RESUMO
Melanosomes are specific organelles dedicated to melanin synthesis and accumulation in melanocytes. Autophagy is suggestively involved in melanosome degradation, although the potential underlying molecular mechanisms remain elusive. In selective autophagy, autophagy receptors and E3-ligases are the key factors conferring cargo selectivity. In B16F10 cells, ß-mangostin efficiently induced melanosome degradation without affecting other organelles such as mitochondria, peroxisomes, and the endoplasmic reticulum. Among various autophagy receptors, optineurin (OPTN) contributes TANK-binding kinase 1 (TBK1)-dependently to melanosome degradation and its knockdown inhibited ß-mangostin-mediated melanosome degradation. OPTN translocation to melanosomes was dependent on its ubiquitin-binding domain. Moreover, OPTN-mediated TBK1 activation and subsequent TBK1-mediated S187 OPTN phosphorylation were essential for melanosome degradation. ß-mangostin increased K63-linked melanosome ubiquitination. Finally, the E3-ligase RCHY1 knockdown inhibited the melanosome ubiquitination required for OPTN- and TBK1-phosphorylation as well as melanosome degradation. This study suggests that melanophagy, melanosome-selective autophagy, contributes to melanosome degradation, and OPTN and RCHY1 are an essential autophagy receptor and a E3-ligase, respectively, conferring cargo selectivity in melanophagy.
Assuntos
Autofagia , Melanossomas , Melanossomas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Xantonas , Melanoma Experimental , Animais , CamundongosRESUMO
BACKGROUND: Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays an important role in diverse cellular processes by regulating Rho guanosine triphosphate (GTP)ases activity. RhoGDI1 phosphorylation regulates the spatiotemporal activation of Rho GTPases during cell migration. In this study, we identified polo-like kinase 1 (PLK1) as a novel kinase of RhoGDI1 and investigated the molecular mechanism by which the interaction between RhoGDI1 and PLK1 regulates cancer cell migration. METHODS: Immunoprecipitation, GST pull-down assay, and proximity ligation assay (PLA) were performed to analyze the interaction between RhoGDI1 and PLK1. In vitro kinase assay and immunoprecipitation were performed with Phospho-(Ser/Thr) antibody. We evaluated RhoA activation using RhoGTPases activity assay. Cell migration and invasion were analyzed by transwell assays. RESULTS: GST pull-down assays and PLA showed that PLK1 directly interacted with RhoGDI1 in vitro and in vivo. Truncation mutagenesis revealed that aa 90-111 of RhoGDI1 are critical for interacting with PLK1. We also showed that PLK1 phosphorylated RhoGDI1 at Thr7 and Thr91, which induces cell motility. Overexpression of the GFP-tagged RhoGDI1 truncated mutant (aa 90-111) inhibited the interaction of PLK1 with RhoGDI1 and attenuated RhoA activation by PLK1. Furthermore, the overexpression of the RhoGDI1 truncated mutant reduced cancer cell migration and invasion in vitro and suppressed lung metastasis in vivo. CONCLUSIONS: Collectively, we demonstrate that the phosphorylation of RhoGDI1 by PLK1 promotes cancer cell migration and invasion through RhoA activation. This study connects the interaction between PLK1 and RhoGDI1 to the promotion of cancer cell behavior associated with malignant progression, thereby providing opportunities for cancer therapeutic interventions.
