Objective: To investigate the molecular mechanism of the growth inhibitory effect of matrine on K562 cells in JAK/STAT3 mediated signal pathway.
Methods: Western blot analyses were performed to investigate the differential expression of JAK2, STAT3, phosphor-STAT3 (Tyr705 & Ser727) and phosphor-JAK2 proteins after matrine treatment in K562 cells with or without human recombinant interleukin 6 (IL-6) pretreatment. The expression of STAT3 response gene products such as Bcl-xL, Cyclin D1 and c-Myc, were investigated by Western blot and quantitative real time RT-PCR (qRT-PCR). Expression of IL-6, a potent upstream activating factor of JAK/STAT3 pathway, was analyzed by both real time qRT-PCR and ELISA.
Resutls: Western blot revealed that matrine treatment resulted in a strong down-regulation of phosphor-STAT3 both in Tyr705 and Ser727 sites or phosphor-JAK2 proteins expression without significant effects on the total STAT3 and JAK2 proteins. The expression of phosphor-Tyr705 STAT3 and phosphor-Ser727 STAT3 was decreased to 0.370 ± 0.172 in K562 cells treated with 0.5 mg/ml matrine for 48 h, respectively, from 0.690 ± 0.119 and 1.150 ± 0.263 in control cells, accompanied with a dramatical down-regulation of phosphor-JAK2 from 0.670 ± 0.137 to 0.049 ± 0.057 (P<0.05). In addition, it was found that the expression of Bcl-xL, Cyclin D1, c-Myc was decreased both at the transcription and protein level in K562 cells after matrine treatment. Matrine treatment resulted in a significant decrease in the expression level of IL-6 in K562 cells from (35.1 ± 1.93) to (10.74 ± 1.83) and (8.66 ± 1.24) pg/ml at the dose of 0.5 and 0.8 mg/ml, respectively (p<0.05). Matrine treatment could diminish the up-regulation of STAT3, JAK2, phosphor-STAT3 and phosphor-JAK2 protein following pretreatment with IL-6 in K562 cells.
Conclusion: Matrine exerts its anti-leukemia effect by interfering with the JAK2/STAT3 signaling pathway. The inhibition of IL-6 expression may play a pivotal role in the disruption of JAK/STAT pathway by matrine.
目的: 探讨JAK/STAT3介导的信号途径在苦参碱对K562细胞增殖抑制作用中的分子机制。
方法: 以不同浓度苦参碱处理K562细胞，Western blot法分析JAK/STAT3通路分子JAK2、STAT3及其磷酸化蛋白表达的改变；荧光实时定量RT-PCR及Western blot法分析STAT3下游调控基因Bcl-xL、Cyclin D1、c-Myc的表达；荧光实时定量RT-PCR及ELISA法检测K562细胞IL-6表达的改变。以IL-6预处理K562细胞，Western blot法分析苦参碱对细胞内JAK2、STAT3及其磷酸化蛋白表达的作用。
结果: 0.5 mg/ml苦参碱处理48 h, K562细胞内STAT3及JAK2总蛋白表达无明显变化，磷酸化STAT3(p-Tyr705 STAT3、p-Ser727 STAT3)和磷酸化JAK2 (p-JAK2）表达水平分别由处理前的0.690± 0.119、1.150±0.263和0.670±0.137下降至0.370±0.024、0.700±0.172和0.049±0.057。苦参碱对STAT3下游调控基因Bcl-xL、Cyclin D1及c-Myc的转录及蛋白表达均有抑制作用，并可显著抑制IL-6的表达和分泌，0.5和0.8 mg/ml苦参碱处理后细胞培养上清中IL-6浓度分别为(10.74±1.83) pg/ml和(8.66± 1.24) pg/ml，较处理前[(35.1±1.93) pg/ml]显著降低(P<0.05)。IL-6预处理K562细胞后，STAT3、JAK2、p-STAT3及p-JAK2表达均有增加，苦参碱可抑制IL-6对上述分子表达的上调。
结论: 苦参碱抑制K562细胞增殖与JAK/STAT3介导的信号通路抑制有关，IL-6表达抑制可能是苦参碱调控JAK/STAT3通路活性的始动机制。