RESUMO
Hypoxia occurs under important clinical conditions such as cancers, heart disease, and ischemia. However, the relationship between hypoxia and autophagy in osteocytes is still unclear. The objective of the present study was to uncover the regulatory mechanisms that prevent regulated cell death, such as apoptosis, necrosis, and autophagy, under hypoxia. MLO-Y4 cells, a mouse osteocyte cell line, were exposed to various O2 partial pressures (PO2). Subsequently, the cells underwent apoptosis, autophagy, autophagic cell death, and/or necrosis, and thereby we designated PO2 = 2% as a representative hypoxic condition. Immunofluorescence staining showed an increase of LC3 and a decrease of p62 in MLO-Y4 cells exposed to hypoxia, indicating the induction of autophagy. We then hypothesized that ß-estradiol (E2) and vitamin D play an important role in apoptosis and autophagy of osteocytes under hypoxia. 1,25α-dihydroxyvitamin D3 (VitD) protected MLO-Y4 cells from cell death and induced autophagy. However, E2 showed little effect. Finally, Western blotting for phosphorylated mTOR and Akt was carried out in order to investigate the altered autophagy signaling pathways affected by the addition of VitD and E2. However, neither E2 nor VitD were capable of recovering the decreased phosphorylation of those factors. Our results indicated that the effects of VitD on autophagy under hypoxia were dependent on the Akt and mTOR pathways. Thus, the results of the present study showed that VitD suppresses osteocyte cell death in an mTOR pathway-dependent manner in hypoxic conditions. This suggests the potential of VitD as a therapeutic intervention for diseases in which the cell death of osteocytes mainly occurs via hypoxia.
Assuntos
Autofagia , Osteócitos , Animais , Apoptose , Hipóxia , Camundongos , Transdução de SinaisRESUMO
Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis-acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis-acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-ß and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis-acting element and trans-acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Elementos de Resposta , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Imunoprecipitação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a Poli(A)/antagonistas & inibidores , Proteínas de Ligação a Poli(A)/genética , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/química , RNA Neoplásico/antagonistas & inibidores , RNA Neoplásico/química , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Antígeno-1 Intracelular de Células TRESUMO
BACKGROUND: Osteoporosis (OP) is one of the most serious diseases in the modern world, and OP patients frequently suffer from fragility fractures in the hip, spine and wrist, resulting in a limited quality of life. Although bisphosphonates (BPs) are the most effective class of anti-bone-resorptive drugs currently available and the most commonly prescribed for the clinical treatment of OP, they are known to cause serious side effects such as bisphosphonate-related osteonecrosis of the jaw. Novel therapeutic materials that can replace the use of BPs have therefore been developed. METHODS: We commenced an institutional collaborative project in which candidates of herbal extracts were selected from more than 400 bioactive herbal products for their potential therapeutic effects not only in OP, but also in oral and skeletal diseases. In the present study, we report on 3 Chinese medical herbal extracts from the root barks of Melia azedarach, Corydalis turtschaninovii, and Cynanchum atratum. RESULTS: All of these extracts inhibited osteoclast proliferation and induced apoptosis by up-regulation of caspase activity and increase of mitochondrial pro-apoptotic proteins expression. Furthermore, the extracts enhanced differentiation, but did not affect proliferation of both osteoblasts and chondrocytes. The osteo-inducible effect was also observed in cultured primary bone marrow cells. CONCLUSIONS: Although these extracts have been utilized in traditional Chinese medicine for hundreds of years, there are no reports to our knowledge, on their therapeutic effects in OP. In this study, we elucidate the potency of these herbal extracts as novel candidates for OP therapy.
