Bacoside A downregulates matrix metalloproteinases 2 and 9 in DEN-induced hepatocellular carcinoma.

Cancer metastasis is a complex multi-step process, responsible for a majority of cancer-related deaths by affecting the critical organs and causing complications in therapies. Hepatocellular carcinoma is a multi-factorial disease and is the third most common cause of cancer related mortality worldwide. Clinical and experimental studies have shown that MMP-2 and MMP-9 are involved in tumor invasion and metastases and their elevated expression has been associated with poor prognosis. Our recent studies showed a strong anti-oxidant and hepatoprotective effects of bacoside A (BA) against carcinogen. Nevertheless the effect of BA on the activities and expression of MMP-2 and MMP-9 during hepatocellular carcinoma is not yet recognized. Therefore, the present study was designed to assess the same. Results of gelatin zymography study showed that BA co-treatment significantly decreased the activities of MMP-2 and MMP-9, which is increased during hepatocellular carcinoma. Further immunoblot analysis showed decreased expression of MMP-2 and MMP-9 in rats co-treated with BA compared to DEN-induced hepatocellular carcinoma. Our results reveal that BA exerts its anti-metastatic effect against DEN-induced hepatocellular carcinoma by inhibiting the activities and expressions of MMP-2 and MMP-9.

Chemopreventive effect of bacoside A on N-nitrosodiethylamine-induced hepatocarcinogenesis in rats.

PURPOSE: Chemoprevention is an effective approach to control hepatocarcinogenesis. Bacoside A, the active constituent of Bacopa monniera Linn., is anticipated to play a role in chemoprevention of liver cancer. METHODS: In the present study, we investigated the chemopreventive effect of bacoside A against N-nitrosodiethylamine-induced hepatocarcinogenesis in an animal model. RESULTS: Administration of carcinogen showed a significant elevation in the levels of lipid peroxidation, serum tumor marker enzymes and liver injury marker enzymes with subsequent decrease in the levels of both hemolysate and liver antioxidant status. Bacoside A co-treatment maintained the N-nitrosodiethylamine-induced alterations at near normal level. Histopathological and electron microscopic study of the liver tissue also supports the above biochemical observations. CONCLUSIONS: From our findings we conclude that bacoside A is effective to prevent DEN-induced hepatocellular carcinoma by quenching lipid peroxidation and enhancing antioxidant status through free radical scavenging mechanism and having potential of protecting endogenous enzymatic and non-enzymatic antioxidant activity.

Differential response of Leydig cells in expressing 11beta-HSD type I and cytochrome P450 aromatase in male rats subjected to corticosterone deficiency.

Emerging evidence suggests that the glucocorticoid and estradiol are important for Leydig cell steroidogenesis and are regulated via aromatase for estradiol production and 11beta-HSD for oxidatively inactivating glucocorticoid. Although it is known that corticosterone deficiency impaired Leydig cell steroidogenesis, its effect on the expression of Leydig cell 11beta-HSD type I and aromatase are yet to be recognized. Following metyrapone-induced corticosterone deficiency, serum corticosterone and testosterone levels decrease, whereas serum estradiol remains unaltered. 11beta-HSD type I mRNA and its activity was decreased by corticosterone deficiency, whereas the activity and mRNA of aromatase remains unaltered. Simultaneous administration of corticosterone prevented its deficiency-induced changes of 11beta-HSD type I in Leydig cells. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell 11beta-HSD enzyme activity and 11beta-HSD type I mRNA expression, and the Leydig cells need to maintain their intracellular concentration of corticosterone for a normal function.

Sex steroids enhance insulin receptors and glucose oxidation in Chang liver cells.

BACKGROUND: The present study was designed to assess the effect of sex steroids (testosterone and 17beta-estradiol) on insulin receptor expression, insulin binding and glucose oxidation in human liver cell line. METHODS: Non-malignant Chang liver cells were treated with different concentrations of testosterone and 17beta-estradiol dissolved in serum free medium for 24 h to identify the effective dose of both steroids for further studies. Cells with 70-80% confluency were challenged with testosterone (0.1 micromol/l), 17beta-estradiol (0.1 micromol/l) and their combination along with insulin as a positive control for 24 h. After the treatment period, insulin receptor mRNA expression, cell surface insulin binding and (14)C-glucose oxidation were assessed. RESULTS: Both testosterone and 17beta-estradiol significantly increased the insulin receptor mRNA expression, cell surface insulin binding and (14)C-glucose oxidation compared to basal, but the increase was not at par with the effect of insulin. Compared to individual effects of testosterone and 17beta-estradiol, their combination significantly increased the glucose oxidation similar to that of insulin. CONCLUSION: It is concluded from the present study that testosterone and 17beta-estradiol can directly enhance insulin receptor mRNA expression, insulin binding and glucose oxidation in Chang liver cells and thereby glucose metabolism.

Hepatoprotective activity of bacoside A against N-nitrosodiethylamine-induced liver toxicity in adult rats.

