RESUMO
BACKGROUND: Progression to stage IV disease remains the main cause of breast cancer-related deaths. Increasing knowledge on the hematogenous phase of metastasis is key for exploiting the entire window of opportunity to interfere with early dissemination and to achieve a more effective disease control. Recent evidence suggests that circulating tumor cells (CTCs) possess diverse adaptive mechanisms to survive in blood and eventually metastasize, encouraging research into CTC-directed therapies. METHODS: On the hypothesis that the distinguishing molecular features of CTCs reveal useful information on metastasis biology and disease outcome, we compared the transcriptome of CTCs, primary tumors, lymph-node and lung metastases of the MDA-MB-231 xenograft model, and assessed the biological role of a panel of selected genes, by in vitro and in vivo functional assays, and their clinical significance in M0 and M+ breast cancer patients. RESULTS: We found that hematogenous dissemination is governed by a transcriptional program and identified a CTC signature that includes 192 up-regulated genes, mainly related to cell plasticity and adaptation, and 282 down-regulated genes, involved in chromatin remodeling and transcription. Among genes up-regulated in CTCs, FADS3 was found to increases cell membrane fluidity and promote hematogenous diffusion and lung metastasis formation. TFF3 was observed to be associated with a subset of CTCs with epithelial-like features in the experimental model and in a cohort of 44 breast cancer patients, and to play a role in cell migration, invasion and blood-borne dissemination. The analysis of clinical samples with a panel of CTC-specific genes (ADPRHL1, ELF3, FCF1, TFF1 and TFF3) considerably improved CTC detection as compared with epithelial and tumor-associated markers both in M0 and stage IV patients, and CTC kinetics informed disease relapse in the neoadjuvant setting. CONCLUSIONS: Our findings provide evidence on the potential of a CTC-specific molecular profile as source of metastasis-relevant genes in breast cancer experimental models and in patients. Thanks to transcriptome analysis we generated a novel CTC signature in the MDA-MB-231 xenograft model, adding a new piece to the current knowledge on the key players that orchestrate tumor cell hematogenous dissemination and breast cancer metastasis, and expanding the list of CTC-related biomarkers for future validation studies.
Assuntos
Neoplasias da Mama , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismoRESUMO
Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.
RESUMO
Hematogenous dissemination may occur early in breast cancer (BC). Experimental models could clarify mechanisms, but in their development, the heterogeneity of this neoplasia must be considered. Here, we describe circulating tumor cells (CTCs) and the metastatic behavior of several BC cell lines in xenografts. MDA-MB-231, BT-474, MDA-MB-453 and MDA-MB-468 cells were injected at the orthotopic level in immunocompromised mice. CTCs were isolated using a size-based method and identified by cytomorphological criteria. Metastases were detected by COX IV immunohistochemistry. CTCs were detected in 90% of animals in each model. In MDA-MB-231, CTCs were observed after 5 weeks from the injection and step wisely increased at later time points. In animals injected with less aggressive cell lines, the load of single CTCs (mean ± SD CTCs/mL: 1.8 ± 1.3 in BT-474, 122.2 ± 278.5 in MDA-MB-453, 3.4 ± 2.5 in MDA-MB468) and the frequency of CTC clusters (overall 38%) were lower compared to MDA-MB231 (946.9 ± 2882.1; 73%). All models had lung metastases, MDA-MB-453 and MDA-MB468 had ovarian foci too, whereas lymph nodal involvement was observed in MDA-MB231 and MDA-MB-468 only. Interestingly, CTCs showed morphological heterogeneity and were rarely associated to host cells. Orthotopic xenograft of BC cell lines offers valid models of hematogenous dissemination and a possible experimental setting to study CTC-blood microenvironment interactions.
Assuntos
Neoplasias da Mama/patologia , Neoplasias Pulmonares/secundário , Células Neoplásicas Circulantes , Animais , Neoplasias da Mama/sangue , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Transplante Heterólogo , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer.