Assuntos
Osso e Ossos/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Magnoliopsida , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoporose , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Osso e Ossos/citologia , Caspases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Corydalis , Cynanchum , Difosfonatos/efeitos adversos , Difosfonatos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Técnicas In Vitro , Melia , Osteoporose/prevenção & controle , Fitoterapia , Casca de Planta , Raízes de Plantas , Qualidade de Vida , Regulação para Cima/efeitos dos fármacosRESUMO
Suprabasin (SBSN) is a secreted protein that is isolated as a novel gene expressed in differentiated keratinocytes in mice and humans. It induces various cellular processes such as proliferation, invasion, metastasis, migration, angiogenesis, apoptosis, therapy and immune resistance. The role of SBSN was investigated in oral squamous cell carcinoma (OSCC) under hypoxic conditions using the SAS, HSC3, and HSC4 cell lines. Hypoxia induced SBSN mRNA and protein expression in OSCC cells and normal human epidermal keratinocytes (NHEKs), and this was most prominent in SAS cells. The function of SBSN in SAS cells was analyzed using 3(4,5dimethylthiazol2yl)2,5diphenyltetrazolium bromide (MTT); 5bromo2'deoxyuridine (BrdU); cell cycle, caspase 3/7, invasion, migration, and tube formation assays; and gelatin zymography. Overexpression of SBSN decreased MTT activity, but the results of BrdU and cell cycle assays indicated upregulation of cell proliferation. Western blot analysis for cyclinrelated proteins indicated involvement of cyclin pathways. However, SBSN did not strongly suppress apoptosis and autophagy, as revealed by caspase 3/7 assay and western blotting for p62 and LC3. Additionally, SBSN increased cell invasion more under hypoxia than under normoxia, and this resulted from increased cell migration, not from matrix metalloprotease activity or epithelialmesenchymal transition. Furthermore, SBSN induced angiogenesis more strongly under hypoxia than under normoxia. Analysis using reverse transcriptionquantitative PCR showed that vascular endothelial growth factor (VEGF) mRNA was not altered by the knockdown or overexpression of SBSN VEGF, suggesting that VEGF is not located downstream of SBSN. These results demonstrated the importance of SBSN in the maintenance of survival and proliferation, invasion and angiogenesis of OSCC cells under hypoxia.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Animais , Camundongos , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Caspase 3 , Bromodesoxiuridina , Proliferação de Células/genética , Fatores de Crescimento do Endotélio Vascular , Movimento Celular , Hipóxia/genética , Linhagem Celular Tumoral , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de NeoplasiasRESUMO
CCN2/connective tissue growth factor (CTGF) can be induced by hypoxia and promotes tumor angiogenesis. Our previous studies revealed that hypoxia-induced gene expression of human ccn2 mRNA is regulated post-transcriptionally in human chondrosarcoma-derived cell line, HCS-2/8, in which a minimal cis-element, entitled CAESAR, in the 3'-untranslated region (UTR) of ccn2 mRNA and a 35-kDa protein counterpart play an important role by determining the stability of ccn2 mRNA. In the present study, we identified this corresponding protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by utilizing RNA affinity chromatography combined with mass spectrometry. The results of an RNA binding assay revealed the specific binding of GAPDH to this cis-element. To further characterize the interaction between GAPDH and ccn2 mRNA, we examined the roles of redox conditions and glycolytic coenzyme in the binding of GAPDH to the ccn2 mRNA. An oxidizing agent, diamide, abolished the GAPDH-RNA interaction in a concentration-dependent manner; whereas this effect could be reversed by subsequent treatment with 2-mercaptoethanol (2-ME). In addition, nicotinamide-adenine dinucleotide (NAD), a coenzyme of GAPDH, inhibited the GAPDH-RNA binding. Taken together, these findings suggest that the glycolytic enzyme GAPDH regulates the gene expression of ccn2 mRNA in trans by acting as a sensor of oxidative stress and redox signals, leading to CCN2 overexpression under the condition of hypoxia and promotion of angiogenesis.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Regulação da Expressão Gênica , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Regiões 3' não Traduzidas , Hipóxia Celular/genética , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , NAD/metabolismo , Neovascularização Patológica/genética , Oxidantes/farmacologia , Estresse Oxidativo/genética , RNA Mensageiro/genética , Elementos Reguladores de TranscriçãoRESUMO
BACKGROUND: Tumor protein D52 (TPD52) reportedly plays an important role in the proliferation and metastasis of various cancer cells, including oral squamous cell carcinoma (OSCC) cells, and is expressed strongly at the center of the tumor, where the microenvironment is hypoxic. Thus, the present study investigated the roles of TPD52 in the survival and death of OSCC cells under hypoxia, and the relationship with hypoxia-inducible factor (HIF). We examined the expression of TPD52 in OSCC cells under hypoxic conditions and analyzed the effects of HIF on the modulation of TPD52 expression. Finally, the combinational effects of TPD52 knockdown and HIF inhibition were investigated both in vitro and in vivo. RESULTS: The mRNA and protein levels of TPD52 increased in OSCC cells under hypoxia. However, the increase was independent of HIF transcription. Importantly, the observation was due to upregulation of mRNA stability by binding of mRNA to T-cell intercellular antigen (TIA) 1 and TIA-related protein (TIAR). Simultaneous knockdown of TPD52 and inhibition of HIF significantly reduced cell viability. In addition, the in vivo tumor-xenograft experiments showed that TPD52 acts as an autophagy inhibitor caused by a decrease in p62. CONCLUSIONS: This study showed that the expression of TPD52 increases in OSCC cells under hypoxia in a HIF-independent manner and plays an important role in the proliferation and survival of the cells in concordance with HIF, suggesting that novel cancer therapeutics might be led by TPD52 suppression.
