Measurement of γ-H2AX foci, miRNA-101, and gene expression as a means to quantify radiation-absorbed dose in cancer patients who had undergone radiotherapy.

Radiological accidents and nuclear terrorism pose an increased threat to members of the public who, following such an event, would need to be assessed for medical care by fast triage. Assay methods such as chromosome aberrations (CA), cytokinesis-block micronucleus (CBMN) and fluorescence in situ hybridization (FISH) techniques have been well established for dose estimation and their potential for handling more samples has also been proved with automation. However, culturing of lymphocytes is an inevitable step, which limits the potential of these markers for triage. In vitro analysis of gamma-H2AX (γ-H2AX), gene and microRNA (miRNA) markers do not require culturing of lymphocytes, and as such have been suggested as attractive tools for triage. Despite studies reporting in vitro dose-response curves, limited evidence is available evaluating the suitability of these assays in real situations. In this study, we have measured the absorbed dose using γ-H2AX, gene (GADD45A, FDXR, and CDKN1A) and miRNA-101 expression in blood samples of cancer patients (n = 20) who had undergone partial-body radiotherapy and compared with the derived equivalent whole-body doses (EWBD). The obtained results from all patients showed a significant (p < 0.05) increase of γ-H2AX foci in post-irradiated as compared to pre-irradiated samples. Moreover, estimated doses using γ-H2AX foci showed a correlation with the derived EWBD (r2 = 0.60, p = 0.0003) and was also shown to be dependent on the irradiated body volume. Consistent with γ-H2AX foci frequency, an increase in fold change expression of genes and miRNA-101 was observed. However, the estimated dose significantly varied among the subjects and showed poor correlation (r2 = 0.09, 0.04, 0.01 and 0.03 for GADD45A, FDXR, CDKN1A and miRNA-101, respectively) with EWBD. The overall results suggest that the established in vitro γ-H2AX assay is suitable for the detection of radiation exposure and can also provide an estimate of the dose in in vivo irradiated samples. The genes and miRNA-101 markers showed increased expression; nevertheless, there is a need for further improvements to measure doses accurately using these markers.

Neuroprotective Effect of Melatonin Against PCBs Induced Behavioural, Molecular and Histological Changes in Cerebral Cortex of Adult Male Wistar Rats.

There is ample evidence stating Polychlorinated biphenyls (PCBs) as neurotoxins. In the current study, we have analyzed the behavioural impact of PCBs exposure in adult rats and assessed the simultaneous effect of antioxidant melatonin against the PCBs action. The rats were grouped into four and treated intraperitoneally with vehicle, PCBs, PCBs + melatonin and melatonin alone for 30 days, respectively. After the treatment period the rats were tested for locomotor activity and anxiety behaviour analysis. We confirmed the neuronal damage in the cerebral cortex by molecular and histological analysis. Our data indicates that there is impairment in locomotor activity and behaviour of PCBs treated rats compared to control. The simultaneous melatonin treated rat shows increased motor coordination and less anxiety like behaviour compared to PCBs treated rats. Molecular and histological analysis supports that, the impaired motor coordination in PCBs treated rats is due to neurodegeneration in motor cortex region. The results proved that melatonin treatment improved the motor co-ordination and reduced anxiety behaviour, prevented neurodegeneration in the cerebral cortex of PCBs-exposed adult male rats.

Lactational exposure effect of polychlorinated biphenyl on rat Sertoli cell markers and functional regulators in prepuberal and puberal F1 offspring.

