RESUMO
Vinyl chloride (VC) is an organochlorine mainly used to manufacture its polymer polyvinyl chloride, which is extensively used in the manufacturing of consumer products. Recent studies suggest that chronic low dose VC exposure affects glucose homeostasis in high fat diet-fed mice. Our data suggest that even in the absence of high fat diet, exposure to VC (0.8 ppm, 6 h/day, 5 day/week, for 12 weeks) induces glucose intolerance (1.0 g/kg, i.p.) in male C57BL/6 mice. This was accompanied with the depletion of hepatic glutathione and a modest increase in lung interstitial macrophages. VC exposure did not affect the levels of circulating immune cells, endothelial progenitor cells, platelet-immune cell aggregates, and cytokines and chemokines. The acute challenge of VC-exposed mice with LPS did not affect lung immune cell composition or plasma IL-6. To examine the effect of VC exposure on vascular inflammation and atherosclerosis, LDL receptor-KO mice on C57BL/6 background maintained on western diet were exposed to VC for 12 weeks (0.8 ppm, 6 h/day, 5 day/week). Unlike the WT C57BL/6 mice, VC exposure did not affect glucose tolerance in the LDL receptor-KO mice. Plasma cytokines, lesion area in the aortic valve, and markers of lesional inflammation in VC-exposed LDL receptor-KO mice were comparable with the air-exposed controls. Collectively, despite impaired glucose tolerance and modest pulmonary inflammation, chronic low dose VC exposure does not affect surrogate markers of cardiovascular injury, LPS-induced acute inflammation in C57BL/6 mice, and chronic inflammation and atherosclerosis in the LDL receptor-KO mice.
Assuntos
Doenças Cardiovasculares , Cloreto de Vinil , Animais , Dieta Hiperlipídica , Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cloreto de Vinil/toxicidadeRESUMO
BACKGROUND: Cadmium, an established carcinogen, is a risk factor for prostate cancer. Induction of autophagy is a prerequisite for cadmium-induced transformation and metastasis. The ability of Psoralidin (Pso), a non-toxic, orally bioavailable compound to inhibit cadmium-induced autophagy to prevent prostate cancer was investigated. METHODS: Psoralidin was studied using cadmium-transformed prostate epithelial cells (CTPE), which exhibit high proliferative, invasive and colony forming abilities. Gene and protein expression were evaluated by qPCR, western blot, immunohistochemistry and immunofluorescence. Xenograft models were used to study the chemopreventive effects in vivo. RESULTS: Cadmium-transformed prostate epithelial cells were treated with Pso resulting in growth inhibition, without causing toxicity to normal prostate epithelial cells (RWPE-1). Psoralidin-treatment of CTPE cells inhibited the expression of Placenta Specific 8, a lysosomal protein essential for autophagosome and autolysosome fusion, which resulted in growth inhibition. Additionally, Pso treatment caused decreased expression of pro-survival signalling proteins, NFκB and Bcl2, and increased expression of apoptotic genes. In vivo, Pso effectively suppressed CTPE xenografts growth, without any observable toxicity. Tumours from Pso-treated animals showed decreased autophagic morphology, mesenchymal markers expression and increased epithelial protein expression. CONCLUSIONS: These results confirm that inhibition of autophagy by Pso plays an important role in the chemoprevention of cadmium-induced prostate carcinogenesis.
