RESUMO
In Ayurveda, Withania somnifera is commonly known as Ashwagandha, its roots are specifically used in medicinal and clinical applications. It possesses numerous therapeutic actions which include anti-inflammatory, sedative, hypnotic and narcotic. Extracts from this plant have been reported for its anticancer properties. In this study we evaluated for the first time, the cytotoxic effect of Withania root extract on human malignant melanoma A375 cells. The crude extract of Withania was tested for cytotoxicity against A375 cells by MTT assay. Cell morphology of treated A375 cells was visualized through phase contrast as well as fluorescence microscopy. Agarose gel electrophoresis was used to check DNA fragmentation of the crude extract treated cells. Crude extract of Withania root has the potency to reduce viable cell count in dose as well as time dependent manner. Morphological change of the A375 cells was also observed in treated groups in comparison to untreated or vehicle treated control. Apoptotic body and nuclear blebbing were observed in DAPI stained treated cells under fluorescence microscope. A ladder of fragmented DNA was noticed in treated cells. Thus it might be said that the crude water extract of Withania somnifera has potent cytotoxic effect on human malignant melanoma A375 cells.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Melanoma/tratamento farmacológico , Extratos Vegetais/farmacologia , Raízes de Plantas , Withania , Linhagem Celular Tumoral , Humanos , Ayurveda , Fitoterapia , Extratos Vegetais/uso terapêuticoRESUMO
Tea polyphenols have potent biological activities against human cancer cells. A major causative factor in malignancies is disregulation of cell-cycle kinetics. In this study, we observed that black tea polyphenols, theaflavins (TF) and thearubigins (TR) induced cell-cycle arrest at the G(0) /G(1) phase in human leukemic U937 and K562 cells. Our objective was to understand the underlying molecular mechanism of cell-cycle inhibition by TF and TR. During elucidation, we observed that both TF and TR treatment augmented expression of p19, p21 and p27, while ablating cylcin-dependent kinase (CDK)2, CDK4, CDK6 and cyclin D1 levels. Our experimental results further determined that Akt signaling suppression by TF and TR played a major role in this process. Moreover, suppression of glycogen synthase kinase-3ß, ß-catenin and amplification of forkhead transcription factor 1 (FOXO1) expression were associated with regulation of certain key components of the cell-cycle machinery. In addition, depletion of heat shock protein (Hsp) 90 by TF and TR also had a pivotal role in cell-cycle arrest. More specifically, inhibition of Akt signaling by TF and TR correlated with the depletion of its downstream targets like Wnt/ß-catenin signaling, cyclin D1 and increase of FOXO1, p27 levels. Inhibition of upstream Hsp90 by TF and TR consequently attenuated Akt signaling and reduced the level of CDK2. These results suggest possible mechanisms for the chemopreventive effect of TF and TR on human leukemic cells. To our knowledge, this is the first report of such a detailed molecular mechanism for TF and the less-investigated polyphenol TR-mediated cell-cycle inhibition in human leukemic U937 and K562 cells.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Polifenóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Biflavonoides/farmacologia , Camellia sinensis/química , Catequina/análogos & derivados , Catequina/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/fisiologia , Fase G1/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Células K562 , Fenóis/farmacologia , Células U937 , Proteínas Wnt/fisiologia , beta Catenina/fisiologiaRESUMO
Theaflavins (TF) and thearubigins (TR) are the major polyphenols of black tea. Our previous study revealed that TF- and TR-induced apoptosis of human malignant melanoma cells (A375) is executed via a mitochondria-mediated pathway. In our present study we observed the role of the three most important MAPK (ERK, JNK, and p38) in TF- and TR-induced apoptosis. TF and TR treatment of A375 cells led to sustained activation of JNK and p38 MAPK but not ERK, suggesting that JNK and p38 are the effector molecules in this polyphenol-induced cell death. This idea was further supported by subsequent studies in which JNK and p38 activation was inhibited by specific inhibitors. Significant inhibition was found in TF- and TR-treated A375 cell death pretreated with JNK- or p38-specific inhibitors only. Further, we have found that TF and TR treatment induces a time-dependent increase in intracellular reactive oxygen species generation in A375 cells. Interestingly, treatment with the antioxidant N-acetyl cystein inhibits TF- and TR-induced JNK and p38 activation as well as induction of cell death in A375 cells. We also provide evidence demonstrating the critical role of apoptosis signal-regulating kinase 1 in TF- and TR-induced apoptosis in A375 cells. Taken together our results strongly suggest that TF and TR induce apoptotic death of A375 cells through apoptosis signal-regulating kinase 1, MAPK kinase, and the JNK-p38 cascade, which is triggered by N-acetyl cystein intracellular oxidative stress.