PURPOSE: Polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental toxicants acting as endocrine disruptors. Many researches evidenced that PCBs affect the male reproductive system in adult rats and it can transfer from mother to offspring through milk. We investigated whether the lactational exposure to PCBs affects the Sertoli cell function in F1 offspring. METHODS: Dams were orally treated with different doses of PCB-Aroclor 1254 (1, 2 and 5 mg/kg bw/day, respectively) from postpartum day 1-20. Male offspring rats were killed on PND 21 and PND 60. Testes were used both for histological study and to isolate Sertoli cell. Serum and testicular interstitial fluid (TIF) levels of testosterone, ABP and estradiol were analyzed by ELISA method. The mRNA and protein expressions of follicle-stimulating hormone (FSHR), androgen-binding protein (ABP), Inhibinß, androgen receptor (AR) and estrogen receptor (ERß) were studied using real-time PCR and immunoblotting, respectively. RESULTS: The testicular architecture was altered in PCB-treated groups of both prepuberal and puberal rats. Testosterone, estradiol and androgen-binding protein levels were altered in both serum and TIF in PCB treated groups. The gene expression level of FSHR, ABP, ERß and AR was decreased in a dose-dependent manner, whereas Inhibinß gene expression level was increased in PCB-treated groups. CONCLUSION: Lactational exposure to PCB affects both the histoarchitecture of testis, Sertoli cell maker and functional regulators in both prepuberal and puberal F1 male progeny.

Lactational exposure of polychlorinated biphenyls downregulates critical genes in Leydig cells of F1 male progeny (PND21).

Polychlorinated biphenyls (PCBs) are environmental contaminants. The present study was aimed to test the effect of lactational exposure of PCBs on Leydig cellular mRNA and protein expressions of 5α-reductase, aromatase and androgen receptor (AR) in F1 male offspring. Lactating dams were orally gavaged with different doses of PCBs (Aroclor 1254) 0, 1, 2 and 5 mg kg b.wt-1  day-1 , respectively, from PND1 to PND21. Male offsprings were sacrificed at PND21. Testes were used to isolate Leydig cells. Blood was collected. Serum testosterone (T) and oestradiol (E2 ) were measured. Anogenital distance was measured. Dams' milk lipid and serum lipids of male pups were estimated. PCB (Aroclor 1254) concentration of dams' milk and serum of male pups were analysed by GC-ECD. Leydig cellular mRNA and protein expressions of 5α-reductase, aromatase and AR were significantly decreased. Our data suggest that lactational exposure of PCBs downregulates selected genes in Leydig cells of F1 generation on post-natal day 21.

Effect of Melatonin on Glutamate: BDNF Signaling in the Cerebral Cortex of Polychlorinated Biphenyls (PCBs)-Exposed Adult Male Rats.

Various epidemiological survey suggests that the central nervous system is the target for many environmental contaminants. One among them is Aroclor 1254, a mixture of polychlorinated biphenyls (PCBs) which explore a spectrum of biochemical and neurotoxic responses in humans and laboratory animals. Learning and motor coordination deficits are the profound effects of PCBs which may be related to cerebral dysfunction. The aim of the study is to elicit the protective effect of melatonin (Mel), a potent, blood brain permeable antioxidant against the effect of Aroclor 1254 on the signaling of glutamate-principal excitatory neurotransmitter and brain derived neurotrophic factor (BDNF) in the cerebral cortex of adult rats which plays a key role in brain functions. Adult male Wistar rats were grouped into four and treated intraperitonealy (i.p) Group I with corn oil (Control), Group II with PCBs (2 mg/kg/bwt), Group III with PCBs + Mel (2 mg/kg/bwt + 5 mg/kg/bwt) and Group IV with Mel (5 mg/kg/bwt). The protein expression of glutamate signaling molecules and mRNA expressions of GLAST, BDNF signaling molecules were analyzed. The results suggest that simultaneous melatonin treatment significantly attenuated the NMDA receptor mediated glutamate excitotoxicity and protects the inhibition of BDNF signaling caused by PCBs exposure in cerebral cortex of adult male rats. Schematic pathway illustrating the proposed mechanism by which melatonin protects against A1254 mediated glutamate induced neurodegeneration in the cerebral cortex of adult male rats. PCBs induced neurodegeneration is caused by the overactivation of NMDAR, followed by the activation of voltage dependent calcium channels leading to the increase in intracellular Ca(2+) that stimulates calpain. Calpain inturn inhibits the PKA α and neurtrophin BDNF, its receptor and downstream signaling MAPK pathway leading to neurodegeneration. Melatonin had scavenged the ROS produced by PCBS and decreased the NMDAR expression which inturn protected the cells from neurodegeneration.