RESUMO
BACKGROUND: The exosome-mediated extracellular secretion of miRNAs occurs in many cancers, and RAB27A is a potent regulator of exosome secretion. For metastatic renal cell carcinoma (RCC), this study examines the mechanisms of cancer metastasis via the RAB27A-regulated secretion of specific miRNAs. METHODS: RAB27A knockdown (KD) and overexpressing (OE) RCC cells were used to examine cell migration and adhesion. The particle counts and sizes of exosomes in RAB27A OE cells were analyzed using Exoview, and those of intraluminal vesicles (ILV) and multivesicular bodies (MVB) were measured using an electron microscope. Analysis of RNA sequences, protein-protein interaction networks, and the competing endogenous RNA (ceRNA) network were used to identify representative downregulated miRNAs that are likely to undergo cargo-sorting into exosomes and subsequent secretion. A molecular beacon of miR-137-3p, one of the most representatively downregulated genes with a fold change of 339, was produced, and its secretion was analyzed using Exoview. RAB27A OE and control cells were incubated in an exosome-containing media to determine the uptake of tumor suppressor miRNAs that affect cancer cell metastasis. RESULTS: Migration and cell adhesion were higher in RAB27A OE cells than in RAB27A KD cells. Electron microscopy revealed that the numbers of multivesicular bodies and intraluminal vesicles per cell were higher in RAB27A OE cells than in control cells, suggesting their secretion. The finding revealed that miR-127-3p was sorted into exosomes and disposed of extracellularly. Protein-protein interaction analysis revealed MYCN to be the most significant hub for RAB27A-OE RCC cells. ceRNA network analysis revealed that MAPK4 interacted strongly with miR-127-3p. CONCLUSION: The disposal of miR-127-3p through exosome secretion in RAB27A overexpressing cells may not inhibit the MAPK pathway to gain metastatic potential by activating MYCN. The exosomes containing miRNAs are valuable therapeutic targets for cancer treatment.
RESUMO
In this study, we investigated the depigmentation effect of Amorpha fruticosa L. root extract (RE), an herbal medicine. A. fruticosa RE significantly induced depigmentation in α-MSH-treated B16F10 cells at noncytotoxic concentrations. Further, the RE decreased the protein levels of the melanosomal proteins Tyr and Pmel without decreasing their transcript levels. We found that MG132, a proteasome complex inhibitor, was unable to rescue the protein levels, but PepA/E-64D (a lysosomal enzyme inhibitor), 3-MA (a representative autophagy inhibitor), and ATG5 knockdown effectively rescued the protein levels and inhibited the depigmentation effect following RE treatment. Among rotenoids, amorphigenin composed in the RE was identified as a functional chemical that could induce depigmentation; whereas rapamycin, an mTOR inhibitor and a nonselective autophagy inducer, could not induce depigmentation, and amorphigenin effectively induced depigmentation through the degradation of melanosomal proteins. Amorphigenin activated AMPK without affecting mTOR, and knockdown of AMPK offset the whitening effect through degradation of melanosome proteins by amorphigenin. Results from this study suggested that amorphigenin can induce degradation of the melanosome through an AMPK-dependent autophagy process, and has the potential to be used as a depigmentation agent for the treatment of hyperpigmentation.
RESUMO
Melanin pigment produced in melanocytes plays a protective role against ultraviolet radiation. Selective destruction of melanocytes causes chronic depigmentation conditions such as vitiligo, for which there are very few specific medical treatments. Here, we found that fraxinol, a natural coumarin from Fraxinus plants, effectively stimulated melanogenesis. Treatment of B16-F10 cells with fraxinol increased the melanin content and tyrosinase activity in a concentration-dependent manner without causing cytotoxicity. Additionally, fraxinol enhanced the mRNA expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. Fraxinol also increased the expression of microphthalmia-associated transcription factor at both mRNA and protein levels. Fraxinol upregulated the phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). Furthermore, H89, a cAMP-dependent protein kinase A inhibitor, decreased fraxinol-induced CREB phosphorylation and microphthalmia-associated transcription factor expression and significantly attenuated the fraxinol-induced melanin content and intracellular tyrosinase activity. These results suggest that fraxinol enhances melanogenesis via a protein kinase A-mediated mechanism, which may be useful for developing potent melanogenesis stimulators.
