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1.
J Cell Physiol ; 235(2): 629-637, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31313842

RESUMO

The present study shows that nuclear factor erythroid 2-related factor 2 (NRF2) and miR-29b-1-5p are two opposite forces which could regulate the fate of MDA-MB-231 cells, the most studied triple-negative breast cancer (TNBC) cell line. We show that NRF2 activation stimulates cell growth and markedly reduces reactive oxygen species (ROS) generation, whereas miR-29b-1-5p overexpression increases ROS generation and reduces cell proliferation. Moreover, NRF2 downregulates miR-29b-1-5p expression, whereas miR-29b-1-5p overexpression decreases p-AKT and p-NRF2. Furthermore, miR-29b-1-5p overexpression induces both inhibition of DNA N-methyltransferases (DNMT1, DNMT3A, and DNMT3B) expression and re-expression of HIN1, RASSF1A and CCND2. Conversely, NRF2 activation induces opposite effects. We also show that parthenolide, a naturally occurring small molecule, induces the expression of miR-29b-1-5p which could suppress NRF2 activation via AKT inhibition. Overall, this study uncovers a novel NRF2/miR-29b-1-5p/AKT regulatory loop that can regulate the fate (life/death) of MDA-MB-231 cells and suggests this loop as therapeutic target for TNBC.


Assuntos
MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Proteínas Proto-Oncogênicas c-akt/genética , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D2/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Espécies Reativas de Oxigênio/metabolismo , Sesquiterpenos/farmacologia , Transdução de Sinais/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Supressoras de Tumor/metabolismo
2.
J Cell Physiol ; 234(10): 18432-18447, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30912136

RESUMO

Triple-negative breast cancer (TNBC) is a form of BC characterized by high aggressiveness and therapy resistance probably determined by cancer stem cells. MCL1 is an antiapoptotic Bcl-2 family member that could limit the efficacy of anticancer agents as recombinant human tumor necrosis factor related apoptosis-inducing ligand (rh-TRAIL). Here, we investigated MCL1 expression in TNBC tissues and cells. We found MCL1 differentially expressed (upregulated or downregulated) in TNBC tissues. Furthermore, in comparison to the human mammary epithelial cells, we found that MDA-MB-231 cells show similar messenger RNA levels but higher MCL1 protein levels, whereas it resulted downregulated in MDA-MB-436 and BT-20 cells. We evaluated the effects of rh-TRAIL and A-1210477, a selective MCL1 inhibitor, on cell viability and growth of MDA-MB-231 cells. We demonstrated that the drug combination reduced the cell growth and activated the apoptotic pathway. Similar effects were observed on three-dimensional cultures and tertiary mammospheres of MDA-MB-231 cells. In MDA-MB-231 cells, after MCL1 silencing, rh-TRAIL confined the cell population in the sub-G0/G1 phase and induced a drop in the mitochondrial transmembrane potential. To understand the molecular mechanism by which the loss of MCL1 function sensitizes the MDA-MB-231 cells to rh-TRAIL, we analyzed by real-time reverse transcription polymerase chain reaction, the expression of genes related to apoptosis, stemness, cell cycle, and those involved in epigenetic regulation. Interestingly, among the upregulated genes through MCL1 silencing or inhibition, there was TNFRSF10A (DR4). Moreover, MCL1 inhibition increased DR4 protein levels and its cell surface expression. Finally, we demonstrated MCL1-DR4 interaction and dissociation of this complex after A-1210477 treatment. Overall, our findings highlight the potential MCL1-roles in MDA-MB-231 cells and suggest that MCL1 targeting could be an effective strategy to overcome TNBC's rh-TRAIL resistance.


Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Sulfonamidas/farmacologia
3.
J Cell Physiol ; 233(11): 8482-8498, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29797573

RESUMO

The Bcl-2 family, which plays important roles in controlling cancer development, is divided into antiapoptotic and proapoptotic members. The change in the balance between these members governs the life and death of the cells. Mcl-1 is an antiapoptotic member of this family and its distribution in normal and cancerous tissues strongly differs from that of Bcl-2. In human cancers, where upregulation of antiapoptotic proteins is common, Mcl-1 expression is regulated independent of Bcl-2 and its inhibition promotes senescence, a major barrier to tumorigenesis. Cancer chemotherapy determines various kinds of responses, such as senescence and autophagy; however, the ideal response to chemotherapy is apoptosis. Mcl-1 is a potent oncogene that is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Mcl-1 is a short-lived protein that, in the NH2 terminal region, contains sites for posttranslational regulation that can lead to proteasomal degradation. The USP9X Mcl-1 deubiquitinase regulates Mcl-1 and the levels of these two proteins are strongly correlated. Mcl-1 has three splicing variants (the antiapoptotic protein Mcl-1L and the proapoptotic proteins Mcl-1S and Mcl-1ES), each contributing toward apoptosis regulation. In cancers responsible for the most deaths in the world, the presence of Mcl-1 is associated with malignant cell growth and evasion of apoptosis. Mcl-1 is also one of the key regulators of cancer stem cells' self-renewal that contributes to tumor survival. A great number of indirect and selective Mcl-1 inhibitors have been produced and some of these have shown efficacy in several clinical trials. Thus, therapeutic manipulation of Mcl-1 can be a useful strategy to combat cancer.


Assuntos
Carcinogênese/genética , Terapia de Alvo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias/genética , Apoptose/genética , Senescência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/uso terapêutico , Neoplasias/terapia , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética
4.
Cell Oncol (Dordr) ; 41(2): 169-184, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29204978

RESUMO

BACKGROUND: Recently, Glypican-3 (GPC3) has been identified as a potential hepatocellular carcinoma (HCC) diagnostic and/or therapeutic target. GPC3 has been found to be up-regulated in HCC and to be absent in normal and cirrhotic liver. As yet, however, the molecular characteristics of GPC3 and its role in HCC cell physiology and development are still undefined. METHODS: Human hepatocyte cultures were established from 10 HCC patients. Additional liver samples were obtained from 5 patients without cirrhosis and/or HCC. Soft agar colony formation, (co-)immunofluorescence and Western blot assays were used to characterize the hapatocyte cultures. The expression of GPC3 in the hepatocytes was silenced using siRNA, after which, apoptosis, scratch wound migration and transwell invasion assays were performed. RESULTS: We found that in HCC precursor hepatocytes GPC3 is increasingly expressed in different forms and at different locations, i.e., a non-cleaved form (70 kDa) was found to be localized in the cytoplasm while a N-terminal cleaved form (N-GPC3: 40 kDa) was fond to be localized in the cytoplasm and at the extracellular side of hepatocyte membranes. In addition, we found that the non-cleaved form of GPC3 co-localizes with Furin-Convertase in the Golgi apparatus. We also found that, similar to GPC3, Furin-Convertase is expressed in HCC precursor cells, suggesting a role in GPC3 processing. Subsequent siRNA-mediated GPC3 silencing resulted in a temporary inhibition of cell proliferation, migration and ivasion, while inducing apoptosis in transformed hepatocytes. CONCLUSION: Our data reveal new aspects of the role of GPC3 in early hepatocyte transformation. In addition we conclude that GPC3 may serve as a new HCC immune-therapeutic target.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Glipicanas/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Células Cultivadas , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , RNA Interferente Pequeno/metabolismo
5.
Oncotarget ; 8(17): 28939-28958, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28423652

