RESUMEN
Drug resistance is a major challenge for all oncological treatments that involve the use of cytotoxic agents. Recent therapeutic alternatives cannot circumvent the ability of cancer cells to adapt or alter the natural selection of resistant cells, so the problem persists. In neuroblastoma, recurrence can occur in up to 50% of high-risk patients. Therefore, the identification of novel therapeutic targets capable of modulating survival or death following classical antitumor interventions is crucial to address this problem. In this study, we investigated the role of the P2X7 receptor in chemoresistance. Here, we elucidated the contributions of P2X7 receptor A and B isoforms to neuroblastoma chemoresistance, demonstrating that the B isoform favors resistance through a combination of mechanisms involving drug efflux via MRP-type transporters, resistance to retinoids, retaining cells in a stem-like phenotype, suppression of autophagy, and EMT induction, while the A isoform has opposite and complementary roles.
RESUMEN
Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the oncogene BCR-ABL1, which encodes an oncoprotein with tyrosine kinase activity. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor, performs exceptionally well with minimal toxicity in CML chemotherapy. According to clinical trials, however, 20-30% of CML patients develop resistance to imatinib. Although the best studied resistance mechanisms are BCR-ABL1-dependent, P-glycoprotein (P-gp, a drug efflux transporter) may also contribute significantly. This study aimed to establish an imatinib-resistant human CML cell line, evaluate the role of P-gp in drug resistance, and assess the capacity of ketoconazole to reverse resistance by inhibiting P-gp. The following parameters were determined in both cell lines: cell viability (as the IC50) after exposure to imatinib and imatinib + ketoconazole, P-gp expression (by Western blot and immunofluorescence), the intracellular accumulation of a P-gp substrate (doxorubicin) by flow cytometry, and the percentage of apoptosis (by the Annexin method). In the highly resistant CML cell line obtained, P-gp was overexpressed, and the level of intracellular doxorubicin was low, representing high P-gp activity. Imatinib plus a non-toxic concentration of ketoconazole (10 µM) overcame drug resistance, inhibited P-gp overexpression and its efflux function, increased the intracellular accumulation of doxorubicin, and favored greater apoptosis of CML cells. P-gp contributes substantially to imatinib resistance in CML cells. Ketoconazole reversed CML cell resistance to imatinib by targeting P-gp-related pathways. The repurposing of ketoconazole for CML treatment will likely help patients resistant to imatinib.
Asunto(s)
Antineoplásicos , Resistencia a Antineoplásicos , Mesilato de Imatinib , Cetoconazol , Leucemia Mielógena Crónica BCR-ABL Positiva , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib/efectos adversos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Células K562 , Cetoconazol/farmacología , Cetoconazol/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Eliminación de Gen , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xenobióticos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Antifúngicos/metabolismo , Antifúngicos/farmacología , Transporte Biológico , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Proteínas de Transporte de Monosacáridos/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Xenobióticos/farmacologíaRESUMEN
Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), a disease that affects millions of people in the world and that is associated with several human diseases. The bacillus is highly adapted to infect and survive inside the host, mainly because of its cellular envelope plasticity, which can be modulated to adapt to an unfriendly host environment; to manipulate the host immune response; and to resist therapeutic treatment, increasing in this way the drug resistance of TB. The superfamily of ATP-Binding Cassette (ABC) transporters are integral membrane proteins that include both importers and exporters. Both types share a similar structural organization, yet only importers have a periplasmic substrate-binding domain, which is essential for substrate uptake and transport. ABC transporter-type importers play an important role in the bacillus physiology through the transport of several substrates that will interfere with nutrition, pathogenesis, and virulence. Equally relevant, exporters have been involved in cell detoxification, nutrient recycling, and antibiotics and drug efflux, largely affecting the survival and development of multiple drug-resistant strains. Here, we review known ABC transporters from M. tuberculosis, with particular focus on the diversity of their structural features and relevance in infection and drug resistance.
