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1.
Sci Adv ; 10(20): eado1463, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38758782

RESUMO

A ketogenic diet (KD) is a high-fat, low-carbohydrate diet that leads to the generation of ketones. While KDs improve certain health conditions and are popular for weight loss, detrimental effects have also been reported. Here, we show mice on two different KDs and, at different ages, induce cellular senescence in multiple organs, including the heart and kidney. This effect is mediated through adenosine monophosphate-activated protein kinase (AMPK) and inactivation of mouse double minute 2 (MDM2) by caspase-2, leading to p53 accumulation and p21 induction. This was established using p53 and caspase-2 knockout mice and inhibitors to AMPK, p21, and caspase-2. In addition, senescence-associated secretory phenotype biomarkers were elevated in serum from mice on a KD and in plasma samples from patients on a KD clinical trial. Cellular senescence was eliminated by a senolytic and prevented by an intermittent KD. These results have important clinical implications, suggesting that the effects of a KD are contextual and likely require individual optimization.


Assuntos
Senescência Celular , Dieta Cetogênica , Camundongos Knockout , Proteína Supressora de Tumor p53 , Animais , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Camundongos , Humanos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Masculino , Especificidade de Órgãos
2.
Biochim Biophys Acta Gene Regul Mech ; 1866(2): 194939, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37116859

RESUMO

Small cell lung cancer (SCLC) is a neuroendocrine tumor noted for the rapid development of both metastases and resistance to chemotherapy. High mutation burden, ubiquitous loss of TP53 and RB1, and a mutually exclusive amplification of MYC gene family members contribute to genomic instability and make the development of new targeted agents a challenge. Previously, we reported a novel OCT4-induced MYC transcriptional activation pathway involving c-MYC, pOCT4S111, and MAPKAPK2 in progressive neuroblastoma, also a neuroendocrine tumor. Using tumor microarray analysis of clinical samples and preclinical models, we now report a correlation in expression between these proteins in SCLC. In correlating c-MYC protein expression with genomic amplification, we determined that some SCLC cell lines exhibited high c-MYC without genomic amplification, implying amplification-independent MYC activation. We then confirmed direct interaction between OCT4 and DNA-PKcs and identified specific OCT4 and DNA-PKcs binding sites. Knock-down of both POU5F1 (encoding OCT4) and PRKDC (encoding DNA-PKcs) resulted in decreased c-MYC expression. Further, we confirmed binding of OCT4 to the promoter/enhancer region of MYC. Together, these data establish the presence of a DNA-PKcs/OCT4/c-MYC pathway in SCLCs. We then disruptively targeted this pathway and demonstrated anticancer activity in SCLC cell lines and xenografts using both DNA-PKcs inhibitors and a protein-protein interaction inhibitor of DNA-PKcs and OCT4. In conclusion, we demonstrate here that DNA-PKcs can mediate high c-MYC expression in SCLCs, and that this pathway may represent a new therapeutic target for SCLCs with high c-MYC expression.


Assuntos
Neoplasias Pulmonares , Tumores Neuroendócrinos , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , DNA
3.
Biotechnol Bioeng ; 118(5): 1987-2000, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33565603

RESUMO

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds ("hits") that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2-OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111 , the "hits" were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found five compounds that may function as lead compounds for further development.


Assuntos
Técnicas Citológicas/métodos , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Medições Luminescentes/métodos , Linhagem Celular Tumoral , Proteína Quinase Ativada por DNA/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases/metabolismo
4.
Cell Death Dis ; 11(5): 368, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409685

