School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
J Ethnopharmacol. 2021 Feb 10;266:113446. doi: 10.1016/j.jep.2020.113446. Epub 2020 Oct 5.
Hepatocellular carcinoma (HCC) is an aggressive malignancy with increasing mortality in China. Screening and identifying effective anticancer compounds from active traditional Chinese herbs for HCC are in demand. Akebia trifoliata (Thunb) Koidz, with pharmacological anti-HCC activities in clinical, has been shown in previous research. In the present research, we elucidated a potential anticancer effect of Akebia saponin E (ASE), which is isolated from the immature seeds of Akebia trifoliata (Thunb.) Koidz, and revealed that ASE could induce severe expanded vacuoles in HCC cells. But the potential mechanism of vacuole-formation and the anti-HCC effects by ASE remain uncover.
To elucidate the potential mechanism of vacuole-formation and the proliferation inhibition effects by ASE in HCC cell lines.
MTT assay, colony formation assay and flow cytometry were performed to detect cell viability. Immunofluorescence analysis was used to examine the biomarkers of endomembrane. Cells were infected with tandem mRFP-GFP-LC3 lentivirus to assess autophagy flux. RNA-seq was conducted to analyze the genome-wide transcriptional between treatment cell groups. In vitro PIKfyve kinase assay is detected by the ADP-Glo Kinase Assay Kit.
ASE could inhibit the proliferation of HCC with severe expanded vacuoles in vitro, and could significantly reduce the size and weight of xenograft tumor in vivo. Further, the vacuoles induced by ASE were aberrant enlarged lysosomes instead of autophagosome or autolysosomes. With cytoplasmic vacuolation, ASE induced a mTOR-independent TFEB activation for lysosomal biogenesis and a decrement of cholesterol levels in HCC cells. Furthermore, ASE could reduce the activity of PIKfyve (phosphoinositide kinase containing a FYVE-type finger), causing aberrant lysosomal biogenesis and cholesterol dyshomeostasis which triggered the expanded vacuole formation.
ASE can prospectively inhibit the kinase activity of PIKfyve to induce lysosome-associated cytoplasmic vacuolation, and may be utilized as an alternative candidate to treat human HCC.
ETHNOPHARMACOLOGICAL 相关性：肝细胞癌 (HCC) 是一种侵袭性恶性肿瘤，在中国的死亡率不断上升。从具有活性的中草药中筛选和鉴定针对 HCC 的有效抗癌化合物是当务之急。在临床前研究中，已证实三叶木通（Thunb）Koidz 的药理学抗 HCC 活性。在本研究中，我们阐明了从三叶木通（Thunb.）Koidz 的未成熟种子中分离得到的木通皂苷 E (ASE) 的潜在抗癌作用，并表明 ASE 可诱导 HCC 细胞中严重扩大的空泡。但是，空泡形成的潜在机制和 ASE 的抗 HCC 作用仍未被揭示。
阐明 ASE 在 HCC 细胞系中诱导空泡形成和抑制增殖的潜在机制。
通过 MTT 测定、集落形成测定和流式细胞术检测细胞活力。免疫荧光分析用于检测内膜生物标志物。用串联 mRFP-GFP-LC3 慢病毒感染细胞以评估自噬通量。通过 RNA-seq 分析处理细胞组之间的全基因组转录组。通过 ADP-Glo 激酶测定试剂盒检测体外 PIKfyve 激酶活性。
ASE 可在体外抑制 HCC 的增殖，并可显著减少体内异种移植肿瘤的大小和重量。此外，ASE 诱导的空泡是异常扩大的溶酶体，而不是自噬体或自溶体。随着细胞质空泡化，ASE 诱导 mTOR 非依赖性 TFEB 激活以进行溶酶体生物发生，并降低 HCC 细胞中的胆固醇水平。此外，ASE 可降低 PIKfyve（含有 FYVE 型手指的磷酸肌醇激酶）的活性，导致异常的溶酶体生物发生和胆固醇动态平衡失调，从而引发扩大的空泡形成。
ASE 可以抑制 PIKfyve 的激酶活性，从而诱导溶酶体相关的细胞质空泡化，并且可能作为治疗人类 HCC 的替代候选药物。
