Tian Ying-Ying, Ma Bei-Bei, Zhao Xin-Yue, Liu Chuang, Li Yi-Lin, Yu Shang-Yue, Tian Shi-Qiu, Pei Hai-Luan, Lyu Ying-Nan, Zuo Ze-Ping, Wang Zhi-Bin
School of Chinese Materia Medica,Beijing University of Chinese Medicine Beijing 100029,China.
Institute of Scientific Research, Beijing Tong Ren Tang Chinese Medicine Co., Ltd. Beijing 100079,China.
Zhongguo Zhong Yao Za Zhi. 2023 Sep;48(17):4702-4710. doi: 10.19540/j.cnki.cjcmm.20230424.401.
This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.
本研究旨在探讨青藤碱对人肝癌HepG2细胞和SK-HEP-1细胞增殖、凋亡、转移的影响及其分子机制，以及与抑制剂联合应用的效果。通过CCK-8法、集落形成实验和BeyoClick™ EdU-488染色研究青藤碱对HepG2和SK-HEP-1细胞生长能力的影响。通过免疫荧光实验检测青藤碱对DNA损伤的影响，通过Hoechst 33258染色和CellEvent™ Cystein-3/7Green ReadyProbes™试剂检测法阐明青藤碱对人肝癌细胞凋亡的影响。进行细胞侵袭实验和3D肿瘤细胞球侵袭实验，以研究青藤碱对人肝癌细胞体外侵袭能力的影响。通过蛋白质免疫印迹法检测青藤碱对HepG2和SK-HEP-1细胞中蛋白激酶B（Akt）/雷帕霉素靶蛋白（mTOR）/信号转导子和转录激活子3（STAT3）信号通路相关蛋白表达调控的影响。采用分子对接技术评估青藤碱与靶标半胱天冬酶-3（caspase-3）和STAT3的亲和力强度，并结合CCK-8法检测与STAT3抑制剂JSI-124联合应用后细胞活力的变化。结果表明，青藤碱能以浓度和时间依赖性方式显著降低人肝癌细胞的活力，显著抑制人肝癌细胞的克隆形成能力，并削弱人肝癌细胞的体外侵袭能力。此外，青藤碱可上调凋亡标志物聚ADP-核糖聚合酶（PARP）的裂解水平，并下调人肝癌细胞中p-Akt、p-mTOR和p-STAT3的蛋白水平。分子对接结果表明，青藤碱与靶标caspase-3和STAT3具有良好的亲和力，抑制STAT3后青藤碱对肝癌细胞的敏感性降低。因此，青藤碱可抑制人肝癌细胞的增殖和侵袭并诱导凋亡，其机制可能归因于caspase-3信号的激活和Akt/mTOR/STAT3通路的抑制。本研究可为青藤碱的深入研究和临床应用提供新的参考，对进一步推动青藤碱的科学开发利用具有重要意义。