RESUMO

Despite the improvement in clinical outcome with 13-cis-retinoic acid (13-cisRA) + anti-GD2 antibody + cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients die of recurrent disease. MYCN genomic amplification is a biomarker of aggressive tumors in the childhood cancer neuroblastoma. MYCN expression is downregulated by 13-cisRA, a differentiating agent that is a component of neuroblastoma therapy. Although MYC amplification is rare in neuroblastoma at diagnosis, we report transcriptional activation of MYC medicated by the transcription factor OCT4, functionally replacing MYCN in 13-cisRA-resistant progressive disease neuroblastoma in large panels of patient-derived cell lines and xenograft models. We identified novel OCT4-binding sites in the MYC promoter/enhancer region that regulated MYC expression via phosphorylation by MAPKAPK2 (MK2). OCT4 phosphorylation at the S111 residue by MK2 was upstream of MYC transcriptional activation. Expression of OCT4, MK2, and c-MYC was higher in progressive disease relative to pre-therapy neuroblastomas and was associated with inferior patient survival. OCT4 or MK2 knockdown decreased c-MYC expression and restored the sensitivity to 13-cisRA. In conclusion, we demonstrated that high c-MYC expression independent of genomic amplification is associated with disease progression in neuroblastoma. MK2-mediated OCT4 transcriptional activation is a novel mechanism for activating the MYC oncogene in progressive disease neuroblastoma that provides a therapeutic target.


Assuntos
Diferenciação Celular/genética , Neuroblastoma/patologia , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Proteínas Oncogênicas/metabolismo , Ativação Transcricional/fisiologia
5.
Biochim Biophys Acta Mol Basis Dis ; 1866(4): 165432, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30904611

RESUMO

Cancer stem-like cells (CSCs) contribute to the tumorigenicity, progression, and chemoresistance of cancers. It is not known whether CSCs arise from normal stem cells or if they arise from differentiated cancer cells by acquiring self-renewal features. These CSCs share stem cell markers that normal stem cells express. There is a rising interest in octamer-binding transcription factor 4 (OCT4), one of the stem cell factors that are essential in embryogenesis and pluripotency. OCT4 is also overexpressed in CSCs of various cancers. Although the majority of the studies in CSCs reported a positive association between the expression of OCT4 and chemoresistance and an inverse correlation between OCT4 and clinical prognosis, there are studies rebuking these findings, possibly due to the sparsity of stem cells within tumors and the heterogeneity of tumors. In addition, post-translational modification of OCT4 affects its activity and warrants further investigation for its association with chemoresistance and prognosis.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/biossíntese , Processamento de Proteína Pós-Traducional , Animais , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética
6.
Mol Cancer Ther ; 18(12): 2270-2282, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31484706

RESUMO

Recurrent high-risk neuroblastoma is a childhood cancer that often fails to respond to therapy. Fenretinide (4-HPR) is a cytotoxic retinoid with clinical activity in recurrent neuroblastoma and venetoclax (ABT-199) is a selective inhibitor of the antiapoptotic protein B-cell lymphoma-2 (BCL-2). We evaluated activity of 4-HPR + ABT-199 in preclinical models of neuroblastoma. Patient-derived cell lines and xenografts from progressive neuroblastoma were tested. Cytotoxicity was evaluated by DIMSCAN, apoptosis by flow cytometry, and gene expression by RNA sequencing, quantitative RT-PCR, and immunoblotting. 4-HPR + ABT-199 was highly synergistic against high BCL-2-expressing neuroblastoma cell lines and significantly improved event-free survival of mice carrying high BCL-2-expressing patient-derived xenografts (PDX). In 10 matched-pair cell lines [established at diagnosis (DX) and progressive disease (PD) from the same patients], BCL-2 expression in the DX and PD lines was comparable, suggesting that BCL-2 expression at diagnosis may provide a biomarker for neuroblastomas likely to respond to 4-HPR + ABT-199. In a pair of DX (COG-N-603x) and PD (COG-N-623x) PDXs established from the same patient, COG-N-623x was less responsive to cyclophosphamide + topotecan than COG-N-603x, but both DX and PD PDXs were responsive to 4-HPR + ABT-199. Synergy of 4-HPR + ABT-199 was mediated by induction of NOXA via 4-HPR stimulation of reactive oxygen species that induced expression of ATF4 and ATF3, transcription factors for NOXA. Thus, fenretinide + venetoclax is a synergistic combination that warrants clinical testing in high BCL-2-expressing neuroblastoma.


