Zhang Guodong, He Lixiang, Chen Junhao, Xu Botao, Mao Zejun
Department of Cardiothoracic Surgery, Zhuji People's Hospital, Zhuji, Zhejiang 311800, P.R. China.
Exp Ther Med. 2020 Apr;19(4):2913-2922. doi: 10.3892/etm.2020.8543. Epub 2020 Feb 21.
The efficacy of ginsenoside Rh2 (Rh2) in cancer therapy has been reported; however, its function in lung cancer remains unknown. To analyze the role of Rh2 in the inhibition of lung cancer cell proliferation in the present study, protein expression levels of E-cadherin, vimentin, β-catenin, Smo, Gli1, and α-catenin were assessed by western blotting, whilst mRNA expression levels of , , , , and were determined by reverse transcription-quantitative PCR in the A549 cell line. Phosphorylation sites were detected by proteomic methods and cell proliferation was analyzed by MTT assay. The present study revealed that Rh2 treatment significantly inhibited cell proliferation. Western blotting indicated that the expression levels of E-cadherin were increased and vimentin was downregulated in Rh2-treated cells compared with control cells. Treatment of A549 cells with Rh2 suppressed phosphorylation of five distinct proteins and increased phosphorylation of nine proteins. Among them, the phosphorylation of α-catenin at S641 was significantly induced. Rh2 treatment suppressed the expression levels of key genes involved in Wnt (, transcription factor 7 and frizzled class receptor 8) and hedgehog [smoothened, frizzled class receptor (), GLI family zinc finger ()1, , and ] signaling. Immunoblotting results indicated that β-catenin, Smo and Gli1 protein expression levels were also suppressed by treatment with Rh2 compared with control treatment. Expression of α-catenin S641D, a phosphomimetic form of α-catenin, inhibited the accumulation of β-catenin and Gli1 and inhibited cell proliferation and invasion. Furthermore, knockdown of or with specific small interfering RNAs inhibited cell proliferation, whereas overexpression of these genes had an opposite effect. Additionally, overexpression of or activated cell proliferation, even in the presence of Rh2, suggesting that Rh2 affects A549 cell proliferation through inhibition of Wnt and hedgehog signaling by phosphorylation of α-catenin at S641. Together, these data suggested that Rh2 treatment may inhibit the proliferation of A549 lung cancer cells. Further exploration of the underlying mechanism by which Rh2 inhibits cell proliferation is warranted.
人参皂苷Rh2（Rh2）在癌症治疗中的功效已有报道；然而，其在肺癌中的作用仍不清楚。为了分析Rh2在本研究中对肺癌细胞增殖抑制的作用，通过蛋白质印迹法评估E-钙黏蛋白、波形蛋白、β-连环蛋白、Smo、Gli1和α-连环蛋白的蛋白表达水平，同时在A549细胞系中通过逆转录定量PCR测定、、、和的mRNA表达水平。通过蛋白质组学方法检测磷酸化位点，并通过MTT法分析细胞增殖。本研究表明，Rh2处理显著抑制细胞增殖。蛋白质印迹表明，与对照细胞相比，Rh2处理的细胞中E-钙黏蛋白的表达水平升高，波形蛋白下调。用Rh2处理A549细胞可抑制五种不同蛋白的磷酸化，并增加九种蛋白的磷酸化。其中，α-连环蛋白S641位点的磷酸化被显著诱导。Rh2处理抑制了参与Wnt（、转录因子7和卷曲蛋白家族受体8）和刺猬信号通路[ smoothened、卷曲蛋白家族受体（）、GLI家族锌指（）1、、和]的关键基因的表达水平。免疫印迹结果表明，与对照处理相比，用Rh2处理也抑制了β-连环蛋白、Smo和Gli1蛋白的表达水平。α-连环蛋白S641D（α-连环蛋白的磷酸模拟形式）的表达抑制了β-连环蛋白和Gli1的积累，并抑制了细胞增殖和侵袭。此外，用特异性小干扰RNA敲低或可抑制细胞增殖，而这些基因的过表达则有相反的作用。此外，或的过表达激活了细胞增殖，即使在存在Rh2的情况下也是如此，这表明Rh2通过在S641位点磷酸化α-连环蛋白来抑制Wnt和刺猬信号通路，从而影响A549细胞增殖。总之，这些数据表明Rh2处理可能抑制A549肺癌细胞的增殖。有必要进一步探索Rh2抑制细胞增殖的潜在机制。