Assuntos
Fator de Transcrição Associado à MicroftalmiaRESUMO
Evidence suggests that augmented expression of a certain gene can influence the efficacy of targeted and conventional chemotherapies. Here, we tested whether the high expression of enhancer of the rudimentary homolog (ERH), which serves as a prognostic factor in some cancers, can influence the efficacy of anthocyanins isolated from fruits of Vitis coignetiae Pulliat, Meoru in Korea (AIMs) on human gastric cancer cells. The anticancer efficacy of AIMs was augmented in ERH-transfected MKN28 cells (E-MKN28 cells). Molecularly, ERH augmented AIM-induced caspase-dependent apoptosis by activating caspase-3 and -9. The ERH-augmented apoptotic effect was related to mitochondrial depolarization and inhibition of antiapoptotic proteins, XIAP, and Bcl-2. In addition, reactive oxygen species (ROS) generation was augmented in AIMs-treated E-MKN28 cells compared to AIMs-treated naïve MKN28 cells. In conclusion, ERH augmented AIM-induced caspase-dependent mitochondrial-related apoptosis in MKN28 cells. A decrease in expression of Bcl-2 and subsequent excessive ROS generation would be the mechanism for ERH-augmented mitochondrial-related apoptosis in AIMs-treated MKN28 cells. A decrease in expression of XIAP would be another mechanism for ERH-augmented caspase-dependent apoptosis in AIMs-treated MKN28 cells.
Assuntos
Antocianinas/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Neoplasias Gástricas/metabolismo , Fatores de Transcrição/metabolismo , Vitis/química , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that consists of Crohn's disease (CD) and ulcerative colitis (UC). Cytokines are thought to be key mediators of inflammation-mediated pathological processes of IBD. These cytokines play a crucial role through the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathways. Several small molecules inhibiting JAK have been used in clinical trials, and one of them has been approved for IBD treatment. Many anti-inflammatory phytochemicals have been shown to have potential as new drugs for IBD treatment. This review describes the significance of the JAK-STAT pathway as a current therapeutic target for IBD and discusses the recent findings that phytochemicals can ameliorate disease symptoms by affecting the JAK-STAT pathway in vivo in IBD disease models. Thus, we suggest that phytochemicals modulating JAK-STAT pathways are potential candidates for developing new therapeutic drugs, alternative medicines, and nutraceutical agents for the treatment of IBD.
Assuntos
Doenças Inflamatórias Intestinais/tratamento farmacológico , Janus Quinases/metabolismo , Compostos Fitoquímicos/uso terapêutico , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Humanos , Compostos Fitoquímicos/farmacologiaRESUMO
Tumor cell resistance to anti-cancer drugs is a major obstacle in tumor therapy. In this study, we investigated the mechanism of cordycepin-mediated resensitization to cisplatin in T24R2 cells, a T24-derived cell line. Treatment with cordycepin or cisplatin (2 µg/mL) alone failed to induce cell death in T24R2 cells, but combination treatment with these drugs significantly induced apoptosis through mitochondrial pathways, including depolarization of mitochondrial membranes, decrease in anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, and increase in pro-apoptotic proteins Bak and Bax. High expression levels of MDR1 were the cause of cisplatin resistance in T24R2 cells, and cordycepin significantly reduced MDR1 expression through inhibition of MDR1 promoter activity. MDR1 promoter activity was dependent on transcription factor Ets-1 in T24R2 cells. Although correlation exists between MDR1 and Ets-1 expression in bladder cancer patients, active Ets-1, Thr38 phosphorylated form (pThr38), was critical to induce MDR1 expression. Cordycepin decreased pThr-38 Ets-1 levels and reduced MDR1 transcription, probably through its effects on PI3K signaling, inducing the resensitization of T24R2 cells to cisplatin. The results suggest that cordycepin effectively resensitizes cisplatin-resistant bladder cancer cells to cisplatin, thus serving as a potential strategy for treatment of cancer in patients with resistance to anti-cancer drugs.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Desoxiadenosinas/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias da Bexiga Urinária/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Interações Medicamentosas , Humanos , Proteína Proto-Oncogênica c-ets-1/metabolismoRESUMO