Assuntos
Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Fenretinida/uso terapêutico , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Fenretinida/farmacologia , Humanos , Camundongos , Sulfonamidas/farmacologia
7.
Cell Death Dis ; 9(9): 925, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206207

RESUMO

Ceramide synthases (CERS) produce ceramides which are key intermediators in the biosynthesis of complex sphingolipids and play an important role in cell proliferation, differentiation, apoptosis and senescence. CERS6 is an isoform of ceramide synthases known to generate ceramides with C16 acyl chain (C16-Cer). CERS6 and C16-Cer levels were significantly higher in acute lymphoblastic leukemia (ALL) cells in comparison to peripheral blood mononuclear cells and T lymphocytes derived from healthy human volunteers. We investigated the role of CERS6 in chemo-resistance in T-ALL cell lines. Stable knockdown of CERS6 in CCRF-CEM and MOLT-4 cells resulted in increased sensitivity to ABT-737, a pan-BCL-2 inhibitor, while CCRF-CEM cells with exogenous CERS6 expression showed resistance to ABT-737 relative to the vector control. The cytotoxic activity of ABT-737 in CERS6 knockdown cells was significantly reduced by the addition of a caspase-8 inhibitor Z-IETD, suggesting that CERS6 alters the cytotoxicity via extrinsic pathway of apoptosis. By co-immunoprecipitation of CERS6 in CCRF-CEM cells, we identified CD95/Fas, a mediator of extrinsic apoptotic pathway, as a novel CERS6 binding partner. In Fas pull-down samples, FADD (Fas-associated protein with death domain) was detected at higher levels in cells with CERS6 knockdown compared with control cells when treated with ABT-737, and this was reversed by the overexpression of CERS6, demonstrating that CERS6 interferes with Fas-FADD DISC assembly. CERS6 may serve as a biomarker in determining the effectiveness of anticancer agents acting via the extrinsic pathway in T-ALL.


Assuntos
Apoptose/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas de Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Esfingosina N-Aciltransferase/metabolismo , Receptor fas/metabolismo , Compostos de Bifenilo/farmacologia , Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Ceramidas/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Nitrofenóis/farmacologia , Oligopeptídeos/farmacologia , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Esfingolipídeos/metabolismo , Esfingosina N-Aciltransferase/genética , Sulfonamidas/farmacologia
8.
Mol Carcinog ; 57(12): 1698-1706, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30129681

RESUMO

The phytonutrient ursolic acid (UA), present in apples, rosemary, and other plant sources, has anti-cancer properties in a number of systems, including skin cancers. However, few reports have examined upstream mechanisms by which UA may prevent or treat cancer. Recent reports have indicated UA induces death of cancer cell lines via AMP-activated protein kinase (AMPK), an energy-sensing kinase which possesses both pro-metabolic and anti-cancer effects. Other studies have shown UA activates peroxisome proliferator activated receptor α (PPARα) and the glucocorticoid receptor (GR). Here, we found the cytotoxic effect of UA in skin carcinoma cells required AMPK activation. In addition, two inhibitors of PPARα partially reversed the cytotoxic effects of UA, suggesting its effects are at least partially mediated through this receptor. Finally, inhibition of the GR did not reverse the effects of UA nor did this compound bind the GR under the conditions of experiments performed. Overall, studies elucidating the anti-cancer effects of UA may allow for the development of more potent analogues utilizing similar mechanisms. These studies may also reveal the mediators of any possible side effects or resistance mechanisms to UA therapy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , PPAR alfa/metabolismo , Neoplasias Cutâneas/metabolismo , Triterpenos/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Ácido Ursólico
9.
Melanoma Res ; 25(2): 103-12, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25647735

RESUMO

Malignant melanoma is associated with a 5-year survival rate of less than 20% once metastasized. Malignant melanoma cells exhibit increased levels of autophagy, a process of intracellular digestion that allows cells to survive various stresses including chemotherapies, resulting in reduced patient survival. Autophagy can be inhibited by chemicals like chloroquine (CQ), which prevents fusion of autophagosomes to lysosomes, resulting in autophagosome accumulation in most systems. Here, we describe how tested CQ to see whether it could sensitize B16F10 metastatic mouse melanoma cells to the anticancer activities of the natural compounds ursolic acid (UA) and resveratrol (RES). CQ with UA or RES strongly and synergistically reduced the viability of B16F10 mouse melanoma and A375 human melanoma cells. Surprisingly, flow cytometry of acridine orange-stained cells showed that UA or RES in combination with CQ significantly reduced autophagosome levels. Western blotting analysis revealed that CQ plus UA or RES paradoxically increased LC3II, indicative of autophagosome accumulation. In addition, CQ plus RES synergistically decreased the levels of both autophagy initiator beclin-1 and autophagy supporter p62. These results indicate that CQ with UA or RES strongly and synergistically reduces the viability of B16F10 and A375 melanoma cells. However, studies on B16F10 cells have shown that the synergistic effect was not mediated by inhibition of autophagy induced by UA or RES. These compounds are well-tolerated in humans, and CQ has shown promise as an adjuvant therapy. These combinations may be valuable treatment strategies for melanoma.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cloroquina/farmacologia , Melanoma Experimental/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Estilbenos/farmacologia , Triterpenos/farmacologia , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Melanoma Experimental/patologia , Camundongos , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Ácido Ursólico
10.
Protein Cell ; 5(2): 124-40, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24515614

