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1.
Anal Chem ; 92(18): 12407-12414, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32786432

RESUMO

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation (r2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/µg of total protein) between immuno-MRM and immuno-MALDI.


Assuntos
Neoplasias do Colo/diagnóstico , Ensaio de Imunoadsorção Enzimática , Peptídeos/química , Proteínas/análise , Calibragem , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Marcação por Isótopo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas
2.
Breast Cancer Res Treat ; 172(1): 23-32, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30039287

RESUMO

PURPOSE: PARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3, a critical player for efficient mitotic progression, is required for the stabilization of the mitotic spindle by regulation of the mitotic components, NuMA and Tankyrase 1. METHODS: The sensitization effect of vinorelbine on PARP3 inhibition-induced cytotoxicity was assessed by the SRB assay. The contribution of programed cell death and cell cycle arrest to the sensitization effect were determined by assessing changes in Annexin V, a marker of apoptosis. Alterations in cell cycle progression were assessed by cell cycle analysis. We used immunofluorescence to assess the effect of vinorelbine and/or PARP3 inhibitors on tubulin and microtubule depolarization. The PARP3 chemiluminescent assay kit was used for PARP3 activity. RESULTS: PARP3 inhibitors sensitize breast cancer cells to vinorelbine, a vinca alkaloid used in the treatment of metastatic breast cancer. Olaparib which was originally described as a PARP1 and 2 inhibitor has recently been shown to be a potent PARP3 inhibitor while ME0328 is a more selective PARP3 inhibitor. The combination of vinorelbine with nontoxic concentrations of ME0328 or olaparib reduces vinorelbine resistance by 10 and 17 fold, respectively, potentiating vinorelbine-induced arrest at the G2/M boundary. In addition, PARP3 inhibition potentiates vinorelbine interaction with tubulin. Furthermore, olaparib or ME0328 potentiates vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis. CONCLUSION: Our results indicated this approach with PARP3 inhibitors and vinorelbine is unique and promising for breast cancer patients with metastases. This combination could significantly increase the survival of breast cancer patients with metastases.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Vinorelbina/farmacologia , Antígenos Nucleares/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Reparo do DNA/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Proteínas Associadas à Matriz Nuclear/genética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Quinazolinonas/farmacologia , Fuso Acromático/genética , Tanquirases/genética
3.
Genes Chromosomes Cancer ; 52(5): 480-94, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23341105

RESUMO

PALB2/FANCN is a BRCA1- and BRCA2-interacting Fanconi Anemia (FA) protein crucial for key BRCA2 genome caretaker functions. Heterozygous germline mutations in PALB2 predispose to breast cancer and biallelic mutations cause FA. FA proteins play a critical role in the telomere maintenance pathway, with telomeric shortening observed in FA cells. Less is known about telomere maintenance in the heterozygous state. Here, we investigate the roles of PALB2 heterozygous mutations in genomic instability, an important carcinogenesis precursor. Patient-derived lymphoblastoid (LCL) and fibroblast (FCL) cell lines with monoallelic truncating PALB2 mutations were investigated using a combination of molecular imaging techniques including centromeric FISH, telomeric Q-FISH and spectral karyotyping (SKY). Mitomycin C and Cisplatin sensitivity was assayed via cellular metabolism of WST-1. The PALB2 c.229delT FCL showed increases in telomere counts associated with increased mean intensity compared with two wild-type FCLs generated from first-degree relatives (P =1.04E-10 and P =9.68E-15) and it showed evidence of chromosomal rearrangements. Significant differences in centromere distribution were observed in one of three PALB2 heterozygous FCLs analyzed when compared with PALB2 wild-type, BRCA1 and BRCA2 heterozygous FCLs. No significant consistently increased sensitivity to Mitomycin C or Cisplatin was observed in LCLs. Our results are suggestive of an altered centromere distribution profile and a telomere instability phenotype. Together, these may indicate critical nuclear organization defects associated with the predisposition to transformation and early stage development of PALB2-related cancers.


Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Núcleo Celular/metabolismo , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Centrômero/metabolismo , Cisplatino/farmacologia , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Síndrome Hereditária de Câncer de Mama e Ovário/patologia , Heterozigoto , Humanos , Cariótipo , Masculino , Pessoa de Meia-Idade , Mitomicina/farmacologia , Telômero/metabolismo , Células Tumorais Cultivadas
4.
Int J Cancer ; 133(1): 247-52, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23238639

RESUMO

BKM120, a pan class I PI3K inhibitor, was cytotoxic in the majority of primary B-chronic lymphocytic leukemia (CLL) lymphocytes, including samples from patients who have a high-risk for poor response to treatment (patient with del11 and del17) at clinically obtainable concentrations. The PI3Kδ inhibitor Cal-101 is cytotoxic in B-CLL lymphocytes in vitro and is active in the treatment of CLL in vivo. Interestingly, we demonstrated that BKM120 is 3.6 fold more toxic than Cal-101 in malignant B-CLL lymphocytes in vitro. BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway. A protein signature of PI3K pathway proteins predicts the response to BKM120 treatment. In the primary B-CLL lymphocytes tested in vitro, BKM120 decreased the phosphorylation status of molecular biomarkers used as indicators of PI3K pathway inhibition in vivo. Also, BKM120 induced apoptosis in primary B-CLL cells culture in the presence and absence of stromal cell support. Our findings suggest that BKM120 should be tested clinically in CLL.


Assuntos
Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Adulto , Idoso , Apoptose/efeitos dos fármacos , Western Blotting , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Células Tumorais Cultivadas
5.
Invest New Drugs ; 31(2): 461-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23054213

RESUMO

Poly [ADP-ribose] polymerase-1 (PARP-1) localizes rapidly to sites of DNA damage and has been associated with various repair mechanisms including base excision repair (BER) and homologous recombination/non-homologous end joining (HRR/NHEJ). PARP-1 acts by adding poly-ADP ribose side chains to target proteins (PARylation) altering molecular interactions and functions. Recently small molecule inhibitors of PARP-1 have been shown to have significant clinical potential and third generation PARP inhibitors are currently being investigated in clinical trials. These drugs alone or in combination with radio/chemotherapy have resulted in meaningful patient responses and an increase in survival in metastatic breast cancer cases bearing BRCA-deficient or triple negative tumors and BRCA-deficient ovarian cancer patients. ABT-888, a potent PARP-1 inhibitor, sensitizes many cancer cells in-vitro and in-vivo to temozolomide. As such, we hypothesized that colon cancers would be sensitized to the DNA damaging chemotherapeutic agents, oxaliplatin and irinotecan, by ABT-888. Using colon cancer cell lines significant synergy was observed between ABT-888 and irinotecan at concentrations of ABT-888 as low as 0.125 µM. The level of synergy observed correlated with the degree of PARP1 inhibition as measured biochemically in cell lysates. ABT-888 at concentrations of 0.5-4 µM resulted in synergy with oxaliplatin. Furthermore, 24 h post treatment combinations of ABT-888/irinotecan generally resulted in increased G2/M cell cycle arrest and increased levels of DNA damage, followed by increased levels of apoptosis 48 h post treatment. In conclusion this study suggests that ABT-888 may be a clinically effective adjuvant to current colon cancer therapies that include the use of irinotecan and/or oxaliplatin.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Camptotecina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Camptotecina/farmacologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Irinotecano , Células Tumorais Cultivadas
6.
Front Biosci (Landmark Ed) ; 28(3): 52, 2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-37005765

RESUMO

BACKGROUND: Poly(ADP-ribose) polymerases 1 and 2 (PARP1, 2), and 3 mediate protein modifications that facilitate the recruitment of DNA repair factors to single and double strand breaks. PARP3 is unique in that it is also required for efficient mitotic progression and stabilization of the mitotic spindle. Eribulin, an anti-microtubule agent used clinically to treat breast cancer, exerts its cytotoxicity by altering microtubule dynamics resulting in cell cycle arrest and apoptosis. Herein, we hypothesize that the pan PARP inhibitor olaparib has the potential to enhance the cytotoxicity of eribulin by halting mitosis through inhibition of PARP3. METHODS: The effect of olaparib on eribulin cytotoxicity was assessed using the Sulforhodamine (SRB) assay, with two triple negative breast cancer cell lines and an estrogen receptor positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer cell line. Alteration by the treatments on PARP3 activity and microtubule dynamics were assessed utilizing a chemiluminescent enzymatic assay and immunofluorescence, respectively. The effect of the treatments on cell cycle progression and apoptosis induction were assessed by flow cytometry using propidium iodide and Annexin V staining, respectively. RESULTS: Our results demonstrate that non-cytotoxic concentrations of olaparib sensitize breast cancer cells regardless of ER status. Mechanistically, our results indicate that olaparib potentiates eribulin-induced cell cycle arrest at the G2/M boundary, PARP3 inhibition and microtubule destabilizing resulting in mitotic catastrophe and apoptosis. CONCLUSIONS: In breast cancer (regardless of ER status) settings, treatment outcomes could be improved by the incorporation of olaparib in eribulin treatment regimens.


