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1.
Oncotarget ; 8(55): 94711-94725, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29212260

RESUMO

Acquired cisplatin resistance is a common feature of tumours following cancer treatment with cisplatin and also of non-small cell lung cancer (H1299) and mesothelioma (P31) cell lines exposed to cisplatin. To elucidate the cellular basis of acquired cisplatin resistance, a comprehensive bioenergetic analysis was undertaken. We demonstrate that cellular oxygen consumption was significantly decreased in cisplatin resistant cells and that the reduction was primarily due to reduced mitochondrial activity as a result of reduced mitochondrial abundance. The differential mitochondrial abundance was supported by data showing reduced sirtuin 1 (SIRT1), peroxisome-proliferator activator receptor-γ co-activator 1-alpha (PGC1α), sirtuin 3 (SIRT3) and mitochondrial transcription factor A (TFAM) protein expression in resistant cells. Consistent with these data we observed increased reactive oxygen species (ROS) production and increased hypoxia inducible factor 1-alpha (HIF1α) stabilization in cisplatin resistant cells when compared to cisplatin sensitive controls. We also observed an increase in AMP kinase subunit α2 (AMPKα2) transcripts and protein expression in resistant H1299 cells. mRNA expression was also reduced for cisplatin resistant H1299 cells in these genes, however the pattern was not consistent in resistant P31 cells. There was very little change in DNA methylation of these genes, suggesting that the cells are not stably reprogrammed epigenetically. Taken together, our data demonstrate reduced oxidative metabolism, reduced mitochondrial abundance, potential for increased glycolytic flux and increased ROS production in acquired cisplatin resistant cells. This suggests that the metabolic changes are a result of reduced SIRT3 expression and increased HIF-1α stabilization.

2.
Exp Cell Res ; 336(1): 23-32, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26004871

RESUMO

BACKGROUND: Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Ceramide increases in response to chemotherapy, leading to proliferation arrest and apoptosis. However, a tumour stress activation of glucosylceramide synthase (GCS) follows to eliminate ceramide by formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination enhances cell proliferation and apoptosis blockade, thus stimulating tumor progression. GSLs transactivate multidrug resistance 1/P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1) expression which further prevents ceramide accumulation and stimulates drug efflux. We investigated the expression of Gb3, MDR1 and MRP1 in NSCLC and MPM cells with acquired cisplatin resistance, and if GCS activity or MDR1 pump inhibitors would reduce their expression and reverse cisplatin-resistance. METHODS: Cell surface expression of Gb3, MDR1 and MRP1 and intracellular expression of MDR1 and MRP1 was analyzed by flow cytometry and confocal microscopy on P31 MPM and H1299 NSCLC cells and subline cells with acquired cisplatin resistance. The effect of GCS inhibitor PPMP and MDR1 pump inhibitor cyclosporin A for 72h on expression and cisplatin cytotoxicity was tested. RESULTS: The cisplatin-resistant cells expressed increased cell surface Gb3. Cell surface Gb3 expression of resistant cells was annihilated by PPMP whereas cyclosporin A decreased Gb3 and MDR1 expression in H1299 cells. No decrease of MDR1 by PPMP was noted in using flow cytometry, whereas a decrease of MDR1 in H1299 and H1299res was indicated with confocal microscopy. No certain co-localization of Gb3 and MDR1 was noted. PPMP, but not cyclosporin A, potentiated cisplatin cytotoxicity in all cells. CONCLUSIONS: Cell surface Gb3 expression is a likely tumour biomarker for acquired cisplatin resistance of NSCLC and MPM cells. Tumour cell resistance to MDR1 inhibitors of cell surface MDR1 and Gb3 could explain the aggressiveness of NSCLC and MPM. Therapy with GCS activity inhibitors or toxin targeting of the Gb3 receptor may substantially reduce acquired cisplatin drug resistance of NSCLC and MPM cells.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ciclosporina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glucosiltransferases/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Triexosilceramidas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ceramidas/metabolismo , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Imunossupressores/farmacologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Meperidina/análogos & derivados , Meperidina/farmacologia , Mesotelioma/enzimologia , Mesotelioma/patologia , Mesotelioma Maligno , Microscopia Confocal , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células Tumorais Cultivadas
3.
Biochem Pharmacol ; 92(1): 55-61, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25107702

