PD-L1 expression in malignant salivary gland tumors.

BACKGROUND: Programmed death-1 ligand-1 (PD-L1) an important cancer biomarker that can suppress the immune system and its high expression is often reported to be related with increased tumor aggressiveness in some cancers. Here, we examined and evaluated PD-L1 expression in patients with malignant salivary gland tumor. Moreover, the relationship between PD-L1 immunolocalization and clinical pathological features, as well as the prognosis of malignant salivary gland tumors was investigated. METHODS: We examined PD-L1expression in 47 patients with malignant salivary gland tumor by immunohistochemical staining. PD-L1 positivity was defined as ≥5% in tumor cell membrane and evaluated according to three categories (0% = 0, < 5% = 1, ≥5% = 2) in tumor-infiltrating mononuclear cells (TIMCs). Fisher's exact test was used to compare between PD-L1 expression and clinico-pathological features, and Kaplan-Meier method was used to estimate the distribution of OS by PD-L1 positivity. RESULTS: PD-L1 expression was detected in 51.1% of malignant salivary gland tumor tissues. No association was observed between PD-L1 immunolocalization in tumor and patient gender, or age. However, PD-L1 immunodetection of tumor cell membranes was significantly associated to stage, recurrence or metastasis after surgery, and patient outcome. On the other hand, PD-L1 immunodetection of tumor-infiltrating mononuclear cells (TIMCs) was significantly associated to recurrence or metastasis after surgery, and patient outcome. PD-L1 positivity in both tumor cell membrane and TIMCs was associated with shorter overall survival (OS) (p = 0.002 and p = 0.016, respectively). CONCLUSION: These findings suggested that patients with PD-L1 positive tumors or TIMCs appear to have poor clinical outcomes in malignant salivary gland tumors.

Induction of artificial cancer stem cells from tongue cancer cells by defined reprogramming factors.

BACKGROUND: The cancer stem cells (CSCs), a small subpopulation of cells in tumor are responsible for the tumor initiation, growth, recurrence and metastasis of cancer, as well as resistance of cancers to drugs or radiotherapy. CSCs are an important target for the development of novel strategies in cancer treatment. However, CSCs-targeted new anti-cancer drug discovery is currently hindered by the lack of easy and reliable methods for isolating, collecting and maintaining sufficient number of CSCs. Here, we examined whether introduction of defined reprogramming factors (Oct4, shp53, Sox2, Klf4, l-Myc and Lin28) into HSC2 tongue cancer cells could transform the HSC2 into HSC2 with CSCs properties. METHODS: We introduced the defined reprogramming factors into HSC2 tongue cancer cells via episomal vectors by electroporation method to generate transfectant cells. We investigated the malignant properties of the transfectant cells by cell proliferation assay, migration assay, wound healing assay, sphere formation assay, chemosensitivity and radiosensitivity assay in vitro; and also examined the tumorigenic potential of the transfectants in vivo. RESULTS: The transfectant cells (HSC2/hOCT3/4-shp53-F, HSC2/hSK, HSC2/hUL, HSC2/hOCT3/4-shp53-F + hSK, HSC2/hOCT3/4-shp53-F + hUL, HSC2/hSK + hUL, HSC2/hOCT3/4-shp53-F + hSK + hUL) displayed a malignant phenotype in culture and form tumors on the back of nude mice more efficiently than parental HSC2 and control HSC2/EGFP transfectant cells. They exhibited increased resistance to chemotherapeutic agents; 5-fluorouracil, cisplatin, docetaxel, trifluorothymidine, zoledronic acid, cetuximab, bortezomib and radiation when compared with HSC2 and HSC2/EGFP. Among all the transfected cells, HSC2/hOCT3/4-shp53-F + hSK + hUL cell containing all of the reprogramming factors showed the most aggressive and malignant properties and presented the highest number of spheres in the culture medium containing human recombinant fibroblast Growth Factor-2 (FGF-2) and epidermal Growth Factor (EGF). CONCLUSION: These findings suggest that artificial cancer stem cells obtained by the induction of cellular reprogramming may be useful for investigating the acquisition of potential malignancy as well as screening the CSCs-targeting drugs.

