Mariko Mihara

Expression of vascular endothelial growth factor A, B, C, and D in oral squamous cell carcinoma

Vascular endothelial growth factor (VEGF) A is known to play an important role in tumor angiogenesis. The additional members of the VEGF family, VEGF B, C and D have been discovered. VEGF C and D show some selectivity toward lymphatic endothelial cells. However, whether VEGF family members play a role in tumor angiogenesis and lymph node metastasis is largely unknown. The aim of the present study was to explore the role of VEGF family members in oral squamous cell carcinoma (OSCC). We evaluated the expression of VEGF family members by immunohistochemistry, reverse transcription-polymerase chain reaction, and western blotting. All VEGF members were expressed at different levels in OSCC. Immunohistological analysis of VEGF family members expression and microvessel density revealed a correlation between VEGF A and B expression and tumor angiogenesis. Although VEGF A and B expression were detected in both node-positive and node-negative OSCC, VEGF C and D expression was detected frequently in node-positive tumors compared to node-negative tumors. These findings suggest a possible relationship between the expression level of VEGF C and/or D and development of lymphatic tumor spread.

Enhancement of tumor radioresponse by combined treatment with gefitinib (Iressa, ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, is accompanied by inhibition of DNA damage repair and cell growth in oral cancer.

Molecular blockade of EGFR with either an EGFR MAb or an EGFR TKI enhances the radiosensitivity of human SCCs. In the present study, we investigated whether treatment with the EGFR TKI gefitinib (Iressa, ZD1839) improves the response to radiotherapy in the OSCC cell lines HSC2 and HSC3. We examined potential mechanisms that may contribute to the enhanced radiation response induced by gefitinib. Growth inhibition was observed in vitro with radiation or gefitinib. A cooperative antiproliferative effect was obtained when cancer cells were treated with radiation followed by gefitinib. Cells treated with a combination of radiation and gefitinib arrested in G1 and G2–M phases, with a decrease in the S‐phase population. While radiation alone did not significantly affect MEK1/2 and p38 MAPK autophosphorylation, the combination of gefitinib and radiation completely inhibited the downstream signaling of EGFR. Results from DNA damage repair analysis in cultured OSCC cells demonstrated that gefitinib had a strong inhibitory effect on DNA‐PKc pathways after radiation. Tumor xenograft studies demonstrated that the combination of gefitinib and radiation caused growth inhibition and tumor regression of well‐established OSCC tumors in athymic mice; tumor volume was reduced from 1,008.2 to 231.4 mm3 in HSC2 cells (p < 0.01) and from 284.2 to 12.4 mm3 in HSC3 cells (p < 0.01). Immunohistochemical analysis of OSCC xenografts revealed that gefitinib caused a striking decrease in tumor cell proliferation when combined with radiotherapy. Overall, we conclude that gefitinib enhances tumor radioresponse by multiple mechanisms that may involve antiproliferative growth inhibition and effects on DNA repair after exposure to radiation.

Up-regulation of DNA-dependent protein kinase correlates with radiation resistance in oral squamous cell carcinoma.

DNA‐PK is a nuclear protein with serine/threonine kinase activity and forms a complex consisting of the DNA‐PKcs and a heterodimer of Ku70 and Ku80 proteins. Recent laboratory experiments have demonstrated that the DNA‐PK complex formation is one of the major pathways by which mammalian cells respond to DNA double‐strand breaks induced by ionizing radiation. In this study, we evaluated the relationship between expression levels of DNA‐PKcs, Ku70 and Ku80 proteins and radiation sensitivity in oral squamous cell carcinoma (OSCC) cell lines and in OSCC patients treated with preoperative radiation therapy. The OSCC cell lines greatly differed in their response to irradiation, as assessed by a standard colony formation assay. However, the expression levels of the DNA‐PK complex proteins were all similar, and there was no association between the magnitude of their expression and the tumor radiation sensitivity. Expression of DNA‐PK complex proteins increased after radiation treatment, and the increased values correlated with the tumor radiation resistance. Expression of DNA‐PKcs and Ku70 after irradiation was increased in the surviving cells of OSCC tissues irradiated preoperatively. These results suggest that up‐regulation of DNA‐PK complex protein, especially DNA‐PKcs, after radiation treatment correlates to radiation resistance. DNA‐PKcs might be a molecular target for a novel radiation sensitization therapy of OSCC.