RESUMO

The selection of human cancer cell lines in cis-diamminedichloroplatinum(II) (CDDP, best known as cisplatin) is accompanied by stereotyped alterations that contribute to the acquisition of a CDDP-resistant state. Thus, CDDP resistance often leads to the upregulation of the DNA repair enzyme poly (ADP-ribose) polymerase-1 (PARP1) with the consequent intracellular accumulation of poly (ADP-ribose) (PAR)-modified proteins. Here we report another frequent alteration accompanying CDDP resistance, namely upregulation of the antiapoptotic BCL-2 family protein MCL-1. Six out of 8 CDDP resistant cancer cell lines manifested an increase in MCL-1 protein expression level, while only a minority of cell lines overexpressed BCL-2 or BCL-XL. BCL-XL was decreased in six out of 8 cancer cell lines. Importantly, MCL-1 overexpressing, CDDP resistant cells appear to be 'addicted' to MCL-1 because they died upon depletion of MCL-1 by RNA interference or pharmacological inhibition of MCL-1 expression by the BH3 mimetic obatoclax. Knockdown of PARP1 did not succeed in reducing MCL-1 expression, while depletion or inhibition of MCL-1 failed to affect the activity of PARP1. Hence, the two resistance mechanisms are not linked to each other by a direct cause-effect relationship. Importantly, CDDP-resistant, MCL-1 overexpressing human non-small cell lung cancers responded to monotherapy with obatoclax in vivo, in xenotransplanted mice, underscoring the probable therapeutic relevance of these findings.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Humanos , Indóis , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Pirróis/farmacologia , Pirróis/uso terapêutico , Interferência de RNA
4.
Cancer Res ; 73(7): 2271-80, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23554447

RESUMO

Non-small cell lung carcinoma patients are frequently treated with cisplatin (CDDP), most often yielding temporary clinical responses. Here, we show that PARP1 is highly expressed and constitutively hyperactivated in a majority of human CDDP-resistant cancer cells of distinct histologic origin. Cells manifesting elevated intracellular levels of poly(ADP-ribosyl)ated proteins (PAR(high)) responded to pharmacologic PARP inhibitors as well as to PARP1-targeting siRNAs by initiating a DNA damage response that translated into cell death following the activation of the intrinsic pathway of apoptosis. Moreover, PARP1-overexpressing tumor cells and xenografts displayed elevated levels of PAR, which predicted the response to PARP inhibitors in vitro and in vivo more accurately than PARP1 expression itself. Thus, a majority of CDDP-resistant cancer cells appear to develop a dependency to PARP1, becoming susceptible to PARP inhibitor-induced apoptosis.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/patologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cell Rep ; 2(2): 257-69, 2012 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-22854025

RESUMO

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Vitamina B 6/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/administração & dosagem , Estudos de Coortes , Intervalo Livre de Doença , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Piridoxal Quinase/biossíntese , Piridoxal Quinase/genética , Taxa de Sobrevida , Vitamina B 6/genética
6.
Toxins (Basel) ; 2(10): 2467-77, 2010 10.
Artigo em Inglês | MEDLINE | ID: mdl-22069561

