RESUMO
The molecular chaperone Hsp90 is a ubiquitous ATPase-directed protein responsible for the activation and structural stabilization of a large clientele of proteins. As such, Hsp90 has emerged as a suitable candidate for the treatment of a diverse set of diseases, such as cancer and neurodegeneration. The inhibition of the chaperone through ATP-competitive inhibitors, however, was shown to lead to undesirable side effects. One strategy to alleviate this problem is the development of molecules that are able to disrupt specific protein-protein interactions, thus modulating the activity of Hsp90 only in the particular cellular pathway that needs to be targeted. Here, we exploit novel computational and theoretical approaches to design a set of peptides that are able to bind Hsp90 and compete for its interaction with the co-chaperone Cdc37, which is found to be responsible for the promotion of cancer cell proliferation. In spite of their capability to disrupt the Hsp90-Cdc37 interaction, no important cytotoxicity was observed in human cancer cells exposed to designed compounds. These findings imply the need for further optimization of the compounds, which may lead to new ways of interfering with the Hsp90 mechanisms that are important for tumour growth.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Chaperoninas/antagonistas & inibidores , Desenho de Fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Conformação ProteicaRESUMO
We report the engineering of intracellular redox-responsive nanoporous poly(ethylene glycol)-poly(l-lysine) particles (NPEG-PLLs). The obtained particles exhibit no toxicity while maintaining the capability to deliver a small interfering RNA sequence (siRNA) targeting the anti-apoptotic factor, survivin, in prostate cancer cells. The redox-mediated cleavage of the disulfide bonds stabilizing the NPEG-PLL-siRNA complex results in the release of bioactive siRNA into the cytosol of prostate cancer PC-3 cells, which, in turn, leads to the effective silencing (â¼59 ± 8%) of the target gene. These findings, obtained under optimal conditions, indicate that NPEG-PLLs may protect the therapeutic nucleic acid in the extracellular and intracellular environments, thus preventing the occurrence of competitive interactions with serum and cytosolic proteins as well as degradation by RNase. The intracellular trafficking and final fate of the NPEG-PLLs were investigated by a combination of deconvolution microscopy, fluorescence lifetime imaging microscopy, and super-resolution structured illumination microscopy. A significant impairment of cell survival was observed in cells concomitantly exposed to paclitaxel and siRNA-loaded NPEG-PLLs. Overall, our findings indicate that NPEG-PLLs represent a highly loaded depot for the delivery of therapeutic nucleic acids to cancer cells.
Assuntos
Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Nanopartículas/química , Neoplasias da Próstata/metabolismo , RNA Interferente Pequeno/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Paclitaxel/farmacologia , Polietilenoglicóis/química , Polilisina/química , RNA Interferente Pequeno/química , SurvivinaRESUMO
The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133+, epithelial-specific antigen-positive (CD133+ESA+) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133(-) counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133+ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133+ABCG2+ and CD133+CXCR4+ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133+ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.
Assuntos
Antígenos CD/metabolismo , Cisplatino/farmacologia , Glicoproteínas/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeos/metabolismo , Antígeno AC133 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores CXCR4/metabolismo , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Drug resistance limits the achievement of persistent cures for the treatment of melanoma, in spite of the efficacy of the available drugs. The aim of the present study was to explore the involvement of lipid metabolism in melanoma resistance and assess the effects of its targeting in cellular models of melanoma with acquired resistance to the BRAF-inhibitor PLX4032/Vemurafenib. Since transcriptional profiles pointed to decreased cholesterol and fatty acids synthesis in resistant cells as compared to their parental counterparts, we examined lipid composition profiles of resistant cells, studied cell growth dependence on extracellular lipids, assessed the modulation of enzymes controlling the main nodes in lipid biosynthesis, and evaluated the effects of targeting Acetyl-CoA Acetyltransferase 2 (ACAT2), the first enzyme in the cholesterol synthesis pathway, and Acyl-CoA Cholesterol Acyl Transferase (ACAT/SOAT), which catalyzes the intracellular esterification of cholesterol and the formation of cholesteryl esters. We found a different lipid composition in the resistant cells, which displayed reduced saturated fatty acids (SFA), increased monounsaturated (MUFA) and polyunsaturated (PUFA), and reduced cholesteryl esters (CE) and triglycerides (TG), along with modulated expression of enzymes regulating biosynthetic nodes of the lipid metabolism. The effect of tackling lipid metabolism pathways in resistant cells was evidenced by lipid starvation, which reduced cell growth, increased sensitivity to the BRAF-inhibitor PLX4032, and induced the expression of enzymes involved in fatty acid and cholesterol metabolism. Molecular targeting of ACAT2 or pharmacological inhibition of SOAT by avasimibe showed antiproliferative effects in melanoma cell lines and a synergistic drug interaction with PLX4032, an effect associated to increased ferroptosis. Overall, our findings reveal that lipid metabolism affects melanoma sensitivity to BRAF inhibitors and that extracellular lipid availability may influence tumor cell response to treatment, a relevant finding in the frame of personalized therapy. In addition, our results indicate new candidate targets for drug combination treatments.
