Interference with megalin expression/endocytic function by montelukast mitigates gentamicin nephrotoxicity: Downregulation of ClC-5 expression.

Megalin receptor-mediated endocytosis participates a crucial role in gentamicin (GM) uptake, accumulation, and toxicity. In this study, we investigated the potential effects of montelukast (MLK) on megalin expression/endocytic function against GM nephrotoxicity. Male Wistar rats were administered GM (120 mg/kg; i.p.) daily in divided doses along 4 hr; 30 mg/kg/hr; for 7 days. MLK (30 mg/kg/day) was orally administered 7 days before and then concurrently with GM. The protein expressions of megalin and chloride channel-5 (ClC-5); one of the essential regulators of megalin endocytic function; were determined by Western blotting. Besides, the endocytic function of megalin was evaluated by the uptake of bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA) into proximal tubular epithelial cells. Moreover, kidney function biomarkers (Cr, BUN, GFR, KIM-1, cystatin-C) and apoptosis markers (p-AKT1, cleaved caspase-3) were estimated. Co-treatment with MLK downregulated ClC-5 expression leading to reduced recycling of megalin to the plasma membrane, reduced expression, and so impaired endocytic function that was evidenced by reduced uptake of FITC-BSA in proximal tubular epithelial cells. The protein expression of the apoptotic executioner cleaved caspase-3 was significantly reduced, while that of the antiapoptotic p-AKT1 was elevated. These results were confirmed by the improvement of kidney functions and histological findings. Our data suggest that MLK could interfere with megalin expression/endocytic function that could be attributed to downregulation of ClC-5 protein expression. That eventually reduces renal cell apoptosis and improves kidney functions after GM administration without affecting the antibacterial activity of GM. Therefore, reduced expression of ClC-5 and interference with megalin expression/endocytic function by MLK could be an effective strategy against GM nephrotoxicity.

Synthesis and biological studies of new piperidino-1,2,3-triazole hybrids with 3-aryl isoxazole side chains.

A small library of new piperidine-triazole hybrids with 3-aryl isoxazole side chains has been designed and synthesized. Their cytotoxicity against a panel of seven cancer cell lines has been established. For the most promising compound, an IC50 value of 3.8 µM on PUMA/Bcl-xL interaction in live cancer cells was established through BRET analysis. A rationale was proposed for these results through complete molecular modelling studies.

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein.

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic ß loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

In vitro synergistic activity of cisplatin and EGFR-targeted nanomedicine of anti-Bcl-xL siRNA in a non-small lung cancer cell line model.

Apoptosis is an important process that directly affects the response of cancer cells to anticancer drugs. Among different factors involved in this process, the BcL-xL protein plays a critical role in inhibiting apoptosis induced by chemotherapy agents. Henceforth, its downregulation may have a synergistic activity that lowers the necessary dose of anticancer agents. In this study, anti-Bcl-xL siRNA were formulated within an EGFR-targeted nanomedicine with scFv ligands (NM-scFv) and its activity was tested in the non-small cell lung cancer (NSCLC) cell line H460. The obtained NMs-scFv anti-Bcl-xL were suitable for intravenous injection with sizes around 100 nm, a high monodispersity level and good siRNA complexation capacity. The nanocomplex's functionalization with anti-EGFR scFv ligands was shown to allow an active gene delivery into H460 cells and led to approximately 63% of gene silencing at both mRNA and protein levels. The NM-scFv anti-Bcl-xL improved the apoptotic activity of cisplatin and reduced the cisplatin IC50 value in H460 cells by a factor of around three from 0.68 ± 0.12 µM to 2.21 ± 0.18 µM (p < 0.01), respectively, in comparison to that of NM-scFv formulated with control siRNA (p > 0.05).