Assuntos
Proteína Coatomer/metabolismo , Terapêutica com RNAi , Neoplasias da Glândula Tireoide/terapia , Animais , Apoptose , Autofagia , Linhagem Celular Tumoral , Sobrevivência Celular , Proteína Coatomer/genética , Estresse do Retículo Endoplasmático , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Nus , Interferência de RNA , Transdução de Sinais , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Fatores de Tempo , Transfecção , Carga Tumoral , Resposta a Proteínas não Dobradas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR), an active polar metabolite of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR), was shown to exert promising antitumor activity through at least two independent mechanisms of action. Specifically, differently from 4-HPR and other retinoids, 4-oxo-4-HPR targets microtubules and inhibits tubulin polymerization causing mitotic arrest and on the other hand, analogously to the parent drug, it induces apoptosis through the activation of a signaling cascade involving the generation of reactive oxygen species (ROS). However, the potential in vivo use of 4-oxo-4-HPR is impaired by its poor solubility. By chemical modification of 4-oxo-4-HPR, a new class of compounds with improved solubility and in vivo bioavailability was obtained. We demonstrated here that, among them, the most promising molecule, sodium 4-carboxymethoxyimino-(4-HPR), was endowed with in vitro antitumor efficacy and entirely preserved the double mechanism of action of the parent drug in cancer cells of different histotypes. In fact, the retinoid induced the activation of the apoptotic cascade related to the generation of ROS through endoplasmic reticulum stress response and upregulation of phospho c-Jun N-terminal kinases and PLAcental Bone morphogenetic protein, leading to cell death through caspase-3 cleavage. Otherwise, sodium 4-carboxymethoxyimino-(4-HPR) caused a marked mitotic arrest coupled with multipolar spindle formation and tubulin depolymerization. To assess the compound antitumor activity, in vivo experiments were performed in three mouse xenograft models (ovarian and breast cancers and mesothelioma). The in vivo results demonstrated that retinoid administration as single agent significantly increased the survival in ovarian cancer xenografts, induced a statistically significant decrease in tumor growth in breast cancer xenografts, and caused a 30% reduction in tumor growth in a mesothelioma mouse model. Even though further studies investigating sodium 4-carboxymethoxyimino-(4-HPR) toxicity and in vitro and in vivo activities in combination with other drugs are required, the double mechanism of action of the retinoid coupled with its in vivo antitumor efficacy and potential low toxicity suggest a promising therapeutic potential for the compound in different solid tumors.
RESUMO
Melanoma is a highly heterogeneous tumor for which recent evidence supports a model of dynamic stemness. Melanoma cells might temporally acquire tumor-initiating properties or switch from a status of tumor-initiating cells (TICs) to a more differentiated one depending on the tumor context. However, factors driving these functional changes are still unknown. We focused on the role of cyto/chemokines in shaping TICs isolated directly from tumor specimens of two melanoma patients, namely Me14346S and Me15888S. We analyzed the secretion profile of TICs and of their corresponding melanoma differentiated cells and we tested the ability of cyto/chemokines to influence TIC self-renewal and differentiation. We found that TICs, grown in vitro as melanospheres, had a complex secretory profile as compared to their differentiated counterparts. Some factors, such as CCL-2 and IL-8, also produced by adherent melanoma cells and melanocytes did not influence TIC properties. Conversely, IL-6, released by differentiated cells, reduced TIC self-renewal and induced TIC differentiation while IL-10, produced by Me15888S, strongly promoted TIC self-renewal through paracrine/autocrine actions. Complete neutralization of IL-10 activity by gene silencing and antibody-mediated blocking of the IL-10Rα was required to sensitize Me15888S to IL-6-induced differentiation. For the first time these results show that functional heterogeneity of melanoma could be directly influenced by inflammatory and suppressive soluble factors, with IL-6 favoring TIC differentiation, and IL-10 supporting TIC self-renewal. Thus, understanding the tumor microenvironment (TME) role in modulating melanoma TIC phenotype is fundamental to identifying novel therapeutic targets to achieve long-lasting regression of metastatic melanoma. Stem Cells 2016;34:2449-2460.