RESUMO
Reconfiguring the structure and selectivity of existing chemotherapeutics represents an opportunity for developing novel tumor-selective drugs. Here, as a proof-of-concept, the use of high-frequency sound waves is demonstrated to transform the nonselective anthracycline doxorubicin into a tumor selective drug molecule. The transformed drug self-aggregates in water to form ≈200 nm nanodrugs without requiring organic solvents, chemical agents, or surfactants. The nanodrugs preferentially interact with lipid rafts in the mitochondria of cancer cells. The mitochondrial localization of the nanodrugs plays a key role in inducing reactive oxygen species mediated selective death of breast cancer, colorectal carcinoma, ovarian carcinoma, and drug-resistant cell lines. Only marginal cytotoxicity (80-100% cell viability) toward fibroblasts and cardiomyocytes is observed, even after administration of high doses of the nanodrug (25-40 µg mL-1 ). Penetration, cytotoxicity, and selectivity of the nanodrugs in tumor-mimicking tissues are validated by using a 3D coculture of cancer and healthy cells and 3D cell-collagen constructs in a perfusion bioreactor. The nanodrugs exhibit tropism for lung and limited accumulation in the liver and spleen, as suggested by in vivo biodistribution studies. The results highlight the potential of this approach to transform the structure and bioactivity of anticancer drugs and antibiotics bearing sono-active moieties.
Assuntos
Nanopartículas , Neoplasias Ovarianas , Antibióticos Antineoplásicos/química , Doxorrubicina/química , Doxorrubicina/farmacologia , Humanos , Nanopartículas/química , Distribuição TecidualRESUMO
Bis-2,3-heteroarylmaleimides and polyheterocondensed imides joined through nitrogen atoms of the N,N'-bis(ethyl)-1,3-propanediamine linker were prepared from substituted maleic anhydrides and symmetrical diamines in good to satisfactory yields and short reaction times using microwave heating. The novel molecules were shown to inhibit proliferation of human tumor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle cells (SMCs) with variable potencies. Compound 11a, the most potent one of the series, showed IC(50) values comparable to those observed for the leading molecule elinafide in both cell lines, but with a higher selectivity toward human tumor cells. Compound 11a affected G1/S phase transition of the cell cycle, showed in vitro DNA intercalating activity and in vivo antitumor activity. A thorough structural analysis of the 11a-DNA complex was also made by mean of NMR and computational techniques.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Aorta/efeitos dos fármacos , Imidas/síntese química , Imidas/farmacologia , Maleimidas/síntese química , Maleimidas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Antineoplásicos/química , Aorta/citologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Imidas/química , Maleimidas/química , Modelos Moleculares , Estrutura Molecular , Músculo Liso Vascular/citologia , Ratos , Relação Estrutura-AtividadeRESUMO
Metabolic changes promoting cell survival are involved in metastatic melanoma progression and in the development of drug resistance. In BRAF-inhibitor resistant melanoma cells, we explored the role of FASN, an enzyme involved in lipogenesis overexpressed in metastatic melanoma. Resistant melanoma cells displaying enhanced migratory and pro-invasive abilities increased sensitivity to the BRAF inhibitor PLX4032 upon the molecular targeting of FASN and upon treatment with the FASN inhibitor orlistat. This behavior was associated with a marked apoptosis and caspase 3/7 activation observed for the drug combination. The expression of FASN was found to be inversely associated with drug resistance in BRAF-mutant cell lines, both in a set of six resistant/sensitive matched lines and in the Cancer Cell Line Encyclopedia. A favorable drug interaction in resistant cells was also observed with U18666 A inhibiting DHCR24, which increased upon FASN targeting. The simultaneous combination of the two inhibitors showed a synergistic interaction with PLX4032 in resistant cells. In conclusion, FASN plays a role in BRAF-mutated melanoma progression, thereby creating novel therapeutic opportunities for the treatment of melanoma.
