RESUMO
Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all malignancies in USA and unfortunately the recurrence of secondary primary tumors and resistance against conventional treatments decrease the overall 5 year survival rate in HNSCC patients. Thus, additional approaches are needed to control HNSCC. Here, for the first time, employing human HNSCC Detroit 562 and FaDu cells as well as normal human epidermal keratinocytes, we investigate grape seed extract (GSE) efficacy and associated mechanism in both cell culture and nude mice xenografts. GSE selectively inhibited the growth and caused cell cycle arrest and apoptotic death in both Detroit 562 and FaDu cells by activating DNA damage checkpoint cascade, including ataxia telangiectasia mutated/ataxia telangiectasia-Rad3-related-checkpoint kinase 1/2-cell division cycle 25C as well as caspases 8, 9 and 3. Consistent with these results, GSE treatment resulted in a strong DNA damage and a decrease in the levels of DNA repair molecules breast cancer gene 1 and Rad51 and DNA repair foci. GSE-caused accumulation of intracellular reactive oxygen species was identified as a major mechanism of its effect for growth inhibition, DNA damage and apoptosis, which was remarkably reversed by antioxidant N-acetylcysteine. GSE feeding to nude mice decreased Detroit 562 and FaDu xenograft tumor growth by 67 and 65% (P < 0.001), respectively. In immunohistochemical analysis, xenografts from GSE-fed groups showed decreased proliferation but increased DNA damage and apoptosis. Together, these findings show that GSE targets both DNA damage and repair and provide mechanistic insights for its efficacy selectively against HNSCC both in cell culture and mouse xenograft, supporting its translational potential against HNSCC.
Assuntos
Carcinoma de Células Escamosas/patologia , Dano ao DNA , Neoplasias de Cabeça e Pescoço/patologia , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismo , Sementes/química , Vitis/embriologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Divisão Celular/efeitos dos fármacos , Imunofluorescência , Fase G2/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Células Tumorais CultivadasRESUMO
PURPOSE: The 3,3â³-di-O-galloyl ester of procyanidin B2 (B2G2) is a component of grape seed extract that inhibits growth of human prostate carcinoma cell lines. In preparation for studies in mice, its hepatic metabolism was examined in vitro and compared to B2 and the corresponding monomers, epicatechin (EC) and 3-O-galloyl-epicatechin (ECG). METHODS: Compounds were incubated with liver microsomes or cytosol containing cofactors for glucuronidation, sulfation or methylation, and products analyzed by liquid chromatography-mass spectrometry (LC-MS). B2G2 was administered orally to mice and plasma analyzed by LC-MS for unmodified procyanidin and metabolites. RESULTS: Glucuronides and methyl ethers of B2 and B2G2 were formed in small amounts. In contrast, EC and ECG were largely or completely converted to glucuronides, sulfates and methyl ethers under the same incubation conditions. B2G2 given orally to mice was partially absorbed intact; no significant metabolites were detected in plasma. CONCLUSIONS: Glucuronidation and methylation of procyanidins B2 and B2G2 occurred but were minor processes in vitro. B2G2 was partially absorbed intact in mice after oral dosing and did not undergo significant metabolism. Unlike the flavanol monomers EC and ECG, therefore, B2G2 bioavailability should not be limited by metabolism. These results paved the way for ongoing pharmacokinetic and efficacy studies.
