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
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
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.