RESUMO
Speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) is the most commonly mutated gene in prostate cancer (PCa). Recent evidence reports a role of SPOP in DNA damage response (DDR), indicating a possible impact of SPOP deregulation on PCa radiosensitivity. This study aimed to define the role of SPOP deregulation (by gene mutation or knockdown) as a radiosensitizing factor in PCa preclinical models. To express WT or mutant (Y87N, K129E and F133V) SPOP, DU145 and PC-3 cells were transfected with pMCV6 vectors. Sensitivity profiles were assessed using clonogenic assay and immunofluorescent staining of γH2AX and RAD51 foci. SCID xenografts were treated with 5 Gy single dose irradiation using an image-guided small animal irradiator. siRNA and miRNA mimics were used to silence SPOP or express the SPOP negative regulator miR-145, respectively. SPOP deregulation, by either gene mutation or knockdown, consistently enhanced the radiation response of PCa models by impairing DDR, as indicated by transcriptome analysis and functionally confirmed by decreased RAD51 foci. SPOP silencing also resulted in a significant downregulation of RAD51 and CHK1 expression, consistent with the impairment of homologous recombination. Our results indicate that SPOP deregulation plays a radiosensitizing role in PCa by impairing DDR via downregulation of RAD51 and CHK1.
RESUMO
A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 degrees C. Structural models of the complex with d(CGTACG)(2) were developed on the basis of DOSY, 2D NOESY and (31)P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.
Assuntos
Antraquinonas/química , DNA/química , Pironas/química , Inibidores da Topoisomerase I , Antraquinonas/farmacologia , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Humanos , Substâncias Intercalantes , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Pironas/farmacologia , SolubilidadeRESUMO
Because cytotoxic stress elicits various signaling pathways that may be implicated in cell survival or cell death, their alterations may have relevance in the development of platinum-resistant phenotype. Thus, in the present study, we investigated cell response to the epidermal growth factor receptor (EGFR) inhibitor gefitinib of ovarian carcinoma cell lines, including cells selected for resistance to cisplatin (IGROV-1/Pt1) and oxaliplatin (IGROV-1/OHP). Resistant sublines exhibited a marked decrease in sensitivity to gefitinib and resistance to apoptosis. Gefitinib was capable of inhibiting the phosphorylation of EGFR in all the studied cell lines. The Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, which act downstream of EGFR, were constitutively active in the three cell lines, but phospho-ERK1/2 levels were increased in the two resistant sublines. This feature was associated with reduced sensitivity to the MEK1/2 inhibitor U0126. Pretreatment of resistant cells with U0126 resulted in restoration of sensitivity to gefitinib. Gefitinib was more effective in inhibiting ERK1/2 and Akt phosphorylation in IGROV-1 cells than in IGROV-1/OHP and IGROV-1/Pt1 cells. Phospho-p38 was up-regulated in the resistant sublines, indicating the concomitant activation of distinct mitogen-activated protein kinases. The up-regulation of phospho-p38 was associated with a peculiar localization of EGFR, which, in resistant sublines, was mainly internalized. In conclusion, our results indicate that the development of resistance to platinum drugs is associated with multiple alterations including deregulation of survival pathways activated by EGFR resulting in a reduced cellular response to gefitinib.
Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular , Neoplasias Ovarianas/patologia , Compostos de Platina/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , FosforilaçãoRESUMO
BACKGROUND: Radiotherapy is one of the main treatment options for non-metastatic prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a considerable fraction of patients still experience relapse due to the development of resistance. Radioresistance is a complex and still poorly understood phenomenon involving the deregulation of a variety of signaling pathways as a consequence of several genetic and epigenetic abnormalities. In this context, cumulative evidence supports a functional role of microRNAs in affecting radioresistance, suggesting the modulation of their expression as a novel radiosensitizing approach. Here, we investigated for the first time the ability of miR-205 to enhance the radiation response of PCa models. METHODS: miR-205 reconstitution by a miRNA mimic in PCa cell lines (DU145 and PC-3) was used to elucidate miR-205 biological role. Radiation response in miRNA-reconstituted and control cells was assessed by clonogenic assay, immunofluorescence-based detection of nuclear γ-H2AX foci and comet assay. RNAi was used to silence the miRNA targets PKCε or ZEB1. In addition, target-protection experiments were carried out using a custom oligonucleotide designed to physically disrupt the pairing between the miR-205 and PKCε. For in vivo experiments, xenografts generated in SCID mice by implanting DU145 cells stably expressing miR-205 were exposed to 5-Gy single dose irradiation using an image-guided animal micro-irradiator. RESULTS: miR-205 reconstitution was able to significantly enhance the radiation response of prostate cancer cell lines and xenografts through the impairment of radiation-induced DNA damage repair, as a consequence of PKCε and ZEB1 inhibition. Indeed, phenocopy experiments based on knock-down of either PKCε or ZEB1 reproduced miR-205 radiosensitizing effect, hence confirming a functional role of both targets in the process. At the molecular level, miR-205-induced suppression of PKCε counteracted radioresistance through the impairment of EGFR nuclear translocation and the consequent DNA-PK activation. Consistently, disruption of miR-205-PKCε 3'UTR pairing almost completely abrogated the radiosensitizing effect. CONCLUSIONS: Our results uncovered the molecular and cellular mechanisms underlying the radiosensitizing effect of miR-205. These findings support the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa response to radiotherapy.
Assuntos
MicroRNAs/genética , Neoplasias da Próstata/radioterapia , Proteína Quinase C-épsilon/antagonistas & inibidores , Tolerância a Radiação/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Reparo do DNA/genética , Humanos , Masculino , Camundongos , Camundongos SCID , Mimetismo Molecular , Células PC-3 , Proteína Quinase C-épsilon/genética , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
The Schizosaccharomyces pombe rad17 is a checkpoint protein critical for maintenance of genomic stability. Since the loss of checkpoint control is a common feature of tumor cells, we investigated the biological function of the human homolog hRAD17. Expression of hRAD17 in a fission yeast rad17 deleted strain reduced growth of yeast colonies and caused slower progression through cell cycle. Immunoprecipitated hRad17 exhibited exonuclease activity. hRAD17 delayed growth of NIH3T3 fibroblasts transformed by the H-ras oncogene in nude mice. Our results support that hRAD17, similarly to other human genes involved in checkpoint mechanisms, plays a role in control of tumor growth.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Neoplasias Experimentais/etiologia , Animais , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Processos de Crescimento Celular , Linhagem Celular Transformada , Exonucleases/isolamento & purificação , Exonucleases/metabolismo , Feminino , Fase G1 , Deleção de Genes , Humanos , Imunoprecipitação , Camundongos , Camundongos Nus , Células NIH 3T3 , Neoplasias Experimentais/patologia , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
A series of structurally simple analogues of natural topopyrone C were synthesized and tested for cytotoxic and topoisomerase I inhibitory activities. The removal of the hydroxyl groups at the 5 and 9 positions resulted in an increased cytotoxic potency and ability to stabilize topoisomerase-mediated cleavage. In addition, the results suggest that some structural features, such as the pyrone ring and a polar group in position 11, are fundamental for topoisomerase I inhibitory effect. These structural requirements are also consistent with the cytotoxic activity.
Assuntos
Antraquinonas/síntese química , Antraquinonas/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Pironas/síntese química , Pironas/farmacologia , Inibidores da Topoisomerase I , Antraquinonas/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/tratamento farmacológico , Pironas/química , Relação Estrutura-AtividadeRESUMO
A series of novel 9-substituted camptothecins derived from 9-formylcamptothecin were synthesized. The aldehyde was obtained from 10-hydroxycamptothecin or, better, by total synthesis. The compounds showed antiproliferative activity higher than that of the reference compound topotecan. Modelling suggested the possibility of a favourable interaction of small and polar 9-substituents with the topoisomerase I-DNA complex, which is consistent with the higher activity of these derivatives with respect to the corresponding 7-substituted camptothecins.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Antineoplásicos Fitogênicos/síntese química , Camptotecina/análogos & derivados , Camptotecina/síntese química , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
A series of 2,3-heteroarylmaleimides 9 and polyheterocondensed imides 12 were prepared in good yields and short reaction time using a very efficient procedure consisting in the condensation of the corresponding anhydrides and N,N-diethylethylenediamine and microwave heating. The antiproliferative activity of the novel molecules was tested against human tumor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle cells (SMCs). The IC50 values for the novel molecules ranged from 0.08 to 13.9 microM in SMCs, and from 0.84 to 9 microM in the tumor cell line. The activity profile for compounds 9 and 12 is comparable to that obtained for amonafide in NCI-H460, except for fused imides 12b,i which proved to be about 10-fold more potent. Whereas, in rat SMCs, only the compound 12b was shown to be 10-fold more potent than amonafide. Instead 12c is equipotent to amonafide. These results suggest that the extended pi-system and the kind of heteroatom are essential in the binding with the molecular target.
