基于Wnt/β-catenin信号通路研究臭壳虫与环磷酰胺联合用药对肺癌细胞增殖和凋亡的影响及机制

Blaps rynchopetera combined with cyclophosphamide affects proliferation and apoptosis of lung cancer cells via Wnt/β-catenin signaling pathway

研究臭壳虫与环磷酰胺单独以及联合用药对肺癌细胞增殖和凋亡的影响及作用机制。选择SD大鼠制备臭壳虫和环磷酰胺含药血清与空白血清,采用细胞增殖与活性检测细胞计数试剂(cell counting kit-8,CCK-8)法检测臭壳虫与环磷酰胺含药血清单独以及联合用药对肺癌细胞株A549和Lewis细胞增殖的影响,金式公式法评价药物联用效应,根据评价结果选择合适的药物浓度与肺癌细胞株用于后续实验。实验分组为对照组、臭壳虫组、环磷酰胺组、联合组、臭壳虫+Wnt/β-catenin通路激动剂氯化锂(LiCl)组,采用免疫细胞化学法检测不同组别药物干预后Lewis细胞中增殖相关蛋白的表达情况;流式细胞术检测细胞周期变化及凋亡情况;同时采用蛋白免疫印迹法检测细胞中PCNA、cyclinD1、Bcl-2、Bax和Wnt/β-catenin信号通路相关蛋白的表达水平。结果显示,臭壳虫与环磷酰胺单独及联合用药均能显著抑制A549和Lewis肺癌细胞增殖,与单独用药相比,联合用药能够进一步升高对细胞增殖的抑制率,且通过金氏公式评价得知臭壳虫与环磷酰胺联合用药具有协同效应。与对照组相比,臭壳虫组、环磷酰胺组及联合组Lewis细胞G_(0)/G_(1)期比例、凋亡率及Bax蛋白表达均显著升高,PCNA、cyclinD1、Bcl-2、Wnt1及β-catenin蛋白表达均显著降低;与环磷酰胺组相比,联合组细胞G_(0)/G_(1)期比例、凋亡率及Bax蛋白表达进一步增加,PCNA、cyclinD1、Bcl-2、Wnt1及β-catenin蛋白表达进一步降低;与臭壳虫组相比,臭壳虫+LiCl组细胞G_(0)/G_(1)期比例、凋亡率及Bax蛋白表达显著降低,PCNA、cyclinD1、Bcl-2、Wnt1及β-catenin蛋白表达显著升高。结果表明,臭壳虫可能通过抑制Wnt/β-catenin信号通路抑制肺癌细胞增殖,阻滞细胞周期,并诱导其凋亡,且与环磷酰胺联合用药具有协同效应。

This study aims to investigate the effects of Blaps rynchopetera Fairmaire and/or cyclophosphamide on the proliferation and apoptosis of lung cancer cells and decipher the underlying mechanism.B.rynchopetera and cyclophosphamide-containing serum and blank serum were prepared from SD rats.Cell counting kit-8(CCK-8) assay was employed to examine the proliferation of lung cancer cell lines A549 and Lewis treated with corresponding agents.The Jin′s formula method was used to evaluate the combined effect of the two drugs.According to the evaluation results,appropriate drug concentrations and lung cancer cell line were selected for subsequent experiments,which included control,B.rynchopetera,cyclophosphamide,B.rynchopetera + cyclophosphamide,and B.rynchopetera + Wnt/β-catenin pathway agonist lithium chloride(LiCl) groups.Immunocytochemistry was employed to measure the expression of proliferation-related proteins in Lewis cells after drug interventions.Flow cytometry was employed to determine the cell cycle and apoptosis.The expression levels of proliferating cell nuclear antigen(PCNA),cyclinD1,B-cell lymphoma 2(Bcl-2),Bcl-2-assiocated X protein(Bax),Wnt1,and β-catenin were determined by Western blot.The results showed that B.rynchopetera and/or cyclophosphamide significantly inhibited the proliferation of A549 and Lewis cells.Compared with B.rynchopetera alone,the combination increased the inhibition rate on cell proliferation.The combination of B.rynchopetera and cyclophosphamide demonstrated a synergistic effect according to Jin′s formula-based evaluation.Compared with the control group,the B.rynchopetera,cyclophosphamide,and B.rynchopetera + cyclophosphamide groups showed increased proportion of Lewis cells in G_0/G_(1) phase,increased apoptosis rate,up-regulated expression of Bax,and down-regulated expression of PCNA,cyclinD1,Bcl-2,Wnt1,and β-catenin.Compared with the cyclophosphamide group,the combination group showed increased proportion of cells in G_0/G_(1) phase,increased apoptosis rate,up-regulated expression of Bax,and down-regulated expression of PCNA,cyclinD1,Bcl-2,Wnt1,and β-catenin.Compared with the B.rynchopetera group,the B.rynchopetera + LiCl group had deceased proportion of cells in G_0/G_(1) phase,decreased apoptosis rate,down-regulated expression of Bax,and up-regulated expression of PCNA,cyclinD1,Bcl-2,Wnt1,and β-catenin.The results indicated that B.rynchopetera could inhibit the proliferation,arrest the cell cycle,and induce the apoptosis of lung cancer cells by inhibiting the Wnt/β-catenin signaling pathway.Moreover,B.rynchopetera had a synergistic effect with cyclophosphamide.