Assuntos
Antineoplásicos , Neoplasias da Mama , Humanos , Feminino , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico
7.
Invest New Drugs ; 30(3): 1248-56, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21221710

RESUMO

This study sought to measure the degree of synergy induced by specific small molecule inhibitors of DNA-PK [NU7026 and IC486241 (ICC)], a major component of the non-homologous end-joining (NHEJ) pathway, with SN38 or oxaliplatin. Synergy between the DNA damaging drugs and the DNA-PK inhibitors was assessed using the sulforhodamine-B assay (SRB). Effects of drug combinations on cell cycle and DNA-PK activity were determined using flow cytometry and western blot analysis. DNA damage was assessed via comet assay and quantification of γH2AX. The role of homologous recombination repair (HRR) was determined by nuclear Rad51 protein levels and a GFP reporter recombination assay. Significant reductions in the IC(50) values of SN38 were observed at 5 and 10 µM of DNA-PK inhibitors. Moreover, at 1-2 µM (attainable concentrations with ICC in mice) these DNA-PKcs inhibitors demonstrated synergistic reductions in the IC(50) of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with DNA-PKcs inhibitors showed dramatic G2/M arrest at 24 h. Furthermore, reduced phosphorylation of DNA-PKcs and increased DNA damage were observed at this time point with SN38 in combination with DNA-PKcs inhibitors as compared to cells treated with SN38 alone. SN38 alone and in the presence of ICC increased nuclear Rad51 protein levels. Furthermore, inhibition of DNA-PKcs increased HRR suggesting that NHEJ is a negative regulator of HRR. These data indicate that small molecule inhibitors of DNA-PKcs dramatically enhance the efficacy of SN38 in colon cancer cell lines.


Assuntos
Acridonas/farmacologia , Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Cromonas/farmacologia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Camptotecina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias do Colo/patologia , Ensaio Cometa , Sinergismo Farmacológico , Células HCT116 , Células HT29 , Humanos , Irinotecano , Compostos Organoplatínicos/farmacologia , Piridinas/farmacologia , Inibidores da Topoisomerase I/farmacologia
8.
Invest New Drugs ; 30(4): 1736-42, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21567185

RESUMO

The purpose of this study was to determine the degree to which the novel DNA-PKcs inhibitor, IC486241 (ICC), synergizes the cytotoxicity of DNA damaging agents in 3 genetically diverse breast cancer cell lines. The sulforhodamine B (SRB) assay was employed as a primary screening method to determine the in-vitro cytotoxicity and the degree of synergy of ICC in combination with the topoisomerase II inhibitor, doxorubicin, or the DNA cross linking agent, cisplatin. Molecular mechanisms underlying drug toxicity were probed using immunostaining and flow cytometry, as well as, the alkaline comet assay to detect DNA damage. In this study, improved cytotoxicity and significant synergy were observed with both anticancer agents in the presence of nontoxic concentrations of ICC. Moreover, ICC decreased doxorubicin-induced DNA-PKcs autophosphorylation on Ser2056 and increased doxorubicin-induced DNA fragmentation. In conclusion, the novel DNA-PKcs inhibitor, ICC, synergistically sensitized 3 breast cancer cell lines to doxorubicin and cisplatin. Enhanced efficacy of doxorubicin was achieved by inhibiting non-homologous end joining resulting in increased accumulation of DNA damage.


