Review on molecular and chemopreventive potential of nimbolide in cancer.

Cancer is the most dreaded disease in human and also major health problem worldwide. Despite its high occurrence, the exact molecular mechanisms of the development and progression are not fully understood. The existing cancer therapy based on allopathic medicine is expensive, exhibits side effects; and may also alter the normal functioning of genes. Thus, a non-toxic and effective mode of treatment is needed to control cancer development and progression. Some medicinal plants offer a safe, effective and affordable remedy to control the cancer progression. Nimbolide, a limnoid derived from the neem (Azadirachta indica) leaves and flowers of neem, is widely used in traditional medical practices for treating various human diseases. Nimbolide exhibits several pharmacological effects among which its anticancer activity is the most promising. The previous studies carried out over the decades have shown that nimbolide inhibits cell proliferation and metastasis of cancer cells. This review highlights the current knowledge on the molecular targets that contribute to the observed anticancer activity of nimbolide related to induction of apoptosis and cell cycle arrest; and inhibition of signaling pathways related to cancer progression.

Impact of lycopene on epididymal androgen and estrogen receptors' expression in polychlorinated biphenyls-exposed rat.

The aim of the study was to evaluate the androgen (AR) and estrogen receptors' (ER) expression in epididymis of polychlorinated biphenyls (PCBs)-exposed rats. The rats were assigned to groups. Group I controls were treated with corn oil 80 µL/d intraperitoneally (ip), group II were treated with 2 mg/kg/d of A1254 ip; and group III were treated with 2 mg/kg/d of A1254 ip along with simultaneous oral supplementation of 4 mg/kg/d lycopene . The treatment was given daily for 30 days. After 24 hours of treatment, the rats were killed, and the epididymal regions (caput, corpus, and cauda) were dissected out, weighed, and prepared to estimate the levels of sialic acid, glyceryl phosphoryl choline (GPC), hydrogen peroxide (H2O2), and lipid peroxidation (LPO). The messenger RNA (mRNA) expressions of AR, ERα, and ERß were analyzed by reverse transcriptase-polymerase chain reaction, and ERα and ERß protein expressions were analyzed by immunoblotting. The toxicity of PCBs was also confirmed by histology. There was a marked decrease in epididymal weight, sialic acid, and GPC levels, while oxidative stress markers H2O2 and LPO were increased in PCBs-treated rats. The mRNA and protein expression of AR, ERα, and ERß were decreased in PCBs-treated groups, and the histology confirms the cytoplasmic damage in the regions of caput, corpus, and cauda in PCBs-treated rats. Simultaneous supplementation of lycopene to PCBs-exposed rats resulted in significant decrease in the oxidative stress markers as that of control, while the AR, ERα, and ERß gene expressions were near to control. The results suggest that lycopene has ameliorative effect against PCBs-induced toxicity in epididymis.

Effect of Quercetin on Haematobiochemical and Histological Changes in the Liver of Polychlorined Biphenyls-Induced Adult Male Wistar Rats.

Polychlorinated biphenyls exposure damages the rat liver cells. Hematological parameters such as hemoglobin, packed cell volume, red-blood cells, white-blood cells, neutrophils, platelet counts, and RBC indices were significantly decreased. Polymorphs, eosinophil counts, and erythrocyte sedimentation rate were significantly increased. Serum liver enzymes such as aspartate transaminase, alanine transaminase, alkaline phosphatase, and gamma-glutamyl transferase were increased by PCBs treatment. Serum lipid profiles such as cholesterol, triglycerides, low-density lipoproteins and very-low-density lipoproteins were increased in PCBs-treated rats. High-density lipoprotein, total protein, albumin, globulin levels, and albumin/globulin ratio were also decreased after PCB exposure. Then levels of sodium, potassium, chloride, and bicarbonate were also altered. Serum glucose levels were increased along with total bilirubin after PCBs exposure. Simultaneous quercetin supplementation significantly protected the PCBs-induced changes of hematobiochemical parameters. Thus, quercetin shows a protective role against PCBs-induced alterations in the hematological and biochemical parameters.

Protective role of lycopene against Aroclor 1254-induced changes on GLUT4 in the skeletal muscles of adult male rat.

