Yuji Kurihara

Inhibition of cyclooxygenase-2 suppresses the invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 production and activation.

Increased cyclooxygenase (COX-2) expression in tumors is known to be correlated with tumor invasion, angiogenesis, resistance to apoptosis, and suppression of host immunity. We previously reported that the invasiveness of human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 was suppressed by treatment with either NS-398, a selective COX-2 inhibitor, or COX-2 antisense oligonucleotide (AS). In the present study, to explore the effects of COX-2 inhibition on the interaction between cancer cells and fibroblasts, we examined the effects of these anti-COX-2 reagents on the expression of matrix metalloproteinases (MMPs) in fibroblast cell lines WI-38 and MRC-5. Western blotting and enzyme-linked immunosorbent assay revealed that NS-398 and COX-2 AS down-regulated the expression and secretion of MMP-2 and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in human fibroblast cell lines. Furthermore, invasion activity of OSCC cells was down-regulated by the addition of culture supernatant from fibroblasts treated with anti-COX-2 reagents in a Matrigel® invasion assay. These results suggest that selective COX-2 inhibition suppresses the invasion activity of OSCC cells via down-regulation of an MMP-2-activating mechanism involving TIMP-2 and production of the MMP-2 protein by an interaction between cancer cells and stromal fibroblasts. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC.

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.

Telomerase-Specific Virotheranostics for Human Head and Neck Cancer

Purpose: Long-term outcomes of patients with squamous cell carcinoma of the head and neck (SCCHN) remain unsatisfactory despite advances in combination of treatment modalities. SCCHN is characterized by locoregional spread and it is clinically accessible, making it an attractive target for intratumoral biological therapies. Experimental Design: OBP-301 is a type 5 adenovirus that contains the replication cassette in which the human telomerase reverse transcriptase promoter drives expression of the E1 genes. OBP-401 contained the replication cassette and the green fluorescent protein (GFP) gene. The antitumor effects of OBP-301 were evaluated in vitro by the sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay and in vivo in an orthotopic xenograft model. Virus spread into the lymphatics was also orthotopically assessed by using OBP-401. Results: Intratumoral injection of OBP-301 resulted in the shrinkage of human SCCHN tumors orthotopically implanted into the tongues of BALB/c nu/nu mice and significantly recovered weight loss by enabling oral ingestion. The levels of GFP expression following ex vivo infection of OBP-401 may be of value as a positive predictive marker for the outcome of telomerase-specific virotherapy. Moreover, whole-body fluorescent imaging revealed that intratumorally injected OBP-401 could visualize the metastatic lymph nodes, indicating the ability of the virus to traffic to the regional lymphatic area and to selectively replicate in neoplastic lesions, resulting in GFP expression and cell death in metastatic lymph nodes. Conclusions: These results illustrate the potential of telomerase-specific oncolytic viruses for a novel therapeutic and diagnostic approach, termed theranostics, for human SCCHN.

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 effects of telomerase-specific replication-selective oncolytic viruses for adenoid cystic carcinoma cell lines

We evaluated the antitumor effect of a telomerase-specific replication-selective adenovirus (Telomelysin, OBP-301) for adenoid cystic carcinoma (ACC) in vitro and in vivo. Adenovirus E1 gene expression was controlled by human telomerase reverse transcription (hTERT). Infection of ACC cells by OBP-301 induced high E1A mRNA expression and subsequent oncolytic cell death in a dose-dependent manner. Using OBP-401 (TelomeScan), a genetically engineered adenovirus that carries the GFP gene under the control of the cytomegalovirus (CMV) promoter at the deleted E3 region of OBP-301, ACC cells expressed bright GFP fluorescence as early as 12 h after OBP-401 infection. The fluorescence intensity gradually increased in a time-dependent manner, followed by rapid cell death due to the cytopathic effect of OBP-401, as evidenced by the floating, highly light-refractive cells using phase-contrast microscopy. Effects of intratumorally injected OBP-401 against established Acc2 xenograft tumors were seen in BALB/c nu/nu mice. The levels of GFP expression following ex vivo infection of OBP-401 may be of value as a positive predictive marker for the outcome of telomerase-specific virotherapy. Our data clearly indicated that telomerase-specific oncolytic adenoviruses have significant therapeutic potential against human ACC in vitro and in vivo. These results suggest that treatment with OBP-301 and OBP-401 may improve the quality of life of oral cancer patients.

Abstract 4973: Telomerase-specific replication-selective oncolytic viruses for adenoid cystic carcinoma cell lines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL There is no effective treatment of adenoid cystic carcinoma (ACC) in oral region, which low sensitivity of chemotherapy and radiation therapy. We previously showed that telomerase-specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase (hTERT) promoter controls the adenoviral E1 gene expression, induces a selective antitumor effect in oral squamous cell carcinoma (OSCC) cells in vitro and in vivo orthotopic graft model. We conducted to evaluate the effectiveness of OBP301 against ACC. First, we could confirm that the expression of hTERT in two types of ACC cell lines (ACC2, ACCM) by Western blotting. Then, we examined the antitumor effect of OBP301 in human ACC cells in vitro. OBP-301 infection induced cell death in human ACC cells in a dose-dependent manner. We also examined the expression levels of coxsackievirus and adenovirus receptor (CAR) on the cell surface of each type of cell by flow cytometric analysis. Apparent amounts of CAR expression were detected on human ACC cells. Relative hTERT mRNA expression in ACC was analyzed by real-time RT-PCR, which showed high expression time dependent. In addition, OBP-401 is a genetically engineered adenovirus that expresses GFP by inserting the GFP gene under the control of the cytomegalovirus promoter at the deleted E3 region of OBP-301. Human ACC cells expressed bright GFP fluorescence as early as 12 h after OBP-401 infection. The fluorescence intensity gradually increased in a dose-dependent manner, followed by rapid cell death due to the cytopathic effect of OBP-401, as evidenced by floating, highly light-refractile cells under phase-contrast photomicrographs. In conclusion, our data clearly indicate that telomerase-specific oncolytic adenoviruses have a significant therapeutic potential against human ACC in vitro. These results suggested that treatment of OBP-301 and OBP-401 are possible to improvement of QOL of oral cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4973. doi:1538-7445.AM2012-4973

Abstract 4976: Telomerase-specific replication-selective virotherapy combined with radiation therapy for oral squamous cell carcinoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Replication-selective tumor-specific viruses present a novel approach for treating neoplastic disease. These vectors are designed to induce virus-mediated lysis of tumor cells after selective viral propagation within the tumor. Telomerase activation is considered to be a critical step in carcinogenesis, and its activity is closely correlated with human telomerase reverse transcriptase (hTERT) expression. We previously, investigated the antitumor effect of the hTERT-specific replication-competent adenovirus (Telomelysin: OBP-301) on oral squamous cell carcinoma (OSCC) cells in vitro and in vivo orthotopic graft model. OBP-301 replicated efficiently and induced more over 50% cell killing in a panel of human OSCC cell lines, but not in normal human fibroblasts, which were lacking telomerase activity. In orthotopic graft model, intratumoral injection of OBP-301 resulted in a significant inhibition of tumor growth and prolongation of survival. Moreover, OBP-301 in use has limited potency when administered alone; however, combination therapy using these agents and conventional anticancer agents, such as radiation exhibits encouraging levels of efficacy. Here, we describe the mechanistic basis for synergy of irradiation and OBP-301. This combination therapy was more effective compared with OBP-301 alone in OSCC cells. These results illustrate the potential of combining oncolytic virotherapy and ionizing radiation as a promising strategy in the management of human oral cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4976. doi:1538-7445.AM2012-4976