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Dive into the research topics where Makoto Kinouchi is active.

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Featured researches published by Makoto Kinouchi.


Clinical & Experimental Metastasis | 2013

Expression and function of CXCR4 in human salivary gland cancers

Daisuke Uchida; Nobuyuki Kuribayashi; Makoto Kinouchi; Go Ohe; Tetsuya Tamatani; Hirokazu Nagai; Youji Miyamoto

Salivary gland cancers (SGCs) frequently metastasize to cervical lymph nodes and distant organs. Currently, the mechanisms responsible for the metastatic behavior of SGC cells are not fully understood. We previously demonstrated that the stromal cell-derived factor-1 (SDF-1; also known as CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral squamous cell carcinoma. In the present study, we investigated the role of CXCR4 in the metastatic behavior of SGCs. We examined the expression of CXCR4 mRNA and protein in human SGC cell lines by quantitative RT-PCR and western blotting, respectively. The expression of CXCR4 mRNA and protein were frequently upregulated in 5 out of 6 SGC cell lines. Functional CXCR4 expression was demonstrated by the ability of these SGC cell lines to migrate toward an SDF-1 gradient. SDF-1 rapidly activated extracellular signal-regulated kinase (ERK)1/2 in SGC cell lines. Immunohistochemical analysis revealed that CXCR4 protein expression was detected in either the nucleus or cytoplasm of cancer cells in 16 out of 20 tissues of adenoid cystic carcinoma (ACC) and in 4 out of 6 tissues of mucoepidermoid carcinoma, which are representative of SGC. Furthermore, ACC cell lines exhibited dramatic metastasis to the lung following intravenous inoculation, whereas AMD3100, a CXCR4 antagonist, significantly inhibited lung metastasis of the cells, ameliorated body weight loss and improved the survival rate of tumor-bearing nude mice. These results indicate that CXCR4 expression contributes to the metastatic potential of SGCs.


International Journal of Oncology | 2013

Bortezomib-enhanced radiosensitization through the suppression of radiation-induced nuclear factor‑κB activity in human oral cancer cells

Tetsuya Tamatani; Natsumi Takamaru; Kanae Hara; Makoto Kinouchi; Nobuyuki Kuribayashi; Go Ohe; Daisuke Uchida; Kenji Fujisawa; Hirokazu Nagai; Youji Miyamoto

Oral cancer cells have a significantly augmented nuclear factor-κB (NF-κB) activity and the inhibition of this activity suppresses tumor growth. Bortezomib is a proteasome inhibitor and a drug used for molecular-targeted therapy (targets NF-κB). In this study, we investigated whether bortezomib would be effective as an inhibitor of proliferation and a radiosensitizer for the treatment of oral cancer. We demonstrate that bortezomib inhibits NF-κB activity and cell proliferation. The combined treatment with bortezomib and radiation (RT) suppressed NF-κB activity and cell growth in vitro and in vivo compared with RT treatment alone. To investigate the mechanisms by which bortezomib suppresses tumor growth, the expression of signaling molecules downstream of NF-κB were examined by ELISA. The combined treatment significantly inhibited the radiation-induced production of angiogenic factors and decreased the number of blood vessels in the tumor tissues. Although the expression of anti-apoptotic proteins was upregulated by RT, bortezomib downregulated the RT-induced expression of these proteins. Moreover, the expression of cleaved poly(ADP-ribose) polymerase in vitro and in vivo was enhanced by bortezomib, indicating that bortezomib inhibits tumor growth by inducing apoptosis. This study clearly demonstrates that bortezomib significantly inhibits tumor growth and that the combined treatment with bortezomib and RT results in a significant inhibition of tumor growth. The mechanisms underlying the inhibition of tumor growth by bortezomib include the suppression of angiogenesis and the induction of apoptosis. A novel molecular targeting therapy including bortezomib may be effective in the treatment of oral cancer by suppressing NF-κB activity.


International Journal of Oncology | 2012

Antitumor efficacy of sequential treatment with docetaxel and 5-fluorouracil against human oral cancer cells.