Assuntos
Acridonas/farmacologia , Neoplasias da Mama/patologia , Cisplatino/farmacologia , Dano ao DNA , DNA de Neoplasias/metabolismo , Doxorrubicina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Acridonas/química , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Ensaio Cometa , Dimetil Sulfóxido/farmacologia , Doxorrubicina/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Inibidores de Proteínas Quinases/química , Quinazolinas/química
9.
J Exp Clin Cancer Res ; 41(1): 340, 2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36482393

RESUMO

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an important effector of the mTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A). METHODS: We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). RESULTS: eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition. CONCLUSIONS: We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.


Assuntos
Antineoplásicos , Citarabina , Fator de Iniciação 4A em Eucariotos , Leucemia Mieloide Aguda , Humanos , Citarabina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2 , Linhagem Celular Tumoral , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Antineoplásicos/farmacologia
10.
Hematol Oncol ; 28(3): 142-50, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20155735

RESUMO

Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy with a variable response to therapy. We have previously shown that DLBCL cell lines differ in their susceptibility to CD40-mediated cell death, and that resistance to CD40-targeted antibodies correlated with increased expression of markers of immature B-cell and absence of Vav-1 mRNA. We used gene expression profiling to investigate the mechanism of CD40 resistance in these cell lines, and found that resistance correlated with lack of Vav-1 and inability to activate NFκB upon CD40 ligation. Analysis of tissue microarrays of 213 DLBCL cases revealed that Vav-1 expression correlated with a higher proliferative index and the presence of the post-germinal centre marker Irf-4. Our results suggest that Vav-1 expression may be associated with activated B-cell DLBCL origin and higher proliferative activity, and indicate Vav-1 as a potential marker to identify tumours likely to respond to CD40-targeted therapies.


Assuntos
Antígenos CD40/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-vav/biossíntese , Idoso , Antígenos CD40/genética , Antígenos CD40/imunologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Fatores Reguladores de Interferon/biossíntese , Fatores Reguladores de Interferon/metabolismo , Antígeno Ki-67/biossíntese , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/imunologia , Prognóstico , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
11.
Leuk Lymphoma ; 61(7): 1653-1659, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32154751

RESUMO

Buparlisib is an orally available pan-Class I PI3K inhibitor, that is more potent than idelalisib in vitro. Its distinct toxicities include hyperglycemia, hypertension, and mood disturbance. IND216 is a single arm phase II trial of buparlisib in Relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). Fourteen patients were enrolled, 13 were evaluable for response and toxicity. Six of 13 patients had a partial response (46%) with a median duration of response of 15.5 months, all 11 patients with tumor assessment experienced tumor shrinkage. The most common adverse events (≥15%) were hyperglycemia, fatigue, anxiety, and gastrointestinal toxicities; all were < grade 3 except for fatigue. Three patients stopped therapy for alterations in mood. Lower levels of raptor were significantly associated with greater tumor shrinkage, suggesting that raptor could be a biomarker for response. This requires further validation in a larger CLL patient cohort. The clinical activity of buparlisib is comparable to other phosphatidylinositol-3-kinase inhibitors, with a different toxicity profile.Novelty and impactBuparlisib, an oral, pan PI3 kinase inhibitor, is associated with a 46% partial response rate among patients with relapse chronic lymphocytic leukemia (CLL). This is a similar clinical activity to other phosphatidylinositol-3-kinase inhibitors tested. However, buparlisib has a distinct toxicity profile, characterized by hyperglycemia, hypertension, and mood alteration. In agreement with our previous preclinical study, our results suggest that basal raptor expression in CLL correlates with clinical response to buparlisib.


Assuntos
Leucemia Linfocítica Crônica de Células B , Aminopiridinas , Biomarcadores , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Morfolinas , Fosfatidilinositol 3-Quinases , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Regulatória Associada a mTOR , Proteínas Quinases S6 Ribossômicas 70-kDa
12.
Crit Rev Oncol Hematol ; 134: 65-70, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30771875