Assuntos
Antineoplásicos Fitogênicos/metabolismo , Biflavonoides/metabolismo , Catequina/análogos & derivados , Catequina/metabolismo , Glucuronídeos/metabolismo , Microssomos Hepáticos/metabolismo , Proantocianidinas/metabolismo , Animais , Biflavonoides/sangue , Biflavonoides/isolamento & purificação , Catequina/sangue , Catequina/isolamento & purificação , Dimerização , Extrato de Sementes de Uva/química , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Proantocianidinas/sangue , Proantocianidinas/isolamento & purificaçãoRESUMO
Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen-activated protein kinase (MAPK)- and Akt-mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF-1)-induced biological responses and associated signaling cascades in advanced and androgen-independent human PCa PC-3 cells. Treatment of PC-3 cells with 2 mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC-3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand-induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK1/2), and p38 under constitutive and ligand-activated conditions. Under similar condition, IP6 also inhibited AP-1 DNA-binding activity and decreased nuclear levels of both phospho and total c-Fos and c-Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF-1 receptor (IGF-1R) pathway-mediated sustained growth promoting and survival signaling cascades in advanced and androgen-independent human PCa PC-3 cells, which might have translational implications in advanced human PCa control and management.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Ácido Fítico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Humanos , Immunoblotting , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismoRESUMO
Effective medical treatment and preventive measures for chemical warfare agent sulfur mustard (HD)-caused incapacitating skin toxicity are lacking, because of limited knowledge of its mechanism of action. The proliferating basal epidermal cells are primary major sites of attack during HD-caused skin injury. Therefore, employing mouse JB6 and human HaCaT epidermal cells, here, we investigated the molecular mechanism of HD analogue 2-chloroethyl ethyl sulfide (CEES)-induced skin cytotoxicity. As compared to the control, up to 1 mM CEES treatment of these cells for 2, 4, and 24 h caused dose-dependent decreases in cell viability and proliferation as measured by DNA synthesis, together with S and G2-M phase arrest in cell cycle progression. Mechanistic studies showed phosphorylation of DNA damage sensors and checkpoint kinases, ataxia telangiectasia-mutated (ATM) at ser1981 and ataxia telangiectasia-Rad3-related (ATR) at ser428 within 30 min of CEES exposure, and modulation of S and G2-M phase-associated cell cycle regulatory proteins, which are downstream targets of ATM and ATR kinases. Hoechst-propidium iodide staining demonstrated that CEES-induced cell death was both necrotic and apoptotic in nature, and the latter was induced at 4 and 24 h of CEES treatment in HaCaT and JB6 cells, respectively. An increase in caspase-3 activity and both caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage coinciding with CEES-caused apoptosis in both cell lines suggested the involvement of the caspase pathway. Together, our findings suggest a DNA-damaging effect of CEES that activates ATM/ATR cell cycle checkpoint signaling as well as caspase-PARP pathways, leading to cell cycle arrest and apoptosis/necrosis in both JB6 and HaCaT cells. The identified molecular targets, quantitative biomarkers, and epidermal cell models in this study have the potential and usefulness in rapid development of effective prophylactic and therapeutic interventions against HD-induced skin toxicity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Gás de Mostarda/análogos & derivados , Proteínas Serina-Treonina Quinases/metabolismo , Pele/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Células Epidérmicas , Humanos , Camundongos , Gás de Mostarda/efeitos adversos , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Sunscreens partially filter UVB and, therefore, could partially prevent skin cancer; however, efficient approaches are desired to effectively prevent photocarcinogenesis. It is hypothesized that nontoxic pharmacologically active natural compounds can increase photoprotective effects. Our completed studies suggest that silibinin, a bioactive phytochemical, strongly prevents photocarcinogenesis; however, its mechanism is not fully understood. Herein, for the first time, we used a clinically relevant UVB dose (30 mJ/cm(2)/day) to examine the photoprotective effect and associated mechanisms of silibinin in SKH1 hairless mice. Topical or dietary silibinin treatment caused a strong protection against photocarcinogenesis in terms of delay in tumor appearance, multiplicity, and volume. Analyses of normal skin, uninvolved skin from tumor-bearing mice, and skin tumors showed a statistically significant decrease (P < 0.05-0.001) in inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) levels by silibinin. Concomitantly, phospho-signal transducers and activators of transcription 3 (Tyr(705)) and phospho-p65(Ser(536)) were also decreased by silibinin, which are potential up-stream regulators of iNOS and COX-2. Simultaneously, silibinin also decreased UVB-caused increase in cell proliferation and microvessel density. In tumors, hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor protein levels were decreased by silibinin. Further analysis showed that silibinin inhibited UVB-caused phosphorylation and nuclear translocation of STAT3 and p65, as well as nuclear factor kappaB (NF-kappaB) DNA binding activity. Together, these results suggest that silibinin causes a strong protective effect against photocarcinogenesis via down-regulation of inflammatory and angiogenic responses, involving HIF-1alpha, STAT3, and NF-kappaB transcription factors, as well as COX2 and iNOS.
