Nimbolide inhibits 2D and 3D prostate cancer cells migration, affects microtubules and angiogenesis and suppresses B-RAF/p.ERK-mediated in vivo tumor growth

Background: 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.

Purpose: Our work aims to explore the role of nimbolide in regulating the essential tumor-associated processes involved in the metastatic cascade in PCa cells.

Study design: Cytotoxicity assay, wound healing and spheroid invasion assays, western blotting, immunofluorescence, tube-formation assay, in vivo and immunohistochemistry.

Methods: 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.

Results: 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.

Conclusion: 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.