Antitumor effects of bevacizumab in combination with fluoropyrimidine drugs on human oral squamous cell carcinoma.

Vascular endothelial growth factor (VEGF) serves an important role in new blood vessel formation or angiogenesis, which is a critical event in tumor growth and metastasis. Bevacizumab is a humanized monoclonal antibody against VEGF-A, whereas S-1 is a fluoropyrimidine antineoplastic agent that induces apoptosis in various types of cancer cells. The present study evaluated the antitumor effects of bevacizumab in combination with 5-fluorouracil (5-FU) or S-1 against oral squamous cell carcinoma (OSCC) in vitro and in vivo. Two human OSCC cell lines were used, namely the high VEGF-A-expressing HSC-2 cells and the low VEGF-A-expressing SAS cells. MTT assay was used to evaluate the effect of bevacizumab and/or 5-FU against HSC-2 and SAS cell proliferation. Additionally, the antitumor effect of bevacizumab was evaluated alone and in combination with S-1 against HSC-2 tumors in nude mice. S-1 (6.9 mg/kg/day) was administered orally every day for 3 weeks, and bevacizumab (5 ml/kg/day) was injected intraperitoneally twice per week for 3 weeks. Apoptotic cells in mouse tumors were detected using the TUNEL method, and cell proliferation and microvessel density (MVD) were determined by immunohistochemical staining of Ki-67 and CD31, respectively. Bevacizumab alone did not inhibit OSCC cell proliferation in vitro, and did not exhibit any synergistic inhibitory effect in combination with 5-FU in vitro. However, combined bevacizumab and S-1 therapy exerted synergistic and significant antitumor effects in vivo on HSC-2 tumor xenografts, and induced apoptosis in tumor cells. Furthermore, this combination therapy led to decreased MVD and cell proliferative abilities, as well as increased apoptosis in residual tumors. The present findings suggested that the bevacizumab plus S-1 combination therapy may exert antitumor effects in high VEGF-A-expressing OSCC cells.

Elemental diet directly affects chemotherapy-induced dermatitis and raw wound areas.

Elental® is an L-glutamine-rich elemental diet (ED) that has been widely used in Japan as a nutritional supplement for malnourished patients. In addition, Elental® has been successfully used in the management of chemotherapy-induced mucositis in cancer patients. Recently, it was also reported that Elental® can effectively reduce chemotherapy-induced oral mucositis in patients with oral squamous cell carcinoma, and can also reduce mucositis and dermatitis in animal models. However, it is unclear whether oral intake or topical application of Elental® can act directly on chemotherapy-induced oral mucositis or dermatitis. The aim of the present study was to investigate the possible direct healing effect of Elental® on chemotherapy-induced dermatitis and raw wound areas in a mouse model. Dermatitis and raw wounds were induced in nude mice by administration of 5-fluorouracil (5-FU) (via gastric tube) and mechanical injury (using a metal brush or a surgical knife). We then compared the outcome following oral or topical application of Elental® in these mice. The effect of Elental® on the growth and migration ability of the human oral keratinocyte cell line, HOK, was also examined using MTT and migration assays, respectively. In the mouse model, both oral administration and topical application of Elental® reduced 5-FU-induced dermatitis and healed raw wound areas more effectively compared with the topical application of saline. The MTT assay revealed that Elental® exerted a growth-promoting effect on HOKs. In addition, Elental® enhanced the ability of HOKs to migrate, as demonstrated by the migration assay. These findings demonstrated that the topical application as well as the oral intake of Elental® exerted a direct healing effect on chemotherapy-induced dermatitis or raw wound areas. The data also indicated that oral intake of an ED may exert a direct healing effect on chemotherapy-induced oral mucositis.

Gimeracil enhances the antitumor effect of cisplatin in oral squamous cell carcinoma cells in vitro and in vivo.

Gimeracil or 5-chloro-2,4-dihydroxypyridine (CDHP) enhances the antitumor effects of 5-fluorouracil (5-FU) by inhibiting dihydropyrimidine dehydrogenase (DPD), which is involved in the degradation of 5-FU. CDHP, as part of a combination therapy, was also reported to exert a radiosensitizing effect. Therefore, CDHP may have underlying mechanisms of action other than DPD inhibition. The focus of the present study was to investigate the antitumor effects of CDHP and cisplatin (CDDP) combination treatment in vitro and in vivo against oral squamous cell carcinoma (OSCC) tumors. The inhibitory growth effects of CDHP and/or CDDP treatment on SAS and HSC2 cells were examined using an MTT assay. The expression levels of DNA double strand break repair proteins, including Ku70, DNA-dependent-protein kinase catalytic subunit (DNA-PKcs), Rad50 and Rad51 in CDHP and/or CDDP-treated cells were detected using western blotting. Nude mice with SAS or HSC2 tumors were treated with CDHP (administered orally 7 times/week) and/or CDDP (administered by intraperitoneal injection once/week) for 2 weeks. Combined treatment of CDHP and CDDP significantly suppressed the growth of SAS and HSC2 cells in vitro and that of tumors in vivo compared with the effects caused by single drug only or control treatments. Western blotting demonstrated that the expression levels of Ku70, DNA-PKcs, Rad50 and Rad51 were downregulated in cells treated with CDHP and CDDP combination treatment. Immunohistochemistry also identified that the expression of DNA double strand break repair proteins was downregulated in tumors treated with CDHP and CDDP combination treatment compared with that of tumors treated with CDDP alone or control. The results of the current study suggest that CDHP may be responsible for enhancing the antitumor effects of CDDP by suppressing the DNA double strand break repair system. Therefore, the combination of CDHP and CDDP may be a potential effective option for OSCC treatment.