Assuntos
Neoplasias Cutâneas/prevenção & controle , Pele/efeitos da radiação , Animais , Antioxidantes/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos da radiação , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/efeitos da radiação , Ciclo-Oxigenase 2/biossíntese , Feminino , Inflamação/tratamento farmacológico , Inflamação/patologia , Camundongos , Camundongos Pelados , NF-kappa B/metabolismo , Neovascularização Patológica/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/biossíntese , Fosforilação , Fator de Transcrição STAT3/metabolismo , Silibina , Silimarina/farmacologia , Pele/enzimologia , Pele/patologia , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/etiologia , Raios UltravioletaRESUMO
UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non-DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N-(2-mercapto-2-methylpropionyl)-l-cysteine] is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB-induced photodamage using the SKH-1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm(-2)) on the dorsal skin of SKH-1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg(-1) of body weight, administered subcutaneously) markedly attenuated UVB-mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser-15 and Ser-20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.
Assuntos
Antioxidantes/farmacologia , Cisteína/análogos & derivados , Modelos Animais , Protetores contra Radiação/farmacologia , Raios Ultravioleta , Animais , Cisteína/farmacologia , Feminino , Camundongos , Camundongos Pelados , Proteína Supressora de Tumor p53/metabolismoRESUMO
Effective strategies are lacking for the management of urinary bladder cancer for which smoking is a potential risk factor. Herein, we evaluated chemoprevention of urinary bladder cancer by natural chemopreventive agents, silymarin and silibinin, in a preclinical animal (ICR mouse) model of bladder cancer induced by tobacco smoke carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN). Mice were fed p.o. with saline or OH-BBN (0.05%, w/v) in drinking water for 6 weeks or with silymarin or silibinin (200 mg/kg body weight for both) starting 1 week before OH-BBN exposure for 51 weeks. Silymarin and silibinin strongly arrested OH-BBN-induced tumor progression at the stage of mucosal dysplasia with a striking reduction in papillary nodular dysplasia as well as invasive carcinoma. Some silymarin- or silibinin-treated mice developed no urothelial lesions in spite of OH-BBN exposure. Immunohistochemical analyses at study conclusion revealed that silymarin and silibinin decreased cell proliferation by 42% (P < 0.001) and 44% (P < 0.001) and increased apoptosis by 4-fold (P < 0.05) and 6-fold (P < 0.05) in OH-BBN-induced urothelium, respectively. Antiproliferative and apoptotic effects of silymarin and silibinin were associated with decreases in (a) cyclin D1 protein level and extracellular signal-regulated kinase-1/2 phosphorylation and in (b) protein levels of survivin and nuclear phospho-p65 (Ser(276) and Ser(536)), respectively. Together, these results suggest that silymarin and silibinin inhibit chemically induced urinary bladder tumor growth and progression possibly by inhibiting cell proliferation and enhancing apoptosis.
Assuntos
Anticarcinógenos/farmacologia , Butilidroxibutilnitrosamina/toxicidade , Carcinógenos/toxicidade , Silimarina/farmacologia , Neoplasias da Bexiga Urinária/induzido quimicamente , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , SilibinaRESUMO
PURPOSE: Accumulating evidences suggest the beneficial effects of fruit-and-vegetable consumption in lowering the risk of various cancers, including colorectal cancer. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of grape seed extract (GSE), a rich source of proanthocyanidins, against colorectal cancer. EXPERIMENTAL DESIGN: Effects of GSE were examined on human colorectal cancer HT29 and LoVo cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral GSE was examined on HT29 tumor xenograft growth in athymic nude mice. Xenografts were analyzed by immunohistochemistry for proliferation and apoptosis. The molecular changes associated with the biological effects of GSE were analyzed by Western blot analysis. RESULTS: GSE (25-100 microg/mL) causes a significant dose- and time-dependent inhibition of cell growth with concomitant increase in cell death. GSE induced G1 phase cell cycle arrest along with a marked increase in Cip1/p21 protein level and a decrease in G1 phase-associated cyclins and cyclin-dependent kinases. GSE-induced cell death was apoptotic and accompanied by caspase-3 activation. GSE feeding to mice at 200 mg/kg dose showed time-dependent inhibition of tumor growth without any toxicity and accounted for 44% decrease in tumor volume per mouse after 8 weeks of treatment. GSE inhibited cell proliferation but increased apoptotic cell death in tumors. GSE-treated tumors also showed enhanced Cip1/p21 protein levels and poly(ADP-ribose) polymerase cleavage. CONCLUSIONS: GSE may be an effective chemopreventive agent against colorectal cancer, and that growth inhibitory and apoptotic effects of GSE against colorectal cancer could be mediated via an up-regulation of Cip1/p21.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Extratos Vegetais/toxicidade , Sementes , Vitis , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Neoplasias Colorretais , Humanos , FitoterapiaRESUMO