Aroclor 1254 is the commercial mixture of highly toxic environmental pollutant, polychlorinated biphenyls (PCBs). Being immensely durable, it is extensively used and widely distributed. Studies show that Aroclor 1254 causes a variety of adverse health effects through free radical generation. The present investigation was designed to check the effect of Aroclor 1254 on the glucose transporter protein, GLUT4, which plays a key role in glucose homeostasis. The protective role of lycopene against the adverse effect of Aroclor 1254 was also tested. Group 1 rats received corn oil as vehicle and served as control. Groups 2, 3, and 4 were administered with Aroclor 1254 [2 mg kg(-1) body weight (b.w.) day(-1)] intraperitoneally for 30 days. Groups 3 and 4 received lycopene (2 and 4 mg kg(-1) b.w. day(-1), respectively) orally in addition to Aroclor 1254. After 30 days, animals were euthanized and the skeletal muscles were dissected to determine the following parameters: GLUT4 messenger RNA (mRNA), GLUT4 protein (both plasma membrane and cytosolic fractions), and (14)C-2-deoxyglucose uptake. Though there was no change in GLUT4 mRNA and fasting plasma glucose levels, Aroclor 1254 significantly decreased the GLUT4 protein level in both the subcellular fractions of the gracilis and triceps muscles. Most important, (14)C-2-deoxyglucose uptake showed a significant decrease in Aroclor 1254 alone treated rats, and Aroclor 1254 plus 4 mg lycopene supplementation treatment maintained the same at par with control. Thus, Aroclor 1254 has adverse effects on GLUT4 translocation and (14)C-2-deoxyglucose uptake, and lycopene administered along with Aroclor 1254 has a protective role over it.

Protective effect of zinc on N-methyl-N-nitrosourea and testosterone-induced prostatic intraepithelial neoplasia in the dorsolateral prostate of Sprague Dawley rats.

Previous studies have suggested that zinc exerts anticarcinogenic and antiproliferative effects against prostate cancer both in vitro and in rat ventral prostate. Zinc accumulation diminishes early in the course of prostate malignancy and it inhibits the growth of several carcinoma cells through induction of cell cycle arrest and apoptosis. In this study, we have investigated the influence of zinc on N-methyl-N-nitrosourea (MNU) and testosterone (T)-induced prostatic intraepithelial neoplasia in the dorsolateral prostate of Sprague Dawley (SD) rats. The results indicate that zinc plays an important role in prostate carcinogenesis. Increased tumor incidence was accompanied by a decrease in prostatic acid phosphatase activity, citrate, zinc, glutathione-S-transferase, reduced glutathione, p53, B-cell lymphoma protein (Bcl-2)-associated X protein and caspase-3 levels in MNU + T-treated rats. On the contrary, significantly increased phase I drug metabolizing enzyme activities, lipid peroxide, hydrogen peroxide, proliferating cell nuclear antigen, Bcl-2 and Bcl-X(L) protein levels were observed in the dorsolateral prostate of MNU + T-treated rats. Simultaneous zinc supplementation significantly reversed these effects in MNU + T-treated rats. Signs of dysplasia, a characteristic of prostatic intraepithelial neoplasia, were evident in the dorsolateral prostatic tissue sections by MNU + T administration. However, zinc supplementation has reversed these effects in the dorsolateral prostatic histoarchitecture. These results suggest that zinc may act as an essential trace element against MNU and testosterone-induced prostatic preneoplastic progression in SD rats.

Induction of apoptosis and inhibition of PI3K/Akt pathway in PC-3 and LNCaP prostate cancer cells by ethanolic neem leaf extract.