RESUMO

A major problem with anti-cancer drug treatment is the development of acquired multidrug resistance (MDR) of the tumor cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the globotriaosylceramide membrane receptor (Gb3), a glycolipid associated with multidrug resistance. Gb3 is overexpressed in many human tumors and tumor cell lines with inherent or acquired MDR. Gb3 is co-expressed and interplays with the membrane efflux transporter P-gp encoded by the MDR1 gene. P-gp could act as a lipid flippase and stimulate Gb3 induction when tumor cells are exposed to cancer chemotherapy. Recent work has shown that apoptosis and inherent or acquired multidrug resistance in Gb3-expressing tumors could be affected by VT-1 holotoxin, a sub-toxic concentration of the holotoxin concomitant with chemotherapy or its Gb3-binding B-subunit coupled to cytotoxic or immunomodulatory drug, as well as chemical manipulation of Gb3 expression. The interplay between Gb3 and P-gp thus gives a possible physiological approach to augment the chemotherapeutic effect in multidrug resistant tumors.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Glicoesfingolipídeos/metabolismo , Toxina Shiga I/farmacologia , Animais , Humanos , Neoplasias/tratamento farmacológico
7.
BMC Cancer ; 9: 67, 2009 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-19245689

RESUMO

BACKGROUND: The prerequisite for the potential use of the bacterial toxin verotoxin-1 in the treatment of breast cancer was investigated by first determining the expression of its receptor Gb3 (CD77) in clinical breast cancer tissue specimens. We then examined the cytotoxicity and mechanism of apoptosis induction of Escherichia coli verotoxin-1 (VT-1) in two human breast cancer cell lines. METHODS: Immunohistochemistry for Gb3 expression was performed on cryostat section from 25 breast cancer specimens. The human breast cancer cell lines T47D and MCF-7 were screened for Gb3 expression by flow cytometry. Fluorescein diacetate and LDH release was used to determine cell viability after VT-1 exposure. Apoptosis was studied by measuring caspase activity and DNA-fragmentation. Signal transduction studies were performed on T47D cells with immunoblotting. RESULTS: Gb3 expression was detected in the vascular endothelial cells of all tumours specimens, and in tumour cells in 17 of the specimens. We found no associations between tumour cell Gb3-expression and age, tumour size, TNM-classification, histological type, hormone receptor expression, or survival time. T47D cells strongly expressed Gb3 and were sensitive to the cytotoxicity, caspase activation and DNA fragmentation by VT-1, whereas MCF-7 cells with faint Gb3-expression were insensitive to VT-1. VT-1 (0.01 - 5 microg/L) exposure for 72 h resulted in a small percentage of viable T47D cells whereas the cytotoxicity of cells pre-treated with 2 micromol/L D, L-treo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, an inhibitor of glucosylceramide synthesis) was eliminated (< or = 0.1 microg/L VT-1) or reduced (0.5 - 5 microg/L VT-1). VT-1 did not cause cellular LDH-release or cell cycle arrest. VT-1 induction of caspase-3 (0.1, 1, and 5 microg/L VT-1), -8, and -9 (1 and 5 microg/L VT-1) activity and DNA fragmentation of T47D cells was blocked by PPMP. Key components of MAP kinase signalling pathways that control mitochondrial function were investigated. VT-1 0.1 - 5 microg/L induced phosphorylation of JNK as well as MKK3/6 suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. CONCLUSION: The high specificity and apoptosis-inducing properties of verotoxin-1 indicates that the toxin potentially may be used for treatment of Gb3-expressing breast cancer.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Toxina Shiga I/farmacologia , Transdução de Sinais/efeitos dos fármacos , Triexosilceramidas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Triexosilceramidas/metabolismo
8.
Cell Physiol Biochem ; 22(1-4): 45-56, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18769031

