RESUMO
Prostate cancer is most common malignancy among men in the world. PI3K-Akt signaling appears to be critical to prostate cancer cell proliferation and survival. Our earlier study reveals that nimbolide (2 µM) prevents cell survival via IGF signaling pathway through PI3K/Akt and induces apoptosis in PC-3 cell line. Akt mediates the phosphorylation and activation of mTOR that plays a critical role in the regulation of protein translation and synthesis, angiogenesis, and cell cycle progression. The present study was aimed to investigate the effect of nimbolide on tPI3K, tAkt, pAkt, tmTOR, GSK3ß, pGSK3ß, PCNA, c-Myc, Cyclin D1, and Survivin protein levels by western blot analysis. Apoptosis was visualized by Ao/EtBr dual staining (20×), and protein expression of PCNA by immunocytochemistry was performed. Molecular docking was performed to understand the possible interaction between nimbolide and Akt, PCNA, and Cyclin D1. Nimbolide altered the PI3K-Akt-mediated cell survival and proliferative molecules. Thus, nimbolide exerted anticancer effects in vitro by representing the PI3K-Akt-mTOR pathway in PC-3 cells. Thereby, it acts as a potent anticancer drug for prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Limoninas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismoRESUMO
Prostate cancer becomes an ideal target for chemoprevention because of its high incidence and extended natural history. The consumption of quercetin (plant flavonoid) in diet is associated with decreased risk of disease and many cancers but then this was not elucidated in prostate malignancy. Hence, a study in which the male Sprague-Dawley rats were induced prostate cancer by hormone (testosterone) and carcinogen (MNU) and simultaneously supplemented with quercetin (200 mg/Kg body weight) thrice a week, was conducted. After the treatment period, rats were killed; ventral and dorsolateral lobes of the prostate were dissected. Histology and oxidative stress markers LPO, H2O2, and antioxidant GSH level were measured in both lobes. The lipid peroxidation, H2O2, in (MNU+T) treated rats were increased and GSH level was decreased, whereas simultaneous quercetin-treated rats reverted back to normal level in both ventral and dorsolateral regions. The different patterns of PIN were observed with associated hyperplasia and dysplasia; changes in these regions and the occurrence of this lesion were reduced in simultaneous quercetin-treated rats. The study concluded that dietary quercetin prevented MNU + T-induced prostate carcinogenesis on both ventral and dorsolateral lobes of Sprague-Dawley rats.
Assuntos
Anticarcinógenos/farmacologia , Neoplasias da Próstata/prevenção & controle , Quercetina/farmacologia , Testosterona/efeitos adversos , Animais , Carcinógenos/toxicidade , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , Masculino , Próstata/efeitos dos fármacos , Próstata/patologia , Neoplasias da Próstata/induzido quimicamente , Ratos , Ratos Sprague-DawleyRESUMO
The insulin-like growth factor I (IGF-I) signalling pathway contributes a major role on various cancer cell proliferation, survival and cell cycle. The present study was aimed to investigate the effect of nimbolide on IGF signalling and cell cycle arrest in MCF-7 and MDA-MB-231 breast cancer cell lines. The protein expression of IGF signalling molecules and cell cycle protein levels was assessed by western blot analysis. In order to study the interaction of nimbolide on IGF-1 signalling pathway, IGF-I and phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) were used to treat MCF-7 and MDA-MB-231 cells. Further, the cell cycle arrest was analysed by flow cytometry. The protein expression of IGF signalling molecules was significantly decreased in nimbolide-treated breast cancer cells. PI3K inhibitor and IGF-I with nimbolide treatment notably inhibited phosphorylated Akt. The cell cycle arrest was observed at the G0/G1 phase, and accumulation of apoptotic cells was observed in nimbolide-treated breast cancer cell lines. Nimbolide also increased the protein expression of p21 and decreased the cyclins in both the cell lines. Nimbolide decreases the proliferation of breast cancer cells by modulating the IGF signalling molecules, which could be very useful for the breast cancer treatment.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Limoninas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromonas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Células MCF-7 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor IGF Tipo 1/metabolismo , beta Catenina/metabolismoRESUMO
Prostate cancer is responsible for major deaths globally after lung cancer. Nimbolide is an important constituent of neem, and it acts as a potent inhibitor for many cancer cells. The present study was designed to evaluate the effects of nimbolide on apoptosis and insulin-like growth factor (IGF) signalling molecules in androgen-independent prostate cancer (PC-3) cells line. Nimbolide (0.5-2 µM) treatment resulted in 50% inhibition at a dose of 2 µM in the PC-3 cell line. The mRNA expression of Fas ligand, Fas-associated death domain receptor (FADDR), Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter (Bad), phosphatidylinositide 3-kinases (PI3K), Akt, IGF1, IGF1 receptor (IGF1R) and IGF binding protein 3 were quantified by reverse transcription polymerase chain reaction and protein expression of Bax, cytochrome c, X-linked inhibitor of apoptosis protein (XIAP), B-Cell Lymphoma 2 (Bcl-2), caspases -8, -9, -10 and -3, poly(ADP-ribose) polymerase (PARP), cleaved PARP, IGF1R, PI3K, Akt, p-Akt was determined by western blot analysis, in nimbolide-treated PC-3 cell line. Nimbolide-induced apoptosis by activating DNA fragmentation in PC-3 cells. Nimbolide treatment increased the mRNA of Fas ligand, FADDR, Bax, Bad and IGF binding protein 3, decreased PI3K, Akt, IGF1 and IGF1R, increased protein expression of caspases 8, 3, 10, 9, Bax and cytochrome c and decreased the expression of XIAP, Bcl2, cleaved PARP, p-Akt and IGF1R. The results suggest that nimbolide acts as a potent anti-cancer agent by inducing apoptosis and inhibiting cell proliferation via PI3K/Akt pathway in PC-3 cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Limoninas/farmacologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/metabolismo , Somatomedinas/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Neoplasias da Próstata/patologia , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Polychlorinated biphenyls (PCBs) exposure produces neurodegeneration and induces oxidative stress. Neuroprotective role of quercetin, on PCBs induced apoptosis in hippocampus has not yet been studied. The present study is focused to see whether quercetin supplementation precludes against PCBs induced oxidative stress and hippocampal apoptosis. The results have shown that quercetin at 50 mg/kg bwt/30 days has protected oxidative stress in hippocampus of adult male rats. Quercetin, a free radical scavenger decreased the levels of oxidative stress markers in the hippocampus of simultaneous PCB+quercetin treated rats. The pro-apoptotic and anti-apoptotic molecules such as Bad, Bid, Bax and Bcl2 were altered in the hippocampus of experimental animals. PCBs increased the DNA damage and induced neurodegeneration were assessed by histological studies. PCB induced ROS may be linked to increased hippocampal neuronal apoptosis. Quercetin supplementation decreased the neuronal damage and scavenged the free radicals induced by PCBs and protects PCBs induced apoptosis and oxidative stress.