RESUMO
AIM: We identified chemical components that exhibited antitumor activity against oral squamous cell carcinoma (OSCC) cells and examined their effective concentrations and additive and/or synergistic effects in combinational usage on the proliferation, apoptosis and cell cycle of OSCC cells. MATERIALS AND METHODS: Using high-performance liquid chromatography, nuclear magnetic resonance spectroscopy and electrospray ionization-mass spectrometry, we identified the main chemical components of the methanol extracts from Paeonia lutea. We investigated the pharmaceutical effects of those components on the proliferation, apoptosis, and cell cycle of an OSCC cell line, SAS, using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and caspase assays, as well as flow cytometry cell cycle analysis. We also examined the effects of those components on the mitogen-activated protein kinase signal transduction pathway by western blotting. Finally, the effects on normal human epidermal keratinocyte cells were also examined in similar experiments. RESULTS: Three chemicals have been identified in P. lutea leaves using high performance liquid chromatography: gallic acid methyl ester (GAME), pentagalloyl glucose (PGG) and paeoniflorin (PF). Both GAME and PGG significantly suppressed cell proliferation, and their combined effects were synergistic, while the effect of PF was minimal. However, those chemicals did not induce apoptosis. Cell cycle and western blotting analysis showed that the suppressive effects on cell proliferation resulted from G2 arrest and the suppression of phosphorylation of Akt/PKB. No effect was identified on normal human epidermal keratinocyte cells. CONCLUSION: These results indicate that GAME and PGG are the main chemical components of P. lutea leaves that have potential anti-cancer therapeutic effects.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Paeonia/química , Extratos Vegetais/química , Folhas de Planta/química , HumanosRESUMO
CCN1, a member of the CCN family of proteins, plays important physiological or pathological roles in a variety of tissues. In the present study, we initially found a highly guanine-cytosine (GC)-rich region of approximately 200 bp near the 5'-end of the open reading frame, which was always truncated by amplification of the corresponding cDNA region through the conventional polymerase chain reaction. An RNA in vitro folding assay and selective ribonuclease digestion of the corresponding segment of the ccn1 mRNA confirmed the involvement of a stable secondary structure. Subsequent RNA electromobility-shift assays demonstrated the specific binding of some cytoplasmic factor(s) in chicken embryo fibroblasts to the RNA segment. Moreover, the corresponding cDNA fragment strongly enhanced the expression of the reporter gene in cis at the 5'-end, but did not do so at the 3'-end. According to the results of a ribosomal assembly test, the effect of the mRNA segment can predominantly be ascribed to the enhancement of transport and/or entry of the mRNA into the ribosome. Finally, the minimal GC-rich mRNA segment that was predicted and demonstrated to form a secondary structure was confirmed to be a functional regulatory element. Thus, we here uncover a novel dual-functionality of the mRNA segment in the ccn1 open reading frame, which segment acts as a cis-element that mediates posttranscriptional gene regulation, while retaining the information for the amino acid sequence of the resultant protein.