Assuntos
Linhagem da Célula/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Melanoma/patologia , Células-Tronco Neoplásicas/patologia , Comunicação Autócrina/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Autorrenovação Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Melanoma/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Testes de Neutralização , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Receptores de Quimiocinas/metabolismo , Esferoides Celulares/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A novel series of 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) derivatives were synthesized with the aim of increasing the poor solubility of the parent compound in biological fluids, while maintaining the cytotoxic activity and the dual mechanism of action. The most promising compound 13a showed antiproliferative/apoptotic activity. The analysis of its mechanism of action revealed that it retained the particular characteristic of 4-oxo-4-HPR which is able to induce cell cycle arrest during the mitotic phase, coupled with the formation of aberrant mitotic spindles.
Assuntos
Apoptose/efeitos dos fármacos , Fenretinida/síntese química , Fenretinida/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fenretinida/análogos & derivados , Fenretinida/química , Humanos , Solubilidade , Água/químicaRESUMO
Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 µM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Melanoma/tratamento farmacológico , Mutação/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos SCID , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CONTEXT: Papillary thyroid carcinoma (PTC) is the most frequent thyroid tumor and is responsible for the overall increase in thyroid cancer incidence. S100A11 (calgizzarin), a member of the S100 Ca(2+)-binding protein family, is involved in several different biological processes. S100A11 has been found up-regulated in PTC, both at the mRNA and protein levels. OBJECTIVE: Through a combination of expression analysis and functional in vitro and in vivo studies, we have attempted to gain insight into the relevance of S100A11 overexpression in PTC biology. DESIGN: The expression of the S100A11 gene in PTC was investigated in several gene expression data sets. The effect of S100A11 silencing on the hallmarks of the malignant phenotype of several PTC-derived cell lines was investigated. In NIH3T3 cells, the cooperation of S100A11 with the different PTC-specific oncogenes was assessed. RESULTS: We found that the S100A11 gene expression is frequently up-regulated in PTC, anaplastic thyroid carcinoma, but not in follicular thyroid carcinoma. S100A11 overexpression was also detected in PTC-derived cell lines, which were then used for functional studies. S100A11 silencing in PTC-derived cell lines did not affect cell proliferation, whereas it reduced the loss of contact inhibition, anchorage-independent growth, and resistance to anoikis. Cotransfection experiments in NIH3T3 cells showed that overexpression of the S100A11 gene was able to enhance the transforming capabilities of the different PTC-associated oncogenes by affecting the loss of contact inhibition, anchorage-independent growth, and in vivo tumor formation. CONCLUSION: Our data indicate that S100A11 overexpression exerts a protumoral functional role in PTC pathogenesis.
Assuntos
Carcinoma Papilar/genética , Proteínas S100/genética , Neoplasias da Glândula Tireoide/genética , Regulação para Cima , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/patologia , Animais , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Neoplasias da Glândula Tireoide/patologiaRESUMO
Quantitative characterization of the in vivo effects of vascular-targeted therapies on tumor vessels is hampered by the absence of useful 3D vascular network descriptors aside from microvessel density. In this study, we extended the quantification of planar vessel distribution to the analysis of vascular volumes by studying the effects of antiangiogenic (sorafenib and sunitinib) or antivascular (combretastatin A4 phosphate) treatments on the quantity and spatial distributions of thin microvessels. These observations were restricted to perinecrotic areas of treated human multiple myeloma tumors xenografted in immunodeficient mice and to microvessels with an approximate cross-sectional area lower than 75 µm(2). Finally, vessel skeletonization minimized artifacts due to possible differential wall staining and allowed a comparison of the various treatment effects. Antiangiogenic drug treatment reduced the number of vessels of every caliber (at least 2-fold fewer vessels vs. controls; p<0.001, nâ=â8) and caused a heterogeneous distribution of the remaining vessels. In contrast, the effects of combretastatin A4 phosphate mainly appeared to be restricted to a homogeneous reduction in the number of thin microvessels (not more than 2-fold less vs. controls; p<0.001, nâ=â8) with marginal effects on spatial distribution. Unexpectedly, these results also highlighted a strict relationship between microvessel quantity, distribution and cross-sectional area. Treatment-specific changes in the curves describing this relationship were consistent with the effects ascribed to the different drugs. This finding suggests that our results can highlight differences among vascular-targeted therapies, providing hints on the processes underlying sample vascularization together with the detailed characterization of a pathological vascular tree.