Anti-cancer activity of quercetin in neuroblastoma: an in vitro approach.

Neuroblastoma is a neuroendocrine tumour derived from neural crest cells and it remains a major therapeutic challenge in pediatric oncology. As response rates to chemotherapy are low, surgery remains the only effective treatment but since many tumors have metastasized at the time of diagnosis, curative surgery is rarely achieved. Consequently, a substantial need for new therapeutic options emerges. Quercetin a flavonoid, has been reported to lower the risk of several cancers. This study was designed to investigate its effects on apoptosis induction in the N2a, a mouse neuroblastoma cell line. The cell viability was determined by dimethyl thiazolyl tetrazolium bromide assay and diamidino-2-phenylindole staining was performed to confirm the apoptosis. The gene expression of bcl-w, p53, p27 and protein expression of caspases (3 and 9), bax, cytochrome-c were studied. This in vitro outcome suggests that quercetin can be used as a potent anti-cancer drug in future.

Regulation of insulin-like growth factors and their binding proteins by thyroid stimulating hormone in human osteoblast-like (SaOS2) cells.

Thyroid stimulating hormone (TSH) is shown to have definite anabolic effects on skeletal metabolism. Previous studies have demonstrated that Insulin-like growth factors (IGF-I and IGF-II) and their six high affinity binding proteins (IGFBPs 1-6) regulate proliferation and differentiation of bone-forming osteoblasts. The current study was intended to determine whether the anabolic effects of TSH on human osteoblastic (SaOS2) cells are mediated through insulin-like growth factor system components. TSH given at 0.01 ng to 10 ng/ml dose levels for 24 and 48 h significantly increased human osteoblastic (SaOS2) cell proliferation and alkaline phosphatase activity, the differentiation marker. TSH significantly increased IGFs (IGF-I and IGF-II) mRNA expression after 6 and 24 h and their protein levels after 24 and 48 h of treatment, respectively. Unlike the IGFs, the IGFBPs responded differently to TSH treatment. Though there were some inconsistencies in the regulation of stimulatory IGF binding protein-3 and -5 by TSH treatment, there was an overall increase at the mRNA abundance and protein levels. Again, the inconsistency persisted at the regulation of the inhibitory IGFBPs 2, 4, and 6 especially at the level of mRNA expression due to TSH treatment, there is an overall decrease in the levels of IGFBP-2, 4, and 6 in the conditioned media (CM) of SaOS2 cell cultures. The IGFBP proteases which control the availability of IGFs are also regulated by hormones. Pregnancy-Associated Plasma Protein-A (PAPP-A) is responsible for the proteolysis of IGFBP-4. TSH treatment significantly unregulated the expression of PAPP-A both at mRNA and protein levels. In conclusion, TSH promotes human osteoblastic (SaOS2) cell proliferation and differentiation by upregulating IGFs and their stimulatory IGF binding proteins and down regulating the inhibitory IGF binding proteins.

Impact of Lactational Exposure to Polychlorinated Biphenyl Causes Epigenetic Modification and Impairs Sertoli Cells Functional Regulators in F1 Progeny.