Assuntos
Apoptose/fisiologia , Hipocampo/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/fisiologia , Bifenilos Policlorados/toxicidade , Quercetina/farmacologia , Fatores Etários , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Urokinase-type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis including prostate cancer. uPA activation is mediated by transactivation of uPAR and epidermal growth factor receptor (EGF-R) in prostate cancer progression. Prostate cancer (PC-3) cells have highly invasive capacity and they express uPA and uPAR gene. PC-3 cells are treated with quercetin, which inhibits invasion and migration of PC-3 cells. Quercetin downregulates uPA, uPAR and EGF, EGF-R mRNA expressions. Quercetin inhibits cell survival factor ß-catenin, NF-κB and also proliferative signalling molecules such as p-EGF-R, N-Ras, Raf-1, c.Fos c.Jun and p-c.Jun protein expressions. But quercetin increased p38 mitogen-activated protein kinase protein expression. Our results suggest that quercetin inhibit migration and invasion of prostate cancer cells. It shows the value for treatment of invasive and metastasis type of prostate cancer.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Neoplasias da Próstata/fisiopatologia , Quercetina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Invasividade Neoplásica , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRß, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.
Assuntos
Androgênios/fisiologia , Apoptose/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Quercetina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Somatomedinas/metabolismo , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Microscopia de Fluorescência , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Antioxidantes/farmacologia , Carotenoides/farmacologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Poluentes Ambientais/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Fosfoproteínas/metabolismo , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Quimioprevenção , Células Intersticiais do Testículo/enzimologia , Licopeno , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar , Testículo/efeitos dos fármacos , Testículo/patologia , Testosterona/sangueRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Azadirachta/química , Inibidores de Fosfoinositídeo-3 Quinase , Extratos Vegetais/farmacologia , Folhas de Planta/química , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Sequência de Bases , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Primers do DNA , Etanol/química , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Neoplasias da Próstata/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Carcinógenos/farmacologia , Metilnitrosoureia/farmacologia , Neoplasias da Próstata/induzido quimicamente , Testosterona/farmacologia , Compostos de Zinco/farmacologia , Fosfatase Ácida , Animais , Western Blotting , Carcinógenos/antagonistas & inibidores , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Próstata/efeitos dos fármacos , Próstata/patologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/prevenção & controle , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Ratos Sprague-Dawley , Testosterona/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Prostate cancer is one of the most frequently diagnosed cancers in men. Progression of these tumors is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. Among these, insulin-like growth factors (IGFs) signaling pathway contributes a major role. In this study, we examined the effect of zinc on insulin-like growth factors signaling in prostate cancer cells. METHODS: Human androgen-independent prostatic carcinoma (PC-3) cells were treated with different concentrations of zinc (20-100micromol/l) for 24 and 48h. Cell viability was performed by 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Insulin-like growth factor binding protein-3 (IGFBP-3), insulin-like growth factor-I receptor (IGF-IR), insulin receptor substrate-1 (IRS-1) and IRS-2, phosphatidylinositol-3 kinase (PI-3 K), protein kinase B or Akt, phosphorylated Akt (p-Akt), extracellular regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), and cyclin D1 protein levels were assessed by Western blot analysis. Apoptosis was confirmed by 4',6'-diaminido-2-phenylindole dihydrochloride (DAPI) staining, and mitochondrial membrane potential was performed using rhodamine-123 staining method. RESULTS: Zinc significantly reduces the cell viability of PC-3 cells. It decreases the protein levels of IGF-IR, IRS-1, and IRS-2 and increases the level of IGFBP-3. Zinc reduces the levels of PI-3 K, Akt, ERK1/2, and cyclin Dl. Loss of mitochondrial membrane potential and apoptotic cell death were also observed in zinc-treated cells. CONCLUSION: This study suggests that zinc decreases the survival of androgen-independent prostate cancer cells by modulating the expression of IGF system components and its signaling molecules. Thus, zinc may be qualified as a potential agent for the treatment of prostate cancer.