BMP4增强结直肠癌耐药细胞化疗敏感性的作用及机制

BMP4 Enhances the Chemosensitivity of Drug-resistant Colorectal Cancer Cells

目的研究骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)对结直肠癌耐药细胞化疗敏感性的影响及机制。方法采用5-氟尿嘧啶(5-Fu)处理结直肠癌细胞株LoVo,建立耐药细胞株LoVo/5-Fu;通过CCK-8法检测LoVo/5-Fu细胞和亲本细胞对5-Fu、奥沙利铂或伊立替康的敏感性,并检测不同浓度BMP4对LoVo/5-Fu细胞生长的抑制效应;用5-Fu联合BMP4处理LoVo/5-Fu细胞3d,采用Annexin-Ⅴ/PI法检测5-Fu联合BMP4处理后LoVo/5-Fu细胞的凋亡情况;通过裸鼠体内成瘤实验观察BMP4在体内对LoVo/5-Fu细胞化疗敏感性的影响;采用Western blot检测BMP4处理后LoVo/5-Fu细胞中AKT信号通路活性和Bcl-2的蛋白水平。结果 5-Fu诱导形成的耐药细胞株LoVo/5-Fu可以在含5μg/mL 5-Fu的培养液中稳定生长,并对奥沙利铂和伊立替康有交叉耐药;BMP4可以抑制LoVo/5-Fu细胞的生长,抑制效应呈时间和浓度依赖性;细胞凋亡实验显示BMP4可以促进培养在含5μg/mL 5-Fu培养液中的LoVo/5-Fu细胞发生凋亡;裸鼠体内成瘤实验显示BMP4可以有效增强LoVo/5-Fu细胞在体内的对5-Fu的敏感性,抑制其在裸鼠体内的生长;Western blot检测发现BMP4可以抑制LoVo/5-Fu细胞中AKT的磷酸化,并抑制抗凋亡蛋白Bcl-2的表达。结论 BMP4可以增强结直肠癌耐药细胞对化疗的敏感性;其机制有可能是通过抑制细胞中AKT通路的活性和Bcl-2表达,降低细胞抵抗凋亡的能力。

Objective To investigate the influence of bone morphogenetic protein 4（BMP4）on the chemosensitivity of drugresistant colorectal cancer cells.Methods Human drug-resistant colon cancer cell line,LoVo/5-Fu,was established by continuous exposure to 5-Fu.CCK-8assay was used to evaluate the chemosensitivity of drug-resistant and parent cells to 5-Fu,oxaliplatin or irinotecan,and the growth inhibitory effect of gradient concentrations of BMP4 on LoVo/5-Fu cells.Annexin V/PI assay was employed to detect the apoptosis of LoVo/5-Fu cells treated with BMP4 and 5-Fu for 3days.Tumorigenicity assay was used to investigate the effect of BMP4 on chemosensitivity of LoVo/5-Fu cells in vivo.Western blot was performed to detect the activity of AKT pathway and the expression of Bcl-2.Results LoVo/5-Fu cells could stably grow in the culture medium containing 5μg/mL 5-Fu,and showed a cross resistance to oxaliplatin and irinotecan.CCK-8assay revealed that BMP4 inhibited cell proliferation in a concentration-and time-dependent manner.Moreover,BMP4 could induce the apoptosis of LoVo/5-Fu cells treated by 5 μg/mL 5-Fu.It enhanced chemotherapeutic sensitivity of LoVo/5-Fu cells in vivo and suppressed tumor growth.Western blot revealed that BMP4 inhibited the activity of AKT pathway and the expression of Bcl-2.Conclusion BMP4 enhances the chemosensitivity of drug-resistant colorectal cancer cells possibly by inhibiting the activity AKT pathway and the expression of Bcl-2and reducing the ability of cells against apoptosis.