Polychlorinated biphenyl (PCB) is an endocrine-disrupting chemical. Sertoli cells (SCs) provide physical and nutritional support for developing germ cells. Dysfunction in SCs has adverse effects on spermatogenesis. Previously, we found that the lactational exposure of PCBs (1, 2, and 5 mg/kg birth weight/day, orally from postnatal days 1 to 20) decreased the follicle-stimulating hormone receptor (FSHR) and androgen receptor (AR) expression in SCs of F1 progeny. Transcription factors initiate and regulate the transcription of genes. DNA methylation plays an important role in epigenetic gene regulation. Hence, this study was aimed to identify the level of transcription factors regulating FSHR, AR gene expression, and DNA methylation in the promoter of these genes in SCs of both F1 prepuberal and puberal offspring. DNA methylation in the promoter of FSHR and AR genes was examined by sodium bisulfite conversion technique. The protein levels of transcription factors (steroidogenic factor 1 [SF1], upstream stimulatory factors 1 and 2, c-fos, c-jun, and CREB-binding protein) and enzymes DNA methyltransferases (Dnmt1, Dnmt3ab, Dnmt3l, and histone deacetylase 1 [HDAC1]) were analyzed by Western blotting. The transcription factors that regulate the FSHR and AR gene in SCs were decreased in both the PCB-exposed F1 progeny. Methylation was observed in the promoter of FSHR, AR, and SF1. The protein levels of Dnmt1, Dnmt3ab, Dnmt3l, and HDAC1 were increased in the PCBs-treated groups. Subsequently, it leads to transcriptional repression of the genes in SCs. Our finding suggests that PCBs caused epigenetic change in SCs, thereby it impaired SCs function in F1 progeny.

Effect of lycopene on insulin-like growth factor-I, IGF binding protein-3 and IGF type-I receptor in prostate cancer cells.

PURPOSE: Prostate cancer is the second most common cancer that leads to death in elderly men. The risk of prostate cancer prevalence is often associated with the elevated level of insulin-like growth factor-I (IGF-I) and decreased level of IGF-binding protein 3 (IGFBP-3). Lycopene, a carotenoid, reduces the proliferation of cancer cells and induces apoptosis. Hence, higher intake of lycopene can be associated with the lower risk of prostate cancer. However, the mechanism of action of lycopene in the prevention of prostate cancer is still unclear. The present study was carried out to study the effects of lycopene on the components of IGF system and apoptosis in androgen-independent prostate cancer cells (PC-3 cells). METHODS: PC-3 cells were treated with various concentrations of lycopene, (20, 40 and 60 microM) for 24, 48, 72 and 96 h. IGF-I, IGFBP-3 and IGF-I receptor (IGF-IR) levels in lycopene-treated cells were evaluated. Annexin V and propidium iodide (PI) binding studies were done to assess apoptosis. RESULTS: PC-3 cells treated with lycopene showed a significant decrease in cell proliferation. Lycopene, at a dose of 40 microM, significantly increased the level of IGFBP-3. Lycopene-induced apoptosis was confirmed by annexin V and PI binding. Lycopene-induced DNA fragmentation was absent after 24 h treatment whereas the same was observed after 48 h treatment. There was a significant decrease in the IGF-IR expression after the cells were treated with lycopene and IGF-I. CONCLUSION: The data obtained suggest that the components of the IGF system may act as a positive regulator of lycopene-induced apoptosis in PC-3 cells. Thus, the observed lycopene-induced biological effects and their associated mechanisms are encouraging and may lead to the development of a highly successful drug for the treatment of prostate cancer.

Effect of 17beta-estradiol on apoptosis, IGF system components and gelatinases A and B in prostate cancer cells (PC-3).

BACKGROUND: Previous studies have indicated that estrogen administration in the advanced stage of prostate cancer provide some benefits to the patients. Estrogen action was thought to be mediated via the blockade of the pituitary-testicular axis that effectively lowered the circulating levels of androgen and, thus, results in tumor regression; however, the effect of estrogens on prostate epithelial cells is still unclear. We investigated the effects of estradiol on insulin-like growth factor type I receptor (IGF-IR), IGF-binding protein 3 (IGFBP-3), IGFBP-4, and matrix metalloproteinase 2 (MMP-2) and MMP-9 in androgen-independent prostate cancer cells (PC-3). METHODS: The cells were treated with different concentrations of estradiol (1, 10 and 100 nmol/l) for different time periods (24, 48, 72 and 96 h). Cell proliferation was assessed using MTT assay, and IGFBP-3 and IGFBP-4 were assessed using immunoradiometric and enzyme immunoassays, respectively. MMP-2, MMP-9 and IGF-IR expression levels were analyzed using western-blot analysis, and MMP-2 and MMP-9 activities were analyzed using gelatin zymography. Apoptosis was confirmed by Annexin V-FITC and acridine orange and ethidium bromide staining methods. DNA fragmentation studies were also performed. RESULTS: Cell proliferation assay revealed that 10 and 100 nmol/l estradiol concentrations inhibit the proliferation of PC-3 cells when incubated for 48-96 h. The secretory levels of IGFBP-3 and IGFBP-4 were increased significantly. The western-blot results showed that estradiol is capable of decreasing the expression of MMP-2 and MMP-9 significantly. Gelatin zymography showed that activities of MMP-2 and MMP-9 are decreased in estradiol-treated cells. Estradiol-induced apoptosis was studied using annexin V-binding and propidium iodide influx. Estradiol also induced nuclear fragmentation in higher doses (100 nmol/l) in PC-3 cells. CONCLUSION: Inhibition of MMPs in cancer cells and increased levels of IGFBP-3 and IGFBP-4 associated with apoptosis may be one of the targets for anticancer function of estradiol. Estradiol inhibits the proliferation of prostate cancer cells by inducing apoptosis.