Efficacy of elemental diet on prevention for chemoradiotherapy-induced oral mucositis in patients with oral squamous cell carcinoma.

PURPOSE: Oral mucositis induced by radiation or chemoradiation can compromise the quality of life of oral squamous cell carcinoma (OSCC) patients. The present study was designed to evaluate the preventive effects of elemental diet (ED), Elental®, on radiotherapy- or chemoradiotherapy-induced mucositis in OSCC patients. PATIENTS AND METHODS: Seventy-four patients who underwent radiation (60-70 Gy) with/without chemotherapy [S-1, cisplatin (CDDP), CDDP plus S-1] were enrolled in this retrospective study; 37 had received Elental® during treatment (Elental® group) and 37 had not (control group). Factors related to alleviation of oral mucositis were identified by multivariate logistic regression analysis. Rates of completion of chemoradiation treatments were compared between Elental® and control groups according to the treatment regimen. The comparison of the nutritional status between groups was also performed. RESULTS: Multivariate analysis indicated that the administration of Elental® and no combined chemotherapy (radiation alone) were significant factors associated with the degree of oral mucositis, i.e., most of the patients who consumed Elental® suffered from a lower degree of mucositis compared to the control group. Elental® was associated with a significantly improved rate of completion of chemoradiation (no interruption). There was no significant difference between Elental® group and control group in terms of mean change of body weight or total protein and albumin levels in blood serum before and after (chemo)radiation. CONCLUSIONS: The present study indicates that Elental® is effective for ameliorating oral mucositis induced by (chemo)radiation in OSCC patients. Elental® was also associated with improved completion rates of (chemo)radiotherapy.

Amino Acids May Have Protective Effects on Salivary Glands of 5-FU-administered Mice.

BACKGROUND/AIM: This study aimed to identify the most useful components of Elental® in the treatment of 5-fluorouracil (FU)-induced mucositis and salivary gland atrophy in mice. MATERIALS AND METHODS: Mice (except the control group) were intraperitoneally injected with 5-FU. The mice received saline (control group and 5-FU group), dextrin (Dextrin group), amino acids (17AA group), or Elental® (Elental® group). RESULTS: The volume and weight of salivary glands was higher in 17AA and Elental® groups compared to 5-FU group. The number of mucous glands was higher, whereas the number of damaged granular ductal epithelial cells was lower in the salivary glands of all groups except the 5-FU group. Salivation was also decreased in the 5-FU group compared to the other groups. CONCLUSION: Amino acids could be the most effective components of Elental® for protecting mouse salivary glands from 5-FU-induced atrophic changes, and might be useful in the treatment of oral mucositis in cancer patients.

An elemental diet protects mouse salivary glands from 5-fluorouracil-induced atrophy.

An elemental diet (ED) reduces adverse effects of chemotherapy, including oral mucositis, in patients with cancer. However, the detailed mechanism(s) of the healing effects of an ED remains unclear. In the present study, the protective effects of the ED, Elental®, were examined against 5-fluorouracil (5-FU)-induced oral mucositis and salivary gland atrophy in mice. Mucositis was induced in female ICR mice by injection of 5-FU. The mice were orally administered Elental® (ED group) or saline (control group). After treatment, the mice body weight, salivary gland weight and the histological changes in the salivary gland granular duct area were monitored. The mice body weight remained stable in the ED group, but was significantly decreased in the control group. Moreover, the salivary gland weight was higher in the ED group compared with the control group. In addition, the salivary gland granular duct area cells were larger in the ED group compared with the control group. Whole transcriptome analysis and network analysis were conducted to understand the mechanisms of action of Elental® against oral mucositis. Whole transcriptome analysis and Ingenuity Pathways Analysis data suggested that Elental® contributed to the recovery of mitochondrial function in 5-FU-damaged salivary glands. Immunohistochemical analysis of salivary gland tissue demonstrated that the expression of cytochrome c oxidase subunit 4 and epidermal growth factor were higher in the ED group compared with the control group. Next, the rate of apoptosis in the salivary glands was examined using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assays. The number of TUNEL-positive cells in the salivary glands was lower in the ED group compared with the control group. These findings suggested that Elental® may protect mouse salivary glands from 5-FU-induced atrophic changes, which suggests that ED treatment may improve xerostomia and alleviate oral mucositis in patients with cancer receiving 5-FU-based chemotherapy.