AIM OF THE STUDY: The present study is aimed to delineate the effect of ethanolic neem leaf extract on PI3K/Akt and apoptotic pathway in prostate cancer cell lines (PC-3 and LNCaP). MATERIALS AND METHODS: To test the hypothesis, two different prostate cancer cell lines LNCaP (androgen dependent) and PC-3 (androgen independent) were taken. Cells were exposed to various concentrations of ethanolic neem leaf extract (ENLE) (25-125 µg/ml). The doses were fixed by cell viability (MTT) assay. For apoptotic detection in situ apoptosis assay, caspase-3 activity and protein expression of cytochrome c and Poly-ADP Ribose Polymerase (PARP) were analysed as well as mRNA expression of Bcl-2 family proteins was studied by RT-PCR. The phosphoinositide 3-kinase (PI3K) and p-Akt were analysed by western blotting and mRNA expression of Akt 1 and 2, PTEN was performed by RT-PCR. Immunoblotting of cyclin D1 and p21 was done to access the inhibition of cell proliferation. RESULTS: ENLE gives 50% inhibition at a dose of 100 µg/ml in both PC-3 and LNCaP cells and considered as effective dose. ENLE decreased the protein expression of PI3K as well as p-Akt and the mRNA expression of Akt 1and 2 in both the cells. There was a significant decrease in mRNA expression of PTEN in LNCaP cells. ENLE induced apoptosis and inhibited cell proliferation by inhibiting PI3K/Akt pathway. Decrease in p-Akt leads to increase in the protein level of Bad, p21 and decrease in the cyclin D1, respectively. ENLE treatment increased the cytochrome c expression and caspase-3 activity as well as regulated the mRNA expression of Bcl-2 family proteins thereby inducing apoptosis to both the cell lines. In situ apoptosis assay showed increased red fluorescence in 100 µg/ml of ENLE in both PC-3 and LNCaP cell lines. CONCLUSIONS: The results suggested that ENLE induces apoptosis and inhibits cell proliferation through inhibiting PI3K/Akt pathway in both PC-3 and LNCaP cells.

Effect of melatonin on PCB (Aroclor 1254) induced neuronal damage and changes in Cu/Zn superoxide dismutase and glutathione peroxidase-4 mRNA expression in cerebral cortex, cerebellum and hippocampus of adult rats.

Polychlorinated biphenyls (PCBs) are one of the environmental toxicants and neurotoxic compounds which induce the production of free radicals leading to oxidative stress. Free radicals represent a class of biologically generated species that pose a potential threat to neuronal survival. Cu/Zn superoxide dismutase (SOD) and glutathione peroxidase-4 (GPx-4) are the key cellular antioxidant enzymes by which neurons and other cells detoxify free radicals and protect themselves from damage. Melatonin, an indoleamine plays an important role in neurodegenerative diseases as an antioxidant and neuroprotector. The aim was to carry out to investigate the effect of melatonin on PCB (Aroclor 1254) induced changes in histomorphology and Cu/Zn SOD, GPx-4 mRNA expression in selected brain regions of adult rats. Group I: rats intraperitoneally (i.p.) administered with corn oil (vehicle) for 30 days. Group II: rats injected (i.p.) with Aroclor 1254 (PCB) at 2mg/kgbw/day for 30 days. Groups III and IV: rats (i.p.) received melatonin (5 or 10mg/kgbw/day) simultaneously with PCB for 30 days. Groups V and VI: rats (i.p.) received melatonin (5 or 10mg/kgbw/day) alone for 30 days. After 30 days, rats were sacrificed and the brain regions were dissected to cerebral cortex, cerebellum and hippocampus. Activities of enzymatic antioxidants such as total SOD, Cu/Zn SOD, Mn SOD, glutathione peroxidase (GPx) were estimated. mRNA expressions of Cu/Zn SOD and GPx-4 were quantified by reverse transcriptase polymerase chain reaction (RT-PCR) method. Histological study was also observed. Specific activities of all antioxidant enzymes and mRNA expression of Cu/Zn SOD and GPx-4 were decreased in brain regions of PCB exposed animals. Neuronal damages were observed in all the brain regions. Exogenous melatonin supplementation retrieved all the parameters. These results suggest that melatonin protects PCB-induced oxidative stress and prevents neuronal damage in brain regions.

Effects of diallyl disulfide (DADS) on expression of apoptosis associated proteins in androgen independent human prostate cancer cells (PC-3).

Prostate cancer is a leading cause of death among the aging men. Surgical or radiotherapy is effective when the cancer is confined to the prostate gland but once the cancer spreads beyond the pelvis even chemotherapy and hormonal ablation therapy fails in curing this disease. Our previous studies have shown that diallyl disulfide (DADS) induces cell cycle arrest and also induces apoptosis in PC-3 cells. And now the present study is focused to see whether there is an activation of caspase cascade pathway. Hence, in the present study the apoptotic effect of DADS is studied by Western blot analysis of caspase-3, -9, -10 and Bcl-2, Bad, and Bax protein. The Apoptotic cells were assessed by Hoechst 33342 staining with 25 and 40 microM concentrations of DADS for 24 h. The results have shown that DADS at 25 and 40 microM concentrations has induced the activation of caspases. There is a significant increase in the expression of caspases (3, 9, and 10). The proapoptotic protein Bax has significantly increased at 40 microM of DADS treatment and there is significant increase of Bad protein at both the concentration. Bcl-2 protein has significantly decreased in DADS treated cells. Therefore, the present investigation serves as evidence that DADS may be a therapeutic drug in the treatment of prostate cancer.