Effects of Aroclor 1254 on femoral bone metabolism in adult male Wistar rats.

Environmental pollutants that disrupt endocrine system might also affect the modeling and remodeling of bone. Environmental factors, irrespective of age and sex contribute for the development of secondary osteoporosis. Polychlorinated biphenyls have adverse effects on various organs including bone. The present study was designed to investigate the effects of PCB (Aroclor 1254) on femur bone and the ameliorative role of vitamin C or E. In this regard, four groups of adult male albino rats were used as control, PCB (2mg/kgb.wt.), PCB+vitamin C (100mg/kgb.wt.) and PCB+vitamin E (50mg/kgb.wt.). The bone formation markers (ALP, Collagen), bone resorption marker (TRAP), antioxidant enzymes (SOD, GPX and GST) and lipid peroxidation in the femur were studied. Aroclor 1254 treatment decreased the ALP activity and collagen, but increased the TRAP activity and lipid peroxidation. While it decreased the SOD and GPX activity, GST was unaltered. Interestingly, simultaneous administration of vitamin C or E prevented the adverse effects of Aroclor 1254 in the femur. In conclusion, the present investigation suggests that Aroclor 1254 induced oxidative stress affects femoral bone metabolism. However, vitamin C or vitamin E protected the femur from the oxidative stress.

Oxidative stress modulates membrane bound ATPases in brain regions of PCB (Aroclor 1254) exposed rats: protective role of alpha-tocopherol.

Polychlorinated biphenyls (PCBs) are a class of widely dispersed and environmentally persistent organic compounds. PCBs exhibit a wide range of toxicological effects including neurotoxicity. Vitamin E (alpha-tocopherol) is an important lipid soluble antioxidant placed in a special region of membranes. Large amounts of energy are required to maintain the signaling activities of the cells in the central nervous system (CNS). Membrane proteins that control ion gradients across organellar and plasma membranes appear to be particularly susceptible to oxidation-induced changes. The aim of this study was to determine the protective role of vitamin E on Aroclor 1254 induced modulation in membrane bound ATPases in brain regions of rats. One group of rats received corn oil as vehicle for 30days as control. The other group of rats were administered Aroclor 1254 at a dose of 2mgkg(-1) bwday(-1) intraperitoneally for 30days. One group of rats received vitamin E (50mgkg(-1) bwday(-1)) orally simultaneously with Aroclor 1254 for 30days. After 30days, the animals were euthanized and the brain was dissected to hypothalamus and hippocampus to determine the following parameters. Hydrogen peroxide (H2O2), Lipid peroxidation (LPO) and the activities of Na+K+-ATPase, Ca2+-ATPase and Mg2+-ATPase were determined. Reduced glutathione (GSH) level was also determined. Activities of all the enzymes were decreased while an increase in H2O2 and LPO were observed in selected brain regions of PCB treated animals. Simultaneous vitamin E treatment in PCB exposed animals restored all the parameters significantly. These results suggest that oxidative stress is involved in the inhibitory effect of PCB (Aroclor 1254) on membrane bound ATPases in selected brain regions. alpha-tocopherol acts against PCB induced neurotoxicity by decreasing oxidative stress.