Calreticulin is a novel independent prognostic factor for oral squamous cell carcinoma.

We focused on the expression of Calreticulin (CALR) in oral squamous cell carcinoma (OSCC) on the basis of proteomic differential display analysis data. We used QR-32 cells in this study which is a regressive murine fibrosarcoma cell clone; and QRsP-11, a progressive malignant tumor cell clone originated from QR-32. CALR is an endoplasmic reticulum luminal Ca2+-binding chaperone protein, which is thought to affect the tumor behavior of various malignancies. This study was aimed to determine the usefulness of CALR as a prognostic factor in patients with OSCC. We investigated the expression of CALR in tissue samples taken from 111 OSCC patients by immunohistochemistry, and we also analyzed the relationship between CALR expression and patients' clinicopathological characteristics as well as patient survival. Positive immunohistchemical staining of CALR was observed in the cancer cell cytoplasm. Among 111 patients, high expression of CALR was observed in 44 patients (39.6%), whereas low expression was observed in 67 patients (60.4%). Significant association was found between CALR expression and T classification (P=0.0027), N classification (P=0.0219), stage (P=0.0013), and patient outcome (P=0.0014). Log-rank test showed that, there is a significant difference (P<0.0001) in 5-year survival rates between patients showing CALR high-expression (59.1%) and CALR low-expression (86.6%). According to our Multivariate analysis, reduced term survival of patients was correlated to high levels of CALR expression (P<0.0001). Our findings suggest that elevated expression of CALR might play an important role in tumor progression in OSCC, and could be considered as a useful prognostic factor in OSCC patients.

Proteomic analysis showed down-regulation of nucleophosmin in progressive tumor cells compared to regressive tumor cells.

Important strategies against cancer are based on the understanding of the mechanisms of tumor progression. To elucidate alterations regarding tumor progression, we have performed proteomic differential display analysis for the expression of intracellular proteins in the regressive murine fibrosarcoma cell clone QR-32 and the progressive malignant tumor cell clone QRsP-11, derived from QR-32, by means of combination of two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and we have previously reported on relevant results. However, besides the protein spots which we already reported, we identified three more particular spots of interest. In the present study, two-dimensional western blot analysis demonstrated a significantly lower expression of three isoforms of nucleophosmin in progressive, compared to regressive cell clones. These results suggest that the down-regulation of the identified nucleophosmin proteins in QRsP-11 cells compared to QR-32 cells is possibly related to tumor malignant progression.

Effects of lentinan alone and in combination with fluoropyrimidine anticancer agent on growth of human oral squamous cell carcinoma in vitro and in vivo.

Chemotherapy has shown little antitumor activity against advanced oral squamous cell carcinoma (OSCC) patients. Therefore, there is an urgent need to develop more effective therapeutic methods for patients with advanced OSCC. Lentinan, beta-(1-->3)-D-glucan, an extract from the edible mushroom, Lentinus edodes, has been reported to show direct antitumor effects and various immunomodulatory effects. S-1 is an oral antineoplastic agent that can induce apoptosis in various types of cancer cells, including OSCC. Hence, combined treatment of cancer cells with Lentinan and S-1 might exert dramatic antitumor effects on OSCC cells. In this study, the response of human OSCC cells to Lentinan alone and in combination with S-1 was examined using nude mouse xenograft models. S-1 (6.9 mg/kg/day, 7 times/week) was administered orally and Lentinan (0.1 mg/kg/day, 2 times/week) was injected into peritumoral tissue for three weeks. Apoptotic cells were detected by a TUNEL method. The gene expression level of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT) was determined using microdissection and RT-PCR, and their protein levels were determined using ELISA. Combined therapy of Lentinan and S-1 markedly exerted antitumor effects on human OSCC xenografts and significantly induced apoptotic cells in tumors treated with Lentinan plus S-1. Microdissection and RT-PCR revealed that the expression of TS and DPD mRNA was down-regulated and that expression of OPRT mRNA was up-regulated in tumors administered the combined treatment. Moreover, ELISA indicated that the protein levels of TS and DPD were down-regulated, and that OPRT was up-regulated in tumors treated with the combined therapy. During the experimental period, no loss of body weight was observed in mice treated with the combined therapy. These findings demonstrate that the combination of Lentinan and S-1 is effective against OSCC and has the potential of being a new therapeutic tool for future treatment of these tumors.