UVB radiation-induced DNA damage in skin activates cellular pathways involved in DNA repair, cell cycle regulation, and apoptosis, important events that prevent conversion of damaged skin cells into cancer. We reported recently the efficacy of silibinin against photocarcinogenesis along with altered molecular events in tumors (Cancer Research, 64:6349-56, 2004). The molecular and biological events modulated by silibinin in chronically UVB-irradiated skin leading to cancer prevention, however, are not known. Herein, we describe effect of silibinin on skin 15 and 25 weeks after UVB exposure and compared them with molecular alterations in skin tumors. UVB decreased E2F1 but increased E2F2 and E2F3 protein levels in skin, and these were reversed by silibinin treatment. Silibinin-induced E2F1 was accompanied by an inhibition of apoptosis and decreases in p53 and cyclin-dependent kinase inhibitors. Silibinin-caused decrease in E2F2 and E2F3 was accompanied by reduced levels of cyclin-dependent kinases, cyclins, CDC25C, and mitogen-activated protein kinases and Akt signaling and inhibition of cell proliferation. In tumorigenesis protocols, topical or dietary silibinin significantly inhibited tumor appearance and growth. As opposed to UVB-exposed skin, UVB-induced tumors showed elevated levels of E2F1, but these were reduced in silibinin-treated tumors without any effect on E2F2 and E2F3. Contrary to the inhibition of apoptosis and p53 expression in UVB-exposed skin cells, silibinin increased these variables in tumors. These differential effects of silibinin on E2F1 versus E2F2 and E2F3 and their associated molecular alterations and biological effects in chronic UVB-exposed skin suggest their role in silibinin interference with photocarcinogenesis.
Assuntos
Fatores de Transcrição E2F/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p27/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/efeitos da radiação , Fatores de Transcrição E2F/metabolismo , Feminino , Camundongos , Camundongos Pelados , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Silibina , Silimarina/farmacologia , Pele/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/efeitos da radiaçãoRESUMO
Solar radiation is the causal etiologic factor in the development of nonmelanoma skin cancer (NMSC). Depletion of the stratospheric ozone layer leads to an increase in ambient UV radiation loads, which are expected to further raise skin cancer incidence in many temperate parts of the world, including the United States, suggesting that skin cancer chemopreventive approaches via biomarker efficacy studies or vice versa are highly warranted. Based on our recent study reporting strong efficacy of silibinin against photocarcinogenesis, we assessed here the protective effects of its dietary feeding on UVB-induced biomarkers involved in NMSC providing a mechanistic rationale for an early-on silibinin efficacy in skin cancer prevention. Dietary feeding of silibinin at 1% dose (w/w) to SKH-1 hairless mice for 2 weeks before a single UVB irradiation at 180 mJ/cm(2) dose resulted in a strong and significant (P < 0.001) decrease in UVB-induced thymine dimer-positive cells and proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase (P < 0.001) in p53 and p21/cip1-positive cell population in epidermis. These findings suggest that dietary feeding of silibinin affords strong protection against UVB-induced damages in skin epidermis by (a) either preventing DNA damage or enhancing repair, (b) reducing UVB-induced hyperproliferative response, and (c) inhibiting UVB-caused apoptosis and sunburn cell formation, possibly via silibinin-caused up-regulation of p53 and p21/cip1 as major UVB-damage control sensors.
Assuntos
Apoptose/efeitos dos fármacos , Suplementos Nutricionais , Epiderme/efeitos dos fármacos , Neoplasias Cutâneas/prevenção & controle , Raios Ultravioleta/efeitos adversos , Animais , Proliferação de Células/efeitos dos fármacos , Quimioprevenção , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Epiderme/efeitos da radiação , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Pelados , Modelos Animais , Dímeros de Pirimidina , Silibina , Silimarina/administração & dosagem , Neoplasias Cutâneas/etiologia , Proteína Supressora de Tumor p53/biossínteseRESUMO
The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.
