Pepper-derived phytochemicals modulate metastatic processes in triple-negative breast cancer cells: a nanoparticle-based delivery approach

Abstract

Metastasis and disease relapse are the primary determinant of cancer prognosis. The process of epithelial to mesenchymal transition (EMT) plays a crucial role in the induction of metastasis. Novel therapeutics are urgently needed to decrease breast cancer mortality by preventing epithelial-to-mesenchymal transition (EMT)-associated metastasis. Transcription factors such as β-catenin, ZEB1, and Slug, along with epigenetic machinery including histone deacetylases (HDAC) and DNA methyltransferases (DNMT) are well-studied regulators of EMT. Piperine (PIP) and piperlongumine (PL), major alkaloids in pepper spices, inhibit breast cancer cell growth in vivo and in vitro; however, their lipophilicity has restricted possible clinical application. The purpose of this study was to increase the water solubility of PIP and PL using nanoparticles (NPs) as drug carrier and investigate the anti-metastatic potential of PIP- and PL-NPs in the context of EMT regulation in triple-negative breast cancer cells. The thin-film hydration method was used to encapsulate PIP and PL into biodegradable methoxy poly(ethylene glycol)-poly(lactic-co-glycolic) acid (mPEG-PLGA) copolymer. Colorimetric MTT and Annexin-V-FLUOS/propidium iodide staining assays were performed on MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cells to determine the effect of PIP- and PL-NPs on cell growth and viability. The invasiveness of MDA-MB-231 cells was also tested in the presence of PL and PL-NPs, using gap closure and trans-well invasion assays. Western blotting and quantitative reverse transcription PCR (qRT-PCR) were used to evaluate the relative expression of EMT markers and associated transcription factors following treatment with PL and PL-NPs. At cytotoxic doses, PIP- and PL-NPs decreased the viability of MDA-MB-231, MDA-MB-468, and BT-549 breast cancer cells to a similar extent as free PIP and PL. Noncytotoxic doses of PL and PL-NPs inhibited breast cancer cell migration and invasion in vitro. Both free PL and PL-NPs inhibited the expression of EMT markers, ZEB1, β-catenin, and Slug, while increasing the expression of E-cadherin and NDRG1, inhibitors of EMT and metastasis, in MDA-MB-231 cells. Furthermore, PL and PL-NPs decreased the expression of HDAC1 and DNMT1, both of which are known transcriptional suppressors of E-cadherin and NDRG1. However, overexpression of HDAC-1 and DNMT-1 did not affect PL-induced E-cadherin expression in these cells. These results demonstrate that PL and PL-NPs inhibit metastatic properties of MDA-MB-231 breast cancer cells through inhibition of EMT-associated transcription factors. Ultimately, these findings indicate the potential use of NPs as phytochemical carriers for future in vivo studies to improve the bioavailability and serum solubility of PIP and PL.