Efficacy of a Novel Oral Chemotherapeutic Agent, TAS-102, Against Human Oral Squamous Cell Carcinoma Cells.

BACKGROUND: TAS-102 is effective against unresectable advanced or recurrent colorectal and gastric cancer. However, its effect on oral squamous cell carcinoma (OSCC) is still unknown. Here, we tried to clarify the possible effect of TAS-102 against angiogenesis and proliferation of human OSCC cells. MATERIALS AND METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, migration assay and mice xenograft models were used to determine the effect of TAS-102 on growth and migration of OSCC. The activity of phosphorylated nuclear factor kappa light-chain-enhancer of activated B-cells (NF-κB) (p-p65) in cells was detected by immunocytochemistry. The expression of p-AKT serine/threonine kinase 1 (p-AKT), p-p65, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF2) and CD31 in mouse tumors were detected by immunohistochemistry. RESULTS: TAS-102 significantly inhibited growth and migration of OSCC both in vitro and in vivo. It suppressed the activity of NF-κB in cells. TAS-102 down-regulated the expression of p-AKT, VEGF, FGF2 and CD31, which was associated with reduced vascularization of HSC2 tumor lesions. CONCLUSION: These findings suggest that TAS-102 might inhibit angiogenesis and proliferation of OSCC cells.

Effects of an elemental diet, Elental®, may differ between healthy oral cells and oral cancer cells.

The effectiveness of an elemental diet (ED), Elental®, against radiotherapy­ or chemoradiotherapy­induced oral mucositis was previously reported. However, the administration of additional nutrition or an ED in patients with oral cancer may also provide extra nutrition for cancer cells, which could result in cancer development. At present, it remains unclear whether the beneficial effects of an ED are likely to surpass its potential harmful effects on oral cancer treatment. In the present study, we aimed to clarify whether Elental® has different effects on a healthy human oral keratinocyte (HOK) cell line compared with its effects on oral squamous cell carcinoma (OSCC) cell lines (HSC2, HSC3, HSC4). The efficacy of Elental® was compared in relation to the growth and migration ability of HOK and OSCC cell lines using MTT assay and migration assay, respectively. In addition, whole transcriptome analysis and network analysis were performed to determine the difference in the mechanism of action of Elental® between HOK and HSC2 cells. In addition, Elental® promoted growth and migration ability of­malnourished and 5­fluorouracil (5­FU)­treated damaged HOK cells cultured in low nutrition medium (0% growth supplement). However, Elental® did not affect the growth ability of 5­FU­treated damaged HSC2 cell line in low nutrition medium (0 or 1% fetal bovine serum (FBS), as well as the growth ability of HSC3 and HSC4 cell lines in medium containing 0% FBS. Elental® pre­treatment also enhanced the apoptosis­inducing effect of anticancer agents against OSCC cells. In addition, whole transcriptome analysis and Ingenuity Pathways Analysis (IPA) data suggested that Elental® may help in the proliferation and survival of HOK through the induction of ERK. Moreover, Elental® added stress to HSC2 cells through the induction of the endoplasmic reticulum stress response marker, BiP and GRP 94. The results showed that Elental® may add stress to HSC2 cells and provide growth stimulation to HOK. These findings suggest that the effects of Elental® on healthy oral cells and oral cancer cells may differ.

