Ashok Sharma

Claudin Family of Proteins and Cancer: An Overview

Tight junctions are the apical cell-cell adhesion that regulate paracellular permeability and are critical for epithelial cell polarity. Molecular architecture of tight junction has been studied extensively, which has confirmed that claudin family of proteins is integral component of tight junction. Loss of cell-cell adhesion is central to the cellular transformation and acquisition of metastatic potential; however, the role of claudin family of proteins play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Several claudin mouse knockout models have been generated and the diversity of phenotypes observed clearly demonstrates their important roles in the maintenance of tissue integrity in various organs and suggest that claudins also participate in cellular contexts other than tight junctions. The mechanisms of claudin regulation and their exact roles in normal physiology and disease are being elucidated, but much work remains to be done. In this review, we have discussed the conceptual framework concerning claudins and their potential implication in cancer. We predict that next several years will likely witness a boom in our understanding of the potential role of claudins in the regulation of tumorigenesis, which may, in turn, provide new approaches for the targeted therapy.

Preventive effect of crocin of Crocus sativus on hemodynamic, biochemical, histopathological and ultrastuctural alterations in isoproterenol-induced cardiotoxicity in rats

We investigated the effects of crocin, a pharmacologically active constituent of Crocus sativus L., in isoproterenol (ISO)-induced cardiotoxicity with reference to hemodynamic, antioxidant, histopathological and ultrastructural parameters. Rats were administered crocin (5, 10 and 20mg/kg/day) or vehicle orally for 21 days along with ISO (85mg/kg, subcutaneously, at 24h interval) on 20th and 21st day. On 22nd day ISO-control rats showed cardiac dysfunction as indicated by lowering of systolic, diastolic and mean arterial blood pressures. In addition, a significant decrease in maximum positive and negative rate of developed left ventricular pressure (+/-LVdp/dt(max)) and an increase in left ventricular end-diastolic pressure (LVEDP) were observed. Furthermore, a marked reduction in the activities of myocardial creatine kinase-MB (CK-MB) isoenzyme, lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) levels along with an increase in content of malondialdehyde (MDA) were observed. Myocardial necrosis, edema and inflammation were evident from the light microscopic and ultrastructural changes. Crocin at the dose of 20mg/kg/day significantly modulated hemodynamic and antioxidant derangements. The preventive role of crocin on ISO-induced MI was reconfirmed by histopathological and ultrastructural examinations. The effect at the dose of 20mg/kg/day of crocin was more pronounced than that of other two doses (5 and 10mg/kg/day). The results suggest that crocin may have cardioprotective effect in ISO-induced cardiac toxicity through modulation of oxidative stress in such a way that maintains the redox status of the cell.

Up-regulation of PPARγ, heat shock protein-27 and -72 by naringin attenuates insulin resistance, β-cell dysfunction, hepatic steatosis and kidney damage in a rat model of type 2 diabetes

Naringin, a bioflavonoid isolated from grapefruit, is well known to possess lipid-lowering and insulin-like properties. Therefore, we assessed whether naringin treatment ameliorates insulin resistance (IR), β-cell dysfunction, hepatic steatosis and kidney damage in high-fat diet (HFD)-streptozotocin (STZ)-induced type 2 diabetic rats. Wistar albino male rats were fed a HFD (55 % energy from fat and 2 % cholesterol) to develop IR and on the 10th day injected with a low dose of streptozotocin (40 mg/kg, intraperitoneal (ip)) to induce type 2 diabetes. After confirmation of hyperglycaemia (>13·89 mmol/l) on the 14th day, different doses of naringin (25, 50 and 100 mg/kg per d) and rosiglitazone (5 mg/kg per d) were administered orally for the next 28 d while being maintained on the HFD. Naringin significantly decreased IR, hyperinsulinaemia, hyperglycaemia, dyslipidaemia, TNF-α, IL-6, C-reactive protein and concomitantly increased adiponectin and β-cell function in a dose-dependent manner. Increased thiobarbituric acid-reactive substances and decreased antioxidant enzyme activities in the serum and tissues of diabetic rats were also normalised. Moreover, naringin robustly increased PPARγ expression in liver and kidney; phosphorylated tyrosine insulin receptor substrate 1 in liver; and stress proteins heat shock protein (HSP)-27 and HSP-72 in pancreas, liver and kidney. In contrast, NF-κB expression in these tissues along with sterol regulatory element binding protein-1c and liver X receptor- expressions in liver were significantly diminished. In addition, microscopic observations validated that naringin effectively rescues β-cells, hepatocytes and kidney from HFD-STZ-mediated oxidative damage and pathological alterations. Thus, this seminal study provides cogent evidence that naringin ameliorates IR, dyslipidaemia, β-cell dysfunction, hepatic steatosis and kidney damage in type 2 diabetic rats by partly regulating oxidative stress, inflammation and dysregulated adipocytokines production through up-regulation of PPARγ, HSP-27 and HSP-72.