Apoptosis pathways in cells are classified into two pathways: the extrinsic pathway, mediated by binding of the ligand to a death receptor and the intrinsic pathway, mediated by mitochondria. Apoptosis is regulated by various proteins such as Bcl-2 (B-cell lymphoma 2) family and cellular FLICE (Fas-associated Death Domain Protein Interleukin-1ß-converting enzyme)-inhibitory protein (c-FLIP), which have been reported to inhibit caspase-8 activity. In this study, it was found that C5 (3ß-Acetyl-nor-erythrophlamide), a compound of cassaine diterpene amine from Erythrophleum fordii, induced cell apoptosis in a variety of types of cancer cells. Induction of apoptosis in cancer cells by C5 was inversely related to the level of Bcl-2 expression. Overexpression of Bcl-2 into cancer cells significantly decreased C5-induced apoptosis. It was also found that treatment of cancer cells with a caspase-8 inhibitor significantly suppressed C5-induced apoptosis; however, treatment with caspase-9 inhibitors did not affect C5-induced apoptosis, suggesting that C5 may induce apoptosis via the extrinsic pathway by activating caspase-8. It was confirmed that treatment with C5 alone induced an association of FADD with procaspase-8; however, overexpression of c-FLIP decreased C5-induced caspase-8 activation. In conclusion, C5 could be utilized as a new useful lead compound for the development of an anti-cancer agent that has the goal of apoptosis.
Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Alcaloides/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fabaceae/química , Fabaceae/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Linfoma/metabolismo , Linfoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Melanogenesis is a key pathway for the regulation of skin pigmentation and the development of skin-lightening/skin-whitening drugs or cosmetics. In this study, we found that ß-mangostin from seedcases of Garcinia mangostana inhibited α-melanocyte-stimulating hormone (α-MSH)-mediated melanogenesis in B16F10 melanoma cells and a three-dimensional human skin model. ß-Mangostin significantly inhibited the protein level of tyrosinase induced by α-MSH in UPS (ubiquitin proteasome system)-independent and lysosome-dependent manner. The inhibition of autophagy by 3-methyladenine treatment or ATG5 knockdown effectively recovered premelanosome protein as well as tyrosinase degraded by the ß-mangostin treatment. However, rapamycin, a representative non-selective autophagy inducer, triggered autophagy in α-MSH-stimulated cells, which was characterized by a considerable decrease in p62, but it was unable to inhibit melanogenesis. Melanosome-engulfing autophagosomes were observed using transmission electron microscopy. Furthermore, previously formed melanin could be degraded effectively in an autophagy-dependent manner in ß-mangostin-treated cells. Taken together, our results suggest that ß-mangostin inhibits the melanogenesis induced by α-MSH via an autophagy-dependent mechanism, and thus, the depigmentation effect of ß-mangostin may depend on autophagy targeted at the melanosome rather than non-selective autophagy.
Assuntos
Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Xantonas/farmacologia , alfa-MSH/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia , Sobrevivência Celular , Garcinia mangostana , Humanos , Inflamação , Melaninas/metabolismo , Melanócitos/citologia , Melanoma Experimental , Melanossomas/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Monofenol Mono-Oxigenase/metabolismo , Pigmentação , Extratos Vegetais/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Sementes/química , Pele/metabolismo , Ubiquitina/metabolismoRESUMO
Rho GDP dissociation inhibitor 2 (RhoGDI2) expression is correlated with tumor growth, metastasis and chemoresistance in gastric cancer. However, the mechanisms by which RhoGDI2 promotes tumor cell survival and metastasis remain unclear. In this study, we clearly demonstrate that RhoGDI2 upregulates VEGF-C expression and RhoGDI2 expression is positively correlated with VEGF-C expression in human gastric tumor tissues as well as parental gastric cancer cell lines. VEGF-C depletion suppressed RhoGDI2-induced gastric cancer metastasis and sensitized RhoGDI2-overexpressing cells to cisplatin-induced apoptosis in vitro and in vivo. Secreted VEGF-C enhanced gastric cancer cell invasion and conferred cisplatin resistance to RhoGDI2-overexpressing cells. We also show that RhoGDI2 positively regulates Rac1 activity in gastric cancer cells. Inhibition of Rac1 expression suppressed RhoGDI2-induced VEGF-C expression, and this inhibition was associated with decreased invasiveness and increased sensitivity to cisplatin in RhoGDI2-overexpressing cells. Our results indicate that RhoGDI2 might be a potential therapeutic target for simultaneously reducing metastasis risk and enhancing chemotherapy efficacy in gastric cancer.
Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/secundário , Neoplasias Gástricas/patologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Análise Serial de Tecidos , Células Tumorais Cultivadas , Fator C de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/antagonistas & inibidores , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/genéticaRESUMO
Octamer transcription factor-1 (OCT-1) is a well-known transcription factor that is reportedly overexpressed in intestinal metaplasia and gastric carcinoma in the intestine. In this study, we investigated OCT-1 overexpression as a prognostic factor for gastric cancer. The association between OCT-1 overexpression (detected using immunohistochemistry) and clinicopathological features including survival was evaluated. In vitro gain-of-function approaches were utilized to assess the function of OCT-1 in malignancy. Analysis of OCT-1 expression in patients with gastric cancer with well-differentiated carcinoma as per the World Health Organization classification showed that OCT-1 overexpression was correlated with advanced tumor invasion (58.8 % of patients with advanced tumor invasion vs. 21.2 % of patients with early tumor invasion; p<0.01), lymph node metastasis (63.9 % of patients with metastasis vs. 24.1 % of those without; p=0.015), and cancer recurrence (83.3 % of patients with recurrence vs. 25.4 % of those without; p<0.01), as well as a lower survival rate (62.8 vs. 87.9 Mo; p<0.01). However, there were no significant differences in the levels of OCT-1 expression in gastric cancer patients with other carcinoma types (p>0.05). Furthermore, we found that the proliferation rate of OCT-1-overexpressing MKN-45 cells was higher than that of the control cells. OCT-1 overexpression may be a marker for poor prognosis in patients with well-differentiated gastric adenocarcinoma.
Assuntos
Fator 1 de Transcrição de Octâmero/fisiologia , Neoplasias Gástricas/mortalidade , Idoso , Fator de Transcrição CDX2 , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Proteínas de Homeodomínio/genética , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Fator 1 de Transcrição de Octâmero/análise , Fator 1 de Transcrição de Octâmero/genética , Prognóstico , Neoplasias Gástricas/química , Neoplasias Gástricas/patologiaRESUMO
Snail is a key regulator of the epithelial-mesenchymal transition (EMT), the key step in the tumorigenesis and metastasis of tumors. Although induction of Snail transcription precedes the induction of EMT, the post-translational regulation of Snail is also important in determining Snail protein levels, stability, and its ability to induce EMT. Several kinases are known to enhance the stability of the Snail protein by preventing its ubiquitination; however, the precise molecular mechanisms by which these kinases prevent Snail ubiquitination remain unclear. Here, we identified ERK3 as a novel kinase that interacts with Snail and enhances its protein stability. Although ERK3 could not directly phosphorylate Snail, Erk3 increased Snail protein stability by inhibiting the binding of FBXO11, an E3 ubiquitin ligase that can induce Snail ubiquitination and degradation, to Snail. Importantly, functional studies and analysis of clinical samples indicated the crucial role of ERK3 in the regulation of Snail protein stability in pancreatic cancer. Therefore, we conclude that ERK3 is a key regulator for enhancing Snail protein stability in pancreatic cancer cells by inhibiting the interaction between Snail and FBXO11.