Assuntos
Autofagia/efeitos dos fármacos , Benzofuranos/farmacologia , Cádmio/efeitos adversos , Carcinogênese/efeitos dos fármacos , Cumarínicos/farmacologia , Próstata/efeitos dos fármacos , Neoplasias da Próstata/induzido quimicamente , Animais , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Western Blotting , Carcinogênese/induzido quimicamente , Proliferação de Células , Células Cultivadas , Imunofluorescência , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos Nus , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Transplante de Neoplasias , Reação em Cadeia da Polimerase , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Oxidative stress is one causative factor of the pathogenesis and aggressiveness of most of the cancer types, including prostate cancer (CaP). A moderate increase in reactive oxygen species (ROS) induces cell proliferation whereas excessive amounts of ROS promote apoptosis. In this study, we explored the pro-oxidant property of 3,9-dihydroxy-2-prenylcoumestan (psoralidin [pso]), a dietary agent, on CaP (PC-3 and C4-2B) cells. Pso greatly induced ROS generation (more than 20-fold) that resulted in the growth inhibition of CaP cells. Overexpression of anti-oxidant enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase, or pretreatment with the pharmacological inhibitor N-acetylcysteine (NAC) significantly attenuated both pso-mediated ROS generation and pso-mediated growth inhibition in CaP cells. Furthermore, pso administration significantly inhibited the migratory and invasive property of CaP cells by decreasing the transcription of ß-catenin, and slug, which promote epithelial-mesenchymal transition (EMT), and by concurrently inducing E-cadherin expression in CaP cells. Pso-induced ROS generation in CaP cells resulted in loss of mitochondrial membrane potential, cytochrome-c release, and activation of caspase-3 and -9 and poly (ADP-ribose) polymerase (PARP), which led to apoptosis. On the other hand, overexpression of anti-oxidants rescued pso-mediated effects on CaP cells. These findings suggest that increasing the threshold of intracellular ROS could prevent or treat CaP growth and metastasis.
Assuntos
Apoptose/efeitos dos fármacos , Benzofuranos/farmacologia , Cumarínicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Caderinas/biossíntese , Caspase 3/metabolismo , Caspase 9/metabolismo , Catalase/biossíntese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Citocromos c/metabolismo , Ativação Enzimática , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Invasividade Neoplásica , Estresse Oxidativo , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias da Próstata/patologia , Fatores de Transcrição da Família Snail , Superóxido Dismutase/biossíntese , Superóxido Dismutase-1 , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , beta Catenina/genéticaRESUMO
Activation of the serine-threonine protein kinase AKT has emerged as a central feature of epithelial-mesenchymal transition (EMT), which is the initial step for metastasis in many cancer models, including colorectal cancer. The focus of our study was to dissect the role of AKT and its molecular regulation of EMT in colorectal cancer. HCT-116 colorectal cancer cells stably overexpressing AKT (AKT/HCT-116) showed significantly higher cell proliferation compared with vector-transfected cells (pCMV/HCT-116). Elevated expression of important EMT-related transcription factors and genes such as Snail, Slug, ß-catenin, vimentin, and MMP-9 correlated with increased migration and invasion by AKT/HCT-116 cells. Further, in vivo studies confirmed that AKT/HCT-116 xenografts were highly aggressive and angiogenic in nature compared with pCMV/HCT-116 xenografts. Molecular analysis of tumor samples revealed transcriptional regulation of Snail, Slug, ß-catenin, MMP-2, and MMP-9 in AKT/HCT-116 tumors. These results were supported by immunohistochemistry analysis. Low levels of E-cadherin expression with a concomitant increase in and nuclear localization of ß-catenin were evident in AKT/HCT-116 tumors compared with control tumors. Increased microvessel formation coincident with high expression of Factor VIII and increased numbers of reticulocytes confirmed the angiogenic property of AKT/HCT-116 tumors. Our results confirm the potential role of AKT signaling in regulating EMT and angiogenesis in colorectal cancer and suggest that inhibition of AKT can serve as an important therapeutic strategy in modulating EMT in colorectal cancer growth and metastasis.