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.

Effects of cepharanthine alone and in combination with fluoropyrimidine anticancer agent, S-1, on tumor growth of human oral squamous cell carcinoma xenografts in nude mice.

BACKGROUND: 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. Cepharanthine is a biscoclaurine alkaloid extracted from Stephania cepharantha Hayata, which is widely used for the treatment of many acute and chronic diseases, and can exert antitumor effects on several human cancer cells. S-1 is a new oral antineoplastic agent that can induce apoptosis in various types of cancer cells, including OSCC. Hence combined treatment of cancer cells with cepharanthine and S-1 might exert dramatic antitumor effects on OSCC cells. MATERIALS AND METHODS: In this study, the response of human OSCC cells to cepharanthine alone and in combination with S-1 was examined using nude mouse xenograft models. S-1 (10 mg/kg/day, 5 times/week) was administered orally and cepharanthine (20 mg/kg, 5 times/week) was injected into peritumoral tissue for three weeks. Apoptotic cells were detected by a TUNEL method. The protein expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyl transferase (OPRT) were assessed using immunohistochemistry; their gene expression was determined using microdissection and RT-PCR, and their protein levels using ELISA. RESULTS: Combined therapy of cepharanthine and S-1 exerted antitumor effects on human OSCC xenografts markedly and significantly induced apoptotic cells in tumors treated with cepharanthine plus S-1. Immunohistochemistry showed that the expressions of TS and DPD were down-regulated, and that OPRT expression was up-regulated in tumors treated with cepharanthine plus S-1. In the same way, 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. CONCLUSION: These findings demonstrate that the combination of cepharanthine and S-1 is effective against OSCC and has the potential of being a new therapeutic tool for future treatment of these tumors.

Prognostic significance of FOXM1 in oral squamous cell carcinoma patients treated by docetaxel-containing regimens.

Forkhead box protein M1 (FOXM1) is an oncoprotein that is involved in cell proliferation, differentiation and aging, and overexpression of FOXM1 is thought to be associated with the development and progression of various types of cancer. The expression of FOXM1 was retrospectively examined in tumor tissues taken from 56 oral squamous cell carcinoma (OSCC) patients by immunohistochemical staining. All of these patients received docetaxel (Doc)-containing regimens as treatments against OSCC. The association between FOXM1 expression and the clinicopathological characteristics and prognosis of these patients was then examined. FOXM1 was expressed in the nucleus and cytoplasm of OSCC tissues samples. There was a significant association between FOXM1 expression in tumor tissues and N classification (P=0.0395), stage (P=0.004), therapeutic efficacy (P=0.0113) and outcome (P=0.0134) of patients. However, FOXM1 expression had no association with patients' sex, age or T classification. Additionally, high expression of FOXM1 in tumor cells was associated with a shorter overall survival (P=0.0257) of patients. Multivariate analysis also revealed that elevated expression of FOXM1 was a predictor of patients' poor survival (P=0.0327). The results suggested that high expression of FOXM1 in OSCC tumors may result in reduced therapeutic effects and poor clinical outcomes of patients receiving Doc-based treatment regimens.

The Elental® elemental diet for chemoradiotherapy-induced oral mucositis: A prospective study in patients with oral squamous cell carcinoma.