RESUMEN
Chromobacterium violaceum is an environmental Gram-negative bacterium that causes infections in humans. Treatment of C. violaceum infections is difficult and little is known about the mechanisms of antibiotic resistance in this bacterium. In this work, we identified mutations in the MarR family transcription factor EmrR and in the protein GyrA as key determinants of quinolone resistance in C. violaceum, and we defined EmrR as a repressor of the MFS-type efflux pump EmrCAB. Null deletion of emrR caused increased resistance to nalidixic acid, but not to other quinolones or antibiotics of different classes. Moreover, the ΔemrR mutant showed decreased production of the purple pigment violacein. Importantly, we isolated C. violaceum spontaneous nalidixic acid-resistant mutants with a point mutation in the DNA-binding domain of EmrR (R92H), with antibiotic resistance profile similar to that of the ΔemrR mutant. Other spontaneous mutants with high MIC values for nalidixic acid and increased resistance to fluoroquinolones presented point mutations in the gene gyrA. Using DNA microarray, Northern blot and EMSA assays, we demonstrated that EmrR represses directly a few dozen genes, including the emrCAB operon and other genes related to transport, oxidative stress and virulence. This EmrR repression on emrCAB was relieved by salicylate. Although mutation of the C. violaceum emrCAB operon had no effect in antibiotic susceptibility or violacein production, deletion of emrCAB in an emrR mutant background restored antibiotic susceptibility and violacein production in the ΔemrR mutant. Using a biosensor reporter strain, we demonstrated that the lack of pigment production in ΔemrR correlates with the accumulation of quorum-sensing molecules in the cell supernatant of this mutant strain. Therefore, our data revealed that overexpression of the efflux pump EmrCAB via mutation and/or derepression of EmrR confers quinolone resistance and alters quorum-sensing signaling in C. violaceum, and that point mutation in emrR can contribute to emergence of antibiotic resistance in bacteria.
RESUMEN
Metabolic alterations in the tumor microenvironment have a complex effect on cancer progression. Extracellular acidity is a consequence of metabolic switch in cancer and results in cell phenotypes with higher resistance to chemotherapeutics. However, mechanisms underlying the relationship between the extracellular acidity and chemoresistance are not clearly understood. This systematic review was carried out by searching the databases PubMed and EMBASE using the keywords "cancer" and "acidosis" or "acidic" and "chemoresistance" or "drug resistance." In vitro and in vivo studies that evaluated the effects of acidification of the tumor microenvironment on chemotherapeutic treatments were included. Literature reviews, letters to the editor, and articles that were not published in English were excluded. The search resulted in a total of 352 articles. After discarding 75 duplicate references, 277 articles were analyzed by sequentially reading through their titles, abstracts, and finally full-text. A total of 14 articles was selected. Acidification of the tumor microenvironment can trigger resistance through different mechanisms, such as increase in drug efflux transporters, inhibition of proton pumps, induction of the unfolded protein response (UPR), and cellular autophagy.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Microambiente Tumoral/fisiología , Animales , Autofagia/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Transporte de Membrana/metabolismo , Microambiente Tumoral/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defense by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. venezuelae VS678 (ΔjadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not show a significant decrease in production in comparison to the wildtype strain, as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that JadL is not involved in jadomycin efflux. Potentially, other transporters within S. venezuelae ISP5230 may adopt this role upon inactivation of JadL to export jadomycins.
RESUMEN
A series of chalcones substituted by a quinoxaline unit at the B-ring were synthesized and tested as inhibitors of breast cancer resistance protein-mediated mitoxantrone efflux. These compounds appeared more efficient than analogs containing other B-ring substituents such as 2-naphthyl or 3,4-methylenedioxyphenyl while an intermediate inhibitory activity was obtained with a 1-naphthyl group. In all cases, two or three methoxy groups had to be present on the phenyl A-ring to produce a maximal inhibition. Molecular modeling indicated both electrostatic and steric positive contributions. A higher potency was observed when the 2-naphthyl or 3,4-methylenedioxyphenyl group was shifted to the A-ring and methoxy substituents were shifted to the phenyl B-ring, indicating preferences among polyspecificity of inhibition.