Studies on the protective role of lycopene against polychlorinated biphenyls (Aroclor 1254)-induced changes in StAR protein and cytochrome P450 scc enzyme expression on Leydig cells of adult rats.

Polychlorinated biphenyls (PCBs) are ubiquitous and persistent environmental contaminants that disturb normal endocrine functions, including gonadal functions in humans and mammals. PCBs (Aroclor 1254) - induced toxic manifestations are associated with the production of free radicals. Lycopene belongs to the group of natural carotenoids, which are found in many fruits, vegetables and other green plants. Lycopene, the most potent antioxidant protects against oxidative damage. The present study was conducted to elucidate the protective role of lycopene against Aroclor 1254-induced changes in Leydig cellular steroidogenic acute regulatory (StAR) protein, cytochrome P450 side chain cleavage (P450 scc) enzyme expression and 3beta-hydroxy steroid dehydrogenase (3beta-HSD) activity. The rats were divided into four groups. Each group consists of six animals. Group I rats were administered with corn oil intraperitoneally (i.p.) for 30 days. Group II rats were treated with Aroclor 1254 (i.p.) 2mgkg(-1)body weight (bwt)day(-1) for 30 days. Group III rats were treated with Aroclor 1254 (i.p.) 2mgkg(-1)bwtday(-1) along with simultaneous supplementation of lycopene 4mgkg(-1)bwtday(-1) (gavage) for 30 days. Group IV rats administered with lycopene alone at the dose of 4mgkg(-1)bwt day(-1) (gavage) for 30 days. After 24h of the last treatment, animals were decapitated, blood was collected and serum testosterone level was estimated by radioimmunoassay (RIA). Testes were removed and Leydig cells were isolated in aseptic condition. StAR protein, cytochrome P450 scc enzyme expression were studied by Western blot analysis and 3beta-HSD activity was estimated spectrophotometrically. Aroclor 1254 treatment significantly reduced the serum testosterone level. Simultaneous supplementation of lycopene maintained the serum testosterone to near normal. Aroclor 1254 exposure decreased Leydig cellular StAR protein, cytochrome P450 scc enzyme expression and activity of 3beta-HSD. However, simultaneous supplementation of lycopene improved Leydig cellular StAR protein, cytochrome P450 scc expression and activity of 3beta-HSD. These results suggested that lycopene have ameliorative role against Aroclor 1254 induced Leydig cell dysfunction.

Protective role of melatonin on PCB (Aroclor 1,254) induced oxidative stress and changes in acetylcholine esterase and membrane bound ATPases in cerebellum, cerebral cortex and hippocampus of adult rat brain.

Polychlorinated biphenyls (PCBs) are one of the environmental toxicants and neurotoxic compounds which induce the production of free radicals leading to oxidative stress. Membrane proteins that control ion gradients across organellar and plasma membranes appear to be particularly susceptible to oxidation induced changes. Melatonin plays an important role in neurodegenerative diseases as an antioxidant and neuroprotector. The aim of this study was to determine the protective role of melatonin on PCB (Aroclor 1254) induced changes in activities of membrane bound ATPases and acetylcholine esterase in selected brain regions of adult rats. Group I: rats intraperitoneally (i.p.) administered corn oil (vehicle) for 30 days. Group II: rats injected i.p. with Aroclor 1,254 (PCB) at 2mg/kg bw/day for 30 days. Groups III and IV: rats intraperitoneally received melatonin (5 or 10mg/kg bw/day) simultaneously with Aroclor 1,254 for 30 days. Groups V and VI: rats intraperitoneally received melatonin (5 or 10mg/kg bw/day) alone for 30 days. After 30 days, rats were sacrificed and the brain regions were dissected to cerebral cortex (Cc), cerebellum (C) and hippocampus (H). Lipid peroxidation (LPO), hydrogen peroxide (H(2)O(2)), hydroxyl radical (OH) and the activities of Na(+)K(+) ATPase, Ca(2+) ATPase, Mg(2+) ATPase and acetyl cholinesterase were determined. Reduced glutathione (GSH) level was also determined. Melatonin levels in serum was measured by enzyme labeled immunosorbent assay (ELISA). Activities of all the enzymes and GSH level were decreased while an increase in H(2)O(2), OH and LPO were observed in brain regions of PCB treated animals. Melatonin levels in serum was decreased in PCB exposed animals. Exogenous melatonin supplementation retrieved all the parameters, significantly. These results suggest that PCB alters membrane bound ATPases and cholinergic function by inducing oxidative stress in brain regions, which can be protected by melatonin.