RESUMO

MiR-29 family dysregulation occurs in various cancers including breast cancers. We investigated miR-29b-1 functional role in human triple negative breast cancer (TNBC) the most aggressive breast cancer subtype. We found that miR-29b-1-5p was downregulated in human TNBC tissues and cell lines. To assess whether miR-29b-1-5p correlated with TNBC regenerative potential, we evaluated cancer stem cell enrichment in our TNBC cell lines, and found that only MDA-MB-231 and BT-20 produced primary, secondary and tertiary mammospheres, which were progressively enriched in OCT4, NANOG and SOX2 stemness genes. MiR-29b-1-5p expression inversely correlated with mammosphere stemness potential, and miR-29b-1 ectopic overexpression decreased TNBC cell growth, self-renewal, migration, invasiveness and paclitaxel resistance repressing WNT/ßcatenin and AKT signaling pathways and stemness regulators. We identified SPINDLIN1 (SPIN1) among predicted miR-29b-1-5p targets. Consistently, SPIN1 was overexpressed in most TNBC tissues and cell lines and negatively correlated with miR-29b-1-5p. Target site inhibition showed that SPIN1 seems to be directly controlled by miR-29b-1-5p. Silencing SPIN1 mirrored the effects triggered by miR-29b-1 overexpression, whereas SPIN1 rescue by SPIN1miScript protector, determined the reversal of the molecular effects produced by the mimic-miR-29b-1-5p. Overall, we show that miR-29b-1 deregulation impacts on multiple oncogenic features of TNBC cells and their renewal potential, acting, at least partly, through SPIN1, and suggest that both these factors should be evaluated as new possible therapeutic targets against TNBC.


Assuntos
Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Fosfoproteínas/genética , Transdução de Sinais/genética , Neoplasias de Mama Triplo Negativas/genética , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Mama/patologia , Carcinogênese/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , MicroRNAs/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Homeobox Nanog/metabolismo , Invasividade Neoplásica/genética , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Fosfoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia
6.
Cell Death Discov ; 3: 17078, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29354292

RESUMO

Triple-negative breast cancer is a group of aggressive cancers with poor prognosis owing to chemoresistance, recurrence and metastasis. New strategies are required that could reduce chemoresistance and increases the effectiveness of chemotherapy. The results presented in this paper, showing that parthenolide (PN) prevents drug resistance in MDA-MB231 cells, represent a contribution to one of these possible strategies. MDA-MB231 cells, the most studied line of TNBC cells, were submitted to selection treatment with mitoxantrone (Mitox) and doxorubicin (DOX). The presence of resistant cells was confirmed through the measurement of the resistance index. Cells submitted to this treatment exhibited a remarkable increment of NF-E2-related factor 2 (Nrf2) level, which was accompanied by upregulation of catalase, MnSOD, HSP70, Bcl-2 and P-glycoprotein. Moreover, as a consequence of overexpression of Nrf2 and correlated proteins, drug-treated cells exhibited a much lower ability than parental cells to generate ROS in response to a suitable stimulation. The addition of PN (2.0 µM) to Mitox and DOX, over the total selection time, prevented both the induction of resistance and the overexpression of Nrf2 and correlated proteins, whereas the cells showed a good ability to generate ROS in response to adequate stimulation. To demonstrate that Nrf2 exerted a crucial role in the induction of resistance, the cells were transiently transfected with a specific small interfering RNA for Nrf2. Similarly to the effects induced by PN, downregulation of Nrf2 was accompanied by reductions in the levels of catalase, MnSOD, HSP70 and Bcl-2, prevention of chemoresistance and increased ability to generate ROS under stimulation. In conclusion, our results show that PN inhibited the development of the resistance toward Mitox and DOX, and suggest that these effects were correlated with the prevention of the overexpression of Nrf2 and its target proteins, which occurred in the cells submitted to drug treatment.