N-Nitrosodiethylamine (DEN) is a notorious carcinogen, present in many environmental factors. DEN induces oxidative stress and cellular injury due to enhanced generation of reactive oxygen species; free radical scavengers protect the membranes from DEN-induced damage. The present study was designed to evaluate the protective effect of bacoside A (the active principle isolated from Bacopa monniera Linn.) on carcinogen-induced damage in rat liver. Adult male albino rats were pretreated with 15 mg/kg body weight/day of bacoside A orally (for 14 days) and then intoxicated with single necrogenic dose of N-nitrosodiethylamine (200 mg/kg bodyweight, intraperitonially) and maintained for 7 days. The liver weight, lipid peroxidation (LPO), and activity of serum marker enzymes (aspartate transaminases, alanine transaminases, lactate dehydrogenase, alkaline phosphatase, and gamma-glutamyl transpeptidase) were markedly increased in carcinogen-administered rats, whereas the activities of marker enzymes were near normal in bacoside A-pretreated rats. Activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutatione-S-transferase, and reduced glutathione) in liver also decreased in carcinogen-administered rats, which were significantly elevated in bacoside A-pretreated rats. It is concluded that pretreatment of bacoside A prevents the elevation of LPO and activity of serum marker enzymes and maintains the antioxidant system and thus protects the rats from DEN-induced hepatotoxicity.

Differential effect of corticosterone deficiency on the expression of LH, prolactin and insulin receptors on rat Leydig cells.

The adverse effects of glucocorticoid deficiency on the expression of genes encoding Leydig cell surface receptors and the response to LH/prolactin/insulin to produce testosterone production are yet to be recognized. Following metyrapone-induced corticosterone deficiency, serum corticosterone, testosterone and insulin levels decrease, whereas serum prolactin exhibits a significant increase and serum LH remains unaltered. LH binding and LH receptor mRNA expression were not altered, but a significant decrease in PRL and insulin binding and in the mRNA expressions of their receptors were observed in corticosterone-deficient rats in vivo. Corticosterone deficiency significantly decreases the Leydig cellular basal as well as hormone-stimulated testosterone production in vitro. Simultaneous administration of corticosterone prevented its deficiency-induced changes in Leydig cells both in vivo and in vitro. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell insulin and prolactin receptors, and their mRNA expression and the response of Leydig cells to LH/PRL/insulin on testosterone production.

Assessment of in vitro effects of metyrapone on Leydig cell steroidogenesis.

Metyrapone, a specific inhibitor of 11beta-hydroxylase inhibits glucocorticoid production and it is used in the diagnosis/treatment of hypercortisolism and also to test the functional integrity of hypothalamo-pituitary-adrenal axis. To assess the impact of glucocorticoid deficiency, this drug is preferred over adrenalectomy, which eliminates all the hormonal secretions of the adrenal cortex and medulla. However, whether metyrapone has any direct effect on the extra-adrenocortical cellular or tissue functions remains to be resolved. Our previous study showed a depressed testicular Leydig cell testosterone production in rats treated with metyrapone. Therefore, the present study was designed to examine the possible direct effect of metyrapone on testicular Leydig cell steroidogenesis in vitro. Leydig cell viability and the reactive oxygen species (ROS) concentration were not altered by any of the concentration of metyrapone tested. The efficacy of Leydig cell testosterone production under basal as well as LH-stimulated condition was not altered by metyrapone treatment. Further, Leydig cellular (14)C-glucose oxidation, the activity and mRNA levels of cytochrome side chain cleavage (P(450)scc), 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were not altered in metyrapone-treated cells. Therefore, it is concluded from the present study that metyrapone has no direct effect on Leydig cell testosterone production and, therefore, changes recorded in the in vivo studies are exclusively due to corticosterone deficiency.

Diethylhexyl phthalate impairs insulin binding and glucose oxidation in Chang liver cells.

The present study examined the dose-dependent effects of diethylhexyl phthalate (DEHP) on insulin receptor concentration and glucose oxidation in Chang liver cells. Chang liver cells (5 x 10(5) cells) were exposed to different concentrations (0, 50, 100, 200 and 400 microM) of DEHP for 24h. At the end of exposure, cells were utilized for assessing insulin receptor concentration and glucose oxidation. Both insulin receptor concentration and glucose oxidation in Chang liver cells were significantly reduced by high doses (200 and 400 microM) of phthalate exposure. The present study is first of its kind to report the direct adverse effects of DEHP on insulin receptor and glucose oxidation in Chang liver cells and suggests that DEHP exposure may have a negative influence on glucose homeostasis.

Metyrapone-induced corticosterone deficiency impairs glucose oxidation and steroidogenesis in Leydig cells of adult albino rats.

The present study was designed to identify the effects of metyrapone-induced corticosterone deficiency on Leydig cell steroidogenesis in adult male rats. Adult Wistar rats (200-250 g body weight) were treated with metyrapone, an inhibitor of corticosterone synthesis (10 mg/100 g body weight, s.c., twice daily) for 10 days. Experimental animals were killed along with controls, blood was collected, and sera separated for testosterone and estradiol assays. Testes were removed and Leydig cells were isolated, purified and used for estimating the specific activity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 14C-glucose oxidation. Serum testosterone (p < 0.05), Leydig cellular 14C-glucose oxidation (p < 0.001) and the specific activity of 17beta-HSD (p < 0.01) were significantly decreased in metyrapone treated rats. However, serum estradiol was not markedly altered compared to control. In addition to this, a set of in vitro experiments were also performed to identify the effects of metyrapone-induced corticosterone deficiency on hCG and prolactin-induced Leydig cell testosterone production. Metyrapone treatment significantly (p < 0.05) decreased the Leydig cellular basal as well as hCG and its combination with prolactin stimulated testosterone production in vitro. It is concluded from the present study that the inhibitory effects of metyrapone-induced corticosterone deficiency on Leydig cell steroidogenesis are mediated through impaired glucose oxidation and 17beta-HSD activity. In vitro studies showed that corticosterone deficiency impairs not only hCG action but also the potentiating effect of prolactin on Leydig cell steroidogenesis.