Oral mucositis is a common adverse effect of cancer treatment that can increase the risk for local and systemic infection. This prospective study was designed to evaluate the preventive effects of an amino-acid-rich elemental diet (ED), Elental®, on radiotherapy- or chemoradiotherapy-induced mucositis in oral squamous cell carcinoma (OSCC) patients. Fifty patients were enrolled in this prospective study, who had received radiation (60-70 Gy) with/without chemotherapy [S-1, UFT, cisplatin (CDDP), docetaxel (DOC) plus CDDP, or Cetuximab]. The Elental® group (25 patients) had received Elental® during treatment, and the control group (25 patients) had not. Multivariate logistic regression analysis was used to identify the factors related to abatement of oral mucositis. A comparison of the rates of completion of chemoradiation treatments as well as the nutritional or inflammatory status between Elental® and control groups was performed. Multivariate analysis indicated that most of the patients who received Elental® suffered from a lower degree of mucositis and showed significantly improved rate of completion of chemoradiation (no interruption) compared to the control group. There was a significant difference between the Elental® group and the control group in terms of the mean change of C-reactive protein (CRP) levels in blood serum; however, there was no significant difference in terms of a mean change of body weight and total protein level in blood serum before and after chemoradiation. Our study shows that the Elental® elemental diet could be useful for the treatment of oral mucositis induced by chemoradiation. Elental® might also promote improved completion rates of chemoradiotherapy in OSCC patients.

Elemental Diet Accelerates the Recovery From Oral Mucositis and Dermatitis Induced by 5-Fluorouracil Through the Induction of Fibroblast Growth Factor 2.

Mucositis and dermatitis induced by anticancer agents are common complications of anticancer therapies. In this study, we evaluated the efficacy of Elental (Ajinomoto Pharmaceutical Ltd, Tokyo, Japan), an elemental diet with glutamine in the treatment of 5-fluorouracil (5-FU)-induced oral mucositis and dermatitis in vivo and tried to clarify the underlying mechanisms of its action. Oral mucositis and dermatitis was induced through a combination of 5-FU treatment and mild abrasion of the cheek pouch in hamsters and the dorsal skin in nude mice respectively. These animals received saline, dextrin or Elental suspension (18 kcal/100 g) by a gastric tube daily until sacrifice. Elental reduced oral mucositis and dermatitis more effectively than dextrin in the animal model. Moreover, growth facilitating effects of Elental on HaCaT cells were examined in vitro. MTT assay, wound healing assay, and migration assay revealed that Elental could enhance the growth, invasion, and migration ability of HaCaT. ELISA and Western blotting showed upregulated FGF2 in Elental-treated HaCaT. These findings suggest that Elental is effective for the treatment of mucositis and dermatitis, and may accelerate mucosal and skin recovery through FGF2 induction and reepithelization.

Low carbonyl reductase 1 expression is associated with poor prognosis in patients with oral squamous cell carcinoma.

Carbonyl reductase 1 (CBR1) is an enzyme that catalyzes the reduction of numerous compounds by using NADPH-dependent oxidoreductase activity. Decreased expression of CBR1 is associated with disease progression and an unfavorable outcome in several types of malignancies. The purpose of the current study was to determine whether CBR1 expression could be a useful prognostic factor in patients with oral squamous cell carcinoma (OSCC). Therefore, its mechanisms of action were investigated in order to understand how CBR1 affects cancer cell behavior in vitro. CBR1 expression was evaluated using immunohistochemistry and tissue samples obtained from 90 patients with OSCC. The associations between CBR1 expression, clinicopathological characteristics and patient survival were also analyzed. In addition, the role of CBR1 in cancer cell invasion and metastasis was examined, along with its underlying molecular mechanisms, via transfecting CBR1-siRNA into the HSC2 human OSCC cell line. Immunohistochemical analysis revealed that biopsy tissue samples of 71.1% of the patients with OSCC were positive for CBR1. In addition, CBR1 expression status was correlated with the N classification (P<0.0001), stage (P=0.0018) and outcome (P=0.0095). Furthermore, a statistical correlation was determined between the protein expression status and overall survival (P=0.0171). In vitro studies indicated that the suppression of CBR1 by CBR1-siRNA increased cancer cell proliferative, wound healing and migratory abilities. These findings suggest that low expression levels of CBR1 may affect cancer prognosis, and that CBR1 may have potential as a prognostic factor for patients with OSCC.