7.
Int J Oncol ; 48(6): 2339-48, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27082853

RESUMO

Triple-negative breast cancer (TNBC) is a clinically aggressive form of breast cancer that is unresponsive to endocrine agents or trastuzumab. TNBC accounts for ~10-20% of all breast cancer cases and represents the form with the poorest prognosis. Patients with TNBC are at higher risk of early recurrence, mainly in the lungs, brain and soft tissue, therefore, there is an urgent need for new therapies. The present study was carried out in MDA-MB-231 cells, where we assessed the role of caspase-8 (casp-8), a critical effector of death receptors, also involved in non­apoptotic functions. Analysis of casp-8 mRNA and protein levels indicated that they were up-regulated with respect to the normal human mammalian epithelial cells. We demonstrated that silencing of casp-8 by small interfering-RNA, strongly decreased MDA-MB-231 cell growth by delaying G0/G1- to S-phase transition and increasing p21, p27 and hypo-phosphorylated/active form of pRb levels. Surprisingly, casp-8-knockdown, also potently increased both the migratory and metastatic capacity of MDA-MB­231 cells, as shown by both wound healing and Matrigel assay, and by the expression of a number of related-genes and/or proteins such as VEGFA, C-MYC, CTNNB1, HMGA2, CXCR4, KLF4, VERSICAN V1 and MMP2. Among these, KLF4, a transcriptional factor with a dual role (activator and repressor), seemed to play critical roles. We suggest that in MDA-MB­231 cells, the endogenous expression of casp-8 might keep the cells perpetually cycling through downregulation of KLF4, the subsequent lowering of p21 and p27, and the inactivation by hyperphosphorylation of pRb. Simultaneously, by lowering the expression of some migratory and invasive genes, casp-8 might restrain the metastatic ability of the cells. Overall, our findings showed that, in MDA-MB-231 cells, casp-8 might play some unusual roles which should be better explored, in order to understand whether it might be identified as a molecular therapeutic target.


Assuntos
Caspase 8/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Caspase 8/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Fase G1/fisiologia , Humanos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Fase de Repouso do Ciclo Celular/fisiologia , Transfecção , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
8.
Int J Oncol ; 49(1): 352-60, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27121069

RESUMO

Breast cancer stem cells seem to play important roles in breast tumor recurrence and endocrine therapy resistance, although the underlying mechanisms have not been well established. Moreover, in some tumor systems the immunosurveillance failure against cancer cells has been related to the presence of the granzyme B inhibitor PI-9. This study explored the status of PI-9 in tumorspheres isolated from estrogen receptor-α positive (ERα+) breast cancer MCF7 cells. Studies were performed in tertiary tumorspheres which possess high levels of stemness markers (Nanog, Oct3/4 and Sox2) and self-renewal ability. The exposure to estrogens (17-ß estradiol and genistein) increased the number and sizes of tumorspheres, promoting cell proliferation as demonstrated by the increase in the proliferating cell nuclear antigen (PCNA). The study of the three isoforms (66, 46 and 36 kDa) of ERα disclosed that tertiary tumorspheres exhibit a marked increase in ERα36, while the level of ERα66, which is highly expressed in MCF7 cells, declines. Although it is known that PI-9 is a transcriptional target of ERα66, surprisingly in tertiary tumorspheres, despite the reduced level of ERα66, the protein and mRNA content of PI-9 is higher than in MCF7 cells. Treatment with estrogens further increased PI-9 level while decreased that of ERα66 isoform thus excluding the involvement of this receptor isoform in the event. Moreover, our studies also provided evidence that tertiary tumorspheres express elevated levels of CXCR4 and phospho-p38, suggesting that the high PI-9 content might be ascribed to the activation of the proliferative CXCR4/phospho-p38 axis. Taken together, these events could supply a selective advantage to breast cancer stem cells by interfering with immunosurveillance systems and open up the avenue to new possible targets for breast cancer treatment.


Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/biossíntese , Recidiva Local de Neoplasia/genética , Células-Tronco Neoplásicas/metabolismo , Serpinas/biossíntese , Neoplasias da Mama/patologia , Proliferação de Células/genética , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genisteína/administração & dosagem , Granzimas/antagonistas & inibidores , Humanos , Células MCF-7 , Recidiva Local de Neoplasia/fisiopatologia , Células-Tronco Neoplásicas/patologia , Receptores CXCR4/biossíntese , Serpinas/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese
9.
PLoS One ; 11(4): e0153613, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27074018

RESUMO

Isolation of hepatocytes from cirrhotic human livers and subsequent primary culture are important new tools for laboratory research and cell-based therapeutics in the study of hepatocellular carcinoma (HCC). Using such techniques, we have previously identified different subpopulations of human hepatocytes and among them one is showing a progressive transformation of hepatocytes in HCC-like cells. We have hypothesized that increasing the distance from the neoplastic lesion might affect hepatocyte function and transformation capacity. However, limited information is available in comparing the growth and proliferation of human hepatocytes obtained from different areas of the same cirrhotic liver in relation to their distance from the HCC lesion. In this study, hepatocytes from 10 patients with cirrhosis and HCC undergoing surgical resections from specimens obtained at a proximal (CP) and distal (CD) distance from the HCC lesion were isolated and placed in primary culture. CP hepatocytes (CP-Hep) were isolated between 1 to 3 cm (leaving at least 1cm margin to avoid cancer cells and/or satellite lesions), while CD hepatocytes (CD-Hep) were isolated from more than 5 cm or from the contralateral-lobe. A statistical model was built to analyze the proliferation rates of these cells and we evaluated expression of HCC markers (Glypican-3 (GPC3), αSmooth Muscle Actin (α-SMA) and PCNA). We observed a significant difference in proliferation and in-vitro growth showing that CP-Hep had a proliferation pattern and rate significantly different than CD-Hep. Based on these data, this model can provide information to predict growth of human hepatocytes in primary culture in relation to their pre-cancerous state with significant differences in the HCC markers expression. This model provides an important innovative tool for in-vitro analysis of HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Proliferação de Células , Hepatócitos/patologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células Tumorais Cultivadas
10.
Int J Oncol ; 48(3): 1205-17, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26717856

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Currently, there is limited knowledge of neoplastic transformation of hepatocytes in HCC. In clinical practice, the high rate of HCC local recurrence suggests the presence of different hepatocyte populations within the liver and particularly in the tumor proximity. The present study investigated primary human hepatocyte cultures obtained from liver specimens of patients affected by cirrhosis and HCC, their proliferation and transformation. Liver samples were obtained from seven HCC cirrhotic patients and from three patients with normal liver (NL). Immediately after surgery, cell outgrowth and primary cultures were obtained from the HCC lesion, the cirrhotic tissue proximal (CP, 1-3 cm) and distal (CD, >5 cm) to the margin of the neoplastic lesion, or from NL. Cells were kept in culture for 16 weeks. Morphologic analyses were performed and proliferation rate of the different cell populations compared over time. Glypican-3, Heppar1, Arginase1 and CD-44 positivity were tested. The degree of invasiveness of cells acquiring neoplastic characteristics was studied with a transwell migration assay. We observed that HCC cells maintained their morphology and unmodified neoplastic characteristics when cultured. Cells isolated from CP, showed a progressive morphologic transformation in HCC-like cells accompanied by modification of markers expression with signs of invasiveness. Absence of HCC contamination in the CP isolates was confirmed. In CD samples some of these characteristics were present and at significantly lower levels. With the present study, we are the first to have identified and describe the existence of human hepatocytes near the cancerous lesion that can transform in HCC in vitro.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica , Hepatócitos/citologia , Neoplasias Hepáticas/metabolismo , Idoso , Antígenos de Neoplasias/metabolismo , Arginase/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Citometria de Fluxo , Glipicanas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Imuno-Histoquímica , Cirrose Hepática/fisiopatologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica
11.
Int J Oncol ; 48(3): 1039-44, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26698404