RESUMO

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the western world. CLL consists of the accumulation of malignant B-cells in the blood stream and homing tissues. Although treatable, this disease is not curable, and resistance or relapse is often present. In many cancers, the study of metabolic reprograming has uncovered novel targets that are already being exploited in the clinic. However, CLL metabolism is still poorly understood. The ability of CLL lymphocytes to adapt to diverse microenvironments is accompanied by modifications in cell metabolism, revealing the challenge of targeting the CLL lymphocytes present in all different compartments. Despite this, the study of CLL metabolism led to an ongoing clinical trial using glucose uptake and mitochondrial respiration inhibitors. In contrast, glutamine and fatty acid metabolism remain to be further exploited in CLL. Here, we summarize the present knowledge of CLL metabolism, as well as the metabolic influence of Myc, ATM and p53 on CLL lymphocytes.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Respiração Celular , Humanos
13.
Sci Rep ; 9(1): 11069, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363127

RESUMO

In this work we explored metabolic aspects of human primary leukemic lymphocytes that hold a potential impact on the treatment of Bruton tyrosine kinase (BTK)-driven diseases. Our results suggest that there is crosstalk between Bruton tyrosine kinase (BTK) signaling and bioenergetic stress responses. In primary chronic lymphocytic leukemia (CLL) lymphocytes, pharmacological interference with mitochondrial ATP synthesis or glucose metabolism affects BTK activity. Conversely, an inhibitor of BTK used clinically (ibrutinib) induces bioenergetic stress responses that in turn affect ibrutinib resistance. Although the detailed molecular mechanisms are still to be defined, our work shows for the first time that in primary B cells, metabolic stressors enhance BTK signaling and suggest that metabolic rewiring to hyperglycemia affects ibrutinib resistance in TP53 deficient chronic lymphocytic leukemia (CLL) lymphocytes.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfócitos/metabolismo , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Humanos , Linfócitos/efeitos dos fármacos , Piperidinas , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
14.
Br J Haematol ; 143(5): 698-706, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19062342

RESUMO

The dual c-abl/Src kinase inhibitor, dasatinib, utilized to treat chronic myeloid leukaemia (CML) when used at clinically attainable sublethal concentrations, synergistically sensitized primary chronic lymphocytic leukaemia (CLL) lymphocytes to chlorambucil and fludarabine. In contrast, dasatinib alone demonstrated toxicity to CLL lymphocytes at concentrations that are generally not clinically attainable. Dasatinib resistance and poorer dasatinib-mediated sensitization to chlorambucil and fludarabine was associated with higher expression of c-abl protein levels. In contrast, chlorambucil and fludarabine resistance correlated with basal p53 protein levels. Moreover, Western blot analysis after in vitro treatment of primary CLL lymphocytes with dasatinib, chlorambucil and/or fludarabine, showed that dasatinib: (i) inhibited c-abl function (e.g. downregulation of c-abl protein levels and decreased the phosphorylation of a c-abl downstream target, Dok2), (ii) decreased chlorambucil/fludarabine induced accumulation of p53 protein levels, (iii) altered the response to chlorambucil/fludarabine induced DNA-damage as evidenced by an increase in chlorambucil/fludarabine-induced H2AX phosphorylation, and (iv) accentuated the c-abl downregulation induced by chlorambucil/fludarabine. Our results suggest that dasatinib in combination with chlorambucil or fludarabine may improve the therapy of CLL.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Tiazóis/uso terapêutico , Western Blotting/métodos , Sobrevivência Celular , Clorambucila/administração & dosagem , Testes Imunológicos de Citotoxicidade , Análise Mutacional de DNA , Dasatinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Modelos Lineares , Proteínas Proto-Oncogênicas c-abl/metabolismo , Estatísticas não Paramétricas , Proteína Supressora de Tumor p53/metabolismo , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados
15.
Front Oncol ; 8: 411, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319974