Elemental diet inhibits pro­inflammatory cytokine production in keratinocytes through the suppression of NF­κB activation.

An elemental diet (ED) has been reported to reduce oral mucositis and dermatitis induced by chemotherapy. However, its molecular mechanism of action as an anti­inflammatory agent is still unknown. The aim of the present study was to clarify whether ED confers its anti­inflammatory action via reduction of pro­inflammatory cytokine production in keratinocytes in vivo and in vitro. We evaluated the efficacy of ED in the treatment of 5­fluorouracil (5­FU)­induced dermatitis of nude mice, and examined the expression of pro­inflammatory cytokines such as tumor necrosis factor­α (TNF­α), interleukin (IL)­1ß and IL­6 using immunohistochemistry. Moreover, we assessed the expression and production of these pro­inflammatory cytokines by western blotting and ELISA assays, respectively, in immortalized human keratinocyte cell line, HaCaT. Furthermore, we investigated the effect of ED on a major inflammation­related factor, nuclear transcription factor­κB (NF­κB), since it controls many genes involved in the inflammation pathway. Our results indicated that ED reduced the expression of TNF­α, IL­1ß and IL­6. It also inhibited the nuclear transition of p65 NF­κB, which is known to regulate inflammatory cytokine expression in keratinocytes suffering from 5­FU­induced dermatitis. In addition, ED reduced the production of TNF­α, IL­1ß and IL­6 in HaCaT cells. Moreover, ED attenuated 5­FU­induced transcriptional activation of NF­κB. These findings revealed that ED suppresses the expression of pro­inflammatory cytokines by suppressing NF­κB in keratinocytes, suggesting the potential usefulness of ED in the treatment of various inflammatory diseases of the dermal region.

Investigation of optimal schedule of concurrent radiotherapy with S-1 for oral squamous cell carcinoma.

In the present study, we examined the appropriate schedule of S-1 medication in the combination with radiation by investigating the safety, the clinical efficacy, and antitumor effects on tumors in nude mice. In the patients with oral squamous cell carcinoma (OSCC), S-1 was given orally according to a 4-week application followed by 2-week rest regimen (4-week regimen), or a 2-week application followed by a 1-week rest regimen (2-week regimen). Radiation was given (2 Gy/day; 5 days/week) for a total of 60 Gy. In nude mouse models, human oral cancer cell lines were used as subcutaneous xenografts in nude mice. The mice were treated by S-1 (10 mg/kg) and radiation (1 Gy) with a 4-week regimen or a 2-week regimen. Apoptotic cells were detected by TUNEL method. In the patients with OSCC, the response rate with the 4-week regimen was 100% and the response rate with the 2-week regimen was 92.3%. However, a high frequency of adverse effect was found in the 4-week regimen when compared to the 2-week regimen. Grade 3 toxicity of leukopenia, neutropenia and stomatitis were seen in 3 cases, grade 3 toxicity of anorexia and nausea were seen in 2 cases, and grade 3 toxicity of decrease of hemoglobin level, heartburn/dyspepsia and increase of bilirubin level were seen in a case of the 4-week regimen. On the other hand, grade 3 toxicity of stomatitis, anorexia, nausea, heartburn/dyspepsia and increase of bilirubin level were seen in a case of the 2-week regimen. In nude mouse models, the 2-week regimen was more effective than the 4-week regimen. In addition, significant increase in the percentage of apoptotic cells was observed in the tumors treated with the 4-week regimen when compared with the tumors treated with the 2-week regimen. No loss of body weight was observed in mice treated with the 2-week regimen during the experimental period. These results suggested that the 2-week regimen might reduce adverse effects, and enhance therapeutic effects compared to the 4-week regimen. Briefly, this 2-week regimen may be a useful concurrent chemo-radiotherapy improving the quality of life (QOL) of patients with OSCC.