Assuntos
Flavonolignanos/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Silybum marianum/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Flavonolignanos/química , Humanos , Masculino , Camundongos , Camundongos Nus , Silibina , Silimarina/análogos & derivados , Silimarina/química , Silimarina/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Alterations in Smad4 signaling and its loss cause genomic instability and head and neck squamous cell carcinoma (HNSCC), suggesting that agents that target both Smad4-dependent and -independent pathways could control HNSCC. EXPERIMENTAL DESIGN: Resveratrol efficacy was evaluated against the HNSCC cells FaDu, Cal27, Det562, and Cal27-Smad4 for viability, DNA damage, cell-cycle progression, and apoptosis, as well as γ-H2AX expression, and focus formation (γ-H2AX and Brca1). Resveratrol efficacy was also examined in nude mice for FaDu xenograft growth. Xenografts were analyzed for γ-H2AX and cleaved caspase-3. RESULTS: Resveratrol (5-50 µmol/L) suppressed viability and induced DNA damage in FaDu and Cal27 cells but not in normal human epidermal keratinocytes and human foreskin fibroblasts, showing its selectivity toward HNSCC cells; however, Det562 cells were resistant to resveratrol even at 100 µmol/L. Cal27 cells stably transfected with Smad4 showed similar resveratrol effects as parental Cal27, indicating that a lack of resveratrol effect in Det562 cells was independent of Smad4 status in these cells. Furthermore, resveratrol caused S-phase arrest and apoptotic death of FaDu and Cal27 cells together with induction of Brca1 and γ-H2AX foci. Resveratrol (50 mg/kg body weight) treatment also inhibited FaDu tumor growth in nude mice, and γ-H2AX and cleaved caspase-3 were strongly increased in xenografts from resveratrol-treated mice compared with controls. CONCLUSION: Our findings for the first time showed antiproliferative, DNA damaging, and apoptotic effects of resveratrol in HNSCC cells independent of Smad4 status, both in vitro and in vivo, suggesting that more studies are needed to establish its potential usefulness against HNSCC.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Dano ao DNA , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteína Smad4/metabolismo , Estilbenos/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína BRCA1/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio Cometa , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Histonas/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Mutação , Resveratrol , Proteína Smad4/genética , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bifunctional alkyalating agent, sulfur mustard (SM)-induced cutaneous injury is characterized by inflammation and delayed blistering. Our recent studies demonstrated that 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM that can be used in laboratory settings, induces oxidative stress. This could be the major cause of the activation of Akt/MAP kinase and AP1/NF-κB pathways that are linked to the inflammation and microvesication, and histopathological alterations in SKH-1 hairless mouse skin. To further establish a link between CEES-induced DNA damage and signaling pathways and inflammatory responses, skin samples from mice exposed to 2 mg or 4 mg CEES for 9-48 h were subjected to molecular analysis. Our results show a strong CEES-induced phosphorylation of H2A.X and an increase in cyclooxygenase-2 (COX-2), inducible NOS (iNOS), and matrix metalloproteinase-9 (MMP-9) levels, indicating the involvement of DNA damage and inflammation in CEES-induced skin injury in male and female mice. Since, our recent studies showed reduction in CEES-induced inflammatory responses by glutathione (GSH), we further assessed the role of oxidative stress in CEES-related DNA damage and the induction of inflammatory molecules. Oral GSH (300 mg/kg) administration 1h before CEES exposure attenuated the increase in both CEES-induced H2A.X phosphorylation (59%) as well as expression of COX-2 (68%), iNOS (53%) and MMP-9 (54%). Collectively, our results indicate that CEES-induced skin injury involves DNA damage and an induction of inflammatory mediators, at least in part via oxidative stress. This study could help in identifying countermeasures that alone or in combination, can target the unveiled pathways for reducing skin injury in humans by SM.
