RESUMO
The antiapoptotic protein Bcl-2 contributes to a more chemoresistant phenotype of nonsmall cell lung cancer and therefore serves as an important target for novel anticancer strategies. Interestingly, docetaxel as a standard of care for treatment of nonsmall cell lung cancer has been shown to inactivate the Bcl-2 function by phosphorylation. We investigated the Bcl-2 expression status of nonsmall cell lung cancer cells in response to cisplatin or docetaxel and its effect on sensitizing nonsmall cell lung cancer cells by Bcl-2 downregulation employing a small interfering RNA approach. Bcl-2 expression was assessed by Western blotting and RT-PCR. Cell proliferation and apoptosis of nonsmall cell lung cancer cells were measured by an MTS-based assay and Annexin V/7-Aminoactinomycin, respectively. Combination treatment of Bcl-2 small interfering RNA with cisplatin resulted in a synergistic activity. By contrast, Bcl-2 downregulation did not sensitize nonsmall cell lung cancer cells to docetaxel. Of note, docetaxel treatment resulted in Bcl-2 phosphorylation of nonsmall cell lung cancer cells, whereas cisplatin increased the Bcl-2 overall expression and abrogated Bcl-2 phosphorylation. On the basis of our findings, a Bcl-2 silencing approach appears to be a suitable strategy for sensitizing nonsmall cell lung cancer to cisplatin, but not to docetaxel.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Taxoides/farmacologia , Apoptose , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Docetaxel , Regulação para Baixo , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Inativação Gênica , Humanos , Neoplasias Pulmonares/patologia , Fosforilação , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Gastric cancer is the second most common cause of death from cancer worldwide and resistant to various chemotherapeutic regimens. In gastric cancer, the anti-apoptotic Mcl-1 protein is expressed in up to 75% of all cases and associated with poor prognosis. The biological relevance of Mcl-1 expression in gastric cancer is unclear. Thus, we investigated the functional significance and potential role of Mcl-1 as a molecular target in gastric cancer by using an antisense strategy. Gastric cancer cell lines (NCI-N87, MKN-28, MKN-45) were treated with Mcl-1 antisense oligonucleotides. Protein expression, cell growth and apoptosis were assessed for single-agent Mcl-1 AS oligonucleotide treatment and for combinations with docetaxel or cisplatin. Treatment by Mcl-1 AS oligonucleotides resulted in approximately 50% reduction in Mcl-1 protein levels in all gastric cancer cell lines examined. Surprisingly, dose-dependent Mcl-1 downregulation produced a significant increase in apoptosis and up to 60% decrease in cell growth. Moreover, combination of Mcl-1 AS oligonucleotide with docetaxel or cisplatin displayed synergistic anti-tumor activity. In conclusion, the impressive single-agent anti-tumor activity and the synergistic effect of Mcl-1 AS oligonucleotides in combination with chemotherapy might qualify Mcl-1 as a promising molecular target for AS oligonucleotide based treatment strategies for gastric cancer in the future.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Neoplasias/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Neoplasias Gástricas/patologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Docetaxel , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Taxoides/farmacologiaRESUMO
Gene silencing by siRNA (short interfering RNA) is a still developing field in biology and has evolved as a novel post-transcriptional gene silencing strategy with therapeutic potential. With siRNAs, virtually every gene in the human genome contributing to a disease becomes amenable to regulation, thus opening unprecedented opportunities for drug discovery. Besides the well-established role for siRNA as a tool for target screening and validation in vitro, recent progress of siRNA delivery in vivo raised expectations for siRNA drugs as the up-and-coming 'magic bullet'. Whether siRNA compounds will make it as novel chemical entities from 'bench to bedside' will probably depend largely on improving their pharmacokinetics in terms of plasma stability and cellular uptake. Whereas locally administered siRNAs have already entered the first clinical trials, strategies for successful systemic delivery of siRNA are still in a preclinical stage of development. Irrespective of its therapeutic potential, RNAi (RNA interference) has unambiguously become a valuable tool for basic research in biology and thereby it will continue to have a major impact on medical science. In this review, we will give a brief overview about the history and current understanding of RNAi and focus on potential applications, especially as a therapeutic option to treat human disease.
