AGEs-Induced Calcification and Apoptosis in Human Vascular Smooth Muscle Cells Is Reversed by Inhibition of Autophagy.

Vascular calcification (VC) in macrovascular and peripheral blood vessels is one of the main factors leading to diabetes mellitus (DM) and death. Apart from the induction of vascular calcification, advanced glycation end products (AGEs) have also been reported to modulate autophagy and apoptosis in DM. Autophagy plays a role in maintaining the stabilization of the external and internal microenvironment. This process is vital for regulating arteriosclerosis. However, the internal mechanisms of this pathogenic process are still unclear. Besides, the relationship among autophagy, apoptosis, and calcification in HASMCs upon AGEs exposure has not been reported in detail. In this study, we established a calcification model of SMC through the intervention of AGEs. It was found that the calcification was upregulated in AGEs treated HASMCs when autophagy and apoptosis were activated. In the country, AGEs-activated calcification and apoptosis were suppressed in Atg7 knockout cells or pretreated with wortmannin (WM), an autophagy inhibitor. These results provide new insights to conduct further investigations on the potential clinical applications for autophagy inhibitors in the treatment of diabetes-related vascular calcification.

Dicoumarol, an NQO1 inhibitor, blocks cccDNA transcription by promoting degradation of HBx.

BACKGROUND & AIMS: Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. METHODS: We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model. RESULTS: Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. CONCLUSIONS: Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability. LAY SUMMARY: Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.

HM30181A, a potent P-glycoprotein inhibitor, potentiates the absorption and in vivo antitumor efficacy of paclitaxel in an orthotopic brain tumor model.

Objective: Delivery of chemotherapeutic drugs to the brain has remained a major obstacle in the treatment of glioma, owing to the presence of the blood-brain barrier and the activity of P-gp, which pumps its substrate back into the systemic circulation. The aim of the present study was to develop an intravenous formulation of HM30181A (HM) to inhibit P-gp in the brain to effectively deliver paclitaxel (PTX) for the treatment of malignant glioma. Methods: Two formulations of solubilized HM were designed on the basis of different solid dispersion strategies: i) spray-drying [polyvinlypyrrolidone (PVP)-HM] and ii) solvent evaporation [HP-ß-cyclodextrin (cyclodextrin)-HM]. The P-gp inhibition of these 2 formulations was assessed on the basis of rhodamine 123 uptake in cancer cells. Blood and brain pharmacokinetic parameters were also determined, and the antitumor effect of cyclodextrin-HM with PTX was evaluated in an orthotopic glioma xenograft mouse model. Results: Although both PVP-HM and cyclodextrin-HM formulations showed promising P-gp inhibition activity in vitro, cyclodextrin-HM had a higher maximum tolerated dose in mice than did PVP-HM. Pharmacokinetic study of cyclodextrin-HM revealed a plasma concentration plateau at 20 mg/kg, and the mice began to lose weight at doses above this level. Cyclodextrin-HM (10 mg/kg) administered with PTX at 10 mg/kg showed optimal antitumor activity in a mouse model, according to both tumor volume measurement and survival time (P < 0.05). Conclusions: In a mouse orthotopic brain tumor model, the intravenous co-administration of cyclodextrin-HM with PTX showed potent antitumor effects and therefore may have potential for glioma therapy in humans.

Overexpression of ubiquitin-conjugating enzyme E2 L3 in hepatocellular carcinoma potentiates apoptosis evasion by inhibiting the GSK3ß/p65 pathway.

