RESUMO
BACKGROUND: Upregulation of PIM kinase expression has been reported in many malignancies, suggesting that inhibition of PIM kinase activity may be an attractive therapeutic strategy. We hypothesised that inhibition of PIM kinase activity with SGI-1776, a novel small molecule inhibitor of PIM kinase activity, would reduce the viability of renal cell carcinoma (RCC) cells and enhance the activity of sunitinib. METHODS: Immunoblotting, qRT-PCR, and gene expression arrays were carried out to identify genes modulated by SGI-1776 treatment. The anticancer activity of SGI-1776 and sunitinib was determined by viability and apoptosis assays and in tumour xenografts in vivo. RESULTS: Treatment with SGI-1776 led to a decrease in phosphorylated and total c-Myc levels, which resulted in the modulation of c-Myc target genes. SGI-1776 in combination with sunitinib induced a further reduction in c-Myc levels, which was associated with enhanced anticancer activity. siRNA-mediated knockdown of c-Myc demonstrated that its expression has a key role in regulating the sensitivity to the combination of SGI-1776 and sunitinib. Importantly, the combination significantly reduced tumour burden in two RCC xenograft models compared with single-agent therapy and was very well tolerated. CONCLUSION: These data indicate that targeting PIM kinase signalling is a promising treatment strategy for RCC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/patologia , Imidazóis/farmacologia , Indóis/farmacologia , Neoplasias Renais/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Piridazinas/farmacologia , Pirróis/farmacologia , Animais , Carcinoma de Células Renais/enzimologia , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Renais/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , SunitinibeRESUMO
Heat-shock proteins (HSPs) are molecular chaperones that regulate protein folding to ensure correct conformation and translocation and to avoid protein aggregation. Heat-shock proteins are increased in many solid tumours and haematological malignancies. Many oncogenic proteins responsible for the transformation of cells to cancerous forms are client proteins of HSP90. Targeting HSP90 with chemical inhibitors would degrade these oncogenic proteins, and thus serve as useful anticancer agents. This review provides an overview of the HSP chaperone machinery and the structure and function of HSP90. We also highlight the key oncogenic proteins that are regulated by HSP90 and describe how inhibition of HSP90 could alter the activity of multiple signalling proteins, receptors and transcriptional factors implicated in carcinogenesis.
Assuntos
Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Progressão da Doença , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/fisiologia , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Humanos , Modelos Biológicos , Relação Estrutura-AtividadeRESUMO
17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a new anticancer agent currently in clinical trials. The ability of 17-AAG to abrogate the function of heat-shock protein Hsp90 and modulate cellular sensitivity to anticancer agents has prompted recent research to use this compound in drug combination therapy. Here we report that 17-AAG has striking opposite effects on the activity of arsenic trioxide (ATO) and ara-C. Combination of 17-AAG with ATO exhibited a synergistic effect in leukemia cells, whereas coincubation of 17-AAG and ara-C showed antagonistic activity. Mechanistic studies revealed that ATO exerted cytotoxic action by reactive oxygen species generation, and activated Akt survival pathway. 17-AAG abrogated Akt activation and enhanced the activity of ATO. In contrast, treatment of leukemia cells with 17-AAG caused a G1 arrest, a decrease in DNA synthesis and reduced ara-C incorporation into DNA, leading to antagonism. The ability of 17-AAG to enhance the antileukemia activity of ATO was further demonstrated in primary leukemia cells isolated from patients with acute myeloid leukemia and chronic lymphocytic leukemia, including cells from refractory patients. Our data suggest that combination of 17-AAG and ATO may be an effective therapeutic regimen. Caution should be exercised in using 17-AAG together with ara-C, as their combination effects are schedule dependent.