Ameliorative effect of vitamins (alpha-tocopherol and ascorbic acid) on PCB (Aroclor 1254) induced oxidative stress in rat epididymal sperm.

The aim of this study was to investigate the possible protective role of vitamins on PCB (Aroclor 1254)-induced spermiotoxicity using qualitative, quantitative and biochemical approaches. Adult male albino rats of Wistar strain were randomly divided into four groups, each group consists of six animals. The control group received corn oil, the second group of rats were administered Aroclor 1254 at a dose of 2 mg/kg bw/day intraperitoneally for 30 days. The third group of rats were treated with Aroclor 1254 along with alpha-tocopherol (50 mg/kg of bw/day) for 30 days, while the fourth group of rats were treated with Aroclor 1254 along with ascorbic acid (100 mg/kg bw/day) orally for 30 days. Twenty-four hours after the last treatment, control and experimental animals were killed by decapitation. Sperm was collected from the cauda epididymal region and its count and motility were detected. Sperm was sonicated and used for the estimation of reactive oxygen species (ROS) [hydroxyl radical (HO(*)) and hydrogen peroxide (H(2)O(2))], non-enzymic antioxidants [alpha-tocopherol, ascorbic acid and reduced glutathione (GSH)], activity of enzymic antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) glutathione reductase (GR) and glutathione-S-transferase (GST)] and lipid peroxidation (LPO). The result of this experiment shows that PCB significantly decreases the level of alpha-tocopherol, ascorbic acid and GSH and the activities of SOD, CAT, GPx, GR and GST with elevated levels of ROS and LPO. In addition, decreased epididymal sperm motility and count were observed. Simultaneous supplementation with alpha-tocopherol and ascorbic acid restored these parameters to that of normal range. In conclusion, alpha-tocopherol and ascorbic acid exhibited protective effect on sperm by inhibiting PCB-induced ROS generation.

Effects of quercetin on insulin-like growth factors (IGFs) and their binding protein-3 (IGFBP-3) secretion and induction of apoptosis in human prostate cancer cells.

BACKGROUND: Quercetin, the predominant flavonoid, has been reported to lower the risk of several cancers. This flavonoid found in onion, grapes, green vegetables, etc. has been shown to possess potent antiproliferative effects against various malignant cells. This study was designed to investigate its effects on insulin-like growth factors (IGFs) and their binding protein-3 (IGFBP-3) proteins secretion and also apoptosis induction in the human prostate cancer cell line, PC-3. METHODS: We evaluated the secretion of IGF-I, -II and IGFBP-3 in quercetin treated cells by immunoradiometric (IRMA) method. Apoptosis was studied in quercetin treated cells by TUNEL and DNA fragmentation. Protein expressions of Bcl-2, Bcl-xL, Bax and caspase-3 were studied by western blot. RESULTS: At a dose of 100 microM concentration, we observed increased IGFBP-3 accumulation in PC-3 cells conditioned medium with a dose dependent increase with 2 fold over a base line, and significantly reduced the both IGF-I and IGF-II levels. Apoptosis induction was also confirmed by TUNEL assay. Bcl-2 and Bcl-xL protein expressions were significantly decreased and Bax and caspase-3 were increased. CONCLUSION: These results suggest that the decreased level of IGFs could be due to the increased levels of IGFBP-3, because of the high binding affinity towards IGFs, thereby decreasing the cell proliferation. The increased level of IGFBP-3 was associated with increased pro-apoptotic proteins and apoptosis in response to quercetin, suggesting it may be a p53-independent effector of apoptosis in prostate cancer cells.

Antioxidant effect of ascorbic acid on PCB (Aroclor 1254) induced oxidative stress in hypothalamus of albino rats.

