胞分为对照组、C21甾体单用组、紫杉醇单用组、C21甾体联合紫杉醇组(1:1),MTT法检测细胞增殖能力,中效原理分析两药联用效应(Fa)与合用指数(CI)的关系;划痕实验观察细胞迁移能力;流式细胞术及Hoechst 33258染色检测细胞周期和凋亡情况;RT-PCR、Western blot法分别检测ABCB1、SLC4A2、SLCO1A2 mRNA及P-gp表达水平;药物外排实验检测A2780/T细胞内紫杉醇及罗丹明123的蓄积量。结果 4种C21甾体单用或与紫杉醇联用均能抑制A2780/T细胞增殖,且呈剂量依赖效应。通关藤苷G、通关藤苷H与紫杉醇联用48 h具有协同效应,能够显著抑制A2780/T细胞迁移、阻滞细胞于G2/M期、促进细胞凋亡,显著抑制ABCB1、SLC4A2、SLCO1A2 mRNA水平,下调P-gp表达,显著促进了紫杉醇及罗丹明123在A2780/T细胞内的蓄积。结论 通关藤苷G、通关藤苷H与紫杉醇联用均产生协同效应,可逆转人卵巢癌A2780/T细胞对紫杉醇的耐药性,其机制可能与降低基因ABCB1、SLC4A2、SLCO1A2及P-gp的表达水平,促进紫杉醇在A2780/T细胞内蓄积有关。
Abstract
OBJECTIVE To evaluate four C21 steroids in Marsdenia tenacissima, tenacissoside I (Tsd-I), tenacissoside G (Tsd-G), tenacissoside H (Tsd-H), 17β-tenacigenin B (17β-ten-B), on reversing paclitaxel resistance of the human paclitaxel-resistant ovarian cancer cells A2780/Taxol (A2780/T), and explore the underlying mechanism. METHODS Human ovarian cancer cells A2780/T were divided into four groups: control group, C21 steroid monotherapy group, PTX monotherapy group, and C21 steroid combined with PTX group (1∶1). Cell proliferation ability was detected by MTT assay. The relationship between combined action (Fa) and combination index (CI) of the two agents was analyzed by the median-effect principle. Furthermore, cell migration was observed by cell scratch experiment. Flow cytometry and Hoechst 33258 staining were used to observe the effects of drugs on cell cycle, apoptosis and apoptosis morphology. Simultaneously, the mRNA expressions of ABCB1, SLC4A2 and SLCO1A2 was detected by RT-PCR, and the P-gp level was detected by Western blot.Drug efflux assay was performed to detect the accumulation of PTX and rhodamine 123 in A2780/T cells. RESULTS C21 steroid alone or in combination with PTX could suppress the proliferation of A2780/T cells with a dose-dependent effect. Tsd-G, Tsd-H combined with PTX for 48 h had synergistic effect, and could significantly inhibit A2780/T cells migration, arrest cells in G2/M phase, promote cell apoptosis, reduce ABCB1, SLC4A2, SLCO1A2 mRNA level, and further decrease P-gp expression level which significantly promoted PTX and Rhodamine 123 accumulation in A2780/T cells. CONCLUSION The combination of Tsd-G and Tsd-H with PTX respectively can reverse the paclitaxel-resistance of human ovarian cancer A2780/T cells.The mechanism may be related to the inhibition of the expression levels of ABCB1, SLC4A2, SLCO1A2 mRNA and P-gp, and the promotion of PTX accumulation in A2780/T cells.
关键词
通关藤 /
C21甾体化合物 /
紫杉醇 /
卵巢癌 /
中效原理 /
逆转耐药
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Key words
Marsdenia tenacissima /
C21 steroid /
paclitaxel /
ovarian cancer /
median-effect principle /
reversing drug-resistance
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脚注
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基金
国家自然科学基金项目资助(817734949,82003987);上海市自然科学基金项目资助(20ZR142400);上海市浦东新区科技发展基金专项基金(医疗卫生)资助(PKJ2020-Y08,PKJ2020-Y11);上海市浦东新区卫健委卫生行业专项资助(PW2021E-03)
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