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Benzoquinonas/farmacologia , Citarabina/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Mieloide/metabolismo , Óxidos/farmacologia , Doença Aguda , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Arsenicais/antagonistas & inibidores , Citarabina/antagonistas & inibidores , DNA/biossíntese , DNA/efeitos dos fármacos , DNA/metabolismo , Esquema de Medicação , Antagonismo de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Quimioterapia Combinada , Fase G1/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/fisiologia , Células HeLa , Humanos , Células Jurkat , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Mieloide/tratamento farmacológico , Óxidos/antagonistas & inibidores , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries and is currently incurable due, in part, to difficulty in eliminating the leukemia cells protected by stromal microenvironment. Based on previous observations that CLL cells exhibit mitochondrial dysfunction and altered lipid metabolism and that carnitine palmitoyltransferases (CPT) have a major role in transporting fatty acid into mitochondria to support cancer cell metabolism, we tested several clinically relevant inhibitors of lipid metabolism for their ability to eliminate primary CLL cells. We discovered that perhexiline, an antiangina agent that inhibits CPT, was highly effective in killing CLL cells in stromal microenvironment at clinically achievable concentrations. These effective concentrations caused low toxicity to normal lymphocytes and normal stromal cells. Mechanistic study revealed that CLL cells expressed high levels of CPT1 and CPT2. Suppression of fatty acid transport into mitochondria by inhibiting CPT using perhexiline resulted in a depletion of cardiolipin, a key component of mitochondrial membranes, and compromised mitochondrial integrity, leading to rapid depolarization and massive CLL cell death. The therapeutic activity of perhexiline was further demonstrated in vivo using a CLL transgenic mouse model. Perhexiline significantly prolonged the overall animal survival by only four drug injections. Our study suggests that targeting CPT using an antiangina drug is able to effectively eliminate leukemia cells in vivo, and is a novel therapeutic strategy for potential clinical treatment of CLL.
Assuntos
Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Perexilina/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Cardiolipinas/metabolismo , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Glucose/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Consumo de Oxigênio , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mitochondrial DNA (mtDNA) codes for 13 respiratory chain subunits and is more vulnerable to damage than nuclear DNA due, in part, to a lack of histone protection and a weak repair capacity. While mtDNA alterations have been observed in human cancer, their roles in oncogenesis and chemosensitivity remain unclear. We investigated the relationship between mtDNA mutations, reactive oxygen species (ROS) generation, and clinical outcomes in chronic lymphocytic leukemia (CLL) patients. An analysis of mtDNA from 20 CLL patients revealed that primary CLL cells from patients with prior chemotherapy had a significantly higher frequency of heteroplasmic mutations than did those from untreated patients. Overall, mtDNA mutations appeared to be associated with increased ROS generation. Patients refractory to conventional therapeutic agents tended to have higher mutation rates than patients who responded to treatment. Analysis of paired blood samples from the same patient led to the identification of a heteroplasmic mutation in the cytochrome c oxidase II gene several months after chemotherapy. The mutation was associated with increased ROS generation. Our results suggest for the first time that chemotherapy with DNA-damaging agents may cause mtDNA mutations in primary leukemia cells, which often exist in heteroplasmy, and are associated with increased ROS generation.
