Current Molecular Medicine - Volume 18, Issue 7, 2018

Chronic hepatitis C (CHC) virus infection and associated hepatic diseases are still challenging, and the disease burden remains significant around the world. Overall treatment rates for the chronically infected patients have been “dismally poor” and that treatment completion of dual-therapy— pegylated interferon (PEG-IFN) and ribavirin (RBV) is suboptimal in the real-world clinical settings. The approval of first, second and next-generation direct-acting antivirals (DAAs) represents a major breakthrough in hepatitis C virus (HCV) therapeutics to treat CHC infected individuals. Such therapeutic regimens in a fixed dose combination (FDC) or along with RBV have proven their clinical efficacy against different HCV genotypes, and harder-to-treat special populations. We continue to see the development of novel pan-genotypic anti-HCV regimens with very high sustained virologic response (SVR; undetectable viral load at week 12 or at the end of therapy) rates, high barrier to drug resistance, low frequency of adverse events, and fewer drug-drug interactions as compared to some older RBV based triple DAA therapies. Oral interferon-free DAAs seem highly successful strategic treatment approaches against hepatitis C and impulse health policy makers to establish the treatment priorties and policies to reduce the rate of hepatitis C-related morbidity and mortality. This review article comprehensively overviews interferon-free anti-HCV regimens, which have totally shifted the treatment paradigms for hepatitis C with some additional benefits to galvanize our efforts to achieve the global goal of HCV elimination in near future.

Background: Primary hyperoxaluria type 1 (PH1) is an inherited disease caused by mutations in alanine-glyoxylate aminotransferase (AGXT). It is characterized by abnormal metabolism of glyoxylic acid in the liver leading to endogenous oxalate overproduction and deposition of oxalate in multiple organs, mainly the kidney. Patients of PH1 often suffer from recurrent urinary tract stones, and finally renal failure. There is no effective treatment other than combined liver-kidney transplantation. Methods: Microinjection was administered to PH1 rats. Urine samples were collected for urine analysis. Kidney tissues were for Western blotting, quantitative PCR, AGT assays and histological evaluation. Results: In this study, we generated a novel PH1 disease model through CRISPR/Cas9 mediated disruption of mitochondrial localized Agxt gene isoform in rats. Agxt-deficient rats excreted more oxalate in the urine than WT animals. Meanwhile, mutant rats exhibited crystalluria and showed a slight dilatation of renal tubules with mild fibrosis in the kidney. When supplied with 0.4% ethylene glycol (EG) in drinking water, mutant rats excreted greater abundance of oxalate and developed severe nephrocalcinosis in contrast to WT animals. Significantly elevated expression of inflammation- and fibrosisrelated genes was also detected in mutants. Conclusion: These data suggest that Agxt-deficiency in mitochondria impairs glyoxylic acid metabolism and leads to PH1 in rats. This rat strain would not only be a useful model for the study of the pathogenesis and pathology of PH1 but also a valuable tool for the development and evaluation of innovative drugs and therapeutics.

Background: Transforming growth factor-β (TGF-β)/nodal signaling is involved in early embryonic patterning in vertebrates. Nodal modulator (Nomo, also called pM5) is a negative regulator of nodal signaling. Currently, the role of nomo gene in cartilage development in vertebrates remains unknown. Methods: Nomo mutants were generated in a knockout model of zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) targeting of the fibronectin type III domain. The expression of related genes, which are critical for chondrogenesis, was analyzed by whole-mount in situ hybridization and qRT-PCR. Whole-mount alcian staining was performed to analyze the cartilage structure. Results: nomo is highly expressed in various tissues including the cartilage. We successfully constructed a zebrafish nomo knockout model. nomo homozygous mutants exhibited varying degrees of hypoplasia and dysmorphism on 4 and 5 dpf, which is similar to chondrodysplasia in humans. The key genes of cartilage and skeletal development, including sox9a, sox9b, dlx1a, dlx2a, osx, col10a1, and col11a2 were all downregulated in nomo mutants compared with the wildtype. Conclusion: The nomo gene positively regulates the expression of the master regulator and other key development genes involved in bone formation and cartilage development and it is essential for cartilage development in zebrafish.

Background: Madhuca indica belongs to the family sapotaceae, commonly known as Mahua. It is primarily known for alcoholic beverage production and is reported to have anti-inflammatory, analgesic and antipyretic properties. Madhuca indica has also been reported to be effective in several diseases. Objective: This study was undertaken to check the anticancer efficacy and chemopreventive effect of methanolic extract of Mahua flower (ME) on human breast cancer cell lines MCF-7 and MDA-MB-468. Method: The cytotoxic and anti-proliferative effects on MCF-7 and MDA-MB-468 cells were studied by MTT, hexosaminidase and colony formation assay. Expression of caspase 3/7 was assessed by flow cytometry and western blot analysis. Expression of COX-2 was evaluated by western blot analysis, luciferase assay and mRNA analysis. Results: ME inhibited the proliferation of breast cancer cells by inducing apoptosis through up-regulating the expression of Caspase 3/7 (P < 0.0001). Our results showed a decrease in the expression of COX-2 mRNA and COX-2 protein in both MCF-7 and MDA-MB-468 cells with an increase in ME concentration. Furthermore synergistic effect of ME and chemotherapeutic drug paclitaxel was also studied in MCF-7 and MDA-MB- 468 cells which were found to be more effective (P < 0.0001) than treatment of either ME or paclitaxel alone. Results were analyzed by ANOVA and Pearson correlation analysis. Conclusion: All these experiments suggest that ME inhibits breast cancer cell proliferation and apoptosis by inhibiting the expression of COX-2 in MCF-7 and MDAMB- 468 cells. This work further highlighted that ME may enhance the potentiality of paclitaxel in breast cancer treatment.

