Objective: To evaluate the potential anti-prostate cancer effects of Paris polyphylla ethanol extract (PPEE) and its underlying mechanisms. Materials and Methods: The anti-proliferation activity of PPEE was tested on PC3 and DU145 cells using Cell Counting Kit-8 assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by flow cytometry. Apoptosis of prostate cancer cells was induced by PPEE through endogenous and exogenous pathways. A mouse xenograft model was used to examine its anti-prostate cancer effects in vivo. Results: We found that the IC50 of PPEE on PC3 cells was 3.98 µg/ml and the IC50 of PPEE on DU145 cells was 8 µg/ml. PPEE induced prostate cancer cell apoptosis in a concentration dependent manner, through endogenous and exogenous pathways. PPEE induced PC3 cell cycle arrest in G0/G1 and G2/M phases, while in DU145cell it induced cell arrest in the G0/G1 phase. PPEE inhibited the growth of prostate cancer cells in vivo. Conclusion: PPEE could inhibit prostate cancer growth in vitro and in vivo, induce apoptosis of prostate cancer cells, and cause cell cycle arrest, which laid the foundation for further research on the anti-tumor mechanism of PPEE.

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Objectives: Paris polyphylla var. yunnanensis (PPVY), a Chinese herb, has long been used for cancer treatment, and its steroidal saponins are suggested to exert an anti-tumor activity, however, the underlying mechanism is incompletely understood and their effect on bladder cancer (BC) remains unknown. The present study is thus designed to address these issues. Material and Methods: Total steroidal saponins were extracted with ethanol from PPVY and used to treat BC cells (HT1197 and J82 carrying mutant p53). Gene expression was determined using qPCR and immunoblotting and cell cycle analyzed using flow cytometry. DNA damage response activation was assessed using immunofluorescence staining. Results: PPVY saponins treatment led to dose-dependent declines in the number of both HT1197 and J82 cells with IC50 approximately 1.2 μg/ml, which was coupled with strong growth arrest at G2/M phase and the activation of DNA damage response pathway. Moreover, the clonogenic potential of these cells was severely impaired even in the presence of low concentrations of PPVY saponins. Mechanistically, PPVY saponins induced the degradation of mutant p53 while stimulated CDKN1A gene transcription. Phosphorylated AKT was diminished in PPVY saponin-treated cells, but its specific inhibitor LY294002 exhibited significantly weaker efficacy in inducing CDKN1A expression than did PPVY saponins. Conclusion: PPVY saponins activate DNA damage response pathway, degrade mutant p53 and stimulate CDKN1A expression, thereby inhibiting BC cell growth. Given their poor absorption via oral administration, PPVY saponins may be applicable for intravesical instillations in BC treatment.

The interstitial cells in bladder lamina propria (LP-ICs) are believed to be involved in sensing/afferent signaling in bladder mucosa. Transient receptor potential (TRP) cation channels act as mechano- or chemo-sensors and may underlie some of the sensing function of bladder LP-ICs. We aimed to investigate the molecular and functional expression of TRP channels implicated in bladder sensory function and Piezo1/Piezo2 channels in cultured LP-ICs of the human bladder. Bladder tissues were obtained from patients undergoing cystectomy. LP-ICs were isolated and cultured, and used for real-time reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and calcium-imaging experiments. At the mRNA level, TRPA1, TRPV2, and Piezo1 were expressed most abundantly. Immunocytochemical staining showed protein expression of TRPA1, TRPV1, TRPV2, TRPV4, TRPM8, as well as Piezo1 and Piezo2. Calcium imaging using channel agonists/antagonists provided evidence for functional expression of TRPA1, TRPV2, TRPV4, Piezo1, but not of TRPV1 or TRPM8. Activation of these channels with their agonist resulted in release of adenosine triphosphate (ATP) from LP-ICs. Inhibition of TRPV2, TRPV4 and Piezo1 blocked the stretch induced intracellular Ca2+ increase. Whereas inhibition of TRPA1 blocked H2O2 evoked response in LP-ICs. Our results suggest LP-ICs of the bladder can perceive stretch or chemical stimuli via activation of TRPV2, TRPV4, Piezo1 and TRPA1 channels. LP-ICs may work together with urothelial cells for perception and transduction of mechanical or chemical signals in human-bladder mucosa.

Literature documents an age related reduction of bladder sensory function. TRPV1 or TRPV4 channels have been implicated in bladder mechano-transduction. To investigate contributions of TRPV1 or TRPV4 to the age related reduction of bladder sensory function, bladder responses to capsaicin (CAP, TRPV1 agonist) and GSK1016790A (GSK, TRPV4 agonist) in retired breeder (RB, 12-15 months) and young adult (2-3 month) female rats were compared using multiple methods. Metabolic cage and continuous infusion cystometry (CMG) recordings revealed that RB rats exhibit larger bladder capacity and lower voiding frequency. RB rats also have a greater intravesical pressure threshold for micturition, however, the voiding contraction strength was equivalent to that in young rats. CAP (1μM) or GSK (20nM) administered intravesically evoked smaller changes in all CMG parameters in RB rats. In vitro, CAP (1μM) or GSK (20nM) evoked smaller enhancement of bladder strip contractions, while the muscarinic receptor agonist carbachol (at 100, 300 and 1000 nM) elicited greater amplitude contractions in RB rats. Patch clamp recording revealed smaller CAP (100 nM) induced inward currents in bladder primary sensory neurons, and Ca2+ imaging revealed smaller GSK (20 nM) evoked increases in [Ca]i in urothelial cells in RB rats. These results suggest that RB rats have a decreased bladder sensory function commonly observed in elderly women, and could be used as an animal model to study the underling mechanisms. Reduced functional expression of TRPV1 in bladder afferents or reduced functional expression of urothelial TRPV4 may be associated with the diminished sensory function.