RESUMO

Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.


Assuntos
Estresse Oxidativo/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Modificação Traducional de Proteínas/genética , Ubiquitinação/genética , Radicais Livres/metabolismo , Células HeLa , Humanos , Oxirredução , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Ubiquitina/metabolismo
11.
Mol Carcinog ; 53(9): 753-63, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23625588

RESUMO

Glucocorticoids (GCs) are well-known anti-inflammatory compounds, but they also inhibit cell proliferation depending on cell type. Similarly, peroxisome proliferator-activated receptors (PPARα, PPARδ, and PPARγ) also possess anti-proliferation properties beyond their canonical roles as metabolic mediators. In the present study, we investigated the potential additive or synergistic inhibitory effects on cancer cell proliferation by simultaneous application of fenofibrate and budesonide, agonists for PPARα and glucocorticoid receptor, respectively. We observed differential effects on cell proliferation in A549 and SK-MES-1 lung cancer cells by budesonide and fenofibrate. Fenofibrate inhibited cell proliferation in both TP53 wild type and deficient lung cancer cells. The anti-proliferation effect of budesonide in TP53 wild type A549 cells was abolished in SK-MES-1 cells that do not have wild type TP53 protein. An additive effect against cell proliferation by budesonide and fenofibrate combination was observed only in TP53 wild type A549 cancer cells. Analysis of cell cycle distribution and cyclin profile indicated that the inhibition of cell proliferation was associated with G1 cell cycle arrest. The suppression of NF-κB activity and ERK signaling may contribute to the inhibition of cell proliferation by budesonide and or fenofibrate. The additive inhibitory effect on cell proliferation by budesonide and fenofibrate combination suggests that the same or greater therapeutic effect could be achieved with reduced dosage and side effects when the two compounds are applied simultaneously.


Assuntos
Adenocarcinoma/tratamento farmacológico , Budesonida/farmacologia , Proliferação de Células/efeitos dos fármacos , Fenofibrato/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , PPAR alfa/agonistas , Receptores de Glucocorticoides/agonistas , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Anti-Inflamatórios/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Hipolipemiantes/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , PPAR alfa/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
12.
Mol Cancer Res ; 11(12): 1521-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24072817

RESUMO

UNLABELLED: Ursolic acid, present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to ursolic acid treatment. Herein, skin carcinoma cells (Ca3/7) as compared with skin papilloma cells (MT1/2) displayed more resistance to ursolic acid-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in preclinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate rhodamine 123 (Rh123) indicating ursolic acid is transported by P-gp. To determine whether P-gp inhibition can enhance ursolic acid-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil or cyclosporin A. Alternatively, cells were pretreated with the natural compound resveratrol, a known chemotherapy sensitizer. Verapamil and resveratrol enhanced the effects of ursolic acid in both cell lines, whereas cyclosporin A only did so in Ca3/7 cells. Similarly, verapamil inhibited Rh123 efflux in both lines, whereas cyclosporin A only inhibited Rh123 efflux in Ca3/7 cells. Resveratrol did not inhibit Rh123 efflux in either line, indicating the synergistic effects of resveratrol and ursolic acid are not manifest by inhibition of P-gp-mediated efflux of ursolic acid. These results indicate that the anti-skin cancer effects of ursolic acid are enhanced with P-gp inhibitors. In addition, resveratrol and ursolic acid interact synergistically, but not through inhibition of P-gp. IMPLICATIONS: Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos Fitogênicos/farmacologia , Ciclosporina/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Estilbenos/farmacologia , Triterpenos/farmacologia , Verapamil/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Camundongos , Resveratrol , Rodamina 123/metabolismo , Triterpenos/farmacocinética , Ácido Ursólico
14.
Carcinogenesis ; 34(1): 68-78, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23024267

