RESUMO
Huachansu (HCS), a hot water extract of the skin glands of Bufo gargarizans (B. melanostictus), has been used extensively in the treatment of various solid tumors in Asia, particularly in China. However, its effect on the growth of malignancies of hematopoietic origin, particularly lymphomas, is limited. Here we investigated the antiproliferative effect and molecular mechanisms of HCS using non-Hodgkin's lymphoma (NHL) Raji, Ramos, and Namalwa cells and the mantle cell lymphoma cells SP53. HCS inhibited proliferation in these cell lines with an IC50 ranging from 3.1 to 25 µl/ml. At a concentration of 25 µl/ml, HCS triggered a sub-G1 arrest in Ramos cells and induced early to late apoptotic cell death. Cleaved caspase-3 was formed in a concentration-dependent manner in Ramos cells following treatment with HCS for 24 h. Intriguingly, when the Ramos cells were treated with the caspase inhibitor ZDEVD, the apoptotic activity of HCS was partially blocked. Furthermore, HCS also blocked the expression of survivin and pRB proteins in a concentration-dependent manner in Ramos cells. Mechanistically, HCS downregulated both the MAPK gene and proteins in Ramos cells. Collectively, our data suggest that HCS is effective in inducing cell death and apoptosis, in part, by activating caspase-3 activity and suppressing MAP kinase in NHL cells.
Assuntos
Venenos de Anfíbios/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Caspase 3/metabolismo , Linfoma não Hodgkin/enzimologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Linfoma não Hodgkin/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/genética , Inibidores de Proteínas Quinases/farmacologia , SurvivinaRESUMO
The ubiquitin proteasome-proteolytic pathway has emerged as one of the most significant pathways in modulating protein homeostasis under both normal and disease states. The use of proteasome inhibitors (PI) has played a pivotal role in understanding protein turn over. The main objective of this work was to develop a comprehensive, fast, and reliable, yet simple in vitro assay that would allow for the identification and characterization of a wide range of PIs. The assays consist of a 96-well plate high throughput (HTP) method to assess proteasome activity in Hs578T breast cancer cell extracts, purified 20S proteasome, using a fluorogenic substrate, Suc-leu-leu-val-tyr-7-AMC, specific to the chymotrypsin-like enzymatic activity of the proteasome. We showed that the chymotrypsin-like activity of the proteasome was inhibited in the two in vitro systems, albeit to different degrees. The assay system also includes two cell-based assays consisting of a vector expressing a fusion protein of green fluorescent protein (gfp) and Mouse Ornithine Decarboxylase (MODC) in Zs578T (parental Hs578T carrying the vector that expresses the fusion protein). In the cell-based assay analyses (qualitatively by microscopy and quantitatively by flow cytometry), treatment of Zs578T with PIs prevented the degradation of MODC, accumulated gfp, indicative of increased proteasome inhibition. Because no single assay represents a definitive proof of proteasome inhibitory activity, combined, these assays should serve as a comprehensive benchmark for the identification and partial characterization of novel inhibitors. In summary, the four-step assay protocol can easily be adapted into a high throughput format to rapidly screen unknown inhibitors.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Bioensaio , Ácidos Borônicos/farmacologia , Bortezomib , Extratos Celulares , Células Cultivadas , Quimotripsina/metabolismo , Concentração Inibidora 50 , Leupeptinas/farmacologia , Camundongos , Modelos Biológicos , Oligopeptídeos/farmacologia , Ornitina Descarboxilase/metabolismo , Inibidores de Proteases/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
Isoprenoid inhibitors are being evaluated as agents for the treatment of cancer. Their antitumor activity is attributed to inhibition of post-translational modification of Ras, which is crucial for its translocation and attachment to the plasma membrane, and ultimate involvement in signal transduction. However, whether blocking of Ras is solely responsible for the observed antitumor activity is unresolved. In this report, we propose an alternate mechanism. Using breast tumor models, we show that agents possessing a lactone moiety, including statins (such as lovastatin) and the isoprenoid inhibitors (such as FTI-277 and GGTI-298), mediate their cell cycle inhibitory activities by blocking the chymotrypsin activity of the proteasome in vitro. This results in the accumulation of cyclin-dependent kinase inhibitors p21 and p27 with subsequent G(1) arrest. Cells devoid of p21 were refractory to the growth-inhibitory activity of lovastatin, FTI-277, and GGTI-298. However, in these p21 null cells, isoprenylation of key substrates of farnesyl transferase (such as Ras) and of geranylgeranyl transferase (such as RAP-1) were inhibited by FTI-277 and GGTI-298, respectively, suggesting that although both these isoprenoid inhibitors reached and inhibited their intended targets, inhibition of the isoprenylation of Ras and RAP-1A are not sufficient to mediate G(1) arrest. We also show that the cell cycle effects can be attributed to the functional lactone moiety of the aforementioned agents. Collectively, our data suggest that FTI and GGTI and other agents containing an active lactone moiety mediate G(1) arrest via inhibition of the proteasome and up-regulation of p21, independent of the inhibition of isoprenylation of Ras or RAP-1.