RESUMEN
BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are among the most abundant malignancies worldwide. Patients with recurrent/metastatic disease undergo combination chemotherapy containing cetuximab, the monoclonal antibody used against the epidermal growth factor receptor (EGFR). Cetuximab augments the effect of chemotherapy; however, a significant number of patients show therapy resistance. The mechanism of resistance is yet to be unveiled, although extracellular alterations of the receptor have been reported, and their role in cetuximab failure has been proposed. AIMS: Here, we investigate possible effects of the multi-exon deletion variant (EGFRvIII), and the single nucleotide polymorphism EGFR R521K on cetuximab efficacy. RESULTS: Our results show that in HNSCC patients, the EGFRvIII allele frequency is under 1%; therefore, it cannot lead to common resistance. EGFR R521K, present in 42% of the patients, is investigated in vitro in four HNSCC cell lines (two wild-type and two heterozygous for EGFR R521K). While no direct effect is found to be related to the EGFR status, cells harboring R521K show a reduced sensitivity in ADCC experiments and in vivo xenograft experiments. However, this preclinical difference is not reflected in the progression-free or overall survival of HNSCC patients. Furthermore, NK cell and macrophage presence in tumors is not related to EGFR R521K. DISCUSSION: Our results suggest that EGFR R521K, unlike reported previously, is unable to cause cetuximab resistance in HNSCC patients; therefore, its screening before therapy selection is not justifiable.
RESUMEN
Background: Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells. Methods: After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for in vitro proliferation and apoptosis tests, and in vivo animal models using different therapeutic agents. Results: Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter in vitro cell proliferation or apoptosis. In vivo cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Conclusion: Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Mutación , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Cetuximab/administración & dosificación , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib/administración & dosificación , Femenino , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Indoles/administración & dosificación , Ratones , Ratones SCID , Piperazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Sulfonamidas/administración & dosificación , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Ácido Zoledrónico/administración & dosificaciónRESUMEN
Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Transporte Biológico/genética , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Dominio AAA/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Adenosina Trifosfato/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Transporte Biológico Activo/genética , Dominio Catalítico/genética , Ácido Glutámico/genética , Humanos , Hidrólisis , Metionina/genética , Simulación de Dinámica Molecular , Mutación/genética , Nucleótidos/genética , Unión Proteica/genética , Dominios Proteicos/genéticaRESUMEN
Clinical evidence shows that following initial response to treatment, drug-resistant cancer cells frequently evolve and, eventually, most tumors become resistant to all available therapies. We compiled a focused library consisting of >500 commercially available or newly synthetized 8-hydroxyquinoline (8OHQ) derivatives whose toxicity is paradoxically increased rather than decreased by the activity of P-glycoprotein (Pgp), a transporter conferring multidrug resistance (MDR). Here, we deciphered the mechanism of action of NSC297366 that shows exceptionally strong Pgp-potentiated toxicity. Treatment of cells with NSC297366 resulted in changes associated with the activity of potent anticancer iron chelators. Strikingly, iron depletion was more pronounced in MDR cells due to the Pgp-mediated efflux of NSC297366-iron complexes. Our results indicate that iron homeostasis can be targeted by MDR-selective compounds for the selective elimination of multidrug resistant cancer cells, setting the stage for a therapeutic approach to fight transporter-mediated drug resistance. SIGNIFICANCE: Modulation of the MDR phenotype has the potential to increase the efficacy of anticancer therapies. These findings show that the MDR transporter is a "double-edged sword" that can be turned against resistant cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Quelantes del Hierro/farmacología , Hierro/metabolismo , Neoplasias/tratamiento farmacológico , Oxiquinolina/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Quelantes del Hierro/uso terapéutico , Neoplasias/patología , Oxiquinolina/análogos & derivados , Oxiquinolina/uso terapéuticoRESUMEN
ABC multidrug transporters are key players in cancer multidrug resistance and in general xenobiotic elimination, thus their functional assays provide important tools for research and diagnostic applications. In this study we have examined the potential interactions of three key human ABC multidrug transporters with PhenGreen diacetate (PGD), a cell permeable fluorescent metal ion indicator. The non-fluorescent, hydrophobic PGD rapidly enters the cells and, after cleavage by cellular esterases, in the absence of quenching metal ions, PhenGreen (PG) becomes highly fluorescent. We found that in cells expressing functional ABCG2, ABCB1, or ABCC1 transporters, cellular PG fluorescence is strongly reduced. This fluorescence signal in the presence of specific transporter inhibitors is increased to the fluorescence levels in the control cells. Thus the PG accumulation assay is a new, unique tool for the parallel determination of the function of the ABCG2, ABCB1, and ABCC1 multidrug transporters. Since PG has very low cellular toxicity, the PG accumulation assay also allows the selection, separation and culturing of selected cell populations expressing either of these transporters.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Citometría de Flujo , HumanosRESUMEN
