Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China.
Department of Cardiology, The 2nd Affiliated Hospital of Harbin Medical University, Harbin 150001, China.
Phytomedicine. 2019 Jul;60:152957. doi: 10.1016/j.phymed.2019.152957. Epub 2019 May 13.
Inonotus obliquus, namely as Chaga mushroom, is a medicinal and edible fungus, which is widely used in food and medical fields. Inotodiol, a natural lanostane-type triterpenoid with remarkable pharmacological activities, was isolated from Inonotus obliquus, which its potential anti-tumor molecular mechanism was elaborated poorly.
The aim of the present study was to investigate the effect of Inotodiol on HeLa cell migration, invasion and apoptosis through p53-dependent pathway.
The potential mechanisms of Inotodiol on HeLa cell anti-metastatic and pro-apoptosis via wound healing assay, trans-well invasion assay, flow cytometry, caspase-3 activity assay and western blot analysis were studied, as well as the involvement of p53 signaling pathway in anti-metastatic and pro-apoptosis of Inotodiol. Besides, the function of tumor suppressor p53 was further verified by small interfering RNA.
Firstly, the cell viability assay showed that low-concentration of Inotodiol had no cytotoxicity to HeLa cells and whereas the concentration above 25 μM significantly inhibited HeLa cell growth and even induced apoptosis. This result was further demonstrated by cell proliferation and morphology assay. Secondly, in vitro wound healing and trans-well invasion assays reported that low-concentration treatment of Inotodiol significantly inhibited cells migration and invasion in a dose-dependent manner, the western blot analysis of matrix mettalloprotinase-2 (MMP2) and matrix mettalloprotinase-9 (MMP9) levels were also decreased. Moreover, Inotodiol notably induced tumor cell apoptosis by Annexin-V-FITC apoptosis assay, which is associated with activation pro-apoptotic proteins of PARP, cleaved caspase-3 and Bax expression, inhibition anti-apoptotic protein Bcl-2 expression. Finally, the anti-tumor activity of Inotodiol was attenuated by silencing p53 tumor suppressor, the result revealed that pre-treatment with p53-specific small interfering RNA (si-p53) markedly inhibited Intodiol-indeuced HeLa cell apoptosis and decreased the caspase-3 activity. What is more, the inhibitory effect of Inotodiol on tumor migration and invasion was blocked under p53 knockdown.
To sum up, the present study indicated that Inotodiol possessed the potential to prevent malignant tumor migration and invasion, and it might be a natural active compound candidate for clinical treatment of human cervical cancer.
桦褐孔菌，又名恰卡蘑菇，是一种药食两用真菌，广泛应用于食品和医药领域。从桦褐孔菌中分离得到的天然羊毛甾烷型三萜类化合物桦褐孔菌素，具有显著的药理活性，但对其潜在的抗肿瘤分子机制研究甚少。
本研究旨在探讨桦褐孔菌素通过 p53 依赖性途径对 HeLa 细胞迁移、侵袭和凋亡的影响。
通过划痕愈合试验、Transwell 侵袭试验、流式细胞术、caspase-3 活性测定和 Western blot 分析研究桦褐孔菌素对 HeLa 细胞抗转移和促凋亡的潜在机制，以及桦褐孔菌素抗转移和促凋亡过程中 p53 信号通路的参与情况。此外，通过小干扰 RNA 进一步验证肿瘤抑制因子 p53 的功能。
首先，细胞活力测定结果表明，低浓度桦褐孔菌素对 HeLa 细胞无细胞毒性，而浓度高于 25 μM 时则显著抑制 HeLa 细胞生长，甚至诱导细胞凋亡。细胞增殖和形态学测定结果进一步证实了这一点。其次，体外划痕愈合和 Transwell 侵袭试验表明，桦褐孔菌素低浓度处理呈剂量依赖性显著抑制细胞迁移和侵袭，基质金属蛋白酶-2（MMP2）和基质金属蛋白酶-9（MMP9）水平的 Western blot 分析也降低。此外，通过 Annexin-V-FITC 凋亡试验，桦褐孔菌素显著诱导肿瘤细胞凋亡，与 PARP、cleaved caspase-3 和 Bax 表达的促凋亡蛋白激活以及抗凋亡蛋白 Bcl-2 表达的抑制有关。最后，沉默肿瘤抑制因子 p53 减弱了桦褐孔菌素的抗肿瘤活性，结果表明，预先用 p53 特异性小干扰 RNA（si-p53）处理可显著抑制桦褐孔菌素诱导的 HeLa 细胞凋亡，降低 caspase-3 活性。更重要的是，在 p53 敲低的情况下，桦褐孔菌素抑制肿瘤迁移和侵袭的作用被阻断。
综上所述，本研究表明桦褐孔菌素具有预防恶性肿瘤迁移和侵袭的潜力，可能是治疗人宫颈癌的一种天然活性化合物候选药物。
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