RESUMO

Secreted protein acidic and rich in cysteine (SPARC) is a multi-functional protein which modulates cell-cell and cell-matrix interactions. In cancer cells, SPARC behaves as a tumor promoter in a number of tumors, but it can also act as a tumor suppressor factor. Our previous results showed that the synthetic cannabinoid WIN55,212-2 (WIN), a potent cannabinoid receptor agonist, is able to sensitize osteosarcoma MG63 cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis which is accompanied with endoplasmic reticulum (ER)-stress induction and the increase in autophagic markers. In the present investigation, we studied the role of SPARC in WIN/TRAIL-induced apoptosis demonstrating that WIN increased the level of SPARC protein and mRNA in a time-dependent manner. This event was functional to WIN/TRAIL-dependent apoptosis as demonstrated by RNA interfering analysis which indicated that SPARC-silenced cells were less sensitive to cytotoxic effects induced by the combined treatment. Our experiments also demonstrate that SPARC interacts with caspase-8 thus probably favoring its translocation to plasma membrane and the activation of extrinsic apoptotic pathway. In conclusion, to the best of our knowledge, our results are the first to show that WIN-dependent increase in the level of SPARC plays a critical role in sensitizing osteosarcoma cells to TRAIL action.


Assuntos
Neoplasias Ósseas/metabolismo , Osteonectina/metabolismo , Osteossarcoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose , Benzoxazinas/química , Neoplasias Ósseas/patologia , Caspase 8/metabolismo , Morte Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular , Estresse do Retículo Endoplasmático , Inativação Gênica , Humanos , Morfolinas/química , Naftalenos/química , Osteossarcoma/patologia , Domínios Proteicos , Interferência de RNA
12.
J Cell Physiol ; 231(8): 1832-41, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26679758

RESUMO

Osteosarcoma (OS), an aggressive highly invasive and metastatic bone-malignancy, shows therapy resistance and recurrence, two features that likely depend on cancer stem cells (CSCs), which hold both self-renewing and malignant potential. So, effective anticancer therapies against OS should specifically target and destroy CSCs. We previously found that the let-7d microRNA was downregulated in the 3AB-OS-CSCs, derived from the human OS-MG63 cells. Here, we aimed to assess whether let-7d modulation affected tumorigenic and stemness properties of these OS-CSCs. We found that let-7d-overexpression reduced cell proliferation by decreasing CCND2 and E2F2 cell-cycle-activators and increasing p21 and p27 CDK-inhibitors. Let-7d also decreased sarcosphere-and-colony forming ability, two features associated with self-renewing, and it reduced the expression of stemness genes, including Oct3/4, Sox2, Nanog, Lin28B, and HMGA2. Moreover, let-7d induced mesenchymal-to-epithelial-transition, as shown by both N-Cadherin-E-cadherin-switch and decrease in vimentin. Surprisingly, such switch was accompanied by enhanced migratory/invasive capacities, with a strong increase in MMP9, CXCR4 and VersicanV1. Let-7d- overexpression also reduced cell sensitivity to apoptosis induced by both serum-starvation and various chemotherapy drugs, concomitant with decrease in caspase-3 and increase in BCL2 expression. Our data suggest that let-7d in 3AB-OS-CSCs could induce plastic-transitions from CSCs-to-non-CSCs and vice-versa. To our knowledge this is the first study to comprehensively examine the expression and functions of let-7d in OS-CSCs. By showing that let-7d has both tumor suppressor and oncogenic functions in this context, our findings suggest that, before prospecting new therapeutic strategies based on let-7d modulation, it is urgent to better define its multiple functions. J. Cell. Physiol. 231: 1832-1841, 2016. © 2015 Wiley Periodicals, Inc.


Assuntos
Neoplasias Ósseas/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Osteossarcoma/metabolismo , Antineoplásicos/farmacologia , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Autorrenovação Celular , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/patologia , Fenótipo , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
13.
Carcinogenesis ; 36 Suppl 1: S2-18, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26106139

RESUMO

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.


Assuntos
Exposição Ambiental/efeitos adversos , Substâncias Perigosas/efeitos adversos , Neoplasias/induzido quimicamente , Neoplasias/etiologia , Animais , Humanos , Transdução de Sinais/efeitos dos fármacos
14.
J Cell Physiol ; 230(6): 1276-89, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25370819

RESUMO

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy.


