Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition.

Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-ß signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target.

Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in rats.

Flavonoids possess strong anti-oxidant and cancer chemopreventive activities. Chrysin (5,7-dihydroxyflavone) occurs naturally in many plants, honey, and propolis. In vitro, chrysin acts as a general anti-oxidant, causes cell cycle arrest and promotes cell death. However, the mechanism by which chrysin inhibits cancer cell growth and the subcellular pathways activated remains poorly understood. Effect of dietary supplementation with chrysin on proliferation and apoptosis during diethylnitrosamine (DEN)-induced early hepatocarcinogenesis was investigated in male Wistar rats. To induce hepatocarcinogenesis, rats were given DEN injections (i.p., 200 mg/kg) three times at a 15 day interval. An oral dose of chrysin (250 mg/kg bodyweight) was given three times weekly for 3 weeks, commencing 1 week after the last dose of DEN. Changes in the mRNA expression of COX-2, NFkB p65, p53, Bcl-xL and ß-arrestin-2 were assessed by quantitative real-time PCR. Changes in the protein levels were measured by western blotting. Chrysin administration significantly (P<0.001) reduced the number and size of nodules formed. Also, a significant (P<0.01) reduction in serum activities of AST, ALT, ALP, LDH and γGT was noticed. Expression of COX-2 and NFkB p65 was significantly reduced whereas that of p53, Bax and caspase 3 increased at the mRNA and protein levels. Likewise, a decrease in levels of ß-arrestin and the anti-apoptotic marker Bcl-xL was also noted. These findings suggest that chrysin exerts global hepato-protective effect and its chemopreventive activity is associated with p53-mediated apoptosis during early hepatocarcinogenesis.

Nuclear translocation of ß-catenin correlates with CD44 upregulation in Helicobacter pylori-infected gastric carcinoma.

Infection with Helicobacter pylori CagA-positive strains is associated with gastric adenocarcinoma. CagA H. pylori activates the ß-catenin signal by translocation into nucleus which promotes carcinogenesis. Deregulated accumulation of nuclear ß-catenin enhances transcription of ß-catenin target genes including CD44 and promotes malignant transformation. The aim of this study was to investigate whether nuclear translocation of ß-catenin correlates with CD44 expression in CagA H. pylori-infected gastric carcinoma. To address these issues, we examined 140 gastric biopsy specimens by using immunohistochemical and immunofluorescence staining, Western blot, and mutational analysis of the exon 3 ß-catenin gene. The nuclear localization of ß-catenin was significantly (χ(2) = 21.175; P < 0.001) increased in advanced gastric carcinoma and also correlated (χ(2) = 22.857; P < 0.001) with the CagA H. pylori positive specimens. We also observed that tyrosine-phosphorylated ß-catenin was significantly (χ(2) = 14.207; P < 0.001) increased in samples showing nuclear localization of ß-catenin and also it correlated (χ(2) = 43.69; P < 0.03) with the CagA H. pylori positive specimens. Exon 3 ß-catenin gene mutation was not detected in H. pylori-infected gastric carcinoma. CD44 up regulation was significantly associated with tyrosine-phosphorylated ß-catenin (χ(2) = 22.5; P < 0.001), and this change was closely associated with nuclear translocation of ß-catenin (χ(2) = 13.393; P < 0.001) in CagA H. pylori-infected gastric carcinoma. In conclusion, our data suggest that CagA H. pylori infection is responsible for the tyrosine phosphorylation of ß-catenin and its nuclear translocation, which upregulates ß-catenin target gene CD44 in gastric carcinoma.

Regulating RNA Binding Motif 5 Gene Expression- A Novel Therapeutic Target for Lung Cancer.