Assuntos
Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Imidas/farmacologia , Maleimidas/farmacologia , Animais , Antineoplásicos/farmacologia , Aorta , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Imidas/síntese química , Concentração Inibidora 50 , Maleimidas/síntese química , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Relação Estrutura-AtividadeRESUMO
A series of water-soluble camptothecins obtained by linking a spermidine moiety to the 21-position of the open form through an amidic bond have been tested for their biochemical and biological activities. Growth inhibition assay on the human non-small cell lung cancer carcinoma NCI-H460 cell line revealed that the camptothecin analogues were less potent than topotecan and SN38 after 1 hour of treatment. The potency increased after 72 hours of exposure, being similar to that of reference camptothecins. The analysis of topoisomerase I-mediated DNA cleavage using the purified enzyme indicated that the novel camptothecin analogues retained ability to poison topoisomerase I and displayed the same cleavage pattern of SN38. Persistence of the DNA cleavage was comparable with that of SN38. Stabilization of the cleavable complex was not the result of hydrolysis of the N-C bond between polyamine and the drug because no free camptothecin was recovered at the end of DNA cleavage in presence of IDN5174, the analogue selected for detailed studies. IDN5174 exhibited an antitumor activity comparable with that of topotecan and irinotecan against NCI-H460 tumor xenograft. The pharmacokinetics in mice showed a favorable disposition in tumor tissue with low amount of camptothecin detectable in plasma and tumor (around 5-10%), thus supporting the efficacy of intact IDN5174. In conclusion, we found that IDN5174 maintained the biological and antitumor properties, in spite of lack of the closed E ring. The available results support the interpretation that the polyamine linked at the 21-position may allow a favorable drug interaction in the ternary complex.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Animais , Antineoplásicos Fitogênicos/farmacocinética , Camptotecina/farmacocinética , Camptotecina/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA Topoisomerases Tipo I/metabolismo , Humanos , Lactonas/farmacocinética , Lactonas/farmacologia , Camundongos , Espermidina/análogos & derivados , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In previous studies, we have documented the potential therapeutic advantages of camptothecin analogs modified at the 7-position, i.e., 7-oxyiminomethyl derivatives. The present study was performed to explore the therapeutic potential of novel hydrophilic derivatives of this series. With one exception (ST1976), the tested camptothecins exhibited a reduced antiproliferative activity and all compounds retained ability to stabilize the topoisomerase I-mediated cleavable complex. The two analogs (ST1976 and ST1968) characterized by the presence of a free amino group in the side chain also exhibited the formation of persistent cleavable complexes. The most potent compound, ST1976 (7-(4-aminobenzyl)oxyiminomethylcamptothecin), was selected for evaluation of its preclinical profile of antitumor activity in a large panel of human tumor xenografts. As expected on the basis of the introduction of a hydrophilic substituent, the novel camptothecin was a substrate for BCRP. However, in spite of an apparent recognition by BCRP, ST1976 was effective following oral administration. The antitumor activity was evaluated using various schedules and routes of administration (i.v. and p.o.). ST1976 exhibited a remarkable activity in all tested tumors and was effective in a number of tumors which are resistant to irinotecan. The biological and pharmacological profile of ST1976 supports the therapeutic potential of camptothecins containing hydrophilic substituents at the 7-position. On the basis of its excellent activity in preclinical models, ST1976 is a promising candidate for clinical development.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Animais , Antineoplásicos/administração & dosagem , Camptotecina/administração & dosagem , Camptotecina/farmacologia , Linhagem Celular Tumoral , Clivagem do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Irinotecano , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológicoRESUMO
A number of derivatives of camptothecin with a polyamine chain linked to position 7 of camptothecin via an amino, imino, or oxyiminomethyl group were synthesized and tested for their biological activity. All compounds showed marked growth inhibitory activity against the H460 human lung carcinoma cell line. In particular, the iminomethyl derivatives where the amino groups of the chain were protected with Boc groups exhibited a high potency, with IC50 values of approximately 10(-8) M. The pattern of DNA cleavage in vitro and the persistence of the cleavable ternary complex drug-DNA-topoisomerase I observed with polyamine conjugates containing free amino groups support a contribution of specific drug interaction with DNA as a determinant of activity. Modeling of compound 7c in the complex with topoisomerase 1 and DNA is consistent with this hypothesis. The lack of a specific correlation between stabilization of the cleavable complex and growth inhibition likely reflects multiple factors including the cellular pharmacokinetic behavior related to the variable lipophilicity of the conjugate, and the nature and linkage of the polyamine moiety.