Assuntos
Transformação Celular Neoplásica , Imageamento Tridimensional/métodos , Linfoma/patologia , Terapia de Alvo Molecular , Neovascularização Patológica , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Artefatos , Linhagem Celular Tumoral , Feminino , Humanos , Linfoma/tratamento farmacológico , Linfoma/fisiopatologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Neovascularização Patológica/tratamento farmacológico , Estilbenos/farmacologia , Estilbenos/uso terapêuticoRESUMO
The proapoptotic death receptor 5 (DR5) expressed by tumor associated endothelial cells (TECs) mediates vascular disrupting effects of human CD34(+) cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL (+) cells) in mice. Indeed, lack of DR5 on TECs causes resistance to CD34-TRAIL (+) cells. By xenografting in nonobese diabetic/severe combined immunodeficient mice the TRAIL-resistant lymphoma cell line SU-DHL-4V, which generates tumors lacking endothelial DR5 expression, here we demonstrate for the first time that the Akt inhibitor perifosine induces in vivo DR5 expression on TECs, thereby overcoming tumor resistance to the vascular disruption activity of CD34-TRAIL (+) cells. In fact, CD34-TRAIL (+) cells combined with perifosine, but not CD34-TRAIL (+) cells alone, exerted marked antivascular effects and caused a threefold increase of hemorrhagic necrosis in SU-DHL-4V tumors. Consistent with lack of DR5 expression, CD34-TRAIL (+) cells failed to affect the growth of SU-DHL-4V tumors, but CD34-TRAIL (+) cells plus perifosine reduced tumor volumes by 60 % compared with controls. In view of future clinical studies using membrane-bound TRAIL, our results highlight a strategy to rescue patients with primary or acquired resistance due to the lack of DR5 expression in tumor vasculature.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Neovascularização Patológica/fisiopatologia , Fosforilcolina/análogos & derivados , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Análise de Variância , Animais , Antígenos CD34/metabolismo , Western Blotting , Linhagem Celular Tumoral , Primers do DNA/genética , Feminino , Citometria de Fluxo , Imunofluorescência , Transplante de Células-Tronco Hematopoéticas , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia Confocal , Fosforilcolina/farmacologia , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
The anti-lymphoma activity and mechanism(s) of action of the multikinase inhibitor sorafenib were investigated using a panel of lymphoma cell lines, including SU-DHL-4V, Granta-519, HD-MyZ, and KMS-11 cell lines. In vitro, sorafenib significantly decreased cell proliferation and phosphorylation levels of MAPK and PI3K/Akt pathways while increased apoptotic cell death. In vivo, sorafenib treatment resulted in a cytostatic rather than cytotoxic effect on tumor cell growth associated with a limited inhibition of tumor volumes. However, sorafenib induced an average 50% reduction of tumor vessel density and a 2-fold increase of necrotic areas. Upon sorafenib treatment, endothelial and tumor cells from SU-DHL-4V, Granta-519, and KMS-11 nodules showed a potent inhibition of either phospho-ERK or phospho-AKT, whereas a concomitant inhibition of phospho-ERK and phospho-AKT was only observed in HD-MyZ nodules. In conclusion, sorafenib affects the growth of lymphoid cell lines by triggering antiangiogenic mechanism(s) and directly targeting tumor cells.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Linfoma/tratamento farmacológico , Linfoma/enzimologia , Sistema de Sinalização das MAP Quinases , Neovascularização Patológica/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Humanos , Linfoma/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos SCID , Necrose , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Pericitos/efeitos dos fármacos , Pericitos/patologia , Compostos de Fenilureia/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker ß-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ativadores de Enzimas/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/genética , Sequência de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Fenformin/farmacologia , Fosforilação , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/genéticaRESUMO