RNA-binding motif protein 5 (RBM5), also known as LUCA-15/H37, is a gene that maps to human chromosome 3p21.3, a critical region that is deleted in a large number of human cancers, of which the majority are lung cancers, and that is predicted to contain one or more tumor suppressor genes (TSGs). RBM5 is a tumor suppressor gene and is most frequently deleted at the earliest stage of lung cancer development. It represents a significant area of recent progress in cancer genomic, cytogenetic, and molecular biological research because of its role in the induction of cell cycle arrest and apoptosis and the regulation of inhibition of in lung cancer metastasis. RBM5 is involved in the suppression of epidermal growth factor receptor (EGFR) expression, thus preventing proliferation, angiogenesis, invasion, and metastasis of lung cancer. In this way it exhibits its tumor suppressive capacity during lung cancer progression. Exploration of RBM5's potential importance in inhibiting tumor metastasis includes downstream players in the RBM5-mediated metastasis suppressor pathway(s). This review highlights the differential expression of the RBM5 tumor suppressor gene which impacts cell proliferation and apoptosis control during lung cancer progression. Regulating RBM5 expression may be a novel therapeutic target for lung cancer.

Epidermal Growth Factor Receptor Tyrosine Kinase: A Potential Target in Treatment of Non-Small-Cell Lung Carcinoma.

Lung cancer is responsible for 1.6 million deaths. Approximately 80%-85% of lung cancers are of the non-small-cell variety, which includes squamous cell carcinoma, adenocarcinoma, and large-cell carcinoma. Knowing the stage of cancer progression is a requisite for determining which management approach-surgery, chemotherapy, radiotherapy, and/or immunotherapy-is optimal. Targeted therapeutic approaches with antiangiogenic monoclonal antibodies or tyrosine kinase inhibitors are one option if tumors harbor oncogene mutations. Another, newer approach is directed against cancer-specific molecules and signaling pathways and thus has more limited nonspecific toxicities. This approach targets the epidermal growth factor receptor (EGFR, HER-1/ErbB1), a receptor tyrosine kinase of the ErbB family, which consists of four closely related receptors: HER-1/ErbB1, HER-2/neu/ErbB2, HER-3/ErbB3, and HER-4/ErbB4. Because EGFR is expressed at high levels on the surface of some cancer cells, it has been recognized as an effective anticancer target. EGFR-targeted therapies include monoclonal antibodies (mAbs) and small-molecule tyrosine kinase inhibitors. Tyrosine kinases are an especially important target because they play an important role in the modulation of growth factor signaling. This review highlights various classes of synthetically derived molecules that have been reported in the last few years as potential EGFR-TK inhibitors (TKIs) and their targeted therapies in NSCLC, along with effective strategies for overcoming EGFR-TKI resistance and efforts to develop a novel potent EGFR-TKI as an efficient target of NSCLC treatment in the foreseeable future.

Shigella dysenteriae infection activates proinflammatory response through ß-catenin/NF-κB signaling pathway.

Shigella dysenteriae (S.dysenteriae) the causative agent of bacillary dysentery invades the human colonic epithelium resulting in severe intestinal inflammatory response and epithelial destruction. However, the mechanism by which S.dysenteriae infection regulates proinflammatory cytokines during intestinal inflammation is still obscure. In this study, we evaluated whether the interaction of ß-catenin and NF-κB regulates proinflammatory cytokines TNF-α and IL-8 by modulating GSK-3ß activity during S.dysenteriae infection in rat ileal loop model. Here we demonstrated that S.dysenteriae infection stimulate ß-catenin degradation which in turn decreased the association between NF-κB and ß-catenin. Also, we showed that S.dysenteriae infection increased GSK-3ß kinase activity which in turn phosphorylates ß-catenin for its degradation by ubiquitination and upregulates IL-8 through NF-κB activation thereby leading to inflammation. Thus these findings revealed the role of ß-catenin/ NF-κB and GSK-3ß in modulating the inflammatory response during bacterial infection and also showed that ß-catenin acts as a critical regulator of inflammation.

Chronic depletion of glutathione (GSH) and minimal modification of LDL in vivo: its prevention by glutathione mono ester (GME) therapy.