Assuntos
Proliferação de Células , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose , Western Blotting , Adesão Celular , Movimento Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Cell cycle deregulation is strongly associated with the pathogenesis of prostate cancer. Clinical trials of cell cycle regulators that target either the G0/G1 or G2/M phase to inhibit the growth of cancers including prostate cancer are increasing. The present study focused on the cell cycle regulatory potential of the withanolide withaferin A (1) on prostate cancer cells. Compound 1 induced G2/M arrest in both prostate cancer cell lines (PC-3 and DU-145) when treated for 48 h. The G2/M arrest was accompanied by upregulation of phosphorylated Wee-1, phosphorylated histone H3, p21, and Aurora B. On the other hand, downregulation of cyclins (A2, B1, and E2) and a reduction in phosphorylated Cdc2 (Tyr15) were observed in 1-treated prostate cancer cells. In addition, decreased levels of phosphorylated Chk1 (Ser345) and Chk2 (Thr68) were evident in prostate cancer cells on treatment with 1. These results suggest that activation of Cdc2 leads to arrest in the M phase, with abnormal duplication, and initiation of mitotic catastrophe that results in cell death. In conclusion, these results show clearly the potential of 1 as a regulator of the G2/M phase of the cell cycle and as a therapeutic agent for prostate cancer.
Assuntos
Withania/química , Vitanolídeos/farmacologia , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclinas/metabolismo , Fase G2/efeitos dos fármacos , Histonas/metabolismo , Humanos , Masculino , Mitose/efeitos dos fármacos , Estrutura Molecular , Neoplasias da Próstata/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Vitanolídeos/química , Vitanolídeos/isolamento & purificaçãoRESUMO
We recently demonstrated that AKT activation plays a role in prostate cancer progression and inhibits the pro-apoptotic function of FOXO3a and Par-4. AKT inhibition and Par-4 induction suppressed prostate cancer progression in preclinical models. Here, we investigate the chemopreventive effect of the phytonutrient Withaferin A (WA) on AKT-driven prostate tumorigenesis in a Pten conditional knockout (Pten-KO) mouse model of prostate cancer. Oral WA treatment was carried out at two different doses (3 and 5 mg/kg) and compared to vehicle over 45 weeks. Oral administration of WA for 45 weeks effectively inhibited primary tumor growth in comparison to vehicle controls. Pathological analysis showed the complete absence of metastatic lesions in organs from WA-treated mice, whereas discrete metastasis to the lungs was observed in control tumors. Immunohistochemical analysis revealed the down-regulation of pAKT expression and epithelial-to-mesenchymal transition markers, such as ß-catenin and N-cadherin, in WA-treated tumors in comparison to controls. This result corroborates our previous findings from both cell culture and xenograft models of prostate cancer. Our findings demonstrate that the daily administration of a phytonutrient that targets AKT activation provides a safe and effective treatment for prostate cancer in a mouse model with strong potential for translation to human disease.
Assuntos
Carcinogênese/efeitos dos fármacos , Compostos Fitoquímicos/administração & dosagem , Neoplasias da Próstata/dietoterapia , Vitanolídeos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Caderinas/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Masculino , Camundongos , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Exposure to tobacco smoke, which contains several harmful and potentially harmful constituents such as acrolein increases cardiovascular disease (CVD) risk. Although high acrolein levels induce pervasive cardiovascular injury, the effects of low-level exposure remain unknown and sensitive biomarkers of acrolein toxicity have not been identified. Identification of such biomarkers is essential to assess the toxicity of acrolein present at low levels in the ambient air or in new tobacco products such as e-cigarettes. Hence, we examined the systemic effects of chronic (12 weeks) acrolein exposure at concentrations similar to those found in tobacco smoke (0.5 or 1 ppm). Acrolein exposure in mice led to a 2- to 3-fold increase in its urinary metabolite 3-hydroxypropyl mercapturic acid (3-HPMA) with an attendant increase in pulmonary levels of the acrolein-metabolizing enzymes, glutathione S-transferase P and aldose reductase, as well as several Nrf2-regulated antioxidant proteins. Markers of pulmonary endoplasmic reticulum stress and inflammation were unchanged. Exposure to acrolein suppressed circulating levels of endothelial progenitor cells (EPCs) and specific leukocyte subsets (eg, GR-1+ cells, CD19+ B-cells, CD4+ T-cells; CD11b+ monocytes) whilst other subsets (eg, CD8+ cells, NK1.1+ cells, Ly6C+ monocytes) were unchanged. Chronic acrolein exposure did not affect systemic glucose tolerance, platelet-leukocyte aggregates or microparticles in blood. These findings suggest that circulating levels of EPCs and specific leukocyte populations are sensitive biomarkers of inhaled acrolein injury and that low-level (<0.5 ppm) acrolein exposure (eg, in secondhand smoke, vehicle exhaust, e-cigarettes) could increase CVD risk by diminishing endothelium repair or by suppressing immune cells or both.