Assuntos
Proteína Rica em Cisteína 61/genética , RNA Mensageiro/metabolismo , RNA/genética , Ribossomos/metabolismo , Animais , Western Blotting , Embrião de Galinha , Galinhas , Ensaio de Desvio de Mobilidade Eletroforética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cisplatin (cis-diamminedichloroplatinum II [CDDP] ) is a well-known chemotherapeutic drug that has been used for the treatment of various types of human cancers, including head and neck cancer. Cisplatin exerts anticancer effects by causing DNA damage, replication defects, transcriptional inhibition, cell cycle arrest, and the induction of apoptosis. However, drug resistance is one of the most serious problems with cancer chemotherapy, and it causes expected therapeutic effects to not always be achieved. Here, we analyzed global microRNA (miRNA) expression in CD44 standard form (CD44s)-expressing SAS cells, and we identified miR-629-3p as being responsible for acquiring anticancer drug resistance in head and neck cancer. The introduction of miR-629-3p expression inhibited apoptotic cell death under cisplatin treatment conditions, and it promoted cell migration. Among the computationally predicted target genes of miR-629-3p, we found that a number of gene expressions were suppressed by the transfection with miR-629-3p. Using a xenografting model, we showed that miR-629-3p conferred cisplatin resistance to SAS cells. Clinically, increased miR-629-3p expression tended to be associated with decreased survival in head and neck cancer patients. In conclusion, our data suggest that the increased expression of miR-629-3p provides a mechanism of cisplatin resistance in head and neck cancer and may serve as a therapeutic target to reverse chemotherapy resistance.
RESUMO
Connective tissue growth factor (CTGF/CCN2) plays a critical role in endochondral bone formation; however, CCN2 also promotes angiogenesis and bone metastasis in breast cancer. Chondrocytic HCS-2/8 cells and breast cancer MDA231 cells produce over 6 times more CCN2 than any other cell type. In this study, we demonstrate that these cell lines employ different transcriptional strategies for ccn2 gene induction. Four tandem copies of the dominant transcriptional enhancer in chondrocytes (4 x TRENDIC) were chimerically connected to an SV40 promoter-luciferase construct and subsequently analyzed. The enhancement of the promoter activity by 4 x TRENDIC was greater in the HCS-2/8 cells (7-fold) than in the other 4 cell lines (3-4 fold). The TRENDIC-binding protein complex was detected at a higher signal in the HCS-2/8 cells than in the other cell lines. In addition, the HCS-2/8 nuclear factors strongly targeted not only TRENDIC, but also the previously reported basal control element and a novel enhancer element in the ccn2 promoter. In contrast, high-level ccn2 gene induction in MDA231 cells was largely dependent on Smad signaling through the Smad-binding element in the ccn2 promoter. Based on these results, we propose a model of differential transcription of the ccn2 gene between the chondrocytic cell line and the breast cancer cell line, and therefore imply that these cells utilize distinct transcriptional strategies to obtain the enhanced CCN2 production that is not observed in other types of cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Transcrição Gênica , Sequência de Bases , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Condrossarcoma/genética , Condrossarcoma/metabolismo , Condrossarcoma/parasitologia , Fator de Crescimento do Tecido Conjuntivo , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos , Ensaio de Imunoadsorção Enzimática , Feminino , Células HeLa , Humanos , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Genéticos , Mutação , Plasmídeos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Smad/genética , Proteínas Smad/metabolismo , Ativação TranscricionalRESUMO
Connective tissue growth factor (CTGF/CCN2) is a potent angiogenic factor. In this report, we describe for the first time that vascular endothelial growth factor (VEGF)-mediated induction of the ctgf/ccn2 gene was a post-transcriptional event that was inhibited by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells. Steady-state mRNA levels of ctgf/ccn2 were remarkably increased by VEGF in a concentration-dependent manner, whereas the activity of the ctgf/ccn2 promoter was not responsive to VEGF as confirmed by a reporter gene assay and quantitative real-time PCR analysis. By employing a RNA degradation assay, we eventually found that the observed increase in the ctgf/ccn2 mRNA level was due to an increased stability of the mRNA induced by VEGF. DN-9693 at a dose of 0.1 to 2 ng/mL did not affect basal levels of ctgf/ccn2 mRNA; however, enhancement of ctgf/ccn2 mRNA expression by VEGF was specifically inhibited by DN-9693. Of importance, the inhibitory effects could be also ascribed to post-transcriptional regulation, because the VEGF-mediated increase in stability of ctgf/ccn2 mRNA was suppressed by DN-9693. Furthermore, we investigated the effects of DN-9693 on