Skp2 and Jab1 Expression Are Associated with Inverse Expression of p27KIP1 and Poor Prognosis in Oral Squamous Cell Carcinomas

Previous studies have shown that low levels of p27KIP1, an inhibitor of G1 cyclin-dependent kinases (CDK), are associated with high aggressiveness and poor prognosis in a variety of cancers. Decreased levels of p27KIP1 are caused, at least in part, by an acceleration of degradation with Skp2 (S-phase kinase-associated protein 2) and Jab1 (Jun activation domain-binding protein 1). This investigation was undertaken to examine whether the Skp2 and Jab1 expression is correlated with p27KIP1 protein levels, and how it is clinically relevant in oral squamous cell carcinoma (OSCC). The correlations between p27KIP1 and Skp2, and p27KIP1 and Jab1 expression were evaluated by Western blot analysis. Immunohistochemical analysis was done in 75 cases of OSCC. A strongly significant inverse correlation was found between levels of p27KIP1 and Skp2, and p27KIP1 and Jab1 (p < 0.0001). Thus, decreased levels of p27KIP1 were associated with strongly increased levels of Skp2 and Jab1, whereas high levels of p27KIP1 coincided with low levels of Skp2 and Jab1. Reductions of p27KIP1 expression and overexpression of Skp2 and Jab1 were significantly associated with cervical lymph node metastasis and poor prognosis. Overexpression of Skp2 and Jab1 is associated with the reduction of p27KIP1 expression, and may have a role in the progression of OSCC.

Gefitinib ('Iressa', ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, up-regulates p27KIP1 and induces G1 arrest in oral squamous cell carcinoma cell lines.

High expression of epidermal growth factor receptor (EGFR) is frequently observed in many solid tumor types including oral squamous cell carcinomas (OSCC). Recently, the results of preclinical studies and early clinical trials targeting the EGFR have shown evidence of the activity. In this study, gefitinib (Iressa, ZD1839), an EGFR-tyrosine kinase inhibitor, inhibited cell proliferation and upregulated p27KIP1 in OSCC cells. Growth inhibition was observed in OSCC xenografts when mice were treated with gefitinib in vivo. A flow cytometric analysis demonstrated that treatment with gefitinib induced accumulation in G1 phase, accompanied by a decrease in the percentage of cells in S phase. Apoptosis was not seen in this study. Cell growth was inhibited by an increase of the cell cycle inhibitor p27KIP1 and a decrease of its ubiquitin ligase subunit Skp2.

Gefitinib (‘Iressa’), an epidermal growth factor receptor tyrosine kinase inhibitor, mediates the inhibition of lymph node metastasis in oral cancer cells

High expression of epidermal growth factor receptor (EGFR) is frequently observed in many solid tumor types including oral squamous cell carcinomas (OSCC). This study investigated whether treatment with gefitinib (Iressa), an EGFR-tyrosine kinase inhibitor, would inhibit the metastatic spread in OSCC cells. This was evaluated using orthotopic xenografts of highly metastatic OSCC. Metastasis was observed in six of 13 gefitinib treated animals (46.2%), compared with all of 12 control animals (100%). After exposure to gefitinib, OSCC cells showed a marked reduction in cell adhesion ability to fibronectin and in the expression of integrin alpha3, alphav, beta1, beta4, beta5 and beta6.