RESUMO

Chronic Lymphocytic Leukemia (CLL) is an incurable disease, characterized by the accumulation of malignant B-lymphocytes in the blood stream (quiescent state) and homing tissues (where they can proliferate). In CLL, the targeting of B-cell receptor signaling through a Burton's tyrosine kinase inhibitor (ibrutinib) has rendered outstanding clinical results. However, complete remission is not guaranteed due to drug resistance or relapse, revealing the need for novel approaches for CLL treatment. The characterization of metabolic rewiring in proliferative cancer cells is already being applied for diagnostic and therapeutic purposes, but our knowledge of quiescent cell metabolism-relevant for CLL cells-is still fragmentary. Recently, we reported that glutamine metabolism in primary CLL cells bearing the del11q deletion is different from their del11q negative counterparts, making del11q cells especially sensitive to glutaminase and glycolysis inhibitors. In this work, we used our primary CLL lymphocyte bank and compounds interfering with central carbon metabolism to define metabolic traits associated with ibrutinib resistance. We observe a differential basal metabolite uptake linked to ibrutinib resistance, favoring glutamine uptake and catabolism. Upon ibrutinib treatment, the redox balance in ibrutinib resistant cells is shifted toward NADPH accumulation, without an increase in glutamine uptake, suggesting alternative metabolic rewiring such as the activation of fatty acid oxidation. In accordance to this idea, the curtailing of fatty acid oxidation by CPT1 inhibition (etomoxir) re-sensitized resistant cells to ibrutinib. Our results suggest that fatty acid oxidation could be explored as a target to overcome ibrutinib resistance.

16.
Oncotarget ; 8(40): 66758-66768, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28977994

RESUMO

Src family kinases (SFK) are key regulators of cellular proliferation, differentiation, survival, motility and angiogenesis. As such, SFK inhibitors are being tested in clinical trials to prevent metastasis as an alternative to current treatment regimens for a variety of cancers including breast cancer. To contribute to the development of molecular tools improving SFK-targeted therapies, we used the SFK inhibitor dasatinib and a well characterized triple negative breast cancer cell line (BT20). Comparison of the response of BT20 cells with acquired resistance to dasatinib and its' parental counterpart suggest that chronic exposure to SFK inhibition results in increased dependency on TGFß signaling for proliferation, both in the absence or the presence of dasatinib. In addition, we found that acquired (but not de novo) resistance to dasatinib was reduced by non-cytotoxic concentrations compounds hindering on PI3K, mTORC1 signaling, endoplasmic reticulum stress or autophagy.

17.
Cancer Res ; 62(19): 5457-62, 2002 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-12359753

RESUMO

We have recently completed screening of the National Cancer Institute human tumor cell line panel and demonstrated that among four nucleotide excision repair proteins (XPA, XPB, XPD, and ERCC1), only the TFIIH subunit XPD endogenous protein levels correlate with alkylating agent drug resistance. In the present study, we extended this work by investigating the biological consequences of XPD overexpression in the human glioma cell line SK-MG-4. Our results indicate that XPD overexpression in SK-MG-4 cells leads to cisplatin resistance without affecting the nucleotide excision repair activity or UV light sensitivity of the cell. In contrast, in SK-MG-4 cells treated with cisplatin, XPD overexpression leads to increased Rad51-related homologous recombinational repair, increased sister chromatid exchanges, and accelerated interstrand cross-link removal. Moreover, we present biochemical evidence of an XPD-Rad51 protein interaction, which is modulated by DNA damage. To our knowledge, this is the first description of functional cross-talk between XPD and Rad51, which leads to bifunctional alkylating agent drug resistance and accelerated removal of interstrand cross-links.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , DNA Helicases , Reparo do DNA/fisiologia , Endonucleases , Proteínas/fisiologia , Fatores de Transcrição TFII/fisiologia , Fatores de Transcrição , Ciclo Celular/fisiologia , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Glioma/tratamento farmacológico , Glioma/genética , Glioma/metabolismo , Humanos , Melfalan/farmacologia , Testes de Precipitina , Biossíntese de Proteínas , Proteínas/metabolismo , Rad51 Recombinase , Tolerância a Radiação , Fase S/fisiologia , Troca de Cromátide Irmã/efeitos dos fármacos , Troca de Cromátide Irmã/fisiologia , Fator de Transcrição TFIIH , Fatores de Transcrição TFII/biossíntese , Células Tumorais Cultivadas , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A , Proteína Grupo D do Xeroderma Pigmentoso
18.
J Neurosci ; 23(1): 349-57, 2003 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-12514234