RESUMO

AIMS: Resistance mechanisms are important limiting factors in the treatment of solid malignancies with cis-diamminedichloroplatinum(II) (cisplatin). To gain further understanding of the effects of acquired cisplatin-resistance, we compared a human malignant pleural mesothelioma cell line (p31) to a sub-line (p31res1.2) with acquired cisplatin-resistance. METHODS AND RESULTS: The role of Na(+),K(+),2Cl(-)-cotransport (NKCC1) activity in cisplatin-induced morphological changes and acquired cisplatin-resistance was investigated in a time-resolved manner. Acquisition of cisplatin-resistance resulted in markedly reduced NKCC1 activity, absence of cisplatin-induced early membrane blebbing, and increased basal caspase-3 activity. At equitoxic cisplatin concentrations, P31res1.2 cells had a faster activation of caspase-3 than P31 cells, but the end-stage cytotoxicity and number of cells with DNA fragmentation was similar. Bumetanide inhibition of NKCC1 activity in P31 cells repressed cisplatin-induced early-phase membrane blebbing but did not increase P31 cell resistance to cisplatin. CONCLUSIONS: Together, these results suggest that active NKCC1 was necessary for cisplatin-induced early membrane blebbing of P31 cells, but not for cisplatin-resistance. Thus, acquisition of cisplatin-resistance can affect mechanisms that have profound effects on cisplatin-induced morphological changes but are not necessary for the subsequent progression to apoptosis.


Assuntos
Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mesotelioma/metabolismo , Mesotelioma/patologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Bumetanida/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Extensões da Superfície Celular/enzimologia , Fragmentação do DNA/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mesotelioma/enzimologia , Rubídio/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio , Membro 2 da Família 12 de Carreador de Soluto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Cancer Lett ; 265(1): 67-75, 2008 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-18362050

RESUMO

alpha-Toxin (alpha-hemolysin) of Staphylococcus aureus is a pore-forming bacterial toxin which after caveolin-1-dependent assembly induces apoptosis in eukaryotic cells. We investigated if a sub-toxic concentration of staphylococcal alpha-toxin could enhance cisplatin-induced apoptosis and overcome acquired cisplatin-resistance in cultured malignant pleural mesothelioma (MPM) cells. MPM cells (P31wt) and a cisplatin-resistant sub-line (P31res) was incubated with alpha-toxin and/or cisplatin followed by determination of cell viability, apoptosis, and signaling pathways. P31res cells were more sensitive to alpha-toxin than P31 wt cells due to induction of apoptosis. A low-toxic concentration of alpha-toxin re-sensitized cisplatin P31res cytotoxicity by apoptosis-induced through the mitochondrial pathway without detectable activation of common up-stream apoptosis signaling proteins. The toxin/drug combination should be tested for cisplatin-resistant mesothelioma treatment.


Assuntos
Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Hemolisinas/farmacologia , Staphylococcus aureus , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mesotelioma , Neoplasias Pleurais , Transdução de Sinais
10.
Exp Lung Res ; 34(2): 49-67, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18266129

RESUMO

Cisplatin treatment efficacy of malignant pleural mesothelioma (MPM) is aggravated by resistance and adverse effects. In P31 MPM cells, cisplatin induces morphological changes and apoptosis. To determine if very early (10 minutes) morphological responses corresponded to apoptosis-induction, cisplatin effects on P31 morphology were examined with phase-contrast microscopy (PCM), scanning electron microscopy (SEM), and flow cytometry (fluorescence-activated cell sorting [FACS]), and compared to apoptosis-induction over time. Increased membrane protrusions were identified with PCM and SEM, but these were not consistent with the induction of apoptosis. The authors concluded that very early morphological changes can be determined with PCM in MPM, but they did not convincingly correspond to apoptosis induction.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Cisplatino/farmacologia , Mesotelioma/tratamento farmacológico , Microscopia de Contraste de Fase , Neoplasias Pleurais/tratamento farmacológico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Separação Celular , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Mesotelioma/patologia , Microscopia Eletrônica de Varredura , Neoplasias Pleurais/patologia , Reprodutibilidade dos Testes , Fatores de Tempo
11.
Acta Oncol ; 46(7): 951-60, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17917826