Assuntos
Inativação Gênica , RNA Interferente Pequeno/uso terapêutico , Animais , Ensaios Clínicos como Assunto , Marcação de Genes , Terapia Genética/métodos , Humanos , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND/AIMS: Recently, the anti-apoptotic Mcl-1 protein has been reported as a resistance factor in various types of cancer. Here we investigated the presence of Mcl-1 protein in hepatocellular carcinoma (HCC) tissues and its potential role as a molecular drug target for HCC therapy. METHODS: HCC specimens of 149 patients were examined by immunohistochemistry for Mcl-1 expression. Antisense oligonucleotides (ASO) targeting Mcl-1 were evaluated as monotherapy and in combination with cisplatin in the HCC cell lines HepG2 and Snu398. Protein regulation, cell viability, and apoptosis were assessed by western blotting, cell counting, and FACS analysis. RESULTS: Mcl-1 protein is overexpressed in 51% of all cases irrespective of underlying disease. Targeting Mcl-1 by ASO specifically downregulated Mcl-1 protein expression and led to significant dose and time dependent single agent activity in HCC cells characterized by increased apoptosis and decreased cell viability. No significant target regulation or cell death was observed for control oligonucleotide treatment. Upon combination with cisplatin, Mcl-1 ASO revealed a significant chemosensitizing effect. CONCLUSIONS: Mcl-1 is overexpressed in half of HCC-tissues. ASO targeting Mcl-1 revealed a prominent single agent and chemosensitizing activity against HCC in vitro. Targeting Mcl-1 might qualify as a promising novel approach in HCC therapy.
Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Oligonucleotídeos Antissenso/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Neoplásico/genética , Apoptose , Biomarcadores Tumorais , Western Blotting , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Contagem de Células , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Neoplásico/biossíntese , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
PURPOSE: Beta-2 adrenoceptor antagonists such as timolol have been used in the treatment of glaucoma for more than 30 years. Several functionally important polymorphisms for the beta-2 receptor have been described. In the present study we hypothesized that a relation between the intraocular pressure (IOP) lowering effect of timolol and beta-2 adrenoceptor polymorphisms may exist. METHODS: A total of 270 healthy nonsmoking subjects were screened and individuals homozygous for the wild beta-2 Adrenoceptor (Arg16/Gln27) and two polymorphisms (Gly16/Gln27 or Gly16/Glu27) were included. In these subjects the IOP lowering effect of timolol was compared. RESULTS: Twenty-four subjects were included in the group Arg16/Gln27, 18 subjects in the group Gly16/Gln27, and 47 subjects in the group Gly16/Glu27. The ocular hypotensive effect of timolol was between 40 and 45% in all groups, but not significantly different between the three study groups (p=0.979). CONCLUSIONS: The present study indicates that the beta-2 adrenoceptor polymorphism does not influence the ocular hypotensive effects of topical beta adrenoceptor antagonists. Accordingly, other factors appear to be responsible for the intersubject variability seen with timolol in glaucoma subjects.
Assuntos
Antagonistas Adrenérgicos beta/administração & dosagem , Pressão Intraocular/efeitos dos fármacos , Polimorfismo Genético , Receptores Adrenérgicos beta 2/genética , Timolol/administração & dosagem , Administração Tópica , Adulto , Pressão Sanguínea/efeitos dos fármacos , Análise Mutacional de DNA , Genótipo , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Soluções Oftálmicas/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tonometria OcularRESUMO
Cytogenetics has not only precipitated the discovery of several oncogenes, but has also led to the molecular classification of numerous malignancies. The correct identification of aberrations in many tumors has, however, been hindered by extensive tumor complexity and the limitations of molecular cytogenetic techniques. In this study, we have investigated five malignant melanoma (MM) cell lines from at least three different passages using high-resolution R-banding and the recently developed methods of comparative genomic hybridization and multicolor or multiplex fluorescence in situ hybridization. We subsequently detected nine consistent translocations, seven of which were novel: dic(1;11)(p10;q14), der(9)t(3;9)(p12;p11), der(4)t(9;4;7)(q33:p15-q23:q21), der(14)t(5;14)(q12;q32), der(9)t(9;22)(p21;q11), der(19)t(19;20)(p13.3;p11), der(10)t(2;12;7;10)(q31:p12-->pter:q11.2-->q31:q21), der(19)t(10;19)(q23;q13), and der(20)t(Y;20)(q11.23;q13.3). Furthermore, using the human HG-U133A GeneChip, positive expression levels of oncogenes or tumor-related genes located at the regions of chromosomal breakpoints were identified, including AKT1, BMI1, CDK6, CTNNB1, E2F1, GPNMB, GPRK7, KBRAS2, LDB2, LIMK1, MAPK1, MEL, MP1, MUC18, NRCAM, PBX3, RAB22A, RAB38, SNK, and STK4, indicating an association between chromosomal breakpoints and altered gene expression. Moreover, we also show that growth of all five cell lines can be significantly reduced by downregulating CDK6 gene expression with small interfering RNA (siRNA). Because the majority of these breakpoints have been reported previously in MM, our results support the idea of common mechanisms in this disease.