VEGF-induced activation of three subgroups of mitogen-activated protein kinase pathways and found that DN-9693 blocked the activation of these pathways by VEGF. These results suggest that VEGF increases ctgf/ccn2 mRNA stability through mitogen-activated protein kinase-mediated intracellular signaling cascade(s), which can be inhibited posttranscriptionally by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas Imediatamente Precoces/efeitos dos fármacos , Quinazolinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo , Endotélio Vascular , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/efeitos dos fármacos , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The tumor protein D (TPD) family consists of four members, TPD52, TPD53, TPD54, and TPD55. The physiological roles of these genes in normal tissues, including epidermal and mesenchymal tissues, have rarely been reported. Herein, we examined the expression of TPD52 and TPD54 genes in cartilage in vivo and in vitro and investigated their involvement in the proliferation and differentiation of chondrocytes in vitro. TPD52 and TPD54 were uniformly expressed in articular cartilage and trabecular bone and were scarcely expressed in the epiphyseal growth plate. In MC3T3E-1 cells, the expressions of TPD52 and TPD54 were increased in a differentiation-dependent manner. In contrast, their expressions were decreased in ATDC5 cells. In ATDC5 cells, overexpression of TPD52 decreased alkaline phosphatase (ALPase) activity, while knock-down of TPD52 showed little effect. In contrast, overexpression of TPD54 enhanced ALPase activity, Ca2+ deposition, and the expressions of type X collagen and ALPase genes, while knock-down of TPD54 reduced them. The results revealed that TPD52 inhibits and that TPD54 promotes the terminal differentiation of a chondrocyte cell line. As such, we report for the first time the important roles of TPD52 and TPD54, which work oppositely, in the terminal differentiation of chondrocytes during endochondral ossification.
Assuntos
Cálcio/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Regulação da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Osteogênese , Fosfatase Alcalina/biossíntese , Animais , Linhagem Celular , Condrócitos/citologia , Colágeno Tipo X/biossíntese , Colágeno Tipo X/genética , Camundongos , Proteínas de Neoplasias/genética , Células RAW 264.7RESUMO
The tumor protein D52 (TPD52) protein family includes TPD52, -53, -54 and -55. Several reports have shown important roles for TPD52 and TPD53, and have also suggested the potential involvement of TPD54, in D52-family physiological effects. Therefore, we performed detailed expression analysis of TPD52 family proteins in oral squamous cell carcinoma (OSCC). Towards this end, TPD54-overexpressing or knocked-down cells were constructed using OSCC-derived SAS, HSC2 and HSC3 cells. tpd52 or tpd53 was expressed or co-expressed in these cells by transfection. The cells were then analyzed using cell viability (MTT), colony formation, migration, and invasion assays. In OSCC-xenograft experiments, the cells were transplanted into nude mice together with injection of anti-tpd siRNAs. MTT assay of cell monolayers showed little differences in growth of the transfected cells. tpd54 overexpression in SAS cells significantly decreased colony formation in an anchorage-independent manner. Additionally, knock-down of tpd54 enhanced the number of colonies formed and overexpression of tpd52 in tpd54 knock-down cells increased the size of the colonies formed. The chemotaxis assay showed that tpd54 overexpression decreased cell migration. In the OSCC-xenograft in vivo study, tpd54 overexpression slightly attenuated tumor volume in vivo, despite the fact that tumor metastasis or cell survival was not involved. Our results showed that TPD54 not only downregulated anchorage-independent growth and cell migration in vitro, but also attenuated tumor growth in vivo. Based on these results, it is considered that TPD54 might act as a negative regulator of tumor progression in OSCC cells.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Proteínas de Neoplasias/biossíntese , Animais , Apoptose/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Neoplasias Bucais/patologia , Proteínas de Neoplasias/genética , Transdução de Sinais , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors. VEGF165b was recently isolated as the anti-angiogenic VEGF splice variant. In the present study, we examined the association between VEGF165b expression and clinicopathological characteristics in order to determine how VEGF165b produced from oral squamous cell carcinoma (OSCC) affects the stromal cell biological activity. We examined the relationships between the expressions of both VEGF isoforms in normal human dermal fibroblasts (NHDFs) and OSCC cell lines (HSC2, 3, 4 and SAS). Our analyses indicated that both the mRNA and protein expression levels of VEGF165b in the HSC2 and SAS cells were higher than those in the NHDFs. VEGF165b did not promote cell growth or invasive capabilities, but it induced the cell adhesive capabilities to ECM. Although strong expression of the VEGF165 isoforms in tumor cells of OSCC tissues was observed, there was no significant difference in the VEGF165b expression level among the various degrees of malignancy. OSCC cells secrete VEGF165b into the stroma, and this factor may contribute to the process of anti-angiogenesis by inhibiting gelatinase-expressing cells and activating cell adhesive capabilities to ECM, such as that of fibroblasts surrounding tumor cells.