Colorectal carcinomas (CRC) harbor well-defined genetic abnormalities, including aberrant activation of ß-catenin (ß-cat) and KRAS, but independent targeting of these molecules seems to have limited therapeutic effect. In this study, we report therapeutic effects of combined targeting of different oncogenes in CRC. Inducible short hairpin RNA (shRNA)-mediated silencing of ß-cat, ITF2, or KRAS decreased proliferation by 88%, 72%, and 45%, respectively, with no significant apoptosis in any case. In contrast, combined blockade of ß-cat and ITF2 inhibited proliferation by 99% with massive apoptosis. Similar effects occurred after combined shRNA against ß-cat and KRAS. In vivo, single oncogene blockade inhibited the growth of established tumors by up to 30%, whereas dual ß-cat and ITF2 targeting caused 93% inhibition. Similar tumor growth suppression was achieved by double ß-cat/KRAS shRNA in vivo. Our findings illustrate an effective therapeutic principle in CRC based on a combination targeting strategy that includes the ITF2 oncogene, which represents a novel therapeutic target.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Neoplasias Colorretais/terapia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Proteínas ras/antagonistas & inibidores , Animais , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação para Baixo , Doxiciclina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , RNA Interferente Pequeno/genética , Transdução de Sinais , Fator de Transcrição 4 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética , beta Catenina/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.
Assuntos
Antígenos CD34/metabolismo , Membrana Celular/metabolismo , Engenharia Genética , Neoplasias/irrigação sanguínea , Neoplasias/terapia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Transplante de Células , Quimiocina CXCL12/metabolismo , Hemorragia/patologia , Humanos , Camundongos , Necrose , Neoplasias/genética , Neoplasias/patologia , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Ligação Proteica , Distribuição Tecidual , Molécula 1 de Adesão de Célula Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Unbalanced histone deacetylase (HDAC) hyperactivity is a common feature of tumor cells. Inhibition of HDAC activity is often associated with cancer cell growth impairment and death. Valproic acid (VPA) is a HDAC inhibitor used for the treatment of epilepsy. It has recently been recognized as a promising anticancer drug. We investigated the effects of VPA on growth and survival of colon cancer cells. VPA caused growth inhibition and programmed cell death that correlated with histone hyperacetylation. VPA modulated the expression of various factors involved in cell cycle control and apoptosis and induced caspase activation. Interestingly, VPA induced downregulation of c-Src and potentiated the cytotoxic effects of the c-Src inhibitor bosutinib, both in vitro and in vivo. The combination of sublethal doses of VPA and bosutinib led to massive apoptosis of colon cancer cells, irrespective of their genetic background. These results suggest that VPA may be employed as a positive modulator of bosutinib antitumor activity in colorectal cancer.
Assuntos
Compostos de Anilina/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Nitrilas/administração & dosagem , Quinolinas/administração & dosagem , Ácido Valproico/administração & dosagem , Acetilação , Animais , Apoptose , Linhagem Celular Tumoral , Feminino , Histonas/metabolismo , Humanos , Camundongos , Camundongos Nus , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). DESIGN AND METHODS: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. RESULTS: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. CONCLUSIONS: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.