RESUMO

Amplified epidermal growth factor receptor (EGFR) signaling is supposed to contribute to clinical radiation resistance of glioblastoma multiforme (GBM). Therefore, inhibition of EGFR signaling pathways by the selective EGFR tyrosine kinase inhibitor, gefitinib (ZD1839, Iressa), may increase the therapeutic effects of radiotherapy. The effects of different schedules for administration of gefitinib on sensitivity to irradiation of the human glioma cell lines (251MG and SF-767), a rat glioma cell line (BT4C), and an immortalized rat brain endothelial cell line (RBE4) is reported. Differences in effects of the combined treatment on cell toxicity were determined by a fluorometric cytotoxicity assay, and nuclear DNA fragmentation was used for quantification of apoptosis. Pre-administration with gefitinib for 30 min prior to irradiation followed by continuous incubation with gefitinib significantly increased the cytotoxicity of SF-767, BT4C, and RBE4 cells. However, the human glioma cell line 251MG was protected against radiation-induced damage by this treatment schedule, at lower concentrations of gefitinib. Pre-administration with gefitinib for 24 h prior to irradiation without following incubation with gefitinib increased the cytotoxicity of SF-767 and BT4C cells. Post-irradiation treatment with gefitinib significantly increased the cytotoxicity in all cell lines except for 251MG. We demonstrated heterogeneity in the cytotoxic effects of gefitinib between cell lines. Response to gefitinib might be due to other mechanisms than through the EGF receptor as some of the cell lines showed sensitivity to gefitinib despite no or low expression of EGFR. This study also demonstrates the importance of timing of gefitinib administration when this agent is combined with irradiation.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Receptores ErbB/metabolismo , Glioma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Quinazolinas/administração & dosagem , Receptor ErbB-2/metabolismo , Animais , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Terapia Combinada , Fragmentação do DNA , Esquema de Medicação , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/efeitos da radiação , Receptores ErbB/análise , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Glioma/enzimologia , Glioma/radioterapia , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor ErbB-2/análise
12.
Cancer Biol Ther ; 5(9): 1211-7, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16929170

RESUMO

The aim of this study was to examine the cytotoxicity and mechanism of apoptosis induction of verotoxin-1 (VT-1) in human glioma cell lines. VT-1 is a member of the shiga-toxin family expressed by some serotypes of Escherichia coli and Shigella dysenteriae. Shiga-toxins have been shown to induce apoptosis by binding to its membrane receptor Gb3. The human glioma cell lines SF-767, U-343 MG, and U-251 MG were studied together with BT4C, a rat glioma cell line. Cells were first screened for Gb3 expression by flow cytometry. Fluorescein diacetate was used to determine cell viability after VT-1 and irradiation exposure and apoptosis was studied by TUNEL staining, a mitochondrial membrane potential assay, and caspase activity assays. SF-767 and U-343 MG cells were found to express Gb3 and were also sensitive to VT-1-induced cytotoxicity, whereas nonGb3-expressing U-251 MG and BT4C glioma cells were not. VT-1 depolarized the mitochondrial membrane and activated caspase-9 and -3 of SF-767 and U-343 MG cells. VT-1 exposure for 72 h resulted in approx. 60 and 90% TUNEL-stained cells, respectively. D, L-Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) an inhibitor of glucosylceramide synthesis was used to block Gb3 synthesis. Two mumol/L PPMP for 72 h abolished SF-767 and U-343 MG expression of Gb3 and made the cells completely resistant to VT-1 induced apoptosis. Key components of MAP kinase signalling pathways that control BAX and mitochondrial function were investigated. VT-1 induced JNK phosphorylation in both cell lines, suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. Immunohistochemistry of cryostat section from glioma biopsies demonstrated expression of Gb3 was in the vascular endothelial cells as well as tumor cells, but not in astrocytes. The high specificity and apoptosis inducing properties of verotoxin-1 indicates that the toxin may be a potential anti-neoplastic agent for Gb3-expressing gliomas.