RESUMO

The morphological detection of early neoplastic transformation leading to cervical cancer remains problematic. In this work, we have identified deleted in split hand/split foot 1 protein (DSS1) as an early biomarker that is specifically upregulated in premalignant and malignant cervical epithelial cells, but is low or undetectable in non-malignant cells. DSS1 mRNA and protein levels are significantly increased in cultured human cervical carcinoma cell lines originating from primary and metastatic tumors. In fact, > 96% of patient tumor tissues were found to have cells with elevated DSS1 when compared with tumor-adjacent normal cells. In histological sections of cervical tissue containing either invasive cervical carcinoma or its precursor lesions, DSS1 was readily detected in the tumor cells. Steady-state DSS1 expression by immortalized cervical cancer cell lines was found to be necessary for maintenance of their transformed phenotype, since stable shRNA-mediated depletion of DSS1 in HeLa cells inhibited their proliferation and colony-forming activity in monolayer cultures and prevented division of these cells in soft agar. When DSS1 levels are reduced using shRNA, the cells ultimately undergo apoptosis via activation of p53 and the p53 downstream targets, and cleavage of apoptosis-associated proteins including CPP32/caspase-3, poly(ADP-ribose)polymerase and DNA-PKcs. In addition, silencing of DSS1 makes cervical cancer cells sensitive to cell death after treatment with cisplatin. We conclude that the DSS1 protein is critically involved in the maintenance of the transformed phenotype in cervical cancer cells, and that it might be a specific, robust and reliable marker for early detection, diagnosis and treatment.


Assuntos
Biomarcadores Tumorais/genética , Complexo de Endopeptidases do Proteassoma/genética , RNA Mensageiro/genética , Neoplasias do Colo do Útero/diagnóstico , Sequência de Bases , Primers do DNA , Feminino , Técnicas de Silenciamento de Genes , Humanos , Reação em Cadeia da Polimerase
15.
J Biol Chem ; 287(42): 35756-35767, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22927439

RESUMO

Cullin-RING E3 ligases (CRLs) are a class of ubiquitin ligases that control the proteasomal degradation of numerous target proteins, including IκB, and the activity of these CRLs are positively regulated by conjugation of a Nedd8 polypeptide onto Cullin proteins in a process called neddylation. CRL-mediated degradation of IκB, which normally interacts with and retains NF-κB in the cytoplasm, permits nuclear translocation and transactivation of the NF-κB transcription factor. Neddylation occurs through a multistep enzymatic process involving Nedd8 activating enzymes, and recent studies have shown that the pharmacological agent, MLN4924, can potently inhibit Nedd8 activating enzymes, thereby preventing neddylation of Cullin proteins and preventing the degradation of CRL target proteins. In macrophages, regulation of NF-κB signaling functions as a primary pathway by which infectious agents such as lipopolysaccharides (LPSs) cause the up-regulation of proinflammatory cytokines. Here we have analyzed the effects of MLN4924, and compared the effects of MLN4924 with a known anti-inflammatory agent (dexamethasone), on certain proinflammatory cytokines (TNF-α and IL-6) and the NF-κB signaling pathway in LPS-stimulated macrophages. We also used siRNA to block neddylation to assess the role of this molecular process during LPS-induced cytokine responsiveness. Our results demonstrate that blocking neddylation, either pharmacologically or using siRNA, abrogates the increase in certain proinflammatory cytokines secreted from macrophages in response to LPS. In addition, we have shown that MLN4924 and dexamethasone inhibit LPS-induced cytokine up-regulation at the transcriptional level, albeit through different molecular mechanisms. Thus, neddylation represents a novel molecular process in macrophages that can be targeted to prevent and/or treat the LPS-induced up-regulation of proinflammatory cytokines and the disease processes associated with their up-regulation.


