Yuichi Ohnishi

Effects of epidermal growth factor on the invasion activity of the oral cancer cell lines HSC3 and SAS

Epidermal growth factor (EGF) is excreted in a high concentration in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer growth and invasion, we analyzed the Matrigel invasion activity of the cultured oral cancer cell line. Cells grown under the influence of EGF were subjected to Matrigel invasion assays and cells grown in the absence of EGF were used as controls. Gelatin-zymography and Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay (CAT assay) determined the EGF stimulation of matrix metalloproteinase (MMP) expression. EGF increased the number of cells penetrating a Matrigel membrane. Gelatin-zymography and Northern blot analysis revealed that MMP9 and Ets1 expressions correlated with EGF but MMP2 was not changed. a transient transfection assay revealed that EGF increased the promoter activities of the MMP9 genes in HSC3 and SAS cells. These results suggest that EGF increases the invasion activity of oral cancer cells partly by increasing MMP9.

Effects of CRM197, a specific inhibitor of HB-EGF, in oral cancer

CRM197, a nontoxic mutant of diphtheria toxin, is a specific inhibitor of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), which belongs to the EGF family that has been implicated in the increased progression, proliferation, and metastasis of oral cancer. In this study, we analyzed the antitumor effects of CRM197, which represent possible chemotherapeutic agents for oral cancer. In the experiment, we used the oral squamous cell carcinoma cell lines HSC3 and SAS. Cells treated with CRM197 were analyzed based on cell viability, MTT assay, invasion assay, Western blot, and zymography. HSC3 cells were injected subcutaneously into female BALB/c nu/nu mice at 5 weeks of age. CRM197 and/or CDDP were injected intraperitoneally into tumor-bearing mice, and tumor volume was measured over time. HB-EGF expression in HSC3 and SAS cells treated with CRM197 was significantly reduced and cell proliferation was inhibited. The invasiveness of CRM197-treated cells was relatively low. MMP-9 and VEGF were suppressed in HSC3 treated with CRM197 on zymography and Western blot. Further, tumor growth in xenografted mice was suppressed by CRM197 or CDDP at 1 mg/kg/day. Also, the coadministration of CDDP and CRM197 at 1 mg/kg/day completely inhibited tumor formation. These results suggest that HB-EGF is a target for oral cancer and that CRM197 is effective in oral cancer therapy.

Heparin-binding epidermal growth factor-like growth factor is a potent regulator of invasion activity in oral squamous cell carcinoma

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) has been shown to stimulate the growth of various cell types in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors when compared with normal tissue, its contribution to cancer progression remains obscure. The objective of this study was to explore the effects of HB-EGF on proliferation, invasion activity and MMP-9 levels of an oral squamous cell carcinoma cell line, HSC3, in vitro. MTT assays, Matrigel invasion assays and RT-PCR in combination with RNA interference (RNAi) were used in this study. An RNAi-mediated decrease in HB-EGF expression reduced invasion activity and MMP-9 mRNA levels, but not proliferation, in HSC3 cells. The addition of purified HB-EGF to cell culture medium upregulated MMP-9 mRNA levels in HSC3 cells. Furthermore, the TACE inhibitor TAPI-2 or EGFR inhibitor AG1478 decreased MMP-9 mRNA levels in HSC3 cells. These data indicate that HB-EGF released from HSC3 cells by TACE stimulates EGFR in an autocrine manner, which in turn activates invasion activity via MMP-9 upregulation.