BACKGROUND: PCBs are one of the environmental toxicants and neurotoxic compounds which induce the production of free radicals leading to oxidative stress. Vitamin C is well known as an outstanding antioxidant. We determined the protective role of ascorbate on hypothalamic antioxidant system of Aroclor 1254 exposed rats. METHODS: The rats were injected Aroclor 1254 at a dose of 2 mg/kg bw/day intraperitoneally for 30 days. One group of rats received vitamin C (100 mg/kg bw/day) orally simultaneously with Aroclor 1254 for 30 days. Twenty-four hours after last treatment, the animals were killed and hypothalamic region was separated from brain tissue. Enzymatic and non-enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and vitamin C were estimated. Hydrogen peroxide (H(2)O(2)), lipid peroxidation (LPO) and acetylcholine esterase (AchE) activity were determined. Serum gonadotropins such as luteinizing hormone (LH) and follicle stimulating hormone (FSH) were also assayed. RESULTS: Activities of SOD, CAT, GPx, GR, AchE and the concentration of vitamin C were decreased while an increase in H(2)O(2) and LPO were observed in hypothalamus of PCB treated animals. LH and FSH concentrations were also decreased in serum of PCB exposed animals. Vitamin C administration retrieved all the parameters significantly except serum hormonal profiles. CONCLUSION: PCB induces oxidative stress in hypothalamus by decreasing the activities of antioxidant enzymes, which can be protected by vitamin C treatment.

Quercetin induces p53-independent apoptosis in human prostate cancer cells by modulating Bcl-2-related proteins: a possible mediation by IGFBP-3.

Quercetin, a flavonoid found in onion, grapes, green vegetables, etc., has been shown to possess potent antiproliferative effects against various malignant cells. We report insulin-like growth factor-binding protein-3 (IGFBP-3) as an effector of quercetin-induced apoptosis in human prostate cancer cell lines in a p53-independent manner. We evaluated the production of IGFBP-3 in quercetin-treated cells. Apoptosis was studied in quercetin-treated cells to study the IGFBP-3-mediated role with flow cytometry and DNA fragmentation. Protein expressions of Bcl-2, Bcl-x(L), and Bax were studied by Western blot. Increased production of IGFBP-3 was associated with the increased ratio of proapoptotic to antiapoptotic members of the Bcl-2 family. In quercetin-treated PC-3 cells, an increase in Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein were observed. As PC-3 is a p53-negative cell line, these modulations of proapoptotic proteins and induction of apoptosis were independent of p53. The level of IGFBP-3 on the response of PC-3 cells to quercetin was examined. There was a twofold increase in IGFBP-3 level in conditioned media of 100 microM quercetin-treated cells. Quercetin also brought a peak at sub-G1 in PC-3 cells. Thus, increased level of IGFBP-3 was associated with increased proapoptotic proteins and apoptosis in response to quercetin, suggesting it may be a p53-independent effector of apoptosis in prostate cancer cells via its modulation of the Bax/Bcl-2 protein ratio.

Anticancer effects of ethanolic neem leaf extract on prostate cancer cell line (PC-3).

Prostate cancer (PC) is the most prevalent cancer and the leading cause of male cancer death. Azadirachta indica (neem tree) has been used successfully centuries to reduce tumors by herbalists throughout Southeast Asia. Here the present study indicated that an ethanolic extract of neem has been shown to cause cell death of prostate cancer cells (PC-3) by inducing apoptosis as evidenced by a dose-dependent increase in DNA fragmentation and a decrease in cell viability. Western blot studies indicated that treatment with neem extract showed decreased level of Bcl-2, which is anti-apoptotic protein and increased the level of Bax protein. So the neem extract could be potentially effective against prostate cancer treatment.

Effect of chromium on vertebrae, femur and calvaria of adult male rats.