Assuntos
Quinases Ciclina-Dependentes/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Translocação Genética , Linhagem Celular , Linhagem Celular Tumoral , Aberrações Cromossômicas , Bandeamento Cromossômico , Quebra Cromossômica , Quinase 6 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Citogenética , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TemperaturaRESUMO
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects. In this report, a new method for synthesizing oligonucleotide conjugates is described. 2'-Amino-2'-deoxy-5'-dimethoxytrityl-uridine was select ively acylated with a succinic acid linker at the 2' position. This compound was incorporated at the 3' end of an oligonucleotide corresponding to the sequence of Oblimersen. The carboxyl group was protected for oligonucleotide synthesis as a benzyl ester, which could be selectively cleaved at the solid phase by a catalytic phase transfer reaction using palladium nanoparticles as catalyst. An oligonucleotide-fluorescein conjugate was prepared by condensation of aminofluorescein. Circular dichroism spectroscopic experiments showed a B-DNA type structure. The melting temperature of the duplex was only slightly lower than that of Oblimersen. Biological activity measured by western blotting resulted in a Bcl-2 target downregulation nearly identical to that of control Oblimersen on human melanoma cells, proving that this method is attractive for the binding of ligands located in the minor groove.
Assuntos
Oligonucleotídeos Antissenso/química , Uridina/análogos & derivados , Uridina/química , Sequência de Bases , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Códon/genética , DNA/química , DNA/genética , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Desnaturação de Ácido Nucleico , Oligonucleotídeos Antissenso/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura de Transição , Uridina/síntese química , Uridina/genéticaRESUMO
Malignant melanoma is a prime example of a treatment-resistant tumor with poor prognosis. Even with innovative treatment regimens, response rates remain low, and the duration of responses is short. More than 90% of all melanomas express the antiapoptotic protein Bcl-2, shown to contribute to a chemoresistant phenotype in melanoma. We previously demonstrated that antisense-mediated inhibition of Bcl-2 sensitizes malignant melanoma to apoptosis-inducing treatment modalities. In the present study, we evaluated synthetic small interfering RNA (siRNA) compounds targeting Bcl-2 as a novel approach to downregulate Bcl-2 expression in melanoma cells. siRNA treatment led up to a 19-fold reduction of bcl-2 mRNA levels and only barely detectable Bcl-2 protein expression at low nanomolar concentrations. Silencing of Bcl-2 in melanoma cells by specific siRNA led to a moderate increase in apoptotic cell death and inhibition of cell growth. However, if siRNA compounds targeting Bcl-2 were combined with the apoptosis-inducing chemotherapeutic agent cisplatin, a massive increase in apoptotic cell death compared with controls was observed. Notably, the combination of Bcl2 siRNA and low-dose cisplatin resulted in a supra-additive effect, with nearly complete suppression of cell growth, whereas cell growth in cisplatin-only-treated cells was only moderately affected (96% vs. 25%, p < 0.001). These findings underline a key role for Bcl-2 in conferring chemoresistance to melanoma and highlight Bcl-2 siRNA strategies as novel and highly effective tools, with the potential for future targeted therapy of malignant melanoma.