Assuntos
Acroleína/administração & dosagem , Biomarcadores/metabolismo , Exposição por Inalação , Nicotiana/química , Acroleína/metabolismo , Acroleína/toxicidade , Acroleína/urina , Animais , Estresse do Retículo Endoplasmático , Células Endoteliais/metabolismo , Resistência à Insulina , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , FumaçaRESUMO
The oncogenic activation of AKT gene has emerged as a key determinant of the aggressiveness of colorectal cancer (CRC); hence, research has focused on targeting AKT signaling for the treatment of advanced stages of CRC. In this study, we explored the anti-tumorigenic effects of withaferin A (WA) on CRC cells overexpressing AKT in preclinical (in vitro and in vivo) models. Our results indicated that WA, a natural compound, resulted in significant inhibition of AKT activity and led to the inhibition of cell proliferation, migration and invasion by downregulating the epithelial to mesenchymal transition (EMT) markers in CRC cells overexpressing AKT. The oral administration of WA significantly suppressed AKT-induced aggressive tumor growth in a xenograft model. Molecular analysis revealed that the decreased expression of AKT and its downstream pro-survival signaling molecules may be responsible for tumor inhibition. Further, significant inhibition of some important EMT markers, i.e., Snail, Slug, ß-catenin and vimentin, was observed in WA-treated human CRC cells overexpressing AKT. Significant inhibition of micro-vessel formation and the length of vessels were evident in WA-treated tumors, which correlated with a low expression of the angiogenic marker RETIC. In conclusion, the present study emphasizes the crucial role of AKT activation in inducing cell proliferation, angiogenesis and EMT in CRC cells and suggests that WA may overcome AKT-induced cell proliferation and tumor growth in CRC.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vitanolídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The activation of AKT governs many signaling pathways and promotes cell growth and inhibits apoptosis in human malignancies including prostate cancer (CaP). Here, we investigated the molecular association between AKT activation and the function of death-associated protein kinase 3 (DAPK3) in CaP. An inverse correlation of pAKT and DAPK3 expression was seen in a panel of CaP cell lines. Inhibition of AKT by wortmannin/LY294002 or overexpression of DAPK3 reverts the proliferative function of AKT in CaP cells. On the other hand, ectopic expression of AKT inhibited DAPK3 function and induced proliferation of CaP cells. In addition, AKT over-expressed tumors exhibit aggressive growth when compared to control vector in xenograft models. The immunohistochemistry results revealed a down-regulation of DAPK3 expression in AKT over-expressed tumors as compared to control tumors. Finally, we examined the expression pattern of AKT and DAPK3 in human CaP specimens - the expected gradual increase and nuclear localization of pAKT was seen in higher Gleason score samples versus benign hyperplasia (BPH). On the contrary, reduced expression of DAPK3 was seen in higher Gleason stages versus BPH. This suggests that inhibition of DAPK3 may be a contributing factor to the carcinogenesis of the prostate. Understanding the mechanism by which AKT negatively regulates DAPK3 function may suggest whether DAPK3 can be a therapeutic target for CaP.