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Adesão Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , RNA Mensageiro/metabolismo , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Squamous cell carcinoma (SCC) is one of the most common cancers of the head and neck region worldwide and is generally treated surgically in combination with radiotherapy and/or chemotherapy. However, anticancer agents have numerous serious side effects, and alternative, less toxic agents that are effective as chemotherapeutics for SCC are required. The Paeoniaceae family is widely used in traditional Chinese medicine. We examined methanol and butanol extracts of Paeonia lutea (P. lutea) leaves for their potential as an anticancer agent. Both extracts decreased the proliferation of SCC cells, induced apoptotic cell death, and modulated migration, adhesion, chemotaxis, and haptotaxis in an extracellular matrix- (ECM-) dependent manner due to altered expression of several integrin subunits. Subsequently, SCC cells were subcutaneously transplanted into athymic nude mice; the extracts reduced the metastasis of SCC cells but had little effect on the volume of the primary tumor or survival or body weight of the mice. The results suggest that the extracts may hold promise for preventing cancer metastasis.
RESUMO
The cis-acting element of structure-anchored repression (CAESAR) is a post-transcriptional regulatory element of gene expression, which is located in the 3'-untranslated region (UTR) of the human ccn2 gene (ctgf/ccn2). In this report, the repression mechanism of CAESAR, as well as the structural requirement, was investigated. Removal of minor stem-loops from CAESAR resulted in proportional attenuation of the repressive function, whereas removal of the single bulge or modification of primary nucleotide sequence did not affect its functionality. In light of functional mechanism, CAESAR exerted no significant effects on stability or nuclear export of the cis-linked mRNA. However, this element significantly interfered with the association of such mRNA on ribosome and slowed down the translation process thereafter in vitro. A translation repression mechanism by RNA secondary structure to determine the basal ctgf/ccn2 expression level was uncovered herein.
Assuntos
Regiões 3' não Traduzidas , Regulação para Baixo , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Pareamento de Bases , Células COS , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Fator de Crescimento do Tecido Conjuntivo , Genes Reporter/genética , Humanos , Luciferases/análise , Luciferases/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , Ribossomos/metabolismoRESUMO
The osteogenic induction of adipose-derived stem cells (ADSCs) has been regarded as an important step in bone tissue engineering. In the present study, we focused on the buccal fat pad (BFP) as a source of adipose tissue, since BFPs are encapsulated by adipose tissue and are often coextirpated during oral surgery. Low-intensity pulsed ultrasound (LIPUS) is effective in the treatment of fractures, and nanohydroxyapatite (NHA) is known as a bone substitute material. Here we investigated the synergistic effects of LIPUS and NHA in the osteogenesis of ADSCs. A combination of LIPUS irritation and NHA as a scaffold significantly increased the osteogenic differentiation of ADSCs in vitro, and in our in vivo study in which ADSCs were transplanted into calvarial bone defects of nude mice, the combinational effect greatly enhanced the new bone formation of the margin of the defects. These results demonstrate that synergistic effects of LIPUS and NHA are capable of effectively inducing the differentiation of ADSCs into osteoblasts, and they suggest a novel therapeutic strategy for bone regeneration by the autotransplantation of ADSCs.
RESUMO
We used high- (ACCM) and low- (ACC2) metastasis cell lines of human adenoid cystic carcinoma (ACC) as an experimental model to study metastatic mechanisms and compare their expression levels for angiogenic-related factor vascular endothelial growth factor (VEGF). By using a series of extensive analyses, hypoxia-inducible factor-1 (HIF-1) α-dependent VEGF expression levels were observed to be higher in ACCM cell lines, increasing the possible development of tumor metastasis, compared to ACC2 cell lines. Our findings provide the novel insight that HIF-1α-dependent VEGF overexpression under hypoxic conditions shows to some extent associations with the metastatic tendency of ACC cells and may function as a potential target for ACC therapy.