HDAC inhibitors regulate claudin-1 expression in colon cancer cells through modulation of mRNA stability

Expression and cellular distribution of claudin-1, a tight junction protein, is dysregulated in colon cancer and its overexpression in colon cancer cells induced dedifferentiation and increased invasion. However, the molecular mechanism(s) underlying dysregulated claudin-1 expression in colon cancer remains poorly understood. Histone deacetylase (HDAC)-dependent histone acetylation is an important mechanism of the regulation of cancer-related genes and inhibition of HDACs induces epithelial differentiation and decreased invasion. Therefore, in this study, we examined the role of HDAC-dependent epigenetic regulation of claudin-1 in colon cancer. In this study, we show that sodium butyrate and Trichostatin A (TSA), two structurally different and widely used HDAC inhibitors, inhibited claudin-1 expression in multiple colon cancer cell lines. Further studies revealed modulation of claudin-1 mRNA stability by its 3′-UTR as the major mechanism underlying HDAC-dependent claudin-1 expression. In addition, overexpression of claudin-1 abrogated the TSA-induced inhibition of invasion in colon cancer cells suggesting functional crosstalk. Analysis of mRNA expression in colon cancer patients, showed a similar pattern of increase in claudin-1 and HDAC-2 mRNA expression throughout all stages of colon cancer. Inhibition of claudin-1 expression by HDAC-2-specific small interfering RNA further supported the role of HDAC-2 in this regulation. Taken together, we report a novel post-transcriptional regulation of claudin-1 expression in colon cancer cells and further show a functional correlation between claudin-1 expression and TSA-mediated regulation of invasion. As HDAC inhibitors are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.

Zingiber officinale exhibits behavioral radioprotection against radiation-induced CTA in a gender-specific manner

At the organismic level, exposure to radiation can produce taste aversion (CTA) learning and emesis, which have been proposed as behavioral endpoints that are mediated by harmful effects of radiations on peripheral systems, primarily the gastrointestinal system. Thus, the aim of the present investigation was to study the gastroprotective action of hydroalcoholic extract of zingiber rhizome (Zingiber officinale Rosc.) against radiation-induced conditioned taste aversion (CTA) in both male and female species of animals, for testing its potential as a behavioral radioprotector. Administration of zingiber extract 1 h before 2-Gy gamma-radiation was significantly effective in blocking the saccharin avoidance response, with 200 and 250 mg/kg b.wt. i.p., being the most effective doses for male and female rats, respectively. A comparison of the efficacy of zingiber extract with two antiemetic drugs, ondansteron and dexamethasone, revealed that the extract rendered comparable protection against radiation-induced CTA. Our experiments also confirmed the existence of sex dichotomy (i.e., the sex of animal greatly influenced response towards radiation exposure) in relation to behavioral responses (CTA) or differential metabolism. The observed gender variations were hypothesized to be a result of hormonal fluctuations and differences in pharmacological parameters in male and female rats. To correlate the mechanism of action, the free-radical-scavenging potential of zingiber extract to scavenge hydroxyl ion and nitric oxide was also tested, in cell-free system and a concentration of 1000 microg/ml, was found to be the most potent, which has been proposed as one the many activities assisting in its overall ability to modulate radiation-induced taste aversion. The results demonstrate that Z. officinale possesses antioxidant, radioprotective and neuromodulatory properties that can be effectively utilized for behavioral radioprotection and for efficiently mitigating radiation-induced CTA in both males and females species.

