Targeted inhibition of mTOR by BML-275 induces mitochondrial-mediated apoptosis and autophagy in prostate cancer.

Prostate cancer (PCa) is the most frequently diagnosed cancer among men and the second leading cause of death in Western countries. Clinically, screening drugs and develop developing new therapeutics to treat PCa is of great significance. In this study, BML-275 was demonstrated to exert potent antitumor effects in PCa by antagonizing mTOR activity. In cultured PCa cells, BML-275 treatment reduced the expression levels of c-Myc and survivin, promoted the activation of p53, and thereby induced p21/cyclin D1/CDK4/6-dependent cell cycle G1/S arrest. As a result, BML-275 inhibited cellular proliferation and induced mitochondrial-mediated apoptosis. In addition, BML-275 treatment triggered autophagy. Interestingly, EACC-mediated suppression of autophagy did not affect BML-275-induced proliferation and apoptosis. Nude mouse tumorigenic experiments also confirmed that BML-275 inhibited PCa growth, induced PCa cell apoptosis and autophagy. Mechanistically, the activities of PI3K/AKT and AMPK pathways were downregulated by BML-275 treatment in vitro and in vivo. Importantly, mTOR, a common downstream negative protein of PI3K/AKT and AMPK signaling, was induced to inactivate, which may be associated with the induction of apoptosis and autophagy. The pharmacological activation of mTOR by MHY1485 abolished the induction of apoptosis and autophagy of BML-275. Molecular docking results showed that BML-275 can bind to the FKRP12-rapamycin binding site on mTOR protein, and thereby may have the same inhibitory activity on mTOR as rapamycin. Thus, these findings indicated that BML-275 induces mitochondrial-mediated apoptosis and autophagy in PCa by targeting mTOR inhibition. BML-275 may be a potential candidate for the treatment of PCa.

Hyperoside protects human kidney­2 cells against oxidative damage induced by oxalic acid.

The majority of renal calculi (kidney stones) are calcium stones. Oxidative damage to renal tubular epithelial cells induced by reactive oxygen species (ROS) is the predominant cause of calcium oxalate stone formation. Hyperoside (Hyp) is a flavonol glycoside extracted from medicinal plants and appears to exhibit potent antioxidant activity in various cells. The aim of the present study was to investigate the protective effect of Hyp on renal cells exposed to oxidative stress simulated by oxalic acid (OA), and to determine whether the underlying mechanism involves the nuclear factor E2­related factor2 (Nrf2)­antioxidative response element signaling pathway. The study determined the indicators of high oxidative stress, including ROS and hydrogen peroxide (H2O2) in human kidney­2 cells and the results demonstrated that the levels of ROS, as evaluated by flow cytometry, and H2O2 were significantly increased following treatment with OA (5 mmol/l) for 24 h (OA group), compared with those in the untreated control group. The increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in these cells explained this observation, as it is a major source of ROS. The results demonstrated that, in the OA group, the adhesion of calcium oxalate crystals and lactate dehydrogenase (LDH) were significantly increased, and MTT assay demonstrated that cell viability was inhibited, compared with the control, which indicated that severe injury of cells was induced by OA. However, when the cells were pre­treated with Hyp prior to treatment with OA (drug group), the levels of ROS and H2O2, and the activities of NADPH oxidase and LD were increased, and the adhesion of calcium oxalate crystals to cells was reduced, compared with the OA group. Western blot analysis and reverse transcription­quantitative polymerase chain reaction demonstrated that the protein and mRNA expression levels of Nrf2, heme oxygenase­1 (HO­1) and NAD(P)H: quinineoxidoreductase 1 (NQO1) in the Hyp groups were significantly increased, compared with those in the OA group, with the exception of Nrf2 mRNA. These results suggested that Hyp had a marked protective effect on renal cells against the oxidative damage and cytotoxicity simulated by OA. This is the first report, to the best of our knowledge, demonstrating that the ability of Hyp to enhance the endogenous functions of antioxidation and detoxification in cells may involve the Nrf2/HO­1/NQO1 pathway.

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-ß), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-ß binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.