Assuntos
Apoptose , Proteínas Quinases Associadas com Morte Celular/metabolismo , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Quinases Associadas com Morte Celular/genética , Ativação Enzimática , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Gradação de Tumores , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Carga TumoralRESUMO
We previously reported that withaferin A (WA), a natural compound, deters prostate cancer by inhibiting AKT while inducing apoptosis. In the current study, we examined its chemopreventive efficacy against carcinogenesis in the prostate using the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Two distinct sets of experiments were conducted. To determine whether WA delays tumor progression, it was given before cancer onset, at week 6, and until week 44. To determine its effect after the onset of prostate cancer, it was given from weeks 12 to 35. In both strategies, oral administration of WA effectively suppressed tumor burden when compared to vehicle-treated animals. No toxicity was seen in treated animals at gross pathological examination. Western blot analysis and immunohistochemistry of tumor sections revealed that in TRAMP controls, AKT and pAKT were highly expressed while nuclear FOXO3a and Par-4 were downregulated. On the contrary, treated mice showed inhibition of AKT signaling and activation of FOX03a-Par-4-induced cell death. They also displayed inhibition of mesenchymal markers such as ß-catenin, vimentin, and snail as well as upregulation of E-cadherin. Because expressions of the angiogenic markers factor VIII and retic were downregulated, an anti-angiogenic role of WA is suggested. Overall, our results suggest that WA could be a promising anti-cancer agent that effectively inhibits carcinogenesis of the prostate.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias da Próstata/patologia , Vitanolídeos/farmacologia , Administração Oral , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
PURPOSE: The diagnosis and treatment of prostate cancer (CaP) continues to be challenging, as prostate-specific antigen (PSA) appears to be overly sensitive and biopsy is the only reliable method for confirmation. Hence, the goal of the study is to identify a biomarker that could distinguish malignant cancer from benign prostatic hyperplasia (BPH) during the early diagnosis of the disease. MATERIALS AND METHODS: A total of 75 formalin fixed paraffin embedded (FFPE) with matching controls, 4 paired metastatic tumors, 6 fresh tumor tissues and BPH (13 cases) with their clinical diagnosis were selected for this study. Prostate cancer cell lines and normal prostate epithelial cell lines were obtained from ATCC and subjected to phenotypic analysis. RESULTS: We observed significant differential expression of miR-301a in CaP samples in comparison to BPH and adjacent benign samples. The overexpression of miR-301a activates the invasion/migration of CaP cells. In contrast, silencing miR-301a expression inhibited the colony-forming ability, adhesion, invasion and migration of CaP cells. Similarly, the overexpression of miR-301a increased cell motility in normal RWPE-1 prostate epithelial cells. Our results suggest that miR-301a is differentially expressed between BPH and CaP specimens and that the expression of miR-301a correlates with biochemical recurrence and/or metastasis in CaP patients. CONCLUSIONS: The expression of miR-301a could be a potential marker for metastasis in CaP patients. Detecting miR-301a expression during diagnosis will avoid wait and watch timelines, thus preventing morbidity.
Assuntos
Biomarcadores Tumorais/metabolismo , MicroRNAs/metabolismo , Neoplasias da Próstata/diagnóstico , Linhagem Celular Tumoral , Movimento Celular , Humanos , Masculino , Prognóstico , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismoRESUMO
The activation of Notch signaling is implicated in tumorigenesis in the colon due to the induction of pro-survival signaling in colonic epithelial cells. Chemoresistance is a major obstacle for treatment and for the complete eradication of colorectal cancer (CRC), hence, the inhibition of Notch is an attractive target for CRC and several groups are working to identify small molecules or monoclonal antibodies that inhibit Notch or its downstream events; however, toxicity profiles in normal cells and organs often impede the clinical translation of these molecules. Dietary agents have gained momentum for targeting several pro-survival signaling cascades, and recent studies demonstrated that agents that inhibit Notch signaling result in growth inhibition in preclinical models of CRC. In this review, we focus on the importance of Notch as a preventive and therapeutic target for colon cancer and on the effect of WA on this signaling pathway in the context of colon cancer.