Tetsuya Tamatani; Tarannum Ferdous; Natsumi Takamaru; Kanae Hara; Makoto Kinouchi; Nobuyuki Kuribayashi; Go Ohe; Daisuke Uchida; Hirokazu Nagai; Kenji Fujisawa; Youji Miyamoto

Docetaxel (DOC) and 5-fluorouracil (5-FU) are important anticancer agents widely used in the treatment of a variety of cancers including oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the antitumor efficacy of the sequential administration of DOC and 5-FU against OSCC cells (B88 and CAL27 cells) in vitro and in vivo. In in vitro growth inhibition assays, sequential treatment with DOC followed by 5-FU was more effective in inhibiting cancer cell growth than 5-FU followed by DOC, single treatment with DOC or 5-FU, or combined treatment with DOC and 5-FU. Furthermore, DOC followed by 5-FU significantly inhibited tumor growth in vivo compared to 5-FU followed by DOC. To understand the mechanisms underlying the enhanced growth inhibitory effect of the administration sequence, DOC followed by 5-FU, we examined the expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), which were known to regulate the antitumor effect of 5-FU, by real-time RT-PCR and western blot analysis. Downregulation of TS and DPD expression and upregulation of OPRT expression were induced by DOC treatment, suggesting that DOC enhanced the efficacy of 5-FU by altering the expression of its metabolic enzymes. These results indicate that sequential treatment with DOC followed by 5-FU could be a promising therapeutic strategy for oral cancer.


PLOS ONE | 2013

The role of metabotropic glutamate receptor 5 on the stromal cell-derived factor-1/CXCR4 system in oral cancer.

Nobuyuki Kuribayashi; Daisuke Uchida; Makoto Kinouchi; Natsumi Takamaru; Tetsuya Tamatani; Hirokazu Nagai; Youji Miyamoto

We have demonstrated that blocking CXCR4 may be a potent anti-metastatic therapy for CXCR4-related oral cancer. However, as CXCR4 antagonists are currently in clinical use to induce the mobilization of hematopoietic stem cells, continuous administration as an inhibitor for the metastasis may lead to persistent leukocytosis. In this study, we investigated the novel therapeutic downstream target(s) of the SDF-1/CXCR4 system, using B88-SDF-1 cells, which have an autocrine SDF-1/CXCR4 system and exhibit distant metastatic potential in vivo. Microarray analysis revealed that 418 genes were upregulated in B88-SDF-1 cells. We identified a gene that is highly upregulated in B88-SDF-1 cells, metabotropic glutamate receptor 5 (mGluR5), which was downregulated following treatment with 1,1’ -[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist. The upregulation of mGluR5 mRNA in the SDF-1/CXCR4 system was predominately regulated by the Ras-extracellular signal-regulated kinase (ERK)1/2 pathway. Additionally, the growth of B88-SDF-1 cells was not affected by the mGluR5 agonist (S)-3,5-DHPG (DHPG) or the mGluR5 antagonists 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP). However, we observed that DHPG promoted B88-SDF-1 cell migration, whereas both MPEP and MTEP inhibited B88-SDF-1 cell migration. To assess drug toxicity, the antagonists were intraperitoneally injected into immunocompetent mice for 4 weeks. Mice injected with MPEP (5 mg/kg) and MTEP (5 mg/kg) did not exhibit any side effects, such as hematotoxicity, allergic reactions or weight loss. The administration of antagonists significantly inhibited the metastasis of B88-SDF-1 cells to the lungs of nude mice. These results suggest that blocking mGluR5 with antagonists such as MPEP and MTEP could prevent metastasis in CXCR4-related oral cancer without causing side effects.


PLOS ONE | 2014

Involvement of miR-518c-5p to Growth and Metastasis in Oral Cancer

Makoto Kinouchi; Daisuke Uchida; Nobuyuki Kuribayashi; Tetsuya Tamatani; Hirokazu Nagai; Youji Miyamoto

We have previously demonstrated that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral cancer. Recently, small non coding RNAs, microRNAs (miRNAs) have been shown to be involved in the metastatic process of several types of cancers. However, the miRNAs that contribute to metastases induced by the SDF-1/CXCR4 system in oral cancer are largely unknown. In this study, we examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system using B88-SDF-1 oral cancer cells, which exhibit functional CXCR4 and distant metastatic potential in vivo. Through miRNA microarray analysis, we identified the upregulation of miR-518c-5p in B88-SDF-1 cells, and confirmed the induction by real-time PCR analysis. Although an LNA-based miR-518c-5p inhibitor did not affect cell growth of B88-SDF-1 cells, it did significantly inhibit the migration of the cells. Next, we transfected a miR-518c expression vector into parental B88 cells and CAL27 oral cancer cells and isolated stable transfectants, B88-518c and CAL27-518c cells, respectively. The anchorage-dependent and -independent growth of miR-518c transfectants was significantly enhanced compared with the growth of mock cells. Moreover, we detected the enhanced migration of these cells. The LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of miR-518c transfectants, indicating that these phenomena were mainly dependent on the expression of miR-518c-5p. Next, we examined the function of miR-518c-5p in vivo. miR-518c transfectants or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with the miR-518c transfectants. These results indicated that miR-518c-5p regulates the growth and metastasis of oral cancer as a downstream target of the SDF-1/CXCR4 system.


