Connexin32 ameliorates epithelial-to-mesenchymal-transition in diabetic renal tubular via inhibiting NOX4.

Renal tubulointerstitial fibrosis (RIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs), is the main cause of diabetic renal fibrosis. Oxidative stress plays a pivotal role in the development of diabetic RIF. Connexin32 (Cx32), prominently expressed in renal TECs, has emerged as an important player in the regulation of oxidative stress. However, the role of Cx32 in diabetic RIF has not been explored yet. Here, we showed that adenovirus-mediated Cx32 overexpression suppressed EMT to ameliorate RIF and renal function in STZ-induced diabetic mice, while knockout (KO) of Cx32 exacerbated RIF in diabetic mice. Moreover, overexpression of Cx32 inhibited EMT and the production of extra cellular matrix (ECM) in high glucose (HG) induced NRK-52E cells, whereas knockdown of Cx32 showed the opposite effects. Furthermore, we showed that NOX4, the main source of ROS in renal tubular, was down-regulated by Cx32. Mechanistically, Cx32 down-regulated the expression of PKC alpha in a carboxyl-terminal-dependent manner, thereby inhibiting the phosphorylation at Thr147 of p22phox triggered by PKC alpha, which ultimately repressed the formation of the p22phox-NOX4 complex to reduce the protein level of NOX4. Thus, we establish Cx32 as a novel target and confirm the protection mechanism in RIF.

Markers of MEK inhibitor resistance in low-grade serous ovarian cancer: EGFR is a potential therapeutic target.

BACKGROUND: Although low-grade serous ovarian cancer (LGSC) is rare, case-fatality rates are high as most patients present with advanced disease and current cytotoxic therapies are not overly effective. Recognizing that these cancers may be driven by MAPK pathway activation, MEK inhibitors (MEKi) are being tested in clinical trials. LGSC respond to MEKi only in a subgroup of patients, so predictive biomarkers and better therapies will be needed. METHODS: We evaluated a number of patient-derived LGSC cell lines, previously classified according to their MEKi sensitivity. Two cell lines were genomically compared against their matching tumors samples. MEKi-sensitive and MEKi-resistant lines were compared using whole exome sequencing and reverse phase protein array. Two treatment combinations targeting MEKi resistance markers were also evaluated using cell proliferation, cell viability, cell signaling, and drug synergism assays. RESULTS: Low-grade serous ovarian cancer cell lines recapitulated the genomic aberrations from their matching tumor samples. We identified three potential predictive biomarkers that distinguish MEKi sensitive and resistant lines: KRAS mutation status, and EGFR and PKC-alpha protein expression. The biomarkers were validated in three newly developed LGSC cell lines. Sub-lethal combination of MEK and EGFR inhibition showed drug synergy and caused complete cell death in two of four MEKi-resistant cell lines tested. CONCLUSIONS: KRAS mutations and the protein expression of EGFR and PKC-alpha should be evaluated as predictive biomarkers in patients with LGSC treated with MEKi. Combination therapy using a MEKi with EGFR inhibition may represent a promising new therapy for patients with MEKi-resistant LGSC.

Indolyl-naphthyl-maleimides as potent and selective inhibitors of protein kinase C-α/ß.

The indolyl-naphthyl maleimide 7 is a potent inhibitor of the classical PKC isotypes α,ß and shows excellent selectivity over the novel PKC isotypes δ,ε,η,θ and other kinases belonging to the AGC family. The SAR around 7 as well as the physico-chemical characteristics of selected derivatives and their activity in T and B cell activation and proliferation assays are discussed.

PKC-alpha modulation by miR-483-3p in platinum-resistant ovarian carcinoma cells.

The occurrence of drug resistance limits the efficacy of platinum compounds in the cure of ovarian carcinoma. Since microRNAs (miRNAs) may contribute to this phenomenon by regulating different aspects of tumor cell response, the aim of this study was to exploit the analysis of expression of miRNAs in platinum sensitive/resistant cells in an attempt to identify potential regulators of drug response. MiR-483-3p, which may participate in apoptosis and cell proliferation regulation, was found up-regulated in 4 platinum resistant variants, particularly in the IGROV-1/Pt1 subline, versus parental cells. Transfection of a synthetic precursor of miR-483-3p in IGROV-1 parental cells elicited a marked up-regulation of the miRNA levels. Growth-inhibition and colony-forming assays indicated that miR-483-3p over-expression reduced cell growth and conferred mild levels of cisplatin resistance in IGROV-1 cells, by interference with their proliferative potential. Predicted targets of miR-483-3p included PRKCA (encoding PKC-alpha), previously reported to be associated to platinum-resistance in ovarian carcinoma. We found that miR-483-3p directly targeted PRKCA in IGROV-1 cells. In keeping with this finding, cisplatin sensitivity of IGROV-1 cells decreased upon molecular/pharmacological inhibition of PKC-alpha. Overall, our results suggest that overexpression of miR-483-3p by ovarian carcinoma platinum-resistant cells may interfere with their proliferation, thus protecting them from DNA damage induced by platinum compounds and ultimately representing a drug-resistance mechanism. The impairment of cell growth may account for low levels of drug resistance that could be relevant in the clinical setting.

The Effect Of Continuously Dutasteride Monotherapy on The Expression Of Protein Kinase C-Alpha Enzyme In BPH Model Of Wistar Strain Rattus Novergicus Rat.

Background: Benign prostate hyperplasia (BPH) is frequently found in the elderly and significantly impacts the quality of life. One of the risk factors that induce BPH is the androgen hormone. One of the effective medications in reducing the severity of Lower Urinary Tract Symptoms caused by BPH is the α-adrenergic receptor 5α-reductase inhibitor. Objective: The study aims to see the effect of long-term dutasteride on the expression of the PKC-α enzyme in prostatic stromal tissue in the BPH Model of Wistar strain Rattus norvegicus rats. Method: This study was an experimental, post-test-only, control group design that used randomization in sample selection. The objective is to measure the expression of PKC-α enzyme from prostate tissue of an adult male Wistar Strain of Rattus Novergicus rat that was given testosterone to induce BPH and given dutasteride in 1,3 and 6 days continuously. Data is shown in mean±SD, and all of the data were analyzed using the software SPSS 21st version with the One Way ANOVA Statistical method after fulfilling the normality test and variant homogeneity test. Data analysis with confidence rate 95% and a=0,05. Results: There was a decrease of PKC-α enzyme and prostate weight in dutasteride monotherapy in 1,3,6 days compared to the positive control, and the lowest value was on the sixth day (SD ± 2876.8). There was a constant decrease of PKC-α enzyme from the first day until the sixth day. Conclusion: In conclusion, long-term dutasteride monotherapy could significantly decrease the level of PKC-α enzyme. There was no upregulation of the PKC-α enzyme in the long term of dutasteride monotherapy.