Purpose: In the light of contradictory results and paucity of information, this comprehensive study examines the activities and levels of key antioxidants and oxidants/pro-oxidants in preeclamptic patients. Methods: Antioxidants including glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase, catalase, reduced glutathione, selenium, zinc, copper and manganese, as well as marker oxidants/pro-oxidants including hydrogen peroxide, superoxide anions, malondialdehyde, protein carbonyls and oxidized glutathione were determined in plasma and placental tissues of nonpregnant, healthy pregnant and preeclamptic subjects. Results: Data indicated that all plasma antioxidants underwent moderate but significant decreases (p< 0.05) in healthy pregnant women, , and much more significant ones (p< 0.0001) in preeclamptic patients, when both were compared to non-pregnant subjects. Furthermore, whereas all plasma antioxidants underwent significant decreases (p< 0.001) in preeclamptic patients compared to healthy pregnant subjects, their placental activities and levels were very significantly decreased (p< 0.0001). However, copper plasma and placental levels were unchanged in all study groups. In contrast, there were increases similar in magnitude and significance of all plasma and placental oxidants/prooxidants compared among the three study groups leading to equally significant decreases in the reduced/oxidized glutathione ratios. In addition, gene transcripts of all antioxidant enzymes underwent marked downregulation (p< 0.0001) in placental tissue of preeclamptic patients compared to healthy pregnant subjects. Conclusion: Data indicated a metabolic shift in favor of oxidative stress more pronounced in placental tissue of preeclamptic patients compared to healthy pregnant/non-pregnant subjects. We postulate that selenium, zinc and manganese supplements could be beneficial for alleviation of the noted oxidative stress in preeclamptic patients.

Background: We have recently reported that epidermal growth factor (EGF) induces migration of osteoblast-like MC3T3-E1 cells through the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK) and Akt. Furthermore, we demonstrated that heat shock protein 70 (HSP70) down-regulates the transforming growth factor-β- stimulated vascular endothelial growth factor synthesis via suppression of p38 MAP kinase in osteoblast-like MC3T3-E1 cells. However, the exact role of HSP70 underlying osteoblast migration is not fully elucidated. Objective: The aim of this study is to investigate the effects of HSP70 inhibitors on the EGF-stimulated osteoblast migration, and the underlying mechanism. Methods: Osteoblast-like MC3T3-E1 cells were treated with two types of HSP70 inhibitors, VER-155008 or YM-08. Transwell cell migration assay and wound-healing assay were analyzed for osteoblast migration. The expression levels of HSP70 and the phosphorylation of p38 MAP kinase, p44/p42 MAP kinase, SAPK/JNK or Akt were evaluated by a Western blot analysis. Results: EGF hardly affected the expression levels of HSP70 at the present or absent of VER-155008. EGF-stimulated migration was significantly reduced by both HSP70 inhibitors, VER-155008 and YM-08, determined by a transwell cell migration assay. The suppressive effects of both HSP70 inhibitors on the migration stimulated by EGF were also observed by a wound-healing assay. VER-155008 inhibited the EGF-induced phosphorylation of p44/p42 MAP kinase and AKT, but not p38 MAP kinase or SAPK/JNK. Conclusion: This study provides new evidence that HSP70 inhibitors reduce the EGFstimulated migration of osteoblasts through the suppression of p44/p42 MAP kinase and Akt.

Objective: This study aims to analyze whether Cystathionine β-synthase (CBS) plays roles in hepatocellular carcinoma (HCC) drug resistance. Methods: MTS assay was used to detect the effect of chemotherapeutic drugs doxorubicin (DOX) and sunitinib on HCC cell viability and cell growth. Intracellular doxorubicin accumulation assay was performed to evaluate the sensitivity of DOX and sunitinib in HCC cells and the function of multidrug resistance-associated protein Pglycoprotein (P-gp). Quantification of H2S production was performed using the methylene blue method. Production of intracellular ROS was quantified using the DCFHDA assay. The scratch wound and transwell assays were used to determine the cell migration and invasion. Expression of proteins was tested by western blot analysis. Results: HepG2 cells with high CBS expression were less sensitive to DOX and sunitinib and knockdown of CBS significantly elevated the sensitivity to DOX and sunitinib in HepG2 cells. In contrast, CBS overexpression increased the resistance of DOX and sunitinib in BEL-7404 cells. Moreover, the overexpression of CBS caused the up-regulation of the expression level of P-gp and the decrease of DOX accumulation in BEL-7404 cells. In further mechanism research, we found that STAT3/Akt/Bcl-2 pathway activation, reactive oxygen species (ROS) inhibition as well as enhancement of the metastatic ability of hepatoma cells were responsible for the HCC drug resistance. Conclusion: CBS overexpression conferred HCC cell resistance.