Role of alpha-tocopherol on antioxidant status in liver, lung and kidney of PCB exposed male albino rats.

Polychlorinated biphenyls (PCB) are widespread, lipophilic environmental pollutants which have been identified as contaminants in almost every component of the global ecosystem including fish, wildlife, and human adipose tissue, breast milk, and serum. Several studies have shown that PCBs can cause oxidative damage to biomolecules, in the form of lipid peroxidation, modulation of antioxidant enzymes, and oxidative stress. In the present study, we have sought to investigate the effects of alpha-tocopherol (vitamin E) on antioxidant status of PCB-induced toxicity in male Wistar rats. The protective effect of alpha-tocopherol (50mg/kg body weight/day) was tested in PCB-induced toxicity in rat liver, lung, and kidney. We report here that the oral supplementation of alpha-tocopherol was found to maintain the cellular redox status by maintaining the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase accompanied with glutathione and vitamin E levels and down regulation in the levels of lipid peroxides, hydroxyl radical and hydrogen peroxides generation in PCB treated rats. Therefore, our present study demonstrates the PCB-induced deficits in antioxidant enzyme activities and increase in reactive oxygen species and lipid peroxidation levels in liver, kidney, and lung which can be overcome through simultaneous supplementation with alpha-tocopherol.

Chemoprevention of rat prostate carcinogenesis by diallyl disulfide, an organosulfur compound of garlic.

Diallyl disulfide (DADS), an important component of garlic (Allium sativam) has been demonstrated to exert a potential chemopreventive activity against human cancers. DADS inhibits proliferation of both androgen dependent and independent prostate cancer cells in vitro. However there is no report available on the role of DADS on prostate cancer initiation in in vivo model. So the present chemoprevention study was conducted to evaluate the activity of diallyl disulfide as an anticancer agent in prostate carcinogenesis of male Sprague-Dawley rats. Testosterone and N-Methyl N-Nitroso Urea (MNU) were used to induce prostate carcinogenesis that involves a multi step process like, hyperplasia, dysplasia and prostatic intraepithelial neoplasia (PIN). The rats were induced prostate carcinogenesis by injection of testosterone and single dose of MNU and again the testosterone was continued throughout the experimental period. Forty percentage of animals carried PIN in dorsolateral prostate, while dysplasia and hyperplasia (55 to 65%) were common in ventral as well as dorsolateral prostates of the hormone and carcinogen treated rats. Rats treated with hormone and carcinogen along with DADS developed PIN at incidence of 10% in the ventral and dorsolateral prostates about 20 to 10%. Dysplasia and hyperplasia were less common in these rats. The results of this study provide evidence that DADS may have chemopreventive activity in rat prostate carcinogenesis.

Growth suppressing effect of garlic compound diallyl disulfide on prostate cancer cell line (PC-3) in vitro.

Prostate cancer is the most predominant cancer in men and prostate cancer related death increases every year. Till date, there is no effective therapy other than androgen ablation therapy. At this stage, induction of apoptosis is considered as a better strategy to control cancer. Previous studies reported that aged garlic extract suppresses cancer growth and enhances immune system against cancer. In the present study, diallyl disulfide, oil soluble organosulfur compound of garlic, was studied for its antiproliferative effect on prostate cancer cells in vitro. The suppression of cell growth was demonstrated by [(3)H]thymidine incorporation assay. Induction of DNA damage was assessed by agarose gel electrophoresis. The results showed that diallyl disulfide inhibited the growth of prostate cancer cells in a dose dependent manner, compared to the control. At 50 microM and 100 microM concentrations, diallyl disulfide induced DNA damage in PC-3 cells. It is concluded that diallyl disulfide, component of aged garlic extract, inhibits proliferation of prostate cancer cells through the induction of apoptosis.