Supplementary materials.docx

Background/Aim: Benign prostatic hyperplasia (BPH) is characterized by the abnormal proliferation of prostate stromal cells, resulting in the enlargement of the prostate gland and the manifestation of troublesome symptoms such as nocturia, urinary retention, and urinary incontinence. Dihydrotestosterone (DHT) and interleukin-17 (IL-17) are known to be key factors in promoting the overproduction of transforming growth factor-beta (TGF-β) in prostate stromal cells, contributing to their over proliferation and becoming BPH. Materials and Methods: In this study, selected plant extracts based on traditional use for alleviating urinary symptoms were subjected to primary screening for their anti-proliferative activity through DHT- and IL-17-induced WPMY-1 prostate stromal cell proliferation, followed by a secondary screening using TGF-β induction. Results: The extract with significantly inhibited cell proliferation was standardized and investigated its anti-proliferation effects through the TGF-β signaling pathway. Results showed that the leaf extract of <i>P. indica</i> significantly inhibited cell proliferation induced by DHT, IL-17, and TGF-β. It was demonstrated that <i>P. indica</i> has anti-proliferative properties via the TGF-β signaling pathways by inhibiting PCNA protein expression and inducing cell accumulation at the G0/G1 phase, while reducing the cell population at the S phase. It also downregulated the expression of both canonical (p-SMAD2/3) and non-canonical (p-ERK1/2) proteins in TGF-β-induced WPMY-1 cells. Conclusion: the standardized leaf extract of <i>P. indica </i>showed notable anti-proliferative activity against TGF-β-induced WPMY-1 cells by arresting the cell cycle at the G0/G1 phase through the SMAD2/3 and ERK1/2 signaling pathways.