Resistance of oral squamous cell carcinoma cells to cetuximab is associated with EGFR insensitivity and enhanced stem cell-like potency

Cetuximab, a specific anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is used in cancer treatment. Although development of resistance to cetuximab is well recognized, the underlying mechanisms remain unclear. In the present study, we characterized cetuximab-resistant oral squamous cell carcinoma (OSCC) cell lines. The human OSCC cell lines HSC3, HSC4 and SAS were used in the present study. Effects of inhibitors including cetuximab on growth in cells were assessed by MTT assays. Southern blotting and immunofluorescence analysis were performed to examine protein expression and localization. Sphere formation was used to characterize stem cell-like properties. Floating aggregation culture was used for anchorage-independent growth. Cetuximab inhibited proliferation of HSC3 and HSC4 cells, but not SAS cells. Proliferation of all three cell lines was inhibited by the EGFR/ErbB2/ErbB4 inhibitor II. The EGFR inhibitor AG1478 strongly inhibited HSC3 and HSC4 proliferation, but that of SAS cells only moderately. EGFR proteins were localized on cell surface and phosphorylated in all three cell lines. SAS cells could proliferate in serum-free monolayer culture and formed spheres from single cells in floating culture. HSC3 and HSC4 could not proliferate under serum-free culture conditions and could not form spheres. Growth of SAS spheres required serum, and was inhibited by both AG1478 and cetuximab. Thus, cetuximab-resistant SAS cells not only engaged in EGFR-independent growth but also exhibited stem cell-like properties. However, growth was EGFR-dependent in aggregation culture, and the SAS cell aggregates became cetuximab-sensitive. This suggests that cetuximab sensitivity is not only cell-type-dependent but is also affected by the growth microenvironment.

Cyclin D1 expression is correlated with cell differentiation and cell proliferation in oral squamous cell carcinomas

The present study conducted an immunohistochemical investigation of cyclin D1 and Ki-67 expression in oral squamous cell carcinoma (SCC) to evaluate the correlations between cell differentiation, cell proliferation and metastasis, and the effect of anticancer drug medication and cyclin D1 expression. Cyclin D1 and Ki-67 were detected clearly in the nuclei of 35 SCC samples. No correlation between cyclin D1 protein expression and oral SCC differentiation was found. By contrast, the majority of metastatic foci (90%) exhibited strong cyclin D1 expression, whereas weak expression was observed in metastatic foci with pre-operative adjuvant therapy. Additionally, cyclin D1 and Ki-67 were expressed in basal to suprabasal cells of well-differentiated oral SCC, whereas cyclin D1-positive and Ki-67-negative cells were present in the highly-differentiated region, according to a double-immunostaining method. These results indicate that the expression of cyclin D1 protein plays a role in cell differentiation and cell proliferation in well-differentiated oral SCC.

SOX4 expression is closely associated with differentiation and lymph node metastasis in oral squamous cell carcinoma.

SOX4 is a member of the SOX family of transcription regulators. In recent years, SOX4 was shown to be overexpressed in cancers of various organs and related to epithelial-mesenchymal transition, which is a metastatic factor. This study was the first to investigate correlations between SOX4 expression levels and the clinicopathologic factors of oral squamous cell carcinoma (OSCC). We analyzed SOX4 expression levels in 50 patients with OSCC using immunohistochemistry. All samples expressed the SOX4 protein and elevated SOX4 expression was significantly correlated with gender, T status, and stage levels. The expression level of SOX4 in primary foci of poorly differentiated OSCC was higher than that of well differentiated OSCC, which indicated that SOX4 expression is associated with the differentiation of OSCC. However, regardless of the differentiation level in the primary focus, SOX4 expression levels were found to be very high in the metastatic focus. Furthermore, SOX4 expression in metastatic foci was significantly suppressed by neoadjuvant therapy. These results indicate that undifferentiated OSCC cells expressing SOX4 are more likely to metastasize and neoadjuvant therapy including chemoradiation therapy may have some effect in metastatic prevention.

Effects of epidermal growth factor on the invasive activity and cytoskeleton of oral squamous cell carcinoma cell lines.