RESUMO

Recent studies have indicated that exogenously administered neurotrophins produce antidepressant-like behavioral effects. We have here investigated the role of endogenous brain-derived neurotrophic factor (BDNF) and its receptor trkB in the mechanism of action of antidepressant drugs. We found that trkB.T1-overexpressing transgenic mice, which show reduced trkB activation in brain, as well as heterozygous BDNF null (BDNF(+/)-) mice, were resistant to the effects of antidepressants in the forced swim test, indicating that normal trkB signaling is required for the behavioral effects typically produced by antidepressants. In contrast, neurotrophin-3(+/)- mice showed a normal behavioral response to antidepressants. Furthermore, acute as well as chronic antidepressant treatment induced autophosphorylation and activation of trkB in cerebral cortex, particularly in the prefrontal and anterior cingulate cortex and hippocampus. Tyrosines in the trkB autophosphorylation site were phosphorylated in response to antidepressants, but phosphorylation of the shc binding site was not observed. Nevertheless, phosphorylation of cAMP response element-binding protein was increased by antidepressants in the prefrontal cortex concomitantly with trkB phosphorylation and this response was reduced in trkB.T1-overexpressing mice. Our data suggest that antidepressants acutely increase trkB signaling in a BDNF-dependent manner in cerebral cortex and that this signaling is required for the behavioral effects typical of antidepressant drugs. Neurotrophin signaling increased by antidepressants may induce formation and stabilization of synaptic connectivity, which gradually leads to the clinical antidepressive effects and mood recovery.


Assuntos
Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Fluoxetina/farmacologia , Imipramina/farmacologia , Receptor trkB/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Cinética , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotrofina 3/genética , Fosforilação , Córtex Pré-Frontal/metabolismo , Receptor trkB/genética , Transdução de Sinais
19.
Front Pharmacol ; 6: 147, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26257651

RESUMO

Enhanced DNA damage repair is one mechanism involved in colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these DNA repair inhibitors utilized alone at nontoxic single agent concentrations resulted in sensitization to SN38 producing a 1.4-3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 in three colon cancer cell lines. When combined together, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5-27 fold reduction in the IC50 of SN38 with the HCT-116 colon cancer cells demonstrating the highest sensitization as compared to LoVo and HT-29 colon cancer cells. Furthermore, the combination of all three agents was associated with maximal G2 -M arrest and enhanced DNA-damage (γH2AX) in all three colon cancer cell lines. The mechanism of this enhanced sensitization was associated with: (a) maximal suppression of SN38 induced PARP activity in the presence of both inhibitors and (b) ABT-888 producing partial abrogation of the VE-821 enhancement of SN38 induced DNA-PK phosphorylation, resulting in more unrepaired DNA damage; these alterations were only present in the HCT-116 cells which have reduced levels of ATM. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help produce sensitization to this drug in colon cancer.

20.
J Pathol Clin Res ; 1(3): 160-72, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27499901

RESUMO

Currently, there is no marker in use in the clinical management of colon cancer to predict which patients will respond efficiently to 5-fluorouracil (5-FU), a common component of all cytotoxic therapies. Our aim was to develop and validate a multigene signature associated with clinical outcome from 5-FU therapy and to determine if it could be used to identify patients who might respond better to alternate treatments. Using a panel of 5-FU resistant and sensitive colon cancer cell lines, we identified 103 differentially expressed genes providing us with a 5-FU response signature. We refined this signature using a clinically relevant DNA microarray-based dataset of 359 formalin-fixed and paraffin-embedded (FFPE) colon cancer samples. We then validated the final signature in an external independent DNA microarray-based dataset of 316 stage III FFPE samples from the PETACC-3 (Pan-European Trails in Alimentary Tract Cancers) clinical trial. Finally, using a drug sensitivity database of 658 cell lines, we generated a list of drugs that could sensitize 5-FU resistant patients using our signature. We confirmed using the PETACC-3 dataset that the overall survival of subjects responding well to 5-FU did not improve with the addition of irinotecan (FOLFIRI; two-sided log-rank test p = 0.795). Conversely, patients who responded poorly to 5-FU based on our 12-gene signature were associated with better survival on FOLFIRI therapy (one-sided log-rank test p = 0.039). This new multigene signature is readily applicable to FFPE samples and provides a new tool to help manage treatment in stage III colon cancer. It also provides the first evidence that a subgroup of colon cancer patients can respond better to FOLFIRI than 5-FU treatment alone.

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