Assuntos
Carcinoma Adenoide Cístico/metabolismo , Carcinoma Adenoide Cístico/patologia , Neoplasias das Glândulas Salivares/metabolismo , Neoplasias das Glândulas Salivares/patologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metástase Neoplásica , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
PURPOSE: Tumor protein D54 (TPD54) belongs to the TPD52 family of proteins and is expressed in several types of cancer, including oral squamous cell carcinoma (OSCC). Here, we investigated relationships between various OSCC-related characteristics and TPD54 expression in vitro. METHODS: The expression of TPD54 in several OSCC-derived cell lines and normal, non-malignant, cells was assessed. Based on the results obtained, OSCC-derived SAS cells were subsequently subjected to exogenous over-expression of alternative splice variants (ASVs) of TPD54 and to TPD54 knock-down, mediated by siRNA. Next, the role of TPD54 in cellular growth, apoptosis, invasion, migration and extracellular-matrix (ECM)-dependent migration and attachment was investigated, as also the concomitant expression of integrins and integrin-related proteins by the OSCC-derived cells. RESULTS: Western blot analysis and RT-PCR revealed that several TPD54 ASVs were expressed in the OSCC-derived cell lines tested. Neither exogenous ASV over-expression nor TPD54 knock-down modulated the proliferation or invasion of SAS cells in a monolayer culture assay. However, exogenous ASV over-expression did decrease anchorage-independent growth and TPD54 knock-down did increase anchorage-independent growth, irrespective of caspase activities. The same effects were observed on ECM-dependent cellular migration and cell attachment to the ECM. The expression levels of the major α and ß integrin subunits, and of E-cadherin, were found to be similar to those observed in the non-transfected control cells, whereas talin1 expression was found to be increased after TPD54 knock-down. Also Akt was found to be activated after TPD54 knock-down, even in the absence of serum stimulation. Very similar effects were observed in the OSCC-derived cell lines HSC 2 and HSC 3. CONCLUSIONS: Our results show that TPD54 affects OSCC cell attachment to the ECM, OSCC cell migration, and Akt/PKB activation by modulating integrin activation via a talin1-mediated inside-out signal of the ECM. Based on these results, we suggest that TPD54 may serve as a novel biomarker for OSCC and as a putative target for OSCC therapy.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Movimento Celular , Matriz Extracelular/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteínas de Neoplasias/metabolismo , Apoptose/genética , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Integrinas/metabolismo , Neoplasias Bucais/enzimologia , Neoplasias Bucais/genética , Proteínas de Neoplasias/genética , Fosforilação , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Soro/metabolismo , Talina/metabolismoRESUMO
Cis-platinum (II) diammine dichloride (CDDP) is a platinum-based anticancer agent, and is often used for chemotherapy for malignant tumors, albeit CDDP has serious side-effects, including xerostomia (dry mouth). Since patients with xerostomia have reduced quality of life, it is urgent and important to identify nontoxic and natural agents capable of reducing the adverse effect of chemotherapy on salivary gland function. Therefore, we commenced an institutional collaborative project in which candidates of herbal extracts were selected from more than 400 bioactive herbal products for their potential therapeutic effects not only on xerostomia, but also on oral diseases. In the present study, we report on two Chinese medical herbal extracts from the root barks of Juncus effusus and Paeonia suffruticosa. The two extracts showed a protective effect in NS-SV-Ac cells from the cytotoxicity and apoptosis caused by CDDP. The effect was dependent on the p53 pathway, protein kinase B/Akt 1 and mitochondrial apoptosis-related proteins (i.e. Bcl-2 and Bax), but was not dependent on nuclear factor κB. Notably, the apoptosis-protective effect of the extracts was not observed in adenocystic carcinoma cell lines. Although these extracts have been utilized in traditional Chinese medicine for hundreds of years, there are no reports to our knowledge, on their therapeutic effects on xerostomia. Thus, in the present study, we elucidated the potency of these herbal extracts as novel candidates for xerostomia to improve the quality of life of patients undergoing chemotherapy.