Wang Zijie, Liu Hui, Hu Qing, Shi Lei, Lü Muhan, Deng Mingming, Luo Gang
Department of Gastroenterology, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China.
Nuclear Medicine and Molecular Imaging Key Laboratory of Sichuan Province, Luzhou, Sichuan, 646000, China.
J Cancer. 2021 Apr 24;12(12):3597-3610. doi: 10.7150/jca.55519. eCollection 2021.
Oesophageal cancer is the most common malignant tumour with a poor prognosis, and the current treatment methods are limited. Therefore, identifying effective treatment methods has become a research hotspot. Cardamonin (CAR) is a natural chalcone compound and has been reported to play an anticancer role in several cancers. However, its function in oesophageal cancer and the possible underlying mechanism are still unclear. The purpose of this study was to demonstrate the anticancer effect of CAR on oesophageal cancer and and to explore the underlying mechanism. MTT, crystal violet, and colony formation assays were used to detect oesophageal cancer cell proliferation. The effects of CAR on oesophageal cancer cell migration and invasion were detected by wound healing assay and Transwell assay. Hoechst 33258 staining and flow cytometry were used to detect cell apoptosis. Protein expression levels were detected by Western blot. A tumour xenograft model was established to further test the effect of CAR on the growth of oesophageal cancer . The results showed that CAR inhibited the proliferation, migration, and invasion of oesophageal cancer cells in a concentration-dependent manner and induced apoptosis. Furthermore, the Western blot assay showed that CAR could suppress metastasis by inhibiting epithelial-mesenchymal transition (EMT) as indicated by downregulated expression of the mesenchymal markers N-cadherin and vimentin, the EMT transcription factor Snail, and matrix metalloproteinases (MMPs) and upregulated expression of the epithelial marker E-cadherin. CAR was associated with upregulation of the pro-apoptotic proteins Bax and Bad and downregulation of the anti-apoptotic protein Bcl-2 and triggered the mitochondrial apoptosis pathway, which in turn promoted caspase-3 activation and subsequent cleavage of PARP; however, the mitochondria-related apoptotic effects induced by CAR were blocked by caspase inhibitor Z-VAD-FMK pretreatment, which prevented programmed cell death triggered by CAR. In addition, CAR reduced the phosphorylation level of downstream effector molecules of phosphatidylinositol 3 kinase (PI3K) in a dose-dependent manner, and treatment with the PI3K agonist 740Y-P could partially reverse the anticancer effect of CAR, demonstrating that CAR played an antitumour role by inhibiting the PI3K/AKT signalling pathway in oesophageal cancer cells. Moreover, the EC9706 xenograft model further confirmed that CAR can significantly inhibit tumour growth . In summary, CAR exhibited a strong anticancer effect on human oesophageal cancer cells and promoted apoptosis by inhibiting the PI3K/AKT signalling pathway, suggesting that CAR can be used as new strategy for oesophageal cancer treatment.
食管癌是预后较差的最常见恶性肿瘤，目前的治疗方法有限。因此，确定有效的治疗方法已成为研究热点。小豆蔻明（CAR）是一种天然查尔酮化合物，据报道在几种癌症中发挥抗癌作用。然而，其在食管癌中的作用及潜在机制仍不清楚。本研究旨在证明CAR对食管癌的抗癌作用并探索其潜在机制。采用MTT法、结晶紫法和集落形成试验检测食管癌细胞增殖。通过伤口愈合试验和Transwell试验检测CAR对食管癌细胞迁移和侵袭的影响。采用Hoechst 33258染色和流式细胞术检测细胞凋亡。通过蛋白质印迹法检测蛋白质表达水平。建立肿瘤异种移植模型以进一步检测CAR对食管癌生长的影响。结果表明，CAR以浓度依赖性方式抑制食管癌细胞的增殖、迁移和侵袭并诱导凋亡。此外，蛋白质印迹分析表明，CAR可通过抑制上皮-间质转化（EMT）来抑制转移，表现为间质标志物N-钙黏蛋白和波形蛋白、EMT转录因子Snail以及基质金属蛋白酶（MMPs）的表达下调，上皮标志物E-钙黏蛋白的表达上调。CAR与促凋亡蛋白Bax和Bad的上调以及抗凋亡蛋白Bcl-2的下调相关，并触发线粒体凋亡途径，进而促进半胱天冬酶-3激活及随后的PARP裂解；然而，CAR诱导的线粒体相关凋亡效应被半胱天冬酶抑制剂Z-VAD-FMK预处理阻断，这阻止了CAR触发的程序性细胞死亡。此外，CAR以剂量依赖性方式降低磷脂酰肌醇3激酶（PI3K）下游效应分子的磷酸化水平，用PI3K激动剂740Y-P处理可部分逆转CAR的抗癌作用，表明CAR通过抑制食管癌细胞中的PI3K/AKT信号通路发挥抗肿瘤作用。此外，EC9706异种移植模型进一步证实CAR可显著抑制肿瘤生长。总之，CAR对人食管癌细胞表现出强大的抗癌作用，并通过抑制PI3K/AKT信号通路促进凋亡，提示CAR可作为食管癌治疗的新策略。