A decline in reduced glutathione (GSH) level is associated with aging and free radical mediated diseases. The objective of this study was to determine whether the chronic depletion of extra cellular GSH causes oxidative damage to the circulating macromolecules such as lipoproteins. Decreased concentrations of plasma glutathione, vitamin E and ascorbic acid were recorded in the rats treated with buthionine sulfoximine (BSO), a selective GSH inhibitor. In LDL isolated from BSO-treated animals, the concentration of malondialdehyde (MDA) and conjugated dienes were significantly increased (P<0.01), whereas the levels of vitamin E were decreased (P<0.01). The analysis of total and LDL cholesterol revealed significant changes between the control and experimental groups. Of interest, altered concentrations of lyso-phosphatidyl choline (Lyso-PC) and phosphatidyl choline (PC) were recorded from the BSO mediated minimally modified LDL. A negative correlation between LDL-BDC/MDA and its antioxidant capacity was noted. Upon in vitro oxidation with CuSO(4), the electrophoretic behavior of purified LDL-apoprotein-B on agarose gel showed an increased mobility in BSO-treated rats, indicative of in vivo modification of LDL to become susceptible for in vitro oxidation. The increased mobility of LDL (after in vitro oxidation) isolated from the BSO-treated animals correlates with a decrease in its amino groups, as determined by the trinitrobenzene sulfonic acid (TNBS) reactants. However, the mobility of LDL molecule was not altered due to BSO treatment in vivo. Interestingly, the minimal modification on LDL does not lead to any vascular damage in the dorsal aorta of the rats injected with BSO. The administration of glutathione monoester (GME), at a dose of 5 mmol/kg body weight, twice a day, for 30 days, to animals treated with l-buthionine-SR-sulfoximine (BSO, 4 mmol/kg body weight, twice a day, for 30 days) normalized the antioxidant status and prevented the minimal modifications on LDL. Thus, increasing the cellular GSH levels may trigger beneficial effects against oxidative stress.

Expression profile of mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium.

BACKGROUND: Helicobacter pylori (H. pylori) causes gastritis and intestinal metaplasia (IM) that may evolve to gastric carcinoma. The objective of this study was to compare the profile of mucins in the progressive stages of H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. We used a panel of monoclonal antibodies with well-defined specificities of MUC2, MUC5AC and MUC6 to characterize the expression pattern of mucins by immunohistochemistry. METHODS: RUT and ELISA were down for H. pylori confirmation. Human gastric biopsy sections were stained using immunohistochemistry with MUC2, MUC5AC and MUC6 antibodies. RESULTS: MUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric glands. MUC2 was expressed in intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the progressive stages of H. pylori infected human gastric epithelium allows the identification of intestinal metaplasia, which is characterized by a decreased expression of the gastric mucins (MUC5AC and MUC6) and de novo expression of MUC2. CONCLUSION: In conclusion, our results suggest that there is altered expression of MUC5AC and MUC6 together with the aberrant expression of MUC2 in intestinal metaplasia, during the process of gastric carcinogenesis. The present study indicates that the MUC2 mucin expression pattern is a reliable marker of intestinal metaplasia, which appears in the context of H. pylori infected individuals.

Cardioprotective effect of the Hibiscus rosa sinensis flowers in an oxidative stress model of myocardial ischemic reperfusion injury in rat.

BACKGROUND: The present study investigates the cardioprotective effects of Hibiscus rosa sinensis in myocardial ischemic reperfusion injury, particularly in terms of its antioxidant effects. METHODS: The medicinal values of the flowers of Hibiscus rosa sinensis (Chinese rose) have been mentioned in ancient literature as useful in disorders of the heart. Dried pulverized flower of Hibiscus rosa sinensis was administered orally to Wistar albino rats (150-200 gms) in three different doses [125, 250 and 500 mg/kg in 2% carboxy methyl cellulose (CMC)], 6 days per week for 4 weeks. Thereafter, rats were sacrificed; either for the determination of baseline changes in cardiac endogenous antioxidants [superoxide dismutase, reduced glutathione and catalase] or the hearts were subjected to isoproterenol induced myocardial necrosis. RESULTS: There was significant increase in the baseline contents of thiobarbituric acid reactive substances (TBARS) [a measure of lipid per oxidation] with both doses of Hibiscus Rosa sinensis. In the 250 mg/kg treated group, there was significant increase in superoxide dismutase, reduced glutathione, and catalase levels but not in the 125 and 500 mg/kg treated groups. Significant rise in myocardial thiobarbituric acid reactive substances and loss of superoxide dismutase, catalase and reduced glutathione (suggestive of increased oxidative stress) occurred in the vehicle treated hearts subjected to in vivo myocardial ischemic reperfusion injury. CONCLUSION: It may be concluded that flower of Hibiscus rosa sinensis (250 mg/kg) augments endogenous antioxidant compounds of rat heart and also prevents the myocardium from isoproterenol induced myocardial injury.