UBE2L3 is a ubiquitin-conjugating protein belonging to the E2 family that consists of 153 amino acid residues. In this study, we found that UBE2L3 was generally upregulated in clinical HCC samples compared to non-tumour samples and that there was a strong association between high UBE2L3 expression and tumour size, clinical grade and prognosis in HCC patients. UBE2L3 depletion inhibited the proliferation and induced the apoptosis of HCC cells. At the molecular level, we observed that UBE2L3 depletion enhanced the protein stability of GSK3ß, thus promoting the expression and activation of GSK3ß. Subsequently, activated GSK3ß phosphorylated p65 and promoted its nuclear translocation to increase the expression of target genes, including PUMA, Bax, Bim, Bad, and Bid. In vivo, knockout of UBE2L3 in HCC cells inhibited tumour growth in orthotopic liver injection nude mouse models. Moreover, inhibition of p65 or GSK3ß significantly restored the effects induced by UBE2L3 knockout in HCC. Together, this study reveals the stimulatory effect of UBE2L3 on HCC cell proliferation, suggesting that UBE2L3 may be an important pro-tumorigenic factor in liver carcinogenesis and a potential therapeutic target of HCC.

The active components derived from Penthorum chinensePursh protect against oxidative-stress-induced vascular injury via autophagy induction.

Oxidative stress-induced damage has been proposed as a major risk factor for cardiovascular disease and is a pathogenic feature of atherosclerosis. Although autophagy was reported to have a protective effect against atherosclerosis, its mechanism for reducing oxidative stress remains un-elucidated. In this study, we have identified 4 novel autophagic compounds from traditional Chinese medicines (TCMs), which activated the AMPK mediated autophagy pathway for the recovery of mitochondrial membrane potential (MMP) to reduce the production of reactive oxygen species (ROS) in Human umbilical vein endothelial cells (HUVECs). In this study, 4 compounds (TA, PG, TB and PG1) identified from Penthorum chinense Pursh (PCP) were demonstrated for the first time to possess binding affinity to HUVECs cell membranes via cell membrane chromatography (CMC) accompanied by UHPLC-TOF-MS analysis, and the 4 identified compounds induce autophagy in HUVECs. Among the 4 autophagic activators identified from PCP, TA (Thonningianin A, Pinocembrin dihydrochalcone-7-O-[3″-O-galloyl-4″,6″-hexahydroxydiphenoyl]-glucoside) is the major chemcial component in PCP, which possesses the most potent autophagy effect via a Ca2+/AMPK-dependent and mTOR-independent pathways. Moreover, TA efficiently reduced the level of ROS in HUVECs induced by H2O2. Additionally, the expression of pro- and cleaved-IL-1ß in the aortic artery of ApoE-KO mice were also alleviated at the transcription and post-transcription levels after the administration of TA, which might be correlated to the reduction of oxidative-stress induced inflammasome-related Nod-like receptor protein3 (NLRP3) in the aortic arteries of ApoE-KO mice. This study has pinpointed the novel autophagic role of TA in alleviating the oxidative stress of HUVECs and aortic artery of ApoE-KO mice, and provided insight into the therapeutic application of TA in treatment of atherosclerosis or other cardiovascular diseases.

NAD(P)H: Quinone oxidoreductase 1 overexpression in hepatocellular carcinoma potentiates apoptosis evasion through regulating stabilization of X-linked inhibitor of apoptosis protein.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an antioxidant enzyme which is associated with poor prognosis in human breast, colon, lung and liver cancers. However, the molecular mechanisms underlying the pro-tumorigenic function of NQO1 remains unclear. This study investigated the function of NQO1 in the context of hepatocellular carcinoma (HCC) development. We found that NQO1 was frequently up-regulated in human liver cancer, and its high expression level was correlated with the tumor stage and low survival rate of HCC patients. Loss-of-function of NQO1 inhibited growth in HCC cells with increased apoptosis in vitro, and suppressed orthotopic tumorigenicity in vivo. Mechanistically, high level of NQO1 in HCC cells enhanced protein stability of X-linked inhibitor of apoptosis protein (XIAP) by increasing its phosphorylation at Ser 87. Reintroduction of wile type XIAP and the phospho-mimic mutants XIAPS87D significantly reversed NQO1 knock-down/out induced growth inhibition and apoptosis. In mouse model with orthotopically implanted hepatocarcinoma, NQO1 suppression and NQO1 inhibitor suppressed tumor growth and induced apoptosis. NQO1 plays an important role in sustaining HCC cell proliferation and may thus act as a potential therapeutic target in HCC treatment.