Assuntos
Anilidas/farmacologia , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Leucemia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Pirimidinas/farmacologia , Anilidas/química , Animais , Antineoplásicos/química , Benzamidas/química , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Resistencia a Medicamentos Antineoplásicos , Humanos , Mesilato de Imatinib , Camundongos , Transplante de Neoplasias , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Pirimidinas/químicaRESUMO
The molecular pathogenesis of tumors arising from the thyroid follicular epithelial cells, including papillary (PTC) and follicular thyroid carcinoma (FTC), is only partially understood, and the role of tumor suppressor genes has not yet been assessed. The metallothionein (MT) gene family encodes a class of metal-binding proteins involved in several cellular processes, and their expression is often deregulated in human tumors. Recently, downregulation of MT gene expression in PTC has been reported, suggesting a possible oncosuppressor role of this gene family in the pathogenesis of thyroid tumors. To further explore this possibility, we performed expression and functional studies. Analysis of microarray data of thyroid tumors of different histologic types showed that several MT genes were downregulated with respect to normal tissue. The microarray data were corroborated by quantitative PCR experiments, showing downregulation of MTs in PTC and FTC, but to a greater extent in papillary carcinoma. The expression of MTs was also investigated at the protein level by immunohistochemistry; the results were consistent with the microarray data, showing general downregulation in tumor samples, which was more evident in PTC. The functional consequence of MT downregulation was addressed employing an experimental model made of the PTC-derived K1 cell line in which MT1G expression is repressed by promoter methylation. Restoration of MT1G expression by cDNA transfection affected growth rate and in vivo tumorigenicity of K1 cells, indicating an oncosuppressor role for MT1G in thyroid papillary tumorigenesis.
Assuntos
Carcinoma Papilar/metabolismo , Metalotioneína/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma Papilar/genética , Linhagem Celular Tumoral/metabolismo , Metilação de DNA , DNA Complementar , Regulação para Baixo , Expressão Gênica , Humanos , Imuno-Histoquímica , Metalotioneína/genética , Análise em Microsséries , Regiões Promotoras Genéticas , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , TransfecçãoRESUMO
To investigate the therapeutic activity of the fully human anti-HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138(+) cells, analyzed the capacity of IFN-gamma to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-gamma. CD138(+)HLA-DR(+) cells were detected in 31 of 60 patients, with 15 of 60 patients having >/=20% CD138(+)HLA-DR(+) cells (median, 50%; range, 23-100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-gamma restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-gamma/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P = 0.0001) or mice receiving 1D09C3 alone (147 versus 92 days, P = 0.03). The better therapeutic activity of IFN-gamma/1D09C3 treatment over 1D09C3 alone was further shown by a 2-fold increase of mice being disease-free at 150 days after xenograft (47% versus 25%). No mice experienced any apparent treatment-related toxicity. Our data show that (a) one fourth of MM patients express HLA-DR on CD138(+) cells and (b) IFN-gamma-induced up-regulation of HLA-DR results in a potent enhancement of the in vivo antimyeloma activity of 1D09C3.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos HLA-DR/imunologia , Interferon gama/farmacologia , Mieloma Múltiplo/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/imunologia , Sinergismo Farmacológico , Feminino , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Plasmócitos/imunologia , Plasmócitos/patologia , Proteínas Recombinantes , Sindecana-1/imunologia , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The complex hematopoietic effects of placental growth factor (PlGF) prompted us to test in mice and nonhuman primates the mobilization of peripheral blood progenitor cells (PBPCs) elicited by recombinant mouse PlGF-2 (rmPlGF-2) and recombinant human PlGF-1 (rhPlGF-1). PBPC mobilization was evaluated by assaying colony-forming cells (CFCs), high-proliferative potential-CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). In mice, both rmPlGF-2 and rhPlGF-1 used as single agents failed to mobilize PBPCs, whereas the combination of rhPlGF-1 and granulocyte colony-stimulating factor (rhG-CSF) increased CFCs and LTC-ICs per milliliter of blood by four- and eightfold, respectively, as compared with rhG-CSF alone. rhPlGF-1 plus rhG-CSF significantly increased matrix metalloproteinase-9 plasma levels over rhG-CSF alone, suggesting a mechanistic explanation for rhPlGF-1/rhG-CSF synergism. In rhesus monkeys, rhPlGF-1 alone had no mobilization effect, whereas rhPlGF-1 (260 microg/kg per day) plus rhG-CSF (100 microg/kg per day) increased rhG-CSF-elicited mobilization of CFCs, HPP-CFCs, and LTC-ICs per milliliter of blood by 5-, 7-, and 15-fold, respectively. No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination. In conclusion, our data demonstrate that rhPlGF-1 significantly increases rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating rhPlGF-1 in the clinical setting.