Assuntos
Antígenos Glicosídicos Associados a Tumores/biossíntese , Apoptose/efeitos dos fármacos , Glioma/tratamento farmacológico , Toxina Shiga I/farmacologia , Animais , Linhagem Celular Tumoral , Terapia Combinada , Glioma/enzimologia , Glioma/imunologia , Glioma/radioterapia , Humanos , Immunoblotting , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ratos
13.
Oncol Res ; 15(9): 423-30, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16555548

RESUMO

The present study examined the possibility to enhance lung cancer cell cytotoxicity and apoptosis of the anticancer drug cisplatin by exposure with adenylate cyclase (AC) toxin from Bordetella pertussis. A malignant mesothelioma cell line (P31) and a small-cell lung cancer cell line (U1690) were exposed to increasing concentrations of cisplatin and AC toxin, alone or in combination. Cytotoxicity was determined by a fluorescein-based assay and apoptosis by flow cytometry quantification of annexin V binding. Caspase-3, -8, and -9 activities were measured by enzyme activity assays. The cytotoxicity of AC toxin was time and dose dependent with an LD50 value at 72 h of 3 and 7 mg/L for P31 cells and U1690 cells, respectively. Cisplatin showed a similar time- and dose-dependent cytotoxicity, which was increased in the presence of a low toxic concentration (1 mg/L) of AC toxin. Furthermore, cisplatin caused a dose-dependent increase of annexin V binding cells of both cell lines after 24-h incubation, which was also enhanced in combination with AC toxin. AC toxin (1 mg/L) increased cisplatin-induced caspase-3, -8, and -9 activities in U1690 cells. Only minor increases of caspase-8 and -9 were noted for P31 cells. The present results, together with the knowledge that bacterial toxins decrease side effects of traditional cancer treatment, suggest a possibility to use them to enhance the therapeutic effect of cancer chemotherapy with reduced clinical adverse effects.


Assuntos
Toxina Adenilato Ciclase/metabolismo , Toxina Adenilato Ciclase/toxicidade , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Bordetella pertussis/enzimologia , Cisplatino/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Antineoplásicos/farmacologia , Bordetella pertussis/metabolismo , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/toxicidade , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Cinética , Dose Letal Mediana , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/metabolismo , Mesotelioma/patologia
14.
Anticancer Res ; 25(4): 2609-16, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16080501

RESUMO

BACKGROUND: Depletion of intracellular potassium ions (K+) is necessary for cells to shrink, induce DNA fragmentation and activate caspases, events which are features of apoptosis. MATERIALS AND METHODS: We used 86Rb+ as a K+ analogue to evaluate the possibility of pharmacologically depleting human pulmonary mesothelioma (P31) and small cell lung cancer (U1690) cells of K+, for future use in studies of apoptosis induction. RESULTS: The Na+, K+, 2CI(-)-cotransport inhibitor bumetanide transiently inhibited 86Rb+ influx, but when combined with the Na+, K+, ATPase pump inhibitor ouabain there was a marked and lasting (up to 6 h) 86Rb+ influx inhibition. Cellular K+ efflux was augmented by amphotericin B, digitonin and nigericin. Amphotericin B was an effective 86Rb+ efflux stimulator with low cytotoxicity, whereas digitonin caused cell detachment and nigericin increased LDH release in the U1690 cell line, indicating considerable toxicity of the drugs. CONCLUSION: It is possible to efficiently reduce intracellular K+ by persistent K+ influx inhibition and simultaneous K+ efflux stimulation with clinically available drugs.