CA10 is associated with HBV-related hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the main threat for the patients infected with hepatitis B virus (HBV), but the oncogenic mechanism of HBV-related HCC is still controversial. Previously, we have found that several HBV surface gene (HBS) non-sense mutations are oncogenic. Among these mutations, sW182* was found to have the most potent oncogenicity. In this study, we found that Carbonic Anhydrase X (CA10) level was specifically increased in sW182* mutant-expressing cells. CA10 overexpression was also associated with HBS nonsense mutation in HBV-related HCC tumor tissues. Transformation and tumorigenesis assays revealed that CA10 had significant oncogenic activity. In addition, CA10 overexpression resulted in dysregulation of apoptosis-related proteins, including Mcl-1, Bcl-2, Bcl-xL and Bad. While searching for the regulatory mechanism of CA10, miR-27b was found to downregulate CA10 expression by regulating its mRNA degradation and its expression was decreased in sW182* mutant cells. Moreover, CA10 overexpression was associated with down-regulation of miR-27b in human HBV-related HCC tumor tissues with sW182* mutation. Therefore, induction of the expression of CA10 through repression of miR-27b by sW182* might be one mechanism involved in HBS mutation-related hepatocarcinogenesis.

Nanoparticles design considerations to co-deliver nucleic acids and anti-cancer drugs for chemoresistance reversal.

Chemoresistance and hence the consequent treatment failure is considerably challenging in clinical cancer therapeutics. The understanding of the genetic variations in chemoresistance acquisition encouraged the use of gene modulatory approaches to restore anti-cancer drug efficacy. Many smart nanoparticles are designed and optimized to mediate combinational therapy between nucleic acid and anti-cancer drugs. This review aims to define a rational design of such co-loaded nanocarriers with the aim of chemoresistance reversal at various cellular levels to improve the therapeutic outcome of anticancer treatment. Going through the principles of therapeutics loading, physicochemical characteristics tuning, and different nanocarrier modifications, also looking at combination effectiveness on chemosensitivity restoration. Up to now, these emerging nanocarriers are in development status but are expected to introduce outstanding outcomes.

Chemical composition and pharmacological bio-efficacy of Parrotiopsis jacquemontiana (Decne) Rehder for anticancer activity.

Consistent STAT3 (Single transducer and activator of transcription 3) activation is observed in many tumors and promotes malignant cell transformation. In the present investigation, we evaluated the anticancer effects of Parrotiopsis jacquemontiana methanol fraction (PJM) on STAT3 inhibition in HCCLM3 and MDA-MB 231 cells. PJM suppressed the activation of upstream kinases i.e. JAK-1/2 (Janus kinase-1/2), and c-Src (Proto-oncogene tyrosine-protein kinase c-Src), and upregulated the expression levels of PIAS-1/3 (Protein Inhibitor of Activated STATs-1/3), SHP-1/2 (Src-homology region 2 domain-containing phosphatase-1/2), and PTP-1ß (Protein tyrosine phosphatase 1 ß) which negatively regulate STAT3 signaling pathway. PJM also decreased the levels of protein products conferring to various oncogenes, which in turn repressed the proliferation, migration, invasion, and induced apoptosis in cancer cell lines. The growth inhibitory effects of PJM on cell-cycle and metastasis were correlated with decreased expression levels of CyclinD1, CyclinE, MMP-2 (Matrix metalloproteinases-2), and MMP-9 (Matrix metalloproteinases-9). Induction of apoptosis was indicated by the cleavage and subsequent activation of Caspases (Cysteine-dependent Aspartate-directed Proteases) i.e. caspase-3, 7, 8, 9, and PARP (Poly (ADP-ribose) polymerase) as well as through the down-regulation of anti-apoptotic proteins. These apoptotic effects of PJM were preceded by inhibition of STAT3 cell-signaling pathway. STAT3 was needed for PJM-induced apoptosis, and inhibition of STAT3 via pharmacological inhibitor (Stattic; SC-203282) abolished the apoptotic effects. Conclusively, our results demonstrate the capability of PJM to inhibit cancer cell-proliferation and induce apoptosis by suppressing STAT3 via upregulation of STAT3 inhibitors and pro-apoptotic proteins whereas the down-regulation of upstream kinases and anti-apoptotic protein expression. In future, one-step advance studies of PHM regarding its role in metastatic inhibition, immune response modulation for reducing tumor, and inducing apoptosis in suitable animal models would be an interesting and promising research area.