Despite significant progress, resistance to chemotherapy is still the main reason why cancer remains a deadly disease. An attractive strategy is to target the collateral sensitivity of otherwise multidrug resistant (MDR) cancer. In this study, our aim was to catalog various compounds that were reported to elicit increased toxicity in P-glycoprotein (Pgp)-overexpressing MDR cells. We show that the activity of most of the serendipitously identified compounds reported to target MDR cells is in fact cell-line specific, and is not influenced significantly by the function of Pgp. In contrast, novel 8-hydroxyquinoline derivatives that we identify in the National Cancer Institute (NCI) drug repository possess a robust Pgp-dependent toxic activity across diverse cell lines. Pgp expression associated with the resistance of the doxorubicin-resistant Brca1-/-;p53-/- spontaneous mouse mammary carcinoma cells could be eliminated by a single treatment with NSC57969, suggesting that MDR-selective compounds can effectively revert the MDR phenotype of cells expressing Pgp at clinically relevant levels. The discovery of new MDR-selective compounds shows the potential of this emerging technology and highlights the 8-hydroxyquinoline scaffold as a promising starting point for the development of compounds targeting the Achilles heel of drug-resistant cancer. Mol Cancer Ther; 16(1); 45-56. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Bases de Datos Farmacéuticas , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Expresión Génica , Humanos , Neoplasias Mamarias Experimentales , Ratones , Ratones Noqueados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico Activo , Dominio Catalítico , Animales , Células Cultivadas , Perros , Ratones , Unión Proteica , Conformación ProteicaRESUMEN
Three novel pyrimidinylhydrazones substituted at either the aromatic moiety or at the imine carbon atom were synthesized and characterized by standard analytical methods. All compounds were found to be toxic in the micro- to submicromolar range against a diverse panel of cancer cell lines including multidrug resistant (MDR) derivatives expressing P-glycoprotein (Pgp). UV-visible spectrophotometry experiments demonstrated that the most active compound (3) forms highly stable complexes with iron(III) and copper(II) in a wide pH range with a stronger preference towards iron(III). The redox activity of the iron and copper complexes of ligand 3 was investigated using cyclic voltammetry and was tested with cellular reductants. The impact of reactive oxygen species (ROS) on the mechanism of toxicity was assessed using the ROS-sensitive cell permeable dye 2',7'-dichlorofluorescin diacetate (DCFDA). Our results demonstrate that the studied pyrimidinylhydrazones form redox-active iron and copper complexes that are capable of producing intracellular ROS, which might lead to cellular damage and cell death in cancer cells regardless of their resistance status.
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Antineoplásicos/síntesis química , Hidrazonas/síntesis química , Hidrazonas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Cobre/química , Perros , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Fluoresceínas , Hidrazonas/química , Hidrazonas/metabolismo , Hierro/química , Ligandos , Células de Riñón Canino Madin Darby , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , EspectrofotometríaRESUMEN
There is a great need for the development of novel chemotherapeutic agents that overcome the emergence of multidrug resistance (MDR) in cancer. We catalogued the National Cancer Institute's DTP drug repository in search of compounds showing increased toxicity in MDR cells. By comparing the sensitivity of parental cell lines with MDR derivatives, we identified 22 compounds possessing MDR-selective activity. Analysis of structural congeners led to the identification of 15 additional drugs showing increased toxicity in Pgp-expressing cells. Analysis of MDR-selective compounds led to the formulation of structure activity relationships and pharmacophore models. This data mining coupled with experimental data points to a possible mechanism of action linked to metal chelation. Taken together, the discovery of the MDR-selective compound set shows the robustness of the developing field of MDR-targeting therapy as a new strategy for resolving Pgp-mediated MDR.
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Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/aislamiento & purificación , Biología Computacional , Humanos , Células Tumorales CultivadasRESUMEN
Targeted drugs inhibit specific pathways that contribute to the malignant phenotype of cancer cells. The initial success of molecularly targeted therapies raised hope that newly developed agents would evade the general mechanisms of resistance that have reduced the efficacy of traditional anticancer drugs. In recent years, ATP-binding cassette (ABC) transporters related to multidrug resistance (MDR), such as P-glycoprotein (P-gp; ABCB1/MDR1) and ABCG2 (breast cancer resistance protein/mitoxantrone resistance protein) have emerged as key factors that regulate the intracellular concentrations of many small-molecule therapeutic inhibitors. Drug transporters may be overexpressed in cancer cells, reducing intracellular drug concentrations, and may allow the evolution of point mutations that confer stronger drug resistance. It is proposed that P-gp, a universally accepted biomarker of drug resistance, should also be considered as a molecular target in multidrug-resistant cancer. By exploiting the paradoxical hypersensitivity of multidrug-resistant cells, MDR1-inverse compounds can selectively eliminate cancer cells that overexpress P-gp. Successful targeting of multidrug-resistant cells would reduce the tumor burden and would also enable the elimination of ABC transporter-overexpressing cancer stem cells that are responsible for the replenishment of tumors.