RESUMO
The preclinical study of the mechanism of action of anticancer small molecules is challenging due to the complexity of cancer biology and the fragmentary nature of available data. With the aim of identifying a protein subset characterizing the cellular activity of anticancer peptides, we used differential mass spectrometry to identify proteomic changes induced by two peptides, LR and [d-Gln(4)]LR, that inhibit cell growth and compared them with the changes induced by a known drug, pemetrexed, targeting the same enzyme, thymidylate synthase. The quantification of the proteome of an ovarian cancer cell model treated with LR yielded a differentially expressed protein data set with respect to untreated cells. This core set was expanded by bioinformatic data interpretation, the biologically relevant proteins were selected, and their differential expression was validated on three cis-platinum sensitive and resistant ovarian cancer cell lines. Via clustering of the protein network features, a broader view of the peptides' cellular activity was obtained. Differences from the mechanism of action of pemetrexed were inferred from different modulation of the selected proteins. The protein subset identification represents a method of general applicability to characterize the cellular activity of preclinical compounds and a tool for monitoring the cellular activity of novel drug candidates.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Peptídeos/farmacologia , Proteínas/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos/química , Western Blotting , Linhagem Celular Tumoral/efeitos dos fármacos , Biologia Computacional/métodos , Feminino , Ácido Fólico/metabolismo , Glutamatos/farmacologia , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Espectrometria de Massas/métodos , Terapia de Alvo Molecular , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Pemetrexede , Peptídeos/química , Proteínas/análise , Reprodutibilidade dos Testes , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismoRESUMO
Melanoma is one of the most common cancers, and its incidence has continued to increase over the past few decades. Chemotherapy resistance and related defects in apoptotic signaling are critical for the high mortality of melanoma. Effective drugs are lacking because apoptosis regulation in this tumor type is not well understood. The folate pathway has been considered an interesting target for anticancer therapies, and approaches targeting this pathway have recently been extended to melanoma treatment. In this study, the intracellular apoptosis signaling pathways of two melanoma cells lines (SK-MEL-2 and SK-MEL-28) were investigated after treatment with a new experimental antifolate substance (MR36) that targets thymidylate synthase. In both melanoma cell lines, apoptosis induction was triggered by a p53-independent mechanism. MR36-induced apoptosis was associated with a loss of both mitochondrial membrane potential and caspase-3 activation. Induction of cell cycle arrest by MR36 was associated with changes in the expression of key cell cycle regulators, such as p21 and cyclin D1, and the hypophosphorylation of pRb. In addition, Fas signaling was also analyzed. These findings suggest that, unlike classical antifolates, MR36 exerted an inhibitory effect on both the enzymatic function and expression of thymidylate synthase, thereby inducing apoptosis through the activation of the extrinsic and intrinsic pathways in the melanoma cell lines. MR36 showed a different mechanism of action from the known antifolates (Nolatrexed and Pemetrexed) that resulted in higher anticancer activity. Therefore, MR36 should be included as a potential new therapeutic treatment in melanoma research.
Assuntos
Ciclo Celular/efeitos dos fármacos , Cumarínicos/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Melanoma/patologia , Ácido Poliglutâmico/metabolismo , Timidilato Sintase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Cumarínicos/química , Cumarínicos/uso terapêutico , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/uso terapêutico , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma/genética , Modelos Biológicos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Timidilato Sintase/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Information on the cellular internalization and stability of the ovarian cancer cell growth inhibitor peptide, LSCQLYQR (LR), is vital for lead optimization. Ad-hoc-synthesized LR/fluorescent-probe conjugates were used to monitor the internalization of the peptide. Mass spectrometry was used to identify adducts resulting from the thiol reactivity of the cysteine residue in LR. A mechanistic model is proposed to explain the observed change in intracellular peptide amount over time. Structural modifications can be foreseen to improve the peptide stability.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Timidilato Sintase/antagonistas & inibidores , Feminino , Fluorescência , Humanos , Microscopia Confocal , Estrutura Molecular , Neoplasias Ovarianas/enzimologia , Espectrometria de Massas em Tandem , Células Tumorais CultivadasRESUMO
Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The d-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.