Assuntos
Mediadores da Inflamação/metabolismo , Interleucina-6/biossíntese , Macrófagos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Ubiquitinas/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular Tumoral , Ciclopentanos/farmacologia , Dexametasona/farmacologia , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Camundongos , Proteína NEDD8 , NF-kappa B/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Pirimidinas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Enzimas Ativadoras de Ubiquitina , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia
16.
Int J Mass Spectrom ; 301(1-3): 12-21, 2011 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-21499536

RESUMO

CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown.Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309-382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34.

17.
Mol Carcinog ; 50(12): 981-91, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21438027

RESUMO

The T-box transcription factor, Tbx1, an important regulatory gene in development, is highly expressed in hair follicle (HF) stem cells in adult mice. Because mouse models of skin carcinogenesis have demonstrated that HF stem cells are a carcinogen target population and contribute significantly to tumor development, we investigated whether Tbx1 plays a role in skin carcinogenesis. We first assessed Tbx1 expression levels in mouse skin tumors, and found down-regulation in all tumors examined. To study the effect of Tbx1 expression on growth and tumorigenic potential of carcinoma cells, we transfected mouse Tbx1 cDNA into a mouse spindle cell carcinoma cell line that did not express endogenous Tbx1. Following transfection, two cell lines expressing different levels of the Tbx1/V5 fusion protein were selected for further study. Intradermal injection of the cell lines into mice revealed that Tbx1 expression significantly suppressed tumor growth, albeit with no change in tumor morphology. In culture, ectopic Tbx1 expression resulted in decreased cell growth and reduced development into multilayered colonies, compared to control cells. Tbx1-transfectants exhibited a reduced proliferative rate compared to control cells, with fewer cells in S and G2/M phases. The Tbx1 transfectants developed significantly fewer colonies in soft agar, demonstrating loss of anchorage-independent growth. Taken together, our data show that ectopic expression of Tbx1 restored contact inhibition to the skin tumor cells, suggesting that this developmentally important transcription factor may have a novel dual role as a negative regulator of tumor growth. © 2011 Wiley Periodicals, Inc.


Assuntos
Neoplasias Cutâneas/patologia , Proteínas com Domínio T/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Inibição de Contato , Regulação Neoplásica da Expressão Gênica , Camundongos , Camundongos Transgênicos , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Transfecção
18.
Neuropsychopharmacology ; 34(10): 2344-57, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19536110

RESUMO

Memantine shows clinically relevant efficacy in patients with Alzheimer's disease and Parkinson's disease. Most in vivo and in vitro studies attribute the neuroprotective effects of memantine to the blockade of N-methyl-D-aspartate (NMDA) receptor on neurons. However, it cannot be excluded that mechanisms other than NMDA receptor blockade may contribute to the neuroprotective effects of this compound. To address this question, primary midbrain neuron-glia cultures and reconstituted cultures were used, and lipopolysaccharide (LPS), an endotoxin from bacteria, was used to produce inflammation-mediated dopaminergic (DA) neuronal death. Here, we show that memantine exerted both potent neurotrophic and neuroprotective effects on DA neurons in rat neuron-glia cultures. The neurotrophic effect of memantine was glia dependent, as memantine failed to show any positive effect on DA neurons in neuron-enriched cultures. More specifically, it seems to be that astroglia, not microglia, are the source of the memantine-elicited neurotrophic effects through the increased production of glial cell line-derived neurotrophic factor (GDNF). Mechanistic studies showed that GDNF upregulation was associated with histone hyperacetylation by inhibiting the cellular histone deacetylase activity. In addition, memantine also displays neuroprotective effects against LPS-induced DA neuronal damage through its inhibition of microglia activation showed by both OX-42 immunostaining and reduction of pro-inflammatory factor production, such as extracellular superoxide anion, intracellular reactive oxygen species, nitric oxide, prostaglandin E(2), and tumor necrosis factor-alpha. These results suggest that the neuroprotective effects of memantine shown in our cell culture studies are mediated in part through alternative novel mechanisms by reducing microglia-associated inflammation and by stimulating neurotrophic factor release from astroglia.


Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Memantina/farmacologia , Neuroglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Antígeno CD11b/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina/métodos , Dinoprostona/metabolismo , Dopamina/metabolismo , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Histona Desacetilases/metabolismo , Óxido Nítrico/metabolismo , Gravidez , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo , Estatísticas não Paramétricas , Superóxidos/metabolismo , Trítio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
19.
J Mol Biol ; 383(3): 693-712, 2008 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-18775730

RESUMO

Deleted in Split hand/Split foot 1 (DSS1) was previously identified as a novel 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible gene with possible involvement in early event of mouse skin carcinogenesis. The mechanisms by which human DSS1 (HsDSS1) exerts its biological effects via regulation of the ubiquitin-proteasome system (UPS) are currently unknown. Here, we demonstrated that HsDSS1 regulates the human proteasome by associating with it in the cytosol and nucleus via the RPN3/S3 subunit of the 19S regulatory particle (RP). Molecular anatomy of HsDSS1 revealed an RPN3/S3-interacting motif (R3IM), located at amino acid residues 15 to 21 of the NH(2) terminus. Importantly, negative charges of the R3IM motif were demonstrated to be required for proteasome interaction and binding to poly-ubiquitinated substrates. Indeed, the R3IM motif of HsDSS1 protein alone was sufficient to replace the ability of intact HsDSS1 protein to pull down proteasome complexes and protein substrates with high-molecular mass ubiquitin conjugates. Interestingly, this interaction is highly conserved throughout evolution from humans to nematodes. Functional study, lowering the levels of the endogenous HsDSS1 using siRNA, indicates that the R3IM/proteasome complex binds and targets p53 for ubiquitin-mediated degradation via gankyrin-MDM2/HDM2 pathway. Most significantly, this work indicates that the R3IM motif of HsDSS1, in conjunction with the complexes of 19S RP and 20S core particle (CP), regulates proteasome interaction through RPN3/S3 molecule, and utilizes a specific subset of poly-ubiquitinated p53 as a substrate.


Assuntos
Sequência de Aminoácidos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Evolução Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Alinhamento de Sequência , Proteína Supressora de Tumor p53/genética , Ubiquitina/metabolismo
20.
J Immunol ; 181(1): 660-8, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18566433

RESUMO

TGF-beta1 is one of the most potent endogenous immune modulators of inflammation. The molecular mechanism of its anti-inflammatory effect on the activation of the transcription factor NF-kappaB has been well-studied; however, the potential effects of TGF-beta1 on other proinflammatory signaling pathways is less clear. In this study, using the well-established LPS and the 1-methyl-4-phenylpyridinium-mediated models of Parkinson's disease, we demonstrate that TGF-beta1 exerts significant neuroprotection in both models via its anti-inflammatory properties. The neuroprotective effects of TGF-beta1 are mainly attributed to its ability to inhibit the production of reactive oxygen species from microglia during their activation or reactivation. Moreover, we demonstrate that TGF-beta1 inhibited LPS-induced NADPH oxidase (PHOX) subunit p47phox translocation from the cytosol to the membrane in microglia within 10 min. Mechanistic studies show that TGF-beta1 fails to protect dopaminergic neurons in cultures from PHOX knockout mice, and significantly reduced LPS-induced translocation of the PHOX cytosolic subunit p47phox to the cell membrane. In addition, LPS-induced ERK phosphorylation and subsequent Ser345 phosphorylation on p47phox were significantly inhibited by TGF-beta1 pretreatment. Taken together, our results show that TGF-beta1 exerted potent anti-inflammatory and neuroprotective properties, either through the prevention of the direct activation of microglia by LPS, or indirectly through the inhibition of reactive microgliosis elicited by 1-methyl-4-phenylpyridinium. The molecular mechanisms of TGF-beta1-mediated anti-inflammatory properties is through the inhibition of PHOX activity by preventing the ERK-dependent phosphorylation of Ser345 on p47phox in microglia to reduce oxidase activities induced by LPS.


Assuntos
Glicoproteínas de Membrana/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NADPH Oxidases/metabolismo , Fosfosserina/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , 1-Metil-4-fenilpiridínio/farmacologia , Animais , Membrana Celular/metabolismo , Citoproteção/efeitos dos fármacos , Citosol/metabolismo , Dopamina/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Ratos , Espécies Reativas de Oxigênio/metabolismo , Técnicas de Cultura de Tecidos
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