RESUMO
Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2-DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-of-function analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Eletroforese em Gel Bidimensional , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metástase Neoplásica , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , Neoplasias Gástricas/tratamento farmacológico , Regulação para Cima , Quinases Ativadas por p21/análise , Quinases Ativadas por p21/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate to form orthophosphate. Pyrophosphate can substitute for ATP under certain circumstances. We previously conducted a proteomic analysis to investigate tumor-specific protein expression in gastric cancer, and PPase was identified as a potential gastric tumor-specific marker; it was therefore selected for further study. Clinicopathological analysis, using proteomic analysis and immunohistochemistry, was used to validate PPase as a prognostic marker in gastric cancers. Proteomic analysis showed that PPase was overexpressed in patients with lymph node (LN) metastases and high tumor node metastasis (TNM) stages (p < 0.05). Based on immunohistochemistry, patients whose tumors overexpressed PPase had higher T stages, LN metastasis, a higher TNM stage, a higher cancer recurrence rate, and shorter survival times than patients whose tumors exhibited PPase underexpression (p < 0.05). Gain-of-function and loss-of-function approaches were employed to examine the malignant phenotypes of PPase-overexpressing or PPase-depleted cells. A decrease in PPase expression caused a significant decrease in gastric cancer cell migration and invasion in vitro, whereas forced overexpression of PPase enhanced migration but not invasion. Our findings indicate that PPase is involved in gastric tumor progression and that PPase may be a useful marker for poor prognosis of human gastric cancers.
Assuntos
Adenocarcinoma Mucinoso/patologia , Biomarcadores Tumorais/metabolismo , Pirofosfatase Inorgânica/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias Gástricas/patologia , Estômago/patologia , Adenocarcinoma Mucinoso/enzimologia , Adenocarcinoma Mucinoso/mortalidade , Western Blotting , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Progressão da Doença , Eletroforese em Gel Bidimensional , Feminino , Humanos , Técnicas Imunoenzimáticas , Pirofosfatase Inorgânica/antagonistas & inibidores , Pirofosfatase Inorgânica/genética , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/mortalidade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estômago/enzimologia , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/mortalidade , Taxa de Sobrevida , Análise Serial de Tecidos , Células Tumorais CultivadasRESUMO
Rho GDP dissociation inhibitor 2 (RhoGDI2), a regulator of Rho family GTPase, has been known to promote tumor growth and malignant progression in gastric cancer. We previously showed that RhoGDI2 positively regulates Rac1 activity and Rac1 activation is critical for RhoGDI2-induced gastric cancer cell invasion. In this study, to identify the precise molecular mechanism by which RhoGDI2 activates Rac1 activity, we performed two-hybrid screenings using yeast and found that RhoGDI2 plays an important role in the interaction between Rac1, Filamin A and Rac1 activation in gastric cancer cells. Moreover, we found that Filamin A is required for Rac1 activation and the invasive ability of gastric cancer cells. Depletion of Filamin A expression markedly reduced Rac1 activity in RhoGDI2-expressing gastric cancer cells. The migration and invasion ability of RhoGDI2-expressing gastric cancer cells also substantially decreased when Filamin A expression was depleted. Furthermore, we found that Trio, a Rac1-specific guanine nucleotide exchange factor (GEF), is critical for Rac1 activation and the invasive ability of gastric cancer cells. Therefore, we conclude that RhoGDI2 increases Rac1 activity by recruiting Rac1 to Filamin A and enhancing the interaction between Rac1 and Trio, which is critical for the invasive ability of gastric cancer cells.
RESUMO
Gadd45b has been known as a positive mediator of apoptosis induced by certain cytokines and oncogenes. Here, we identified Gadd45b as an effector of Fas-induced apoptosis and found that p38-mediated Rb hyperphosphorylation is one of the mechanisms of Fas-induced apoptosis in murine hepatocyte AML12 cells. Gadd45b has been shown to activate p38 through its physical interaction with MTK1 and induce apoptosis. However, in this study, we have showed that the function of Gadd45b during Fas-induced apoptosis in AML12 cells is different from that reported in previous studies. Depletion of Gadd45b expression did not inhibit the phosphorylation of p38, but it suppressed p38-mediated Rb phosphorylation and apoptosis in response to Fas stimulation by reducing the interaction between p38 and Rb. Ectopic expression of Gadd45b was sufficient to enhance this interaction. These findings suggest that Gadd45b mediates p38-induced Rb phosphorylation by enhancing the interaction between p38 and Rb during Fas-induced apoptosis in murine hepatocytes.