Assuntos
Substâncias para a Guerra Química/toxicidade , Dano ao DNA , Dermatite de Contato/metabolismo , Mediadores da Inflamação/metabolismo , Gás de Mostarda/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Pele/efeitos dos fármacos , Alquilantes/administração & dosagem , Alquilantes/toxicidade , Animais , Antídotos/metabolismo , Antídotos/uso terapêutico , Vesícula/induzido quimicamente , Ciclo-Oxigenase 2/metabolismo , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Dermatite de Contato/prevenção & controle , Feminino , Glutationa/metabolismo , Glutationa/uso terapêutico , Histonas/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Pelados , Gás de Mostarda/administração & dosagem , Gás de Mostarda/toxicidade , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Pele/patologiaRESUMO
Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities.
Assuntos
Fibroblastos/efeitos dos fármacos , Gás de Mostarda/análogos & derivados , Pele/efeitos dos fármacos , Animais , Linhagem Celular , Cromanos/farmacologia , Dano ao DNA , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Glutationa/farmacologia , Histonas/metabolismo , Camundongos , Camundongos Pelados , Gás de Mostarda/farmacologia , Oxirredução , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Pele/citologia , Pele/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: Colorectal cancer is the second leading cause of cancer-associated deaths, which suggests that more effort is needed to prevent/control this disease. Herein, for the first time, we investigate in vivo the efficacy of silibinin against azoxymethane-induced colon tumorigenesis in A/J mice. EXPERIMENTAL DESIGN: Five-week-old male mice were gavaged with vehicle or silibinin (250 and 750 mg/kg) for 25 weeks starting 2 weeks before initiation with azoxymethane (pretreatment regime) or for 16 weeks starting 2 weeks after the last azoxymethane injection (posttreatment regime). The mice were then sacrificed, and colon tissues were examined for tumor multiplicity and size, and molecular markers for proliferation, apoptosis, inflammation, and angiogenesis. RESULTS: Silibinin feeding showed a dose-dependent decrease in azoxymethane-induced colon tumorigenesis with stronger efficacy in pretreatment versus posttreatment regimen. Mechanistic studies in tissue samples showed that silibinin inhibits cell proliferation as evident by a decrease (P < 0.001) in proliferating cell nuclear antigen and cyclin D1, and increased Cip1/p21 levels. Silibinin also decreased (P < 0.001) the levels of inducible nitric oxide synthase, cyclooxygenase-2, and vascular endothelial growth factor, suggesting its anti-inflammatory and antiangiogenic potential in this model. Further, silibinin increased cleaved caspase-3 and poly(ADP-ribose) polymerase levels, indicating its apoptotic effect. In other studies, colonic mucosa and tumors expressed high levels of ß-catenin, insulin-like growth factor-1 receptorß, phospho Glycogen synthase kinase-3ß, and phospho protein kinase B/pAkt proteins in azoxymethane-treated mice, which were strongly lowered (P < 0.001) by silibinin treatment. Moreover, azoxymethane reduced insulin-like growth factor binding protein-3 protein level, which was enhanced by silibinin. CONCLUSIONS: Silibinin targets ß-catenin and IGF-1Rß pathways for its chemopreventive efficacy against azoxymethane-induced colon carcinogenesis in A/J mice. Overall, these results support the translational potential of silibinin in colorectal cancer chemoprevention.
Assuntos
Azoximetano , Carcinoma/induzido quimicamente , Carcinoma/prevenção & controle , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/prevenção & controle , Silimarina/farmacologia , Proteínas Angiogênicas/metabolismo , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinógenos , Carcinoma/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Citoproteção/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Silibina , Silimarina/uso terapêuticoRESUMO
Constitutive activation of phosphoinositide 3-kinase (PI3K)-Akt pathway transmits growth-regulatory signals that play a central role in promoting survival, proliferation, and angiogenesis in human prostate cancer cells. Here, we assessed the efficacy of inositol hexaphosphate (IP6) against invasive human prostate cancer PC-3 and C4-2B cells and regulation of PI3K-Akt pathway. IP6 treatment of cells suppressed proliferation, induced apoptosis along with caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, and inhibited constitutive activation of Akt and its upstream regulators PI3K, phosphoinositide-dependent kinase-1 and integrin-linked kinase-1 (ILK1). Downstream of Akt, IP6 inhibited the phosphorylation of glycogen synthase kinase-3alpha/beta at Ser(21/9) and consequently reduced cyclin D1 