Sayaka Yoshiba

Hypoxia Induces Resistance to 5-Fluorouracil in Oral Cancer Cells Via G1 Phase Cell Cycle Arrest

Malignant tumors are exposed to various levels of hypoxic condition in vivo. It has been known that tumor cells under hypoxia are resistant to chemotherapies. To clarify the mechanism of the hypoxia-induced chemoresistance, we evaluated the effects of hypoxia on the resistance of oral squamous cell carcinoma (OSCC) cell lines to 5-fluorouracil (5-FU). OSCC cells were divided to two groups by the proliferation activity under hypoxic condition; hypoxia-resistant (HR) and hypoxia-sensitive (HS) cells. Growth of HS cells were inhibited by hypoxia and introduced to G(1) arrest in cell cycle. 5-FU effect on HS cell viability was markedly reduced in hypoxic condition without an induction of chemoresistant related protein, P-glycoprotein. However, proliferation, cell cycle, and 5-FU sensitivity of HR cells were not affected by hypoxia. Hypoxia-inducible factor (HIF)-1alpha was induced by hypoxia in all OSCC cell lines, but diminished in HS cells within 48h. Expression of p21 and p27 was strongly augmented and CyclinD expression was reduced by hypoxia in HS cells. However, the expression of these proteins was constitutive in HR cells during 48h hypoxic culture. Phosphorylation of mammalian target of rapamycin (mTOR) was reduced by hypoxia in HS cells. From these findings, we concluded that HS OSCC cells acquire 5-FU resistance under hypoxia by G(1)/S transition through an upregulation of cell cycle inhibitors.

Abstract 4809: Epigenomic profiling of oral cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Despite combined surgery and radiation therapy, long-term survival for oral cancer has not improved over the past several decades. This is primary due to poor clinical prognosis of patients with the lymph nodes metastasis. Therefore, deeper understanding of the molecular basis of highly malignant property and patient stratification along with these characters are needed. Although, recent genome-wide analyses of DNA methylation have revealed a large number of aberrantly methylated genes in many cancers, and allowed us to identify the molecularly defined subgroups such as CpG island methylation phenotype (CIMP) in certain tumors, the methylation diversity of clinical oral cancers are still unknown. The purpose of this study is to characterize DNA methylation profiles of oral cancer tissues (six normal oral mucosal swab, three non-cancerous mucosal tissues, and fifty-nine oral cancer tisseus) and to identify the specific methylation pattern related to the clinical features. Genome-wide methylation analysis was performed using Infinium HumanMethylation27 BeadArray system, which can analyze methylation status of 27,000 CpG sites semi-quantitatively, using 500ng of genomic DNA.We found 2,762 aberrantly methylated CpG sites in oral cancers against normal mucosa. By unsupervised clustering analysis, oral cancer could be divided into two methylation epigenotypes that are significantyly correlated with high incidence of lymph node metastasis and poor differentiation. We obtained the highly predictive candidates of classifier genes and they were confirmed by MassARRAY analysis. In summary, we identified two methylation epigenotypes related with clinical outcome in oral cancer, and candidate classifier genes would be useful clues of patient stratification for intensive therapy to overcome the tumor recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4809. doi:10.1158/1538-7445.AM2011-4809

microRNA expression profiles in oral squamous cell carcinoma

microRNAs (miRNAs) are involved in cancer pathogenesis, apoptosis and cell growth, thereby functioning as both tumor suppressors and oncogenes. However, the expression patterns and roles of miRNAs in oral squamous cell carcinoma (OSCC) remain largely unknown. We hypothesized that oral cancer may have a unique miRNA profile, which in turn may play a critical role in oral cancer development, progression, diagnosis and prognosis. We, therefore, investigated the expression profiles of 29 OSCC tumors and 7 normal oral mucosal samples. The miRNA expression patterns in OSCC were examined by TaqMan-based microRNA assays. We were subsequently able to identify the candidates of cancer-related miRNAs through analysis of the miRNA expression profiles. In conclusion, OSCC tissues were shown to have a unique miRNA profile pattern when compared with that in normal tissues. The present study may provide useful information for further investigation of the functional roles of miRNAs in OSCC development, progression, diagnosis and prognosis.

Proteasome inhibitor sensitizes oral squamous cell carcinoma cells to TRAIL-mediated apoptosis

Oral squamous cell carcinoma (OSCC) cells are relatively resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis during culture. We investigated the role of a proteaosome inhibitor in the survival and apoptosis of these cells. We found that the proteasome inhibitor MG132 markedly accelerated TRAIL-mediated apoptosis in OSCC cell lines HSC-2 and HSC-3. Addition of TRAIL to MG132-treated cells resulted in Bid cleavage. Furthermore, the inhibitors of caspase-3, caspase-8 and caspase-9 reduced the accelerative effect of MG132 on TRAIL-mediated apoptosis. These results suggest that the pro-apoptotic effect of a proteasome inhibitor on TRAIL-mediated apoptosis may contribute to both extrinsic and intrinsic pathways. MG132 enhanced the expression of the TRAIL receptors DR4 and DR5, and neutralization of DR5 receptors showed a marked reduction of TRAIL-mediated apoptosis, whereas that of DR4 was a partial reduction. MG132 also markedly reduced cellular FLICE-inhibitory protein (c-FLIP), cellular inhibitor of apoptosis protein-1 (cIAP-1), X-linked IAP (XIAP) and survivin. Therefore, MG132 provides partial regulation of TRAIL-mediated apoptosis in OSCC cells via modulation of DR5, c-FLIP, cIAP-1, XIAP and survivin. The proteasome inhibitor MG132 may therefore represent a novel strategy for overcoming resistance to TRAIL-mediated apoptosis in OSCC cells.

Antitumor activity of suberoylanilide hydroxamic acid against human oral squamous cell carcinoma cell lines in vitro and in vivo

It has been reported recently that histone deacetylase inhibitors (HDACIs) can block the growth of a variety of malignant tumor cells by reversing the silencing of the tumor suppressor genes; these will be the anticancer agents of the next generation. In this study, we evaluated the antitumor effects of the HDACI suberoylanilide hydroxamic acid (SAHA) on oral squamous cell carcinoma (OSCC) and investigated its molecular mechanism. SAHA suppressed the in vitro proliferation of OSCC cell lines in a dose- and time-dependent manner. Flow cytometric analyses showed that treatment with SAHA led to G1 phase cell-cycle arrest of OSCC cells, accompanying a decrease in the percentage of S-phase cells. Western blot analyses demonstrated that the expression of p21 protein was remarkably augmented and hyperacetylation of p53 was induced after SAHA treatment. These results suggest that administration of SAHA suppresses OSCC growth through G1 phase arrest. Additionally, we observed that the growth of xenograft SAS tumors in nude mice was significantly blocked by the administration of SAHA without major adverse effects.