Modulation of rat erythrocyte antioxidant defense system by buthionine sulfoximine and its reversal by glutathione monoester therapy.

The protective effects of glutathione monoester (GME) on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion and its sequel were evaluated in rat erythrocyte/erythrocyte membrane. Animals were divided into three groups (n=6 in each): control, BSO and BSO+GME group. Administration of BSO, at a concentration of 4 mmol/kg bw, to the albino rats resulted in depletion of blood GSH level to about 59%. GSH was elevated several folds in the GME group as compared to the control (P<0.05) and BSO (P<0.001) groups. Decreased concentration of vitamin E was found in the erythrocyte membrane isolated from BSO-administered animals. Antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were also found to be altered due to BSO-induced GSH depletion in blood erythrocytes. The SOD and CAT activities in BSO group were significantly lower (P<0.001) than the other groups. Lipid peroxidation index and malondialdehyde (MDA) levels in erythrocytes and their membranes were increased to about 45% and 40%, respectively. The activities of Ca2+ ATPase, Mg2+ ATPase and Na+K+ ATPase were lower than those of control group (P<0.05), whereas the activities of these enzymes were found to be restored to normal followed by GME therapy (P<0.05). Cholesterol, phospholipid and C/P ratio and some of the phospholipid classes like phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin were significantly (P<0.05) altered in the erythrocyte membranes of BSO-administered rats compared with those of control group. These parameters were restored to control group levels in GME-treated group. Oxidative stress may play a major role in the BSO-mediated gamma glutamyl cysteine synthetase (gamma-GCS) inhibition and hence the depletion of GSH. In conclusion, our findings have shown that antioxidant status decreased and lipid peroxidation increased in BSO-treated rats. GME potentiates the RBC and blood antioxidant defense mechanisms and decreases lipid peroxidation.

Vitamin E prevents buthionine sulfoximine-induced biochemical disorders in the rat.

Antioxidant therapy can improve the protection and metabolic activity of cells and tissues. In this study, the effect of vitamin E administration on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion in the rat lung and liver was investigated. Hepatic GSH was depleted by intraperitoneal administration of BSO (4 mmol kg(-1)), twice a day, for 30 days to rats. We also investigated whether the lung and liver mitochondrial GSH contents were influenced by BSO administration and whether an extracellular supply of vitamin E could prevent the changes caused by BSO-mediated GSH depletion. Glutathione levels in lung and liver tissues were depleted by 47% and 60%, respectively. Depletion of hepatic and pulmonary GSH in turn causes decline in the levels of mitochondrial GSH, leading to impaired antioxidant defence function of mitochondria. Both the cytosolic and mitochondrial glutathione disulfides (GSSG) were altered during BSO treatment, and led to drastic increase in GSSG/GSH redox status. One of the experimental groups was given vitamin E (65 mg (kg diet)(-1)) mixed with rat feed. The rats fed with vitamin E were found to have partially restored GSH levels in liver and lung, diminished levels of TBARS and minimized tissue damage. The current findings suggest that the impaired glutathione and glutathione-dependent enzyme status may be correlated with the elevated lipid peroxidation and mitochondrial membrane damage and that vitamin E therapy to the BSO-administered rats prevents the above changes. However, vitamin E did not have any effect on the activity of gamma-glutamyl cysteine synthetase (gamma-GCS).

Shigella dysenteriae modulates BMP pathway to induce mucin gene expression in vivo and in vitro.