Effects of tumor necrosis factor-related apoptosis-inducing ligand alone and in combination with fluoropyrimidine anticancer agent, S-1, on tumor growth of human oral squamous cell carcinoma xenografts in nude mice.

BACKGROUND: 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. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor ligand family that selectively induces apoptosis of cancer cells. S-1 is a new oral antineoplastic agent that can induce apoptosis in various types of cancer cells, including OSCC. Hence, combined treatment of cancer cells with TRAIL and S-1 might exert dramatic antitumor effects on OSCC cells. MATERIALS AND METHODS: In this study, the response of human OSCC cells to TRAIL alone and in combination with S-1 was examined using nude mouse xenograft models. S-1 (10 mg/kg/day, 5 times/week) was administered orally and TRAIL (1 mg/kg, 5 times/week) was injected into peritumoral tissue for three weeks. Apoptotic cells were detected by a TUNEL method. The protein expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyl transferase (OPRT) were assessed using immunohistochemistry; their gene expression was determined using microdissection and RT-PCR, and their protein levels using ELISA. RESULTS: Combined therapy of TRAIL and S-1 exerted antitumor effects on human OSCC xenografts markedly and significantly induced apoptotic cells in tumors treated with TRAIL plus S-1. Immunohistochemistry showed that the expressions of TS and DPD were down-regulated, and that OPRT expression was up-regulated in tumors treated with TRAIL plus S-1. In the same way, 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. CONCLUSION: These findings demonstrate that the combination of TRAIL and S-1 is effective against OSCC and has the potential of being a new therapeutic tool for future treatment of these tumors.

Therapeutic strategies with oral fluoropyrimidine anticancer agent, S-1 against oral cancer.

Oral cancer has been recognized as a tumor with low sensitivity to anticancer agents. However, introduction of S-1, an oral cancer agent is improving treatment outcome for patients with oral cancer. In addition, S-1, as a main drug for oral cancer treatment in Japan can be easily available for outpatients. In fact, S-1 exerts high therapeutic effects with acceptable side effects. Moreover, combined chemotherapy with S-1 shows higher efficacy than S-1 alone, and combined chemo-radiotherapy with S-1 exerts remarkable therapeutic effects. Furthermore, we should consider the combined therapy of S-1 and molecular targeting agents right now as these combinations were reportedly useful for oral cancer treatment. Here, we describe our findings related to S-1 that were obtained experimentally and clinically, and favorable therapeutic strategies with S-1 against oral cancer with bibliographic considerations.

High expression of stathmin 1 is a strong prognostic marker in oral squamous cell carcinoma patients treated by docetaxel-containing regimens.

Stathmin 1 is an oncoprotein that regulates cell cycle by modulating microtubule dynamics and can cause uncontrolled cell proliferation in mutated state. The present study examined stathmin 1 expression in 49 patients with oral squamous cell carcinoma (OSCC) treated with docetaxel (Doc)-containing regimens by immunohistochemical staining. Moreover, we investigated the relationship between stathmin 1 expression and clinicopathological features, as well as the prognosis of above patients. Stathmin 1 could be detected in the cytoplasm of tumor cells in OSCC tissues though its expression level was variable. There was no correlation between stathmin 1 expression and patient gender, or age in OSCC. However, stathmin 1 expression of tumor cell was significantly correlated with T classification (P = 0.0017), N classification (P = 0.0171), stage (P < 0.0001), therapeutic efficacy (P < 0.0001), and patient outcome (P = 0.0387). In addition, high expression of stathmin 1 in tumor cells was associated with shorter overall survival (OS, P = 0.0017). Multivariate analysis also revealed that high expression of stathmin 1 was a predictor of reduced survival (P = 0.0241). These findings suggest that patients with OSCC tumors showing high expression of stathmin 1 might have poor therapeutic effects and worse clinical outcomes in OSCC treated with Doc-containing regimen.

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.