Assuntos
Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Sesquiterpenos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Inibidores de Histona Desacetilases/metabolismo , Humanos , NF-kappa B/metabolismo , Vorinostat
15.
J Cell Biochem ; 116(3): 418-30, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25335865

RESUMO

ß-Catenin is a central effector of the Wnt pathway and one of the players in Ca(+)-dependent cell-cell adhesion. While many wnts are present and expressed in vertebrates, only one ß-catenin exists in the majority of the organisms. One intriguing exception is zebrafish that carries two genes for ß-catenin. The maternal recessive mutation ichabod presents very low levels of ß-catenin2 that in turn affects dorsal axis formation, suggesting that ß-catenin1 is incapable to compensate for ß-catenin2 loss and raising the question of whether these two ß-catenins may have differential roles during early axis specification. Here we identify a specific antibody that can discriminate selectively for ß-catenin1. By confocal co-immunofluorescent analysis and low concentration gain-of-function experiments, we show that ß-catenin1 and 2 behave in similar modes in dorsal axis induction and cellular localization. Surprisingly, we also found that in the ich embryo the mRNAs of the components of ß-catenin regulatory pathway, including ß-catenin1, are more abundant than in the Wt embryo. Increased levels of ß-catenin1 are found at the membrane level but not in the nuclei till high stage. Finally, we present evidence that ß-catenin1 cannot revert the ich phenotype because it may be under the control of a GSK3ß-independent mechanism that required Axin's RGS domain function.


Assuntos
Proteína Axina/metabolismo , Mutação/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Especificidade de Anticorpos , Proteína Axina/genética , Blástula/efeitos dos fármacos , Blástula/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Dominantes , Imuno-Histoquímica , Cloreto de Lítio/farmacologia , Fenótipo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , beta Catenina/metabolismo
16.
Biochim Biophys Acta ; 1846(2): 539-46, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25444714

RESUMO

In the last decades the approach to cancer patient management has been deeply revolutionized. We are moving from a "one-fits-all" strategy to the "personalized medicine" based on the molecular characterization of the tumor. In this new era it is becoming more and more clear that the monitoring of the disease is fundamental for the success of the treatment, thus there is the need of new biomarker discovery. More precisely in the last years the scientific community has started to use the term "liquid biopsy". A liquid biopsy is a liquid biomarker that can be easily isolated from many body fluids (blood, saliva, urine, ascites, pleural effusion, etc.) and, as well as a tissue biopsy, a representative of the tissue from which it is spread. In this review we will focus our attention on circulating tumor cells, circulating tumor DNA, exosomes and secretomes with the aim to underlie their usefulness and potential application in a clinical setting for lung cancer patient management.


Assuntos
Neoplasias Pulmonares/patologia , Animais , Biomarcadores Tumorais/análise , Biópsia , DNA de Neoplasias/sangue , Exossomos , Humanos , Células Neoplásicas Circulantes
17.
Int J Oncol ; 45(5): 2013-23, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25174983

RESUMO

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. MicroRNAs (miRNAs) have been implicated in the maintenance of the CSC phenotype, thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Downregulation of miRNA-29 family members (miR-29a/b/c; miR­29s) was observed in human OS, however, little is known about the functions of miR-29s in human OS CSCs. Previously, during the characterization of 3AB-OS cells, a CSC line selected from human OS MG63 cells, we showed a potent downregulation of miR-29b. In this study, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated the role of miR-29b-1 in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS cells, in vitro. We found that, miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony-forming ability. In addition, while miR-29b-1 overexpression sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis, it did not influence their migratory and invasive capacities, thus suggesting a context-depending role of miR-29b-1. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We also analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b-1 as a novel therapeutic agent might offer benefits for OS treatment.


