No abstract

No abstract

No abstract

Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blocking therapy has become a major pillar of cancer immunotherapy. Compared with antibodies targeting, small-molecule checkpoint inhibitors which have favorable pharmacokinetics are urgently needed. Here we identified berberine (BBR), a proven anti-inflammation drug, as a negative regulator of PD-L1 from a set of traditional Chinese medicine (TCM) chemical monomers. BBR enhanced the sensitivity of tumour cells to co-cultured T-cells by decreasing the level of PD-L1 in cancer cells. In addition, BBR exerted its antitumor effect in Lewis tumor xenograft mice through enhancing tumor-infiltrating T-cell immunity and attenuating the activation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). BBR triggered PD-L1 degradation through ubiquitin (Ub)/proteasome-dependent pathway. Remarkably, BBR selectively bound to the glutamic acid 76 of constitutive photomorphogenic-9 signalosome 5 (CSN5) and inhibited PD-1/PD-L1 axis through its deubiquitination activity, resulting in ubiquitination and degradation of PD-L1. Our data reveals a previously unrecognized antitumor mechanism of BBR, suggesting BBR is small-molecule immune checkpoint inhibitor for cancer treatment.

BackgroundBerberine (BBR) is a drug with multiple effects on cellular energy metabolism. The present study explored answers to the question of which CYP450 (Cytochrome P450) isoenzymes execute the phase-I transformation for BBR, and what are the bioactivities of its metabolites on energy pathways.MethodsBBR metabolites were detected using LC-MS/MS. Computer-assistant docking technology as well as bioassays with recombinant CYP450s were employed to identify CYP450 isoenzymes responsible for BBR phase-I transformation. Bioactivities of BBR metabolites in liver cells were examined with real time RT-PCR and kinase phosphorylation assay.ResultsIn rat experiments, 4 major metabolites of BBR, berberrubine (M1), thalifendine (M2), demethyleneberberine (M3) and jatrorrhizine (M4) were identified in rat's livers using LC-MS/MS (liquid chromatography-tandem mass spectrometry). In the cell-free transformation reactions, M2 and M3 were detectable after incubating BBR with rCYP450s or human liver microsomes; however, M1 and M4 were below detective level. CYP2D6 and CYP1A2 played a major role in transforming BBR into M2; CYP2D6, CYP1A2 and CYP3A4 were for M3 production. The hepatocyte culture showed that BBR was active in enhancing the expression of insulin receptor (InsR) and low-density-lipoprotein receptor (LDLR) mRNA, as well as in activating AMP-activated protein kinase (AMPK). BBR's metabolites, M1-M4, remained to be active in up-regulating InsR expression with a potency reduced by 50-70%; LDLR mRNA was increased only by M1 or M2 (but not M3 and M4) with an activity level 35% or 26% of that of BBR, respectively. Similarly, AMPK-α phosphorylation was enhanced by M1 and M2 only, with a degree less than that of BBR.ConclusionsFour major BBR metabolites (M1-M4) were identified after phase-I transformation in rat liver. Cell-free reactions showed that CYP2D6, CYP1A2 and CYP3A4 seemed to be the dominant CYP450 isoenzymes transforming BBR into its metabolites M2 and M3. BBR's metabolites remained to be active on BBR's targets (InsR, LDLR, and AMPK) but with reduced potency.

Aberrant expressions of the neuronal protein synuclein gamma (SNCG) in malignant mammary epithelial cells are strongly associated with the progression of breast cancer. SNCG is not expressed in normal breast tissues but abundantly expressed in a high percentage of invasive and metastatic breast carcinomas. Several studies have demonstrated that SNCG expression significantly stimulates proliferation, invasion, and metastasis of breast cancer cells. To elucidate the molecular and cellular mechanisms underlying the tumorigenic functions of SNCG, we investigated the effects of SNCG expression on the mitotic checkpoint function of breast cancer cells. By conducting several different lines of investigations, we now demonstrate that SNCG expression in breast cancer cells overrides the mitotic checkpoint control and confers the cellular resistance to anti-microtubule drug-caused apoptosis. We further show that the inhibitory effects of SNCG on mitotic checkpoint can be overthrown by enforced overexpression of the mitotic checkpoint protein BubR1 in SNCG-expressing cells. These new findings combined with our previous observation that SNCG intracellularly associates with BubR1 together suggest that SNCG expression compromises the mitotic checkpoint control by inhibition of the normal function of BubR1, thereby promoting genetic instability. Genetic instability is recognized as an important contributing factor in tumorigenesis. Hence, our studies gain insight into the mechanisms whereby SNCG expression advances breast cancer disease progression and fasters tumor metastasis.

No abstract

Host heat shock cognate 70 (Hsc70) protein is packaged into hepatitis C viral (HCV) particles as a structural component of the virus in the assembly process. It helps HCV RNA release into the cytoplasm in the next infection cycle. The goal of this study is to investigate whether chemically down-regulating host Hsc70 expression could be a novel strategy to interrupt HCV replication. Compounds were screened with an Hsc70 messenger RNA (mRNA) assay. IMB-DM122 was found to be an effective and safe inhibitor for Hsc70 mRNA/protein expression in human hepatocytes. IMB-DM122 inhibited HCV replication through destabilization of Hsc70 mRNA, and the half-life of host Hsc70 mRNA was reduced by 78% after the compound treatment. The Hsc70 mRNA 3 0 untranslated region sequence is the element responsible for the effect of IMB-DM122 on Hsc70 mRNA. The compound appears to be highly efficient in inhibiting Hsc70-related HCV replication. Treatment of the HCV-infected hepatocytes with IMB-DM122 reduced the virion encapsidation of Hsc70, and therefore disrupted HCV replication and the infection cycle. IMB-DM122 showed considerable good safety in vitro as well as in vivo with no indication of harmful effect on liver and kidney functions. Conclusion: Hsc70 might be a new drug target and mechanism to inhibit HCV proliferation. (HEPATOLOGY 2010;52:845-853) H epatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae family. 1 Current standard therapy for hepatitis C in the clinic is the combination of pegylated-interferon with ribavirin. 2,3 The regimen is effective in 40%-50% of patients infected with HCV genotype 1 and is associated with significant side effects. 3,4 Recently, telaprevir and boceprevir, two peptidomimetic inhibitors of the HCV nonstructural protein 3/4A (NS3/4A) protease, have shown great promise in clinical patients. [5][6][7][8] However, antiviral therapy targeting specific HCV proteases has caused emergence of drug-resistant mutations, 9-12 and thus, new mechanisms for anti-HCV drugs are highly desirable.Human heat shock cognate 70 (Hsc70, or heat shock protein A8) is a cytoplasm adenosine triphosphate-binding protein with 646 amino acids. 13 It is a member of the heat shock protein 70 (Hsp70) family with the gene located in chromosome 11. 14 The functions of Hsc70 protein in normal cells are complicated and remain to be clarified. 15 Virology research has shown that Hsc70 might play a role in regulating virion capsid assembly. 16 A recent study by Parent et al. demonstrated that host Hsc70 is part of the HCV particle. 17 They showed Hsc70 interaction HPD (HisPro-Asp) motif present on the E2 envelope of HCV (the J6/JFH [Japanese fulminant hepatitis] strain) and coexistence of Hsc70 with the HCV core and E2 proteins around lipid droplets in the infected cells.