Epidermal growth factor (EGF) is present at high concentrations in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer proliferation and invasion, the current study analyzed the Matrigel invasion activity of cultured oral cancer cell lines. Cell proliferation under the influence of EGF was subjected to Matrigel invasion assays, and cell proliferation in the absence of EGF was used as control. Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay determined the EGF stimulation of matrix metalloproteinase (MMP) 1 expression. EGF increased the number of cells penetrating the Matrigel membrane. Northern blot analysis revealed that MMP1 and cytokeratin 19 expression correlate with EGF. In addition, the morphology of HSC-3 and SAS cells changed following the addition of EGF to the culture medium. A transient transfection assay revealed that EGF increases the promoter activities of MMP1 in HSC-3 cells. These observations suggested that EGF increases the invasive activity of oral cancer cells, partly by increasing MMP1, and morphological changes may be induced by altering the composition of cytoskeletal proteins.

Infiltrating angiolipoma of the lower lip: A case report and literature review

Infiltrating angiolipoma (IAL) is a rare lesion and is a clinicopathological variant of angiolipoma. IAL occurs most commonly in the trunk and extremities, it is rarely found in the head and neck regions and extremely rare in the oral cavity. This study presents the case of a 74-year-old female with IAL of the lower lip. To the best of our knowledge, this is the first case of IAL arising in the lower lip to be reported. Microscopically, IAL was unencapsulated and mature lipocytes were separated by a branching network of proliferating small vessels that infiltrated the adjacent tissues. Therefore, complete excision was difficult to perform. Magnetic resonance imaging has been reported to be valuable in determining the extent of the tumor and asserting a preoperative diagnosis. According to previous studies, the recurrence rate of IAL following surgical extirpation is 35–50%. Furthermore, the levels of mRNA expression of the vascular endothelial growth factor (VEGF) family members in the tumor were investigated. VEGF-A and -B expression were detected, however, VEGF-C and -D were expressed at extremely low levels. Excisional biopsy was performed under local anesthesia. During four years of follow-up, no evidence of tumor recurrence had been identified. An operating microscope may be utilized for the total removal of an IAL to minimize damage to normal tissues. This report indicates that mast cell-derived VEGF may be responsible for the enhanced vascularity in the tumor. We would therefore consider careful extirpation with no wide safety margin to be the procedure of choice, except when the tumor invades irregularly into the muscles.

Metastasis of mesothelioma to the maxillary gingiva

Malignant mesothelioma predominantly arises from the serosal surfaces of the pleural or peritoneal cavity. There is currently no effective standard treatment for mesothelioma and the prognosis for patients is poor; the majority of patients with malignant mesothelioma succumb between 12 and 17 months following diagnosis. The association of all forms of malignant mesothelioma with asbestos exposure has been well documented. However, metastasis to the oral gingiva is rare, as only four cases have previously been reported; two cases of metastasis to the tongue and four cases to the jaw bone. In the current report, the case of a 62-year-old male with metastatic mesothelioma is presented. To the best of our knowledge, this is the first report regarding the metastasis of this type of neoplasm to the maxillary gingiva.

Effects of Heparin-binding Epidermal Growth Factor on the Invasion Activity of an Oral Cancer Cell Line

Abstract Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), which belongs to the EGF family, has been shown to stimulate the growth of a variety of cells in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors when compared to normal tissue, its contribution to tumor invasion is still not known. In the present study, we analyzed the effects of HB-EGF on the invasion activity of a cultured oral cancer cell line using short interfering RNA (siRNA). Oral squamous carcinoma cell lines, HSC3 and SAS, were transfected with siRNA targeting HBEGF. Expression of HB-EGF was analyzed by real-time PCR. The invasiveness of the transfected cells was determined using a matrigel invasion assay, and MMP-9 production was measured by RTPCR and gelatin zymography. The expression of HB-EGF was reduced in HSC3-siRNA and SAS-siRNA cells. The matrigel invasion assay demonstrated that the invasiveness of HSC3-siRNA and SAS-siRNA cells was reduced. Gelatin zymography demonstrated that in HSC3-siRNA and SAS-siRNA cells, MMP9 production was decreased. These findings suggest that HB-EGF expression is related to the invasion activity of oral cancer, particularly via regulation of MMP9.