Assuntos
Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Biflavonoides/farmacologia , Catequina/análogos & derivados , Ativação Enzimática/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Fenóis/farmacologia , Catequina/farmacologia , Linhagem Celular Tumoral , Flavonoides/farmacologia , Humanos , MAP Quinase Quinase 4/efeitos dos fármacos , MAP Quinase Quinase 4/metabolismo , Melanoma/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxirredução , Polifenóis , Transdução de Sinais/efeitos dos fármacos , Chá , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Theaflavins (TF) and thearubigins (TR) are the most exclusive polyphenols of black tea. Even though few previous reports showed the anticancer effects of TF through apoptosis, the potential effect of TR has not been appraised. This study investigated the induction of apoptosis in human skin cancer cells after treatment of TF and TR. We report that both TF and TR could exert inhibition of A431 (human epidermoid carcinoma) and A375 (human malignant melanoma) cell proliferation without adversely affecting normal human epidermal keratinocyte cells. Growth inhibition of A375 cells occurred through apoptosis, as evident from cell cycle arrest at G(0)/G(1) phase, increase in early apoptotic cells, externalization of phosphatidylserine and DNA fragmentation. In our pursuit to dissect the molecular mechanism of TF- and TR-induced apoptosis in A375 cells, we investigated whether cell death is being mediated by mitochondria. In our system, Bax translocation to mitochondria persuaded depolarization of mitochondrial membrane potential, cytochrome c release in cytosol and induced activation of caspase-9, caspase-3 and poly (ADP-ribose) polymerase cleavage. Our intricate investigations on apoptosis also explained that TF and TR augmented Bax:Bcl2 ratio, up-regulated the expression of p53 as well as p21 and inhibited phosphorylation of the cell survival protein Akt. Furthermore, TF and TR elicited intracellular reactive oxygen species generation in A375 cells. These observations raise speculations that TF as well as TR might exert chemopreventive effect through cell cycle arrest and induction of apoptogenic signals via mitochondrial death cascade in human skin cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Flavonoides/farmacologia , Melanoma/patologia , Mitocôndrias/fisiologia , Fenóis/farmacologia , Neoplasias Cutâneas/patologia , Chá/química , Proteína X Associada a bcl-2/metabolismo , Carcinoma de Células Escamosas/metabolismo , Humanos , Melanoma/metabolismo , Fosfatidilserinas/metabolismo , Polifenóis , Transporte Proteico , Neoplasias Cutâneas/metabolismoRESUMO
Black tea accounts for nearly 80% of total World tea production. It contains dimeric flavanols and polymeric polyphenols known as theaflavins (TF) and thearubigins (TR). TR is exclusively present in black tea. On the basis of our previous potent antimutagenic and anticlastogenic effects of TF and TR in vitro in bacterial system and in vivo in mouse bone marrow cells, we have decided to extend our study in human cells in vitro. This study investigated the anticlastogenic effects of black tea polyphenols TF and TR as measured by chromosomal aberrations (CA) and micronuclei formation (MN) against two known mutagens/carcinogens i.e. benzo[a]pyerne (B[a]P) and aflatoxin B1(AFB1) with S9 activation. A significant decrease in both CA and MN were observed in the human lymphocyte cultures treated with either TF or TR pretreated with either B[a]P or AFB1 (250, 500, 1000 microg/ml) when compared with B[a]P or AFB1 treated cultures alone. TF shows more protective effects than TR in this in vitro system. These results indicate that both TF and TR have significant anticlastogenic effects in vitro in human lymphocytes.
Assuntos
Antimutagênicos/farmacologia , Biflavonoides/farmacologia , Catequina/análogos & derivados , Linfócitos/efeitos dos fármacos , Fenóis/farmacologia , Chá , Animais , Catequina/farmacologia , Células Cultivadas , Aberrações Cromossômicas , Masculino , Testes para Micronúcleos , Polifenóis , RatosRESUMO
This study investigated the antimutagenic and anticlastogenic effects of black tea polyphenols, theaflavins (TF) and thearubigins (TR) in Salmonella assay in vitro and in vivo in bone marrow cells of mice as measured by chromosomal aberrations (CA) and sister chromatid exchange (SCE) against a known carcinogen, benzo[a]pyrene (B[a]P). A significant decrease in mutagenicity in Salmonella assay and both CA and SCE were observed in all the different concentrations of TF and TR plus B[a]P treated series when compared with B[a]P treated group alone. These results indicate that both TF and TR have significant antimutagenic and anticlastogenic effects.