Assuntos
Antígenos de Diferenciação/metabolismo , Proteína do Retinoblastoma/metabolismo , Receptor fas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Imidazóis/farmacologia , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Camundongos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Piridinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor fas/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Rho GDP dissociation inhibitor 2 (RhoGDI2) is a regulator of the Rho family GTPases. Recent work from our laboratory suggests that RhoGDI2 expression potentially enhances resistance to cisplatin as well as promotes tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that phospholipase C-gamma (PLCγ) is required for RhoGDI2-mediated cisplatin resistance and cancer cell invasion in gastric cancer. The levels of phosphorylated PLCγ are markedly enhanced in RhoGDI2-overexpressing SNU-484 cells and, by contrast, repressed in RhoGDI2-depleted MKN-28 cells. Depletion of PLCγ expression or inhibition of its activity not only significantly increases cisplatin-induced apoptosis but also suppresses the invasive ability of RhoGDI2-overexpressing SNU-484 cells. Taken together, our results suggest that PLCγ plays a key role in RhoGDI2-mediated cisplatin resistance and cell invasion in gastric cancer cells.
Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fosfolipase C gama/fisiologia , Neoplasias Gástricas/tratamento farmacológico , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Invasividade Neoplásica , Fosfolipase C gama/genética , Fosforilação , Neoplasias Gástricas/patologia , Proteínas Supressoras de Tumor/genética , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
TREK1 belongs to a family of two-pore-domain K(+) (K(2P)) channels and produce background currents that regulate cell excitability. In the present study, we identified a vesicle transport protein, beta-COP, as an interacting partner by yeast two-hybrid screening of a human brain cDNA library with N-terminal region of TREK1 (TREK1-N) as bait. Several in vitro and in vivo binding assays confirmed the protein-protein interaction between TREK1 and beta-COP. We also found that beta-COP was associated with TREK1 in native condition at the PC3 cells. When RFP-beta-COP was co-transfected with GFP-TREK1 into COS-7 cells, both proteins were found localized to the plasma membrane. In addition, the channel activity and surface expression of GFP-TREK1 increased dramatically by co-transfection with RFP-beta-COP. Surface expression of the TREK1 channel was also clearly reduced with the addition of beta-COP-specific shRNA. Collectively, these data suggest that beta-COP plays a critical role in the forward transport of TREK1 channel to the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Proteína Coatomer/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteína Coatomer/genética , Humanos , Canais de Potássio de Domínios Poros em Tandem/genética , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-HíbridoRESUMO
PURPOSE: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of Rho family GTPase. However, there is currently no direct evidence suggesting whether RhoGDI2 activates or inhibits Rho family GTPase in vivo (and which type), and the role of RhoGDI2 in tumor remains controversial. Here, we assessed the effects of RhoGDI2 expression on gastric tumor growth and metastasis progression. EXPERIMENTAL DESIGN: Proteomic analysis was done to investigate the tumor-specific protein expression in gastric cancer and RhoGDI2 was selected for further study. Immunohistochemistry was used to detect RhoGDI2 expression in clinical samples of primary gastric tumor tissues which have different pathologic stages. Gain-of-function and loss-of-function approaches were done to examine the malignant phenotypes of the RhoGDI2-expressing or RhoGDI2-depleting cells. RESULTS: RhoGDI2 expression was correlated positively with tumor progression and metastasis potential in human gastric tumor tissues, as well as cell lines. The forced expression of RhoGDI2 caused a significant increase in gastric cancer cell invasion in vitro, and tumor growth, angiogenesis, and metastasis in vivo, whereas RhoGDI2 depletion evidenced opposite effects. CONCLUSION: Our findings indicate that RhoGDI2 is involved in gastric tumor growth and metastasis, and that RhoGDI2 may be a useful marker for tumor progression of human gastric cancer.