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.
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.
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.
These results indicate that GAME and PGG are the main chemical components of P. lutea leaves that have potential anti-cancer therapeutic effects.
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 β-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.
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. 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. 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.
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.
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.
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.
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.
Summary Several reports have indicated that nuclear factor-kappa B (NF-κB) is constitutively activated in a variety of cancer cells including human oral squamous carcinoma cells, and play a key role ...in their growth and survival. Recent studies report that NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), inhibits proliferation and induces apoptosis in prostate cancer cell lines. However this anti-tumor effects are still unknown in end human oral squamous carcinoma cells. In the present study, we investigated the effects of DHMEQ on oral squamous carcinoma cell (OSCC) lines in vitro and in vivo . Human OSCC cell lines (HSC-3, SAS) were treated with DHMEQ and examined for cell viability by MTT assay, cell cycle distribution by flow-cytometry, apoptosis by TUNEL assay, and protein expression by western blotting, respectively. In vivo activities were also investigated in a mouse xenograft model. DHMEQ inhibited growth of two OSCC cell lines in a dose-dependent manner measured by MTT assay. A flow cytometric analysis demonstrated that treatment with DHMEQ induced accumulation in sub-G1 phase. TUNEL assay showed that DHMEQ induced DNA fragmentation. Protein expression by western blotting analysis revealed that DHMEQ induced nuclear down regulation of Survivin, cIAP-1, and cIAP-2. In nude mice, DHMEQ inhibited growth of OSCC without major toxic side effects. The present results demonstrated that administration of DHMEQ is suggested to be a novel anti-tumor approach to the treatment of OSCC.
The RNA-binding protein TIA-1 (T-cell-restricted intracellular antigen-1) functions in regulating post-transcriptional mechanisms, including precursor mRNA (pre-mRNA) alternative splicing and mRNA ...translation. Utilizing a mini-gene consisting of part of the type II procollagen gene (COL2A1), we show that TIA-1 interacts with a conserved AU-rich cis element in COL2A1 intron 2 and modulates alternative splicing of exon 2. This unique, highly conserved cis element containing stem-loop secondary structure was previously identified in our laboratory as an essential motif that controls the developmentally regulated exon 2 splicing switch during chondrogenesis (McAlinden, A., Havlioglu, N., Liang, L., Davies, S. R., and Sandell, L. J. (2005) J. Biol. Chem. 280, 32700-32711). In vivo binding of endogenous TIA-1 to the AU-rich cis element in COL2A1 pre-mRNA was confirmed by the ribonucleoprotein immunoprecipitation assay. Importantly, we also show that TIA-1 interacts with the equivalent DNA sequence with a preference for single-stranded rather than double-stranded DNA. Chromatin immunoprecipitation assays (including an additional RNase step) confirmed this interaction in vivo. Competition assays showed that TIA-1 apparently binds with higher affinity to DNA than to RNA. Finally, we show that this strong DNA-TIA-1 interaction can be disrupted by an RNA polymerase during active transcription. This suggests a potentially novel, dual role for TIA-1 in shuttling between DNA and RNA ligands to co-regulate COL2A1 expression at the level of transcription and pre-mRNA alternative splicing.
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