Objective: To study the effects of oxymatrine and vincristine on resistance in HCT-8/VCR cells and its mechanism.
Methods: HCT-8 / VCR cells were cultured in vitro and were divided into blank control group, oxymatrine group, vincristine group, oxymatrine and vincristine combined group, each group had 6 complexes. The drug resistance of HCT-8/VCR cells was investigated by CCK-8 when treated with vincristine alone or in combination with oxymatrine. The autophagy was determined by monodansylcadaverine (MDC) staining. The level of IL-6 was detected by ELISA. The expressions of autophagy-related gene P62, LC3-Ⅱ / LC3-Ⅰ, Beclin-1 protein and TLR4 were detected by Western blot assay.
Results: Oxymatrine combined with vincristine could reduce the drug resistance of HCT-8 / VCR cells by the reversal multiple of 3.23. Compared with the blank control group, the content of autophagosome and the content of IL-6 in the oxymatrine group and the combination group were also decreased significantly (P＜0.01). The content of autophagosome in the vincristine group was increased and the content of IL-6 was also significantly increased (P＜0.01). Compared with the oxymatrine group, the combination group had higher autophagosome content, while IL-6 content was decreased (P＜0.01); Western blot experiments showed that compared with the blank control group, the expression of P62 in the oxymatrine group was decreased (P＜0.05), while the expressions of LC3-Ⅱ / LC3-Ⅰ, Beclin-1 and TLR4 were all increased (P＜0.05). The expression of P62 in the vincristine group and the combined group was increased (P＜0.05), and the expressions of LC3-Ⅱ / LC3-Ⅰ, Beclin-1, and TLR4 were all decreased (P＜0.05). Compared with the vincristine group, the expression of P62 was increased in the combination group (P＜0.05), and the expressions of LC3-Ⅱ / LC3-Ⅰ, Beclin-1, and TLR4 were all decreased (P＜0.05).
Conclusion: Oxymatrine combined with vincristine can reduce the drug resistance of HCT-8/VCR cells, which may be related to the regulation of autophagy activity and TLR4 signal activation.
目的: 探究苦参素复合长春新碱对HCT-8/VCR细胞耐药性的影响及其机制。方法: HCT-8/VCR体外培养,分为空白组、苦参素单药组,长春新碱单药组、苦参素和长春新碱联合组,每组6个复孔。CCK-8法检测各组HCT-8/VCR细胞的耐药性;MDC法检测细胞内自噬小泡的变化;ELISA法检测IL-6的含量;Western blot检测自噬相关基因与免疫因子TLR4蛋白的表达水平。结果: 苦参素复合长春新碱可降低HCT-8/VCR细胞的耐药性,逆转倍数为3.23;与空白组比较,苦参素组和联合组自噬体含量降低、IL-6含量也明显降低(P＜0.01),长春新碱组自噬体含量升高,IL-6含量也明显升高(P＜0.01);联合组与苦参素组比较,自噬体含量升高,而IL-6含量降低(P＜ 0.01);Western blot实验表明,与空白组比较,苦参素组P62的表达降低(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均升高(P＜0.05),长春新碱组和联合组P62的表达升高(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均降低(P＜ 0.05);联合组与长春新碱组比较,P62的表达升高(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均降低(P＜0.05)。结论: 苦参素与长春新碱联合可显著降低HCT-8/VCR的耐药性,其机制与抑制自噬活动和TLR4信号活化有关。.
Keywords: HCT-8/VCR cells; TLR4; autophagy; colorectal cancer; oxymatrine; reversal; vincristine.