Mucosal epithelial cells in the intestine act as the first line of host defense against pathogens by increasing mucin production for clearance. Despite this fact, the underlying molecular mechanisms by which Shigella dysenteriae transduce mucin gene expression remain poorly defined. The goal of this study was to determine the role of Bone morphogenetic protein (BMP) pathway in mucin gene expression during S. dysenteriae infection. In this study we demonstrate that S. dysenteriae activates BMP signaling to induce MUC2 and MUC5AC gene expression in rat ileal loop model and in vitro. We also observed that BMP pathway regulates CDX2 expression which plays a critical role in induction of MUC2 gene during S. dysenteriae infection. In SMAD4 silenced cells S. dysenteriae infection did not abrogate MUC2 and MUC5AC gene expression whereas in CDX2 silenced cells it induces differential expression of MUC5AC gene. These results suggest that SMAD4-CDX2 induces MUC2 gene expression whereas SMAD4 directly influences differential expression of MUC5AC gene. Altogether, our results show that during S. dysenteriae infection the BMP pathway modulates inflammatory transcription factors CDX2 and SMAD4 to induce MUC2 and MUC5AC gene expression which plays a key role in the regulation of host mucosal defense thereby paving a cue for therapeutic application.

Helicobacter pylori detection and genotyping in gastric biopsy specimens from Chennai patients (India).

The aim of the present study was to investigate the prevalence of Helicobacter pylori infection and the correlation between cagA and vacA (mid-region) genotypes with different clinical outcomes from Chennai, India, patients. Biopsies from the antrum were taken to assess the current H. pylori status by histology, rapid urease test (RUT), and PCR. The RUT and PCR analyses were carried out on a single biopsy specimen. Fasting sera were obtained from all patients and H. pylori status was determined by using ELISA. In addition, the correlations between cagA and vacA genotypes and the consequence of H. pylori infection were statistically examined. Prevalence of the cagA gene was found in 96% (90/94) of patients, and the vacA m2 subtype occurred in 60% (56/94), whereas 32% (30/94) showed the vacA m1 subtype. A significant association between the cagA and vacA m2 region (chi2 = 5.556; p < 0.01) was found in ulcer patients. The vacA m2 genotype showed a near-significant value (chi2 = 3.943; p < 0.047) in ulcer patients when compared with vacA m1. These findings suggest that H. pylori strains with the vacA m2 region were predominant in South India, especially in and around Chennai. This study also showed that PCR has a potential value for studying the cagA gene directly from biopsy specimens.

Interaction of MUC1 with beta-catenin modulates the Wnt target gene cyclinD1 in H. pylori-induced gastric cancer.

Beta-catenin can function as an oncogene when it is translocated to the nucleus, binds to T-cell factor (TCF) or lymphoid enhance factor and transactivate its target gene. The mechanism responsible for the activation of Wnt signaling pathway in the Cytotoxin-associated antigen A (CagA) Helicobacter pylori (H. pylori)-infected gastric carcinoma has not been elucidated. We hypothesize that whether interaction of MUC1 with beta-catenin modulates the Wnt signaling and its target gene cyclinD1 in CagA H. pylori-infected gastric carcinoma. The result demonstrate that binding of MUC1 CT with Protein Kinase C delta (PKC delta), tyrosine phosphorylation of MUC1 CT, and CagA are strongly associated with the interaction of MUC1 with beta-catenin in CagA H. pylori-infected gastric carcinoma. A statistically significant difference (chi(2) = 24.49; P < 0.001) was found when the binding of MUC1 CT and beta-catenin was compared to subcellular localization of beta-catenin. We also observed significant statistical correlation (chi(2) = 14.885; P < 0.001) between the cyclinD1 overexpression and the subcellular localization of beta-catenin. The overexpression of cyclinD1 was significantly higher (chi(2) = 13.785; P < 0.002) in advanced gastric carcinoma with CagA H. pylori infection. In addition cyclinD1 overexpression was significantly higher (chi(2) = 37.267; P < 0.001) with the interaction of MUC1 CT with beta-catenin in advanced gastric cancer. These findings indicate that MUC1 CT plays a role in the intracellular signaling through its interaction with beta-catenin and upregulate the Wnt target gene cyclinD1 in CagA H. pylori-infected gastric carcinoma.