Assuntos
Neoplasias Ósseas/genética , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/biossíntese , Osteossarcoma/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Invasividade Neoplásica/genética , Osteossarcoma/patologia
18.
Int J Biol Sci ; 10(5): 466-78, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24795528

RESUMO

The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Benzoxazinas/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Proteínas de Choque Térmico/metabolismo , Morfolinas/farmacologia , Naftalenos/farmacologia , Osteossarcoma/fisiopatologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Laranja de Acridina , Apoptose/efeitos dos fármacos , Cadaverina/análogos & derivados , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Osteossarcoma/tratamento farmacológico , RNA Interferente Pequeno/genética
19.
Oncol Rep ; 32(1): 167-72, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24859613

RESUMO

It has been shown that the sesquiterpene lactone parthenolide lowers the viability of MDA-MB-231 breast cancer cells, in correlation with oxidative stress. The present report examined the different radical species produced during parthenolide treatment and their possible role in the toxicity caused by the drug. Time course experiments showed that in the first phase of treatment (0-8 h), and in particular in the first 3 h, parthenolide induced dichlorofluorescein (DCF) signal in a large percentage of cells, while dihydroethidium (DHE) signal was not stimulated. Since the effect on DCF signal was suppressed by apocynin and diphenyleneiodonium (DPI), two inhibitors of NADPH oxidase (NOX), we suggest that parthenolide rapidly stimulated NOX activity with production of superoxide anion (O2•-), which was converted by superoxide dismutase 1 (SOD1) into hydrogen peroxide (H2O2). In the second phase of treatment (8-16 h), parthenolide increased the number of positive cells to DHE signal. Since this event was not prevented by apocynin and DPI and was associated with positivity of cells to MitoSox Red, a fluorochrome used to detect mitochondrial production of O2•-, we suggest that parthenolide induced production of O2•- at the mitochondrial level independently by NOX activity in the second phase of treatment. Finally, in this phase, most cells became positive to hydroxyphenyl fluorescein (HPF) signal, a fluorescent probe to detect highly reactive oxygen species (hROS), such as hydroxyl radical and peroxynitrite. Therefore, parthenolide between 8-16 h of treatment induced generation of O2•- and hROS, in close correlation with a marked reduction in cell viability.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Neoplasias da Mama/tratamento farmacológico , Peróxido de Hidrogênio/metabolismo , Sesquiterpenos/farmacologia , Superóxidos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
20.
Apoptosis ; 19(6): 1029-42, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24696378

RESUMO

Cannabinoids have been reported to possess anti-tumorigenic activity in cancer models although their mechanism of action is not well understood. Here, we show that the synthetic cannabinoid WIN55,212-2 (WIN)-induced apoptosis in colon cancer cell lines is accompanied by endoplasmic reticulum stress induction. The formation of acidic vacuoles and the increase in LC3-II protein indicated the involvement of autophagic process which seemed to play a pro-survival role against the cytotoxic effects of the drug. However, the enhanced lysosomal membrane permeabilization (LMP) blocked the autophagic flux after the formation of autophagosomes as demonstrated by the accumulation of p62 and LC3, two markers of autophagic degradation. Data also provided evidence for a role for nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in cannabinoid signalling. PPARγ expression, at both protein and mRNA levels, was significantly down-regulated after WIN treatment and its inhibition, either by specific antagonists or by down-regulation via gene silencing, induced effects on cell viability as well as on ER stress and autophagic markers similar to those obtained in the presence of WIN. Moreover, the observation that the increase in p62 level and the induction of LMP were also modified by PPARγ antagonists seemed to indicate that PPARγ down-regulation was crucial to determinate the block of autophagic flux, thus confirming the critical role of PPARγ in WIN action. In conclusion, at our knowledge, our results are the first to show that the reduction of PPARγ levels contributes to WIN-induced colon carcinoma cell death by blocking the pro-survival autophagic response of cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Benzoxazinas/farmacologia , Neoplasias do Colo/patologia , Morfolinas/farmacologia , Naftalenos/farmacologia , PPAR gama/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Regulação para Baixo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , PPAR gama/genética , Transdução de Sinais/efeitos dos fármacos
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