expression. Efficacy studies employing PC-3 tumor xenograft growth in nude mice showed that 2% (w/v) IP6 feeding in drinking water inhibits tumor growth and weight by 52% to 59% (P < 0.001). Immunohistochemical analysis of xenografts showed that IP6 significantly reduces the expression of molecules associated with cell survival/proliferation (ILK1, phosphorylated Akt, cyclin D1, and proliferating cell nuclear antigen) and angiogenesis (platelet endothelial cell adhesion molecule-1 or CD31, vascular endothelial growth factor, endothelial nitric oxide synthase, and hypoxia-inducible factor-1alpha) together with an increase in apoptotic markers (cleaved caspase-3 and PARP). These findings suggest that, by targeting the PI3K-ILK1-Akt pathway, IP6 suppresses cell survival, proliferation, and angiogenesis but induces death in prostate cancer cells, which might have translational potential in preventing and controlling the growth of advanced and aggressive prostate cancer for which conventional chemotherapy is not effective.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Ácido Fítico/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Ciclina D1/biossíntese , DNA de Neoplasias/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico Sintase Tipo III/biossíntese , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/biossíntese , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sulfur mustard (HD) is an alkylating and cytotoxic chemical warfare agent, which inflicts severe skin toxicity and an inflammatory response. Effective medical countermeasures against HD-caused skin toxicity are lacking due to limited knowledge of related mechanisms, which is mainly attributed to the requirement of more applicable and efficient animal skin toxicity models. Using a less toxic analog of HD, chloroethyl ethyl sulfide (CEES), we identified quantifiable inflammatory biomarkers of CEES-induced skin injury in dose- (0.05-2 mg) and time- (3-168 h) response experiments, and developed a CEES-induced skin toxicity SKH-1 hairless mouse model. Topical CEES treatment at high doses caused a significant dose-dependent increase in skin bi-fold thickness indicating edema. Histopathological evaluation of CEES-treated skin sections revealed increases in epidermal and dermal thickness, number of pyknotic basal keratinocytes, dermal capillaries, neutrophils, macrophages, mast cells, and desquamation of epidermis. CEES-induced dose-dependent increases in epidermal cell apoptosis and basal cell proliferation were demonstrated by the terminal deoxynucleotidyl transferase (tdt)-mediated dUTP-biotin nick end labeling and proliferative cell nuclear antigen stainings, respectively. Following an increase in the mast cells, myeloperoxidase activity in the inflamed skin peaked at 24 h after CEES exposure coinciding with neutrophil infiltration. F4/80 staining of skin integuments revealed an increase in the number of macrophages after 24 h of CEES exposure. In conclusion, these results establish CEES-induced quantifiable inflammatory biomarkers in a more applicable and efficient SKH-1 hairless mouse model, which could be valuable for agent efficacy studies to develop potential prophylactic and therapeutic interventions for HD-induced skin toxicity.
Assuntos
Biomarcadores Farmacológicos/metabolismo , Inflamação/induzido quimicamente , Gás de Mostarda/análogos & derivados , Pele/patologia , Administração Tópica , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Feminino , Inflamação/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Pelados , Gás de Mostarda/administração & dosagem , Gás de Mostarda/toxicidade , Infiltração de Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Distribuição Aleatória , Pele/citologia , Pele/lesõesRESUMO
Inositol hexaphosphate (IP6) causes G(1) arrest and increases cyclin-dependent kinase inhibitors p21/Cip1 and p27/Kip1 protein levels in human prostate cancer (PCa) DU145 cells lacking functional p53. However, whether cyclin-dependent kinase inhibitor I induction by IP6 plays any role in its antitumor efficacy is unknown. Herein, we observed that either p21 or p27 knockdown by small interfering RNA has no considerable effect on IP6-induced G(1) arrest, growth inhibition, and death in DU145 cells; however, the simultaneous knockdown of both p21 and p27 reversed the effects of IP6. To further confirm these findings both in vitro and in vivo, we generated DU145 cell variants with knockdown levels of p21 (DU-p21), p27 (DU-p27), or both (DU-p21+p27) via retroviral transduction of respective short hairpin RNAs. Knocking down p21 or p27 individually did not alter IP6-caused cell growth inhibition and G(1) arrest; however, their simultaneous ablation completely reversed the effects of IP6. In tumor xenograft studies, IP6 (2% w/v, in drinking water) caused a comparable reduction in tumor volume (40-46%) and tumor cell proliferation (26-28%) in DU-EV (control), DU-p21, and DU-p27 tumors but lost most of its effect in DU-p21+p27 tumors. IP6-caused apoptosis also occurred in a Cip/Kip-dependent manner because DU-p21+p27 cells were completely resistant to IP6-induced apoptosis both in cell culture and xenograft. Together, these results provide evidence, for the first time, of the critical role of p21 and p27 in mediating the anticancer efficacy of IP6, and suggest their redundant role in the antiproliferative and proapoptotic effects of IP6 in p53-lacking human PCa cells, both in vitro and in vivo.