Pharmacovigilance: Effects of herbal components on human drugs interactions involving Cytochrome P450

Cytochrome P450 (CYP P450) enzymes are a superfamily of mono-oxygenases that are found in all kingdoms of life. The CYP P450 enzymes constitute a large superfamily of haem-thiolate proteins involved in the metabolism of a wide variety of both exogenous and endogenous compounds. The CYP activities have been shown to be involved in numerous interactions especially between drugs and herbal constituents. The majority of serious cases of drug interactions are as a result of the interference of the metabolic clearance of one drug by yet another co-administered drug, food or natural product. Gaining mechanistic knowledge towards such interactions has been accepted as an approach to avoid adverse reactions. The inductions and inhibition of CYP enzymes by natural products in the presence of a prescribed drug has led to adverse effects. Herbal medicines such as St. Johns wort (Hypericum perforatum), garlic (Allium sativa), piperine (from Piper sp.), ginseng (Ginseng sp.), gingko (Gingko biloba), soya beans (Glycine max), alfalfa (Medicago sativa) and grape fruit juice show clinical interactions when co-administered with medicines. This review documents the involvement of CYP enzymes in the metabolism of known available drugs and herbal products. We also document the interactions between herbal constituents & CYP enzymes showing potential drug-herb interactions. Data on CYP450 enzymes in activation (i.e. induction or inhibition) with natural constituents is also reviewed.

Overexpression of high mobility group (HMG) B1 and B2 proteins directly correlates with the progression of squamous cell carcinoma in skin.

High mobility group B (HMGB) chromosomal proteins, which plays important role in cancer and inflammation, were followed at various stages of the squamous cell carcinoma of skin. Present results were analyzed by histopathology, BrdU assay, immunohistochemistry, western blot and RT-PCR, which indicate that at early stages of tumorigenesis, expression of HMGB (B1, B2), raised only by about 20%. However, the advanced (⩾12 weeks) tumors showed significant (⩾80%) increase in HMG levels. Using skin cancer model, we demonstrated that high levels of HMGB directly correlate with the extent of neoplastic changes, and it appears that HMGB is an effective stimulus for cell differentiation, tumor progression, and metastatic invasion.

Caudal Homeobox Protein Cdx-2 Cooperates with Wnt Pathway to Regulate Claudin-1 Expression in Colon Cancer Cells

Dysregulation of tight junctions (TJs) is often associated with human diseases including carcinogenesis and recent studies support role of TJ integral proteins in the regulation of Epithelial-to-Mesenchymal Transition (EMT). In this regard, expression of claudin-1, a key constituent of TJs, is highly increased in colon cancer and is causally associated with the tumor growth and progression. However, mechanism/s underlying regulation of claudin-1 expression in intestinal epithelial cells remains poorly understood. In our studies, we have identified putative binding sites for intestinal transcription factors Cdx1, -2 and GATA4 in the 5′-flanking region of the claudin-1 gene. Our further studies using full length and/or deletion mutant constructs in two different human colon cancer cell lines, SW480 and HCT116, showed key role of Cdx1, Cdx2 and GATA4 in the regulation of claudin-1 mRNA expression. However, overexpression of Cdx2 had the most potent effect upon claudin-1 mRNA expression and promoter activity. Also, in colon cancer patient samples, we observed a significant and parallel correlation between claudin-1 and Cdx2 expressions. Chromatin immunoprecipitation (ChIP) assay confirmed the Cdx2 binding with claudin-1 promoter in vivo. Using Cdx2 deletion mutant constructs, we further mapped the Cdx2 C-terminus domain to be important in the regulation of claudin-1 promoter activity. Interestingly, co-expression of activated β-catenin further induced the Cdx2-dependent upregulation of claudin-1 promoter activity while expression of the dominant negative (dn)-TCF-4 abrogated this activation. Taken together, we conclude that homeodomain transcription factors Cdx1, Cdx2 and GATA4 regulate claudin-1 gene expression in human colon cancer cells. Moreover, a functional crosstalk between Wnt-signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins and GATA-proteins is demonstrated in the regulation of claudin-1 promoter-activation.