Lymph node metastasis of oral cancer visualized in live tissue by green fluorescent protein expression

Here, we report the establishment of a stably transfected cell line which expresses high levels of green fluorescent protein (GFP), thus permitting the detection and visualization of developing tumors and lymph node metastases after injection into nude mice. Cells of the human oral squamous carcinoma cell line (SAS-L1) were transfected with an expression vector containing a cDNA encoding humanized GFP and the neomycin resistance gene. A clone with stable high-level expression of GFP was selected in vitro using G418. To study metastasis formation, GFP-expressing cells were injected orthotopically into the tongue of nude mice. The resultant tumor growth in the tongue and micrometastases in the lymph nodes could be visualized by GFP fluorescence. Therefore a useful model has been developed for the study of oral cancer, firstly to understand the metastatic process and secondly for the evaluation of potential treatments.

Enhancement of radiosensitivity in head and neck cancer cells by ZD1839 ('IRESSA'), a selective epidermal growth factor receptor tyrosine kinase inhibitor.

Overexpression of epidermal growth factor receptor (EGFR) is frequently observed in many solid tumor types, including head and neck squamous cell carcinomas (HNSCC). Recent laboratory experiments have demonstrated that high EGFR levels correlate with increased tumor resistance to radiation. This study investigated the relationship between EGFR expression levels and radiosensitivity in 5 HNSCC cell lines (HSC2, HSC3, HSC4, SCC25, and Ca9–22) and whether treatment with ZD1839 (‘Iressa’), a selective EGFR-tyrosine kinase inhibitor (TKI), would improve tumor cell response to radiotherapy. ZD1839 suppressed the growth of HNSCC cell lines in a dose- and time-dependent manner. Radiosensitivity of these HNSCC cell lines, assessed by a clonogenic survival assay, differed greatly and the expression of EGFR varied. EGFR expression levels (EGFR numbers/cell) correlated with increased tumor resistance to radiation (f[x]= 4.54 X, R2 = 0.715; f[x]: EGFR numbers/cell, X: radiosensitivity; D10). Following exposure of the HNSCC cells to 1.0 &mgr;M ZD1839 and radiation (0–10 Gy), greater than additive growth inhibitory effects were observed. These results suggest that ZD1839 could enhance tumor radiosensitivity and inhibit tumor growth after radiation, indicating that this combination could have clinical potential in the treatment of patients with head and neck cancer.

Flavopiridol, a cyclin dependent kinase (CDK) inhibitor, induces apoptosis by regulating Bcl-x in oral cancer cells.

Flavopiridol is a synthetic flavone that inhibits tumor growth by suppressing cyclin-dependent kinases (CDKs). We have investigated effects of flavopiridol in oral squamous cell carcinoma (OSCC). Flavopiridol was found to inhibit the growth of OSCC cells in a time- and dose-dependent manner. Induction of apoptosis was observed in all cells showing accumulated cells with sub-G(1) DNA contents, DNA fragmentations, and PARP cleavages. While Bcl-2 and Bax expression did not change, Bcl-x(L) was down regulated and Bcl-xs was up-regulated after being exposed to flavopiridol. Flavopiridol treatments also resulted in remarkable reductions of cyclin A, cyclin B, and cyclin D1 expressions. We also found that expression levels of CDK activation kinase and CDC25C were reduced, and p34 inactive form CDK2 were up-regulated. Our data indicate that flavopiridol has growth inhibition activities against OSCC. Flavopiridol not only inhibits CDKs directly, but it also inhibits the CDKs activation pathway and activates the Bcl-x apoptotic pathway.

HER2/neu Expression in Oral Squamous Cell Carcinoma

Abstract Objective: To evaluate the expression of HER2/neu in oral squamous cell carcinoma with a view to determining the usefulness of molecular target therapy by anti-HER2 antibody. Patients and Methods: Oral squamous cell carcinoma cell lines and 69 clinical tumour samples were tested using enzyme-linked immunosorbent assay and immunohistochemistry. Positive and negative controls were utilised. Results: Expression of HER2/neu in oral squamous cell carcinoma cell lines was low. Overexpression was not observed in the clinical samples. Conclusion: Low prevalence of expression of HER2/neu in oral squamous cell carcinoma limits the likely utility of herceptin therapy.