Chemotherapy is a strategy for patients with advanced prostate cancer, especially those with castration-resistant prostate cancer. Prostate cancer stem cells (PCSCs) are believed to be the origin of cancer recurrence following therapy intervention, including chemotherapy. The mechanisms underlying the chemoresistance of PCSCs are still poorly understood. In the present study, fluorescence-activated cell sorting was used to isolate PCSCs from LNCaP and PC3 cell lines. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide was used to measure the cell viability. Quantitative real-time PCR and western blotting were utilized to evaluate the mRNA and protein levels. ShRNA was employed to knock down target gene expression. Chromatin immunoprecipitation (ChIP) was performed to explore the detailed mechanism underlying ABCC1 expression. Our results revealed that the sorted PCSCs showed enhanced chemoresistance ability than matched non-PCSCs. Protein level of activated form of NOTCH1(ICN1) was significantly higher in PCSCs. Inhibition of NOTCH1 with shRNA could decrease ABCC1 expression, and improve chemosensitivity in PCSCs. Finally, ChIP-PCR showed ICN1 could directly bind to the promoter region of ABCC1. In conclusion, NOTCH1 signaling could transactivate ABCC1, resulting in higher chemoresistance ability of PCSCs, which might be one of the important mechanisms underlying the chemoresistance of PCSCs.

Enhancer of zeste homolog 2 (EZH2) plays a crucial role in embryonic and somatic stem cells for their proliferation and differentiation. However, the roles and underlying mechanisms of EZH2 in prostate cancer stem cells (PCSCs) remain unknown. This study aimed to investigate the effects of EZH2 on PCSCs. PCSCs were isolated from the human prostate cancer cell line LNcap by fluorescence activated cell sorting (FACS). EZH2 expression was compared between PCSCs and non-PCSCs. The association between EZH2 function and PCSC growth was investigated using siRNA-mediated knock-down of EZH2. Cell growth was investigated by MTT, cell cycle and apoptosis of PCSCs were explored by flow cytometric analysis. Finally, the upstream pathway miRNA level was determined via a luciferase reporter assay, and the downstream pathway cycle regulators were examined via reverse transcriptase-polymerase chain reaction. The results showed that LNcap cell line comprised a greater proportion of CD44+/CD133+ cells by comparison to the PC-3 cell line. EZH2 was up-regulated in PCSCs compared with non-PCSCs. Silence of EZH2 inhibited cell growth and the cell cycle and promoted the progression of apoptosis. Furthermore, EZH2 was a direct target of miR-101 in PCSCs and EZH2’s mRNA levels were inversely correlated with miR-101 expression and cyclin E2 (a cell-cycle regulator) was suppressed by siEZH2. In conclusion, EZH2 is essential for PCSC growth, partly through a negative regulation by miR-101 and positively regulating cyclin E2.

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Purpose: To investigate the effects of Glechoma longituba on calcium oxalate (CaOx)-induced stress in HK-2 cells as a possible treatment strategy for nephrolithiasis (kidney stones). Methods: Human kidney HK-2 cells were treated with CaOx and Glechoma longituba at different concentrations. The levels of reactive oxygen species (ROS), lactate dehydrogenase (LDH), and malondialdehyde (MDA) were measured. Cell apoptosis and viability were assessed by flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively, while apoptosis-related proteins were determined using western blotting. The levels of the nuclear factorerythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NADPH-quinone-oxidoreductase 1 (NQO-1) genes were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). Using lentivirus, Nrf2 was knocked down in HK-2 cells, and this was confirmed by both qRT-PCR

Purpose: To investigate the effect of forkhead box N3 (FOXN3) protein on doxorubicin (DOX) resistance of urothelial carcinoma (BLCA). Methods: Bioinformatics prediction and immunoblotting were used to evaluate FOXN3 expression in BLCA tissues and cells. The FOXN3 overexpression was achieved by cell transfection. The effects of FOXN3 on DOX resistance and cell apoptosis were determined by immunoblotting, DOX resistance assay, and flow cytometry, while immunoblotting was applied to evaluate SIRT6/PI3K/AKT/mTOR signaling activity. Finally, SIRT6 overexpression and exogenous addition of a PI3K/AKT activator were used to investigate the molecular mechanism by which FOXN3 regulates DOX resistance phenotype. Results: The FOXN3 was downregulated in DOX-resistant BLCA tissues and cells while its overexpression attenuated doxorubicin resistance (p < 0.01). Furthermore, apoptotic cell ratio increased from 7.54 to 26.83 % in J82/DOX cells and from 6.31 to 17.89 % in T24/DOX cells (p < 0.01) after FOXN3 overexpression. Moreover, FOXN3 upregulation inhibited sirtuin 6 (SIRT6) expression and inactivated PI3K/AKT/mTOR signaling pathway. Both SIRT6 overexpression and PI3K/AKT activation abrogated the FOXN3-mediated inhibition of DOX resistance in BLCA cells. Conclusion: The FOXN3 attenuates the DOX resistance of BLCA through SIRT6/PI3K/AKT/mTOR pathway, thus providing a promising therapeutic strategy for the management of BLCA.