RESUMO
BACKGROUND: Despite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin. METHODS: Cytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting. RESULTS: Initial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells. CONCLUSION: In conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Metformina/farmacologia , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Células 3T3 BALB , Carcinógenos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Meios de Cultura/metabolismo , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Glucose/metabolismo , Humanos , Metformina/uso terapêutico , Metilcolantreno/toxicidade , Camundongos , Mitomicina/farmacologia , Mitomicina/uso terapêuticoRESUMO
The BALB/c cell transformation assay (BALB-CTA) considers inter- and intra-tumor heterogeneities and affords the possibility of a direct comparison between untransformed and malignant cells. In the present study, we established monoclonal cell lines that originate from the BALB-CTA and mimic heterogeneous tumor cell populations, in order to investigate phenotype-specific effects of the anti-diabetic drug metformin and the short-chain fatty acid butyrate. Growth inhibitory effects were measured with a ViCell XR cell counter. The BALB/c tumor therapy model (BALB-TTM) was performed, and the extracellular glucose level was measured in the medium supernatant. Using a Seahorse Analyzer, the metabolic phenotypes of four selected clones were characterized, and effects on energy metabolism were investigated. Anti-carcinogenic effects and reduced glucose uptake after butyrate application were observed in the BALB-TTM. Metabolic characterization of the cell clones revealed three different phenotypes. Surprisingly, treatment with metformin or butyrate induced opposite metabolic shifts with similar patterns in all cell clones tested. In conclusion, the BALB-TTM is a relevant model for mechanistic cancer research, and the generation of monoclonal cell lines offers a novel possibility to investigate specific drug effects in a heterogeneous tumor cell population. The results indicate that induced alterations in energy metabolism seem to be independent of the original metabolic phenotype.
Assuntos
Butiratos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Metformina/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Meios de Cultura/química , Humanos , Camundongos , Modelos Biológicos , FenótipoRESUMO
INTRODUCTION: Ewing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells. METHODS: Effects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis. RESULTS: AUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect. CONCLUSION: Our study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.
RESUMO
Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post-translational modifications (PTMs). While the relevance of p53 C-terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB-binding protein/p300 and the presence of C-terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.