Assuntos
Camptotecina/síntese química , Camptotecina/farmacologia , Poliaminas/química , Camptotecina/análogos & derivados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrólise , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Redox-active polymers and carriers are oxidizing nanoagents that can potentially trigger intracellular off-target effects. In the present study, we investigated the occurrence of off-target effects in prostate cancer cells following exposure to redox-active polymer and thin multilayer capsules with different chemical properties. We show that, depending on the intracellular antioxidant capacity, thiol-functionalized poly(methacrylic acid), PMA(SH) triggers cell defense responses/perturbations that result in off-target effects (i.e., induction of autophagy and down-regulation of survivin). Importantly, the conversion of the carboxyl groups of PMA(SH) into the neutral amides of poly(hydroxypropylmetacrylamide) (pHPMA(SH)) nullified the off-target effects and cytotoxicity in tested cell lines. This suggests that the simultaneous action of carboxyl and disulfide groups in PMA(SH) polymer or capsules may play a role in mediating the intracellular off-target effects. Our work provides evidence that the rational design of redox-active carriers for therapeutic-related application should be guided by a careful investigation on potential disturbance of the cellular machineries related to the carrier association.
Assuntos
Acrilamidas , Antioxidantes , Teste de Materiais , Ácidos Polimetacrílicos , Neoplasias da Próstata/metabolismo , Acrilamidas/química , Acrilamidas/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Cápsulas , Linhagem Celular Tumoral , Humanos , Masculino , Oxirredução , Ácidos Polimetacrílicos/química , Ácidos Polimetacrílicos/farmacologia , Neoplasias da Próstata/patologiaRESUMO
The molecular mechanism of cisplatin uptake remains poorly defined and impaired drug accumulation may be implicated in the acquisition of resistance to cisplatin. Thus, we used cell lines of different tumor types (ovarian carcinoma A2780 and IGROV-1, osteosarcoma U2-OS, cervix squamous cell carcinoma A431) and stable cisplatin-resistant sublines, exhibiting variable levels of resistance (between 2.5 and 18.4), to investigate the mechanisms of cellular accumulation of cisplatin. Among the resistant lines we found that reduced cisplatin uptake was a common feature and ranged between 23 and 76%. In an attempt to examine the role of human copper transporter 1 (CTR1) in cisplatin accumulation by human cells, we selected the well characterized A431 cell line and the resistant variant A431/Pt. As compared with A431/Pt cells, A431/Pt transfectants overexpressing CTR1 (3.4-fold) exhibited increased uptake of copper, thereby supporting the expression of a functional transporter. However, no changes in cisplatin uptake and cellular sensitivity to drug were observed. Also overexpression of CTR1 in A431 cells did not produce modulation of cisplatin accumulation. An analysis of the expression of other factors that could affect drug accumulation indicated that A431/Pt cells displayed increased expression of ATPase, Cu(2+) transporting, alfa polypeptide. In conclusion, our results indicate that the overexpression of a functional CTR1 in a human cell line characterized by impaired cisplatin uptake fails (a) to restore cellular drug accumulation to the level of the parental cell line and (b) to modulate cisplatin sensitivity. Our data are consistent with the interpretation that the defects in cellular accumulation by resistant cells are not mediated by expression of CTR1, that plays a marginal role, if any, in cisplatin transport.