Upregulation of PPARγ by Aegle marmelos ameliorates insulin resistance and β-cell dysfunction in high fat diet fed-streptozotocin induced type 2 diabetic rats.

The global epidemic of type 2 diabetes demands the rapid evaluation of new and accessible interventions. This study investigated whether Aegle marmelos fruit aqueous extract (AMF; 250, 500 and 1000 mg/kg) improves insulin resistance, dyslipidemia and β‐cell dysfunction in high fat diet fed‐streptozotocin (HFD‐STZ)‐induced diabetic rats by modulating peroxisome proliferator‐activated receptor‐γ (PPARγ) expression. The serum levels of glucose, insulin, homeostasis model assessment of insulin resistance (HOMA‐IR), homeostasis model assessment of β‐cell function (HOMA‐B), lipid profile, TNF‐α and IL‐6 were evaluated. Further, the TBARS level and SOD activity in pancreatic tissue and PPARγ protein expression in liver were assessed. In addition, histopathological and ultrastructural studies were performed to validate the effect of AMF on β‐cells. The HFD‐STZ treated rats showed a significant increase in the serum levels of glucose, insulin, HOMA‐IR, TNF‐α, IL‐6, dyslipidemia with a concomitant decrease in HOMA‐B and PPARγ expression. Treatment with AMF for 21 days in diabetic rats positively modulated the altered parameters in a dose‐dependent manner. Furthermore, AMF prevented inflammatory changes and β‐cell damage along with a reduction in mitochondrial and endoplasmic reticulum swelling. These findings suggest that the protective effect of AMF in type 2 diabetic rats is due to the preservation of β‐cell function and insulin‐sensitivity through increased PPARγ expression. Copyright

Podophyllum hexandrum (Himalayan mayapple) extract provides radioprotection by modulating the expression of proteins associated with apoptosis.

Podophyllum hexandrum Royale (Himalayan mayapple), a high‐altitude Himalayan plant, has been shown to provide over 80% whole‐body radioprotection in mice. To investigate the radioprotective potential of P. hexandrum at the molecular level, expression patterns of various proteins associated with apoptosis were studied in the spleen of male Swiss albino strain A mice by immunoblotting. Treatment with P. hexandrum [200 mg/kg of body weight; an ethanolic 50% (w/v) extract delivered intraperitoneally] 2 h before irradiation resulted in MAPKAP (mitogen‐activated protein kinase‐activated protein) kinase‐2 activation along with HSF‐1 (heat‐shock transcription factor‐1), leading to up‐regulation of HSP‐70 (heat‐shock protein‐70) as compared with sham‐irradiated (10 Gy) mice. Strong inhibition of AIF (apoptosis‐inducing factor) expression was observed in the mice treated with P. hexandrum 2 h before irradiation as compared with the sham‐irradiated group. Inhibition in the translocation of free NF‐κB (nuclear factor κB) from cytoplasm to nucleus was observed upon P. hexandrum pretreatment 2 h before irradiation when compared with radiation‐treated mice. P. hexandrum pre‐treatment (2 h before irradiation) resulted in inhibition of NF‐κB translocation, and the expression of tumour suppressor protein p53 was observed to be down‐regulated as compared with sham‐irradiated control. An increase in the expression of proteins responsible for cell proliferation [Bcl‐2 (B‐cell chronic lymphocytic lymphoma 2), Ras‐GAP (Ras‐GTPase‐activating protein) and PCNA (proliferating cell nuclear antigen)] was observed in the P. hexandrum‐pretreated irradiated mice as compared with sham‐irradiated controls. Caspase 3 activation resulted PARP [poly(ADP‐ribose) DNA polymerase] cleavage, and DNA degradation was strongly inhibited in the mice treated with P. hexandrm (+/− irradiation) as compared with the mice treated with radiation (+/− heat shock). The present study thus clearly demonstrated that P. hexandrum extract provides protection from γ‐radiation by the modulation of expression of proteins associated with cell death.