Assuntos
Antineoplásicos/efeitos adversos , DNA Mitocondrial/genética , Leucemia Linfocítica Crônica de Células B/patologia , Mutação , Adulto , Idoso , Antineoplásicos/uso terapêutico , Análise Mutacional de DNA , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Resultado do TratamentoRESUMO
B cell chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the Western hemisphere, yet many biological and molecular features of the disease remain undefined. CLL cells generate increased levels of radical species such as superoxide and nitric oxide (NO), which is associated with mitochondrial DNA mutations. Considering that NO levels can affect mitochondrial biogenesis, we hypothesized that the inherent nitrosative stress in CLL cells may lead to hyperactive mitochondrial biogenesis. Here we report that primary CLL cells contained significantly more mitochondria than normal lymphocytes and that their mitochondrial mass was significantly related to endogenous NO levels. Expression of the mitochondrial biogenesis factors nuclear respiratory factor-1 and mitochondrial transcription factor A was elevated in most CLL specimens examined and appeared to be related to cellular NO levels. Treatment of B cells with exogenous NO caused a substantial increase in mitochondrial mass. In vitro sensitivity of CLL cells to fludarabine was highly related to mitochondrial mass in that cells with greater mitochondrial mass were less sensitive to the drug. Taken together, our results suggest that NO is a key mediator of mitochondrial biogenesis in CLL and that modulation of mitochondrial biogenesis by NO may alter cellular sensitivity to fludarabine.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/metabolismo , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , Vidarabina/análogos & derivados , Vidarabina/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfócitos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Fator 1 Nuclear Respiratório , Biogênese de Organelas , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasAssuntos
Antígeno B7-H1/imunologia , Mieloma Múltiplo/terapia , Mieloma Múltiplo/virologia , Vírus Oncolíticos/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Humanos , Camundongos , Mieloma Múltiplo/imunologia , Plasmócitos/imunologia , Plasmócitos/virologiaRESUMO
Activating mutation of KRas is a genetic alteration that occurs in the majority of pancreatic tumors and is therefore an ideal therapeutic target. The ability of reoviruses to preferentially replicate and induce cell death in transformed cells that express activated Ras prompted the development of a reovirus-based formulation for cancer therapy called Reolysin. We hypothesized that Reolysin exposure would trigger heavy production of viral products leading to endoplasmic reticular (ER) stress-mediated apoptosis. Here, we report that Reolysin treatment stimulated selective reovirus replication and decreased cell viability in KRas-transformed immortalized human pancreatic duct epithelial cells and pancreatic cancer cell lines. These effects were associated with increased expression of ER stress-related genes, ER swelling, cleavage of caspase-4, and splicing of XBP-1. Treatment with ER stress stimuli including tunicamycin, brefeldin A, and bortezomib (BZ) augmented the anticancer activity of Reolysin. Cotreatment with BZ and Reolysin induced the simultaneous accumulation of ubiquitinated and viral proteins, resulting in enhanced levels of ER stress and apoptosis in both in vitro and in vivo models of pancreatic cancer. Our collective results demonstrate that the abnormal protein accumulation induced by the combination of Reolysin and BZ promotes heightened ER stress and apoptosis in pancreatic cancer cells and provides the rationale for a phase I clinical trial further investigating the safety and efficacy of this novel strategy.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Vírus Oncolíticos/genética , Orthoreovirus de Mamíferos/genética , Reoviridae , Animais , Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Bortezomib , Brefeldina A/farmacologia , Caspases Iniciadoras/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Terapia Combinada , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Orthoreovirus de Mamíferos/fisiologia , Pâncreas/patologia , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Pirazinas/farmacologia , Tunicamicina/farmacologia , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/biossíntese , Proteínas ras/genéticaRESUMO
Oncolytic virotherapy with reovirus has demonstrated anti-cancer activity and minimal toxicity in clinical trials, but the mechanisms underlying these effects have not been fully elucidated. Reolysin, a proprietary formulation of reovirus for cancer therapy, stimulated selective viral replication and apoptosis in multiple myeloma (MM) cells. Reolysin-mediated apoptosis was associated with an induction of endoplasmic reticular (ER) stress-related gene expression, swelling of the endoplasmic reticulum, increases in intracellular calcium levels and a strong induction of the Bcl-2 homology 3 (BH3)-only pro-apoptotic protein NOXA. Knockdown of NOXA expression by short hairpin RNA significantly reduced the pro-apoptotic effects of Reolysin. We next showed that co-administration of Reolysin and bortezomib resulted in the dual accumulation of viral and ubiquitinated proteins, which led to enhanced ER stress, NOXA induction and apoptosis. Importantly, the combination of reovirus infection and proteasomal inhibition significantly decreased tumor burden in a xenograft and syngeneic bone disease model of MM without exhibiting adverse side effects. Our study establishes ER stress stimulation and NOXA induction as novel mediators of reovirus-induced apoptosis. Furthermore, reovirus infection can be used as a promising approach to augment the anti-myeloma activity of bortezomib by promoting additional stress to the endoplasmic reticulum of MM cells.