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácido Fítico/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27 , Regulação para Baixo/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A monofunctional analog of the chemical warfare agent sulfur mustard (HD), 2-chloroethyl ethyl sulfide (CEES), induces tissue damage similar to HD. Herein we studied the molecular mechanisms associated with CEES-induced skin inflammation and toxicity in SKH-1 hairless mice. Topical CEES exposure caused an increase in oxidative stress as observed by enhanced 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation and an increase in protein oxidation. The CEES-induced increase in the formation of 8-oxo-2-deoxyguanosine indicated DNA oxidation. CEES exposure instigated an increase in the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK1/2, JNK, and p38). After CEES exposure, a significant increase in the phosphorylation of Akt at Ser473 and Thr308 was observed as well as upregulation of its upstream effector, PDK1, in mouse skin tissue. Subsequently, CEES exposure caused activation of AP-1 family proteins and the NF-kappaB pathway, including phosphorylation and degradation of IkappaBalpha in addition to phosphorylation of the NF-kappaB essential modulator. Collectively, our results indicate that CEES induces oxidative stress and the activation of the transcription factors AP-1 and NF-kappaB via upstream signaling pathways including MAPKs and Akt in SKH-1 hairless mouse skin. These novel molecular targets could be supportive in the development of prophylactic and therapeutic interventions against HD-related skin injury.
Assuntos
Dermatite Irritante/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Gás de Mostarda/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Pele/metabolismo , Aldeídos/metabolismo , Animais , Óxidos N-Cíclicos/metabolismo , Dano ao DNA/efeitos dos fármacos , Desoxiadenosinas/metabolismo , Dermatite Irritante/genética , Feminino , Camundongos , Camundongos Pelados , Gás de Mostarda/administração & dosagem , NF-kappa B/metabolismo , Proteína Oncogênica v-akt/biossíntese , Proteína Oncogênica v-akt/genética , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Pele/efeitos dos fármacos , Pele/patologia , Fator de Transcrição AP-1/metabolismo , Ativação TranscricionalRESUMO
Herein, for the first time, we investigated in vivo efficacy and associated molecular biomarkers and mechanisms of a chemopreventive agent, silibinin, against human colorectal carcinoma (CRC) HT29 xenograft growth. Nude mice were implanted with HT29 cells and fed with vehicle (carboxymethyl cellulose or phosphatidylcholine) or 200 mg/kg/d dose of silibinin or 100 and 200 mg/kg/d doses of silybin-phytosome (5 days per week) for 32 days. Silibinin inhibited tumor growth that accounted for 48% (P = 0.002) decrease in tumor volume and 42% (P = 0.012) decrease in tumor weight at the end of the experiment without any adverse health effect. A stronger antitumor efficacy was observed with silybin-phytosome preparation. Silibinin decreased proliferation index by 40% (P < 0.001), increased apoptotic index by approximately 2-fold (P = 0.001), and reduced microvessel density by 36% (P = 0.001) in tumors. Antiproliferative and proapoptotic effects of silibinin were associated with down-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation as well as cyclin D1 expression. Antiangiogenic effect of silibinin was coupled with a strong decrease in inducible nitric oxide synthase (NOS) and NOS3, cyclooxygenase-1 (COX-1) and COX-2, and hypoxia-inducing factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). These findings suggest in vivo antitumor efficacy of silibinin against CRC involving its antiproliferative, proapoptotic, and antiangiogenic activities. The inhibition of ERK1/2 and Akt signaling may account for antiproliferative and proapoptotic effects, whereas down-regulation of NOS, COX, HIF-1 alpha, and VEGF expression could lead to antiangiogenic effect of silibinin against CRC. Overall, potential use of silibinin against human CRC could be suggested.