Assuntos
Carcinoma de Células Pequenas/tratamento farmacológico , Ionóforos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Potássio/metabolismo , Anfotericina B/farmacologia , Bumetanida/farmacologia , Carcinoma de Células Pequenas/metabolismo , Linhagem Celular Tumoral , Digitonina/farmacologia , Interações Medicamentosas , Humanos , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Nigericina/farmacologia , Ouabaína/farmacologia , Potássio/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Radioisótopos de Rubídio/farmacocinética
15.
Basic Clin Pharmacol Toxicol ; 94(5): 245-51, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15125695

RESUMO

The anticancer drug cisplatin induces cell death by apoptosis. Apoptosis is dependent on cellular loss of potassium ions (K+). We have recently shown that the antifungal drug amphotericin B (enhancing K+ efflux), combined with the Na+, K+, 2Cl(-)-cotransport blocker bumetanide (decreasing K+ influx), augmented cisplatin-induced apoptosis in vitro. We therefore quantified K+ fluxes with the K+ analogue rubidium (86Rb+) in cisplatin-induced apoptosis of mesothelioma cells treated with bumetanide and amphotericin B. Bumetanide combined with amphotericin B enhanced cisplatin-induced apoptosis by a pronounced initial reduction of K+ influx due (in addition to Na+, K+, 2Cl(-)-cotransport inhibition) also to Na+, K+, ATPase pump inhibition. As 86Rb+ efflux was initially preserved, combination of the drugs would lead to net K+ loss. Combinations of K+ flux modulators leading to cellular potassium ion deprivation thus augments cisplatin-induced apoptosis and could therefore possibly be used to enhance the antitumour efficacy of cisplatin treatment.


Assuntos
Antineoplásicos , Apoptose , Cisplatino , Potássio/metabolismo , Anfotericina B/farmacologia , Apoptose/efeitos dos fármacos , Bumetanida/farmacologia , Linhagem Celular Tumoral , Fragmentação do DNA , Sinergismo Farmacológico , Inibidores Enzimáticos , Exocitose/efeitos dos fármacos , Humanos , Ouabaína , Potássio/análise , Radioisótopos de Rubídio , Inibidores de Simportadores de Cloreto de Sódio e Potássio , Simportadores de Cloreto de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo
16.
J Cancer Res Clin Oncol ; 128(8): 449-55, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12200602

RESUMO

PURPOSE: Highly 5-HT(3)-receptor-specific antiemetic agents may have different effects on ion transport of tumour cells during treatment with cytotoxic drugs. Cell volume regulation, which is dependent on potassium ion (K(+)) flux, is involved the control of cell growth, proliferation, and apoptosis. K(+)-flux response mechanisms to the antiemetics ondansetron and granisetron were therefore correlated to malignant glioma cell (Mg251) volume response to estramustine phosphate (EMP) in vitro. METHODS: We quantified the influx and efflux of potassium ions (using the K(+) analogue (86)Rb(+)) as well as cell volume changes (with image analysis) of glioma cells incubated with the 5-HT(3)-receptor antagonists ondansetron and granisetron (0.1 micro mol/l) combined with 40 mg/l EMP. RESULTS: The EMP-induced cell volume increase was fully inhibited by ondansetron but not affected by granisetron. Ondansetron retained high cellular K(+)-efflux and reduced Na(+), K(+), 2Cl(-)-cotransport activity, whereas granisetron (0.1 micro mol/l) reduced K(+)-efflux and retained an augmented Na(+), K(+), 2Cl(-)-cotransport activity in the presence of 40 mg/l EMP. CONCLUSIONS: Ondansetron affects K(+) transport with ensuing effects on cell volume of tumour cells treated with EMP, whereas granisetron does not. Since ondansetron and other 5-HT(3)-receptor antagonists are used routinely to prevent nausea during anticancer treatment, an increased awareness of possible interactions with the antitumour efficacy of anticancer drugs seems warranted.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/patologia , Tamanho Celular/efeitos dos fármacos , Estramustina/farmacologia , Glioma/patologia , Transporte de Íons/efeitos dos fármacos , Potássio/metabolismo , Antagonistas da Serotonina/farmacologia , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Granisetron/farmacologia , Humanos , Ondansetron/farmacologia , Radioisótopos de Rubídio , Serotonina/farmacologia , Sódio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Tumorais Cultivadas
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