Mahmoud Nuha, Dawood Mona, Huang Qi, Ng Jerome P L, Ren Fang, Wong Vincent K W, Efferth Thomas
Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, Mainz 55128, Germany.
Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, Mainz 55128, Germany; Faculty of Medical Laboratory Sciences, Al-Neelain University, Khartoum, Sudan.
Phytomedicine. 2022 Jan;94:153826. doi: 10.1016/j.phymed.2021.153826. Epub 2021 Nov 1.
Prostate cancer (PCa) is the most prominent malignancy among men worldwide. PCa cells have a high tendency to metastasize to various distant organs, and this activity is the main cause of PCa mortality. Nimbolide is a promising phytochemical constituent of neem Azadirachta indica (Meliaceae). Previous studies showed that nimbolide exhibited potent anticancer activity however, its role against PCa tumorigenesis has not been fully elucidated.
Our work aims to explore the role of nimbolide in regulating the essential tumor-associated processes involved in the metastatic cascade in PCa cells.
Cytotoxicity assay, wound healing and spheroid invasion assays, western blotting, immunofluorescence, tube-formation assay, in vivo and immunohistochemistry.
The cytotoxicity of nimbolide towards PCa cell lines was assessed by resazurin assays. The cell mobility and migration of nimbolide-treated DU145 cells were determined by wound healing and spheroid invasion assays. Tubulin network was visualized using U2OS cells and DU145 cells. The effect of nimbolide on E-cadherin, β-catenin, acetylated α-tubulin and HDAC6 protein expressions levels were measured by Western blot. The potentiality of nimbolide to inhibit angiogenesis was revealed by HUVEC tube-formation assay. Nimbolide antitumor effect was studied in a syngeneic model of murine prostate cancer.
The current study indicated that nimbolide negatively affected the migratory and invasive capacity of DU145 prostate cancer cells in 2D and three-dimensional (3D) spheroid cultures. Interestingly, nimbolide induced downregulation of E-cadherin without any influence on the expression level of β-catenin. Additionally, we demonstrated that nimbolide influenced the microtubule network which was supported by the upregulation of acetylated α-tubulin and the reduction in HDAC6 protein. Moreover, the inhibitory effect of nimbolide on angiogenesis was clearly observed in HUVEC tube formation assay. In vivo experiments revealed the significant suppression of PCa growth and targeting of the B-RAF/p.ERK signaling pathway by nimbolide.
Our results showed that nimbolide inhibited 2D and 3D prostate cancer cells migration and downregulated E-cadherin protein expression, a marker for metastatic chemoresistance and tumor recurrence. Nimbolide stabilized the microtubules, combated angiogenesis and suppressed B.RAF/ERK-mediated in vivo tumor growth. Nimbolide may be considered as potential therapeutic agent for metastatic and advanced PCa patients and merits further investigations.
前列腺癌（PCa）是全球男性中最常见的恶性肿瘤。PCa细胞极易转移至各种远处器官，这种转移活动是PCa致死的主要原因。印楝素是印楝（楝科）中一种很有前景的植物化学成分。先前的研究表明印楝素具有强大的抗癌活性，然而，其在PCa肿瘤发生中的作用尚未完全阐明。
我们的工作旨在探讨印楝素在调节PCa细胞转移级联中涉及的关键肿瘤相关过程中的作用。
细胞毒性测定、伤口愈合和球体侵袭测定、蛋白质免疫印迹法、免疫荧光、管形成测定、体内实验和免疫组织化学。
通过刃天青测定法评估印楝素对PCa细胞系的细胞毒性。通过伤口愈合和球体侵袭测定法确定印楝素处理的DU145细胞的细胞迁移和移动能力。使用U2OS细胞和DU145细胞观察微管网络。通过蛋白质免疫印迹法测量印楝素对E-钙黏蛋白、β-连环蛋白、乙酰化α-微管蛋白和HDAC6蛋白表达水平的影响。通过人脐静脉内皮细胞（HUVEC）管形成测定法揭示印楝素抑制血管生成的潜力。在小鼠前列腺癌的同基因模型中研究印楝素的抗肿瘤作用。
当前研究表明，在二维和三维（3D）球体培养中，印楝素对DU145前列腺癌细胞的迁移和侵袭能力产生负面影响。有趣的是，印楝素诱导E-钙黏蛋白下调，而对β-连环蛋白的表达水平没有任何影响。此外，我们证明印楝素影响微管网络，这由乙酰化α-微管蛋白的上调和HDAC6蛋白的减少所支持。此外，在HUVEC管形成测定中清楚地观察到印楝素对血管生成的抑制作用。体内实验揭示印楝素对PCa生长有显著抑制作用，并靶向B-RAF/p.ERK信号通路。
我们的结果表明，印楝素抑制二维和三维前列腺癌细胞迁移，并下调E-钙黏蛋白的蛋白表达，E-钙黏蛋白是转移性化疗耐药和肿瘤复发的标志物。印楝素稳定微管，对抗血管生成，并抑制B.RAF/ERK介导的体内肿瘤生长。印楝素可被视为转移性和晚期PCa患者的潜在治疗药物，值得进一步研究。