Alloys of chromium have a long history of success in the surgical treatment of many orthopaedic defects. Nonetheless, prostheses loosening are commonly found around arthoplasties due to corrosion of metals. On this basis, it is hypothesized that chromium accumulation interferes with remodeling of bone. The present study aims to analyse the toxic effects of chromium on bone phosphatases in various regions of the bone in rats. Rats were treated with chromium intraperitoneally (0.5 mg/kg) in the form of potassium dichromate for 5 days. The accumulation of chromium is approximately 5.2-fold in the vertebrae, 8.9-fold in the femur and 8.7-fold in the calvaria, when compared to control. Chromium administration significantly reduced the activity of enzymes, eg, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). The study revealed a significant increase in the concentration of calcium, altered bone formation rate and bone morphology in the femur, vertebrae and calvaria. The interesting findings of the current study suggest altered bone turnover.

Nimbolide inhibits androgen independent prostate cancer cells survival and proliferation by modulating multiple pro-survival signaling pathways.

BACKGROUND: Prostate cancer is the most prominent cancer in men, experiencing a relapse in disease often express high serum TNF-α levels. It has been correlated with increased cell survival and proliferation of prostate cancer cells. Previous studies reported that nimbolide, a terpenoid derived from the leaves and flowers of neem tree inhibits cancer growth through selective modulation of cell signaling pathways linked to inflammation, survival, proliferation, angiogenesis and metastasis. METHODS: The present study aimed to examine the effect of nimbolide at 1 and 2µM concentrations on TNF-α/TNFR1 mediated signaling molecules involved in cell survival and proliferation in PC-3 cell line via NF-κB and MAPK pathways by real time PCR and western blot. Protein and compound interaction were performed by Molecular docking analysis. RESULTS: Our results indicate that nimbolide treatment suppressed expression of TNF-α, SODD, Grb2, SOS mRNA and modulated TNF-α/TNFR1 regulated NF-κB and MAPK signaling molecules in PC-3 cells. Additional molecular dynamics simulation studies confirmed the stability of nimbolide and signaling molecules binding interactions. Binding pose analysis revealed the significance of hydrogen bond interactions for effective stabilization of virtual ligand protein complexes. CONCLUSION: Nimbolide inhibited prostate cancer cell survival and proliferation via NF-κB and MAPK pathways.

Gold nanoparticle-conjugated quercetin inhibits epithelial-mesenchymal transition, angiogenesis and invasiveness via EGFR/VEGFR-2-mediated pathway in breast cancer.

OBJECTIVES: Epidermal growth factor plays a critical role in breast malignancies by enhancing cell proliferation, invasion, angiogenesis and metastasis. Epithelial-mesenchymal transition (EMT) is a crucial process by which epithelial cells lose polarity and acquire migratory mesenchymal properties. Gold nanoparticles are an efficient drug delivery vehicle for carrying chemotherapeutic agents to target cancer cells and quercetin is an anti-oxidative flavonoid known with potent anti-malignant cell activity. MATERIALS AND METHODS: Cell viability was assessed by MTT assay, and protein expression was examined by Western blotting and immunocytochemistry. Cell invasion was monitored using invasion chambers, and cell migration was analysed by scratch wound-healing assay. In vitro and ex vivo angiogenesis studies were performed by capillary-like tube formation assay and chick embryo angiogenesis assay (CEA). 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinoma in Sprague-Dawley rats. RESULTS: We observed a significant reduction in protein expression of vimentin, N-cadherin, Snail, Slug, Twist, MMP-2, MMP-9, p-EGFR, VEGFR-2, p-PI3K, Akt and p-GSK3ß, and enhanced E-cadherin protein expression in response to AuNPs-Qu-5 treatment. AuNPs-Qu-5 inhibited migration and invasion of MCF-7 and MDA-MB-231 cells compared to free quercetin. AuNPs-Qu-5-treated HUVECs had reduced cell viability and capillary-like tube formation. In vitro and in vivo angiogenesis assays showed that AuNPs-Qu-5 suppressed tube and new blood vessel formation. Treatment with AuNPs-Qu-5 impeded tumour growth in DMBA-induced mammary carcinoma in SD rats compared to treatment with free quercetin. CONCLUSION: Our results suggest that AuNPs-Qu-5 inhibited EMT, angiogenesis and metastasis of the breast cancer cells tested by targeting the EGFR/VEGFR-2 signalling pathway.