Genetic or pharmacological reduction of cholangiocyte senescence improves inflammation and fibrosis in the Mdr2 -/- mouse.

BACKGROUND & AIMS: Cholangiocyte senescence is important in the pathogenesis of primary sclerosing cholangitis (PSC). We found that CDKN2A (p16), a cyclin-dependent kinase inhibitor and mediator of senescence, was increased in cholangiocytes of patients with PSC and from a PSC mouse model (multidrug resistance 2; Mdr2 -/-). Given that recent data suggest that a reduction of senescent cells is beneficial in different diseases, we hypothesised that inhibition of cholangiocyte senescence would ameliorate disease in Mdr2 -/- mice. METHODS: We used 2 novel genetic murine models to reduce cholangiocyte senescence: (i) p16Ink4a apoptosis through targeted activation of caspase (INK-ATTAC)xMdr2 -/-, in which the dimerizing molecule AP20187 promotes selective apoptotic removal of p16-expressing cells; and (ii) mice deficient in both p16 and Mdr2. Mdr2 -/- mice were also treated with fisetin, a flavonoid molecule that selectively kills senescent cells. p16, p21, and inflammatory markers (tumour necrosis factor [TNF]-α, IL-1ß, and monocyte chemoattractant protein-1 [MCP-1]) were measured by PCR, and hepatic fibrosis via a hydroxyproline assay and Sirius red staining. RESULTS: AP20187 treatment reduced p16 and p21 expression by ~35% and ~70% (p >0.05), respectively. Expression of inflammatory markers (TNF-α, IL-1ß, and MCP-1) decreased (by 60%, 40%, and 60%, respectively), and fibrosis was reduced by ~60% (p >0.05). Similarly, p16 -/- xMdr2 -/- mice exhibited reduced p21 expression (70%), decreased expression of TNF-α, IL-1ß (60%), and MCP-1 (65%) and reduced fibrosis (~50%) (p >0.05) compared with Mdr2 -/- mice. Fisetin treatment reduced expression of p16 and p21 (80% and 90%, respectively), TNF-α (50%), IL-1ß (50%), MCP-1 (70%), and fibrosis (60%) (p >0.05). CONCLUSIONS: Our data support a pathophysiological role of cholangiocyte senescence in the progression of PSC, and that targeted removal of senescent cholangiocytes is a plausible therapeutic approach. LAY SUMMARY: Primary sclerosing cholangitis is a fibroinflammatory, incurable biliary disease. We previously reported that biliary epithelial cell senescence (cell-cycle arrest and hypersecretion of profibrotic molecules) is an important phenotype in primary sclerosing cholangitis. Herein, we demonstrate that reducing the number of senescent cholangiocytes leads to a reduction in the expression of inflammatory, fibrotic, and senescence markers associated with the disease.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury.

BACKGROUND & AIMS: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. METHODS: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. RESULTS: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. CONCLUSIONS: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. LAY SUMMARY: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

Clinical efficacies, underlying mechanisms and molecular targets of Chinese medicines for diabetic nephropathy treatment and management.

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN.

Strikingly Different Atheroprotective Effects of Apolipoprotein A-I in Early- Versus Late-Stage Atherosclerosis.

Preclinical studies have shown benefit of apolipoprotein A-I (apoA-I)/high-density lipoprotein (HDL) raising in atherosclerosis; however, this has not yet translated into a successful clinical therapy. Our studies demonstrate that apoA-I raising is more effective at reducing early-stage atherosclerosis than late-stage disease, indicating that the timing of HDL raising is a critical factor in its atheroprotective effects. To date, HDL-raising clinical trials have only been performed in aged patients with advanced atherosclerotic disease. Our findings therefore provide insight, related to important temporal aspects of HDL raising, as to why the clinical trials have thus far been largely neutral.

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies.

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.

DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer.

Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL(wt)), TRAIL-R1 (sTRAIL(DR4)) and TRAIL-R2 (sTRAIL(DR5)) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL(DR4)) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL(wt). Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.