Cancer Research | 2014

Abstract 1446: Isolation of a novel metastasis-related microRNA, miR-518c-5p, induced by the stromal cell-derived factor (SDF)-1/CXCR4 system in oral cancer

Makoto Kinouchi; Daisuke Uchida; Nobuyuki Kuribayashi; Tetsuya Tamatani; Hirokazu Nagai; Youji Miyamoto

We have demonstrated that stromal-cell derived factor (SDF)-1/CXCR4 system is involved in the metastatic process of oral cancer. Recently, small non coding RNAs, microRNAs (miRNAs) are involved in the metastatic process of several types of cancers. However, the miRNAs contributed to metastases induced by the SDF-1/CXCR4 system in oral cancer are largely unknown. Thus, we examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system, using oral cancer cells, B88-SDF-1 which exhibits functional CXCR4 and distant metastatic potential, in vivo. We examined metastasis-related miRNAs induced by the SDF-1/CXCR4 system using miRNA microarray analysis. Among these miRNAs, we confirmed the upregulation of miR-518c-5p in B88-SDF-1 cells by the real-time PCR analysis. Although LNA-based miR518c-5p inhibitor did not affect the cell growth, but did significantly inhibit the migration of B88-SDF-1 cells. Next, we transfected miR-518c expression vector into parental B88 cells, and isolated stable transfectant, B88-518c. The anchorage dependent and independent growth of B88-518c cells were significantly enhanced in compared with those of mock cells. Moreover, we could detect the enhanced migration of B88-518c cells. LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of B88-518c cells, indicating that these phenomenon were dependent on the expression of miR-518c-5p, not on that of miR-518c-3p. Next, we examined the function of miR-518c-5p in vivo. B88-518c cells or mock cells were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with B88-518c cells. These results indicate that SDF-1/CXCR4 system regulates the metastases of oral cancer via induction of miR-518c-5p. Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Tetsuya Tamatani, Hirokazu Nagai, Youji Miyamoto. Isolation of a novel metastasis-related microRNA, miR-518c-5p, induced by the stromal cell-derived factor (SDF)-1/CXCR4 system in oral cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1446. doi:10.1158/1538-7445.AM2014-1446


International Journal of Cancer | 2018

Determination of the origin of oral squamous cell carcinoma by microarray analysis: Squamous epithelium or minor salivary gland?: Oral SCC originating from minor salivary glands

Makoto Kinouchi; Sayaka Izumi; Koh-ichi Nakashiro; Yasuo Haruyama; Gen Kobashi; Daisuke Uchida; Tomonori Hasegawa; Hitoshi Kawamata

More than 90% of oral cancers are histopathologically squamous cell carcinomas (SCCs). According to clinical behavior and histopathological features, we hypothesize that oral SCC can originate from either oral squamous epithelium or minor salivary glands. Here, we examined whether some oral SCCs originate from minor salivary glands, and investigated whether these tumors show particularly aggressive biological behavior. The mRNA expression profiles of samples obtained from six patients with oral floor SCC (five men, one woman; mean age, 62.7 years) were analyzed using a microarray containing 32,878 probes. The six samples were divided into two groups by clustering of expression levels of 845 probes differentially expressed in normal oral squamous epithelium and normal salivary glands. The expression profile in four cases was similar to that of normal oral squamous epithelium, and in two cases was similar to that of normal salivary glands. Furthermore, we identified nine genes that reveal the origin of the oral SCC. Subsequently, we examined the expression levels of these nine marker genes by reverse transcriptase‐polymerase chain reaction to determine the origin of 66 oral SCCs. Twelve of the 66 oral SCCs were considered to originate from minor salivary glands, and these tumors showed high metastatic potential (p = 0.044, Chi‐square test). Furthermore, SCC derived from minor salivary glands showed a poor event‐free survival rate (p = 0.017, Kaplan–Meier analysis). In conclusion, determination of the origin of oral SCC is helpful in planning treatment for patients with oral SCC.


Cancer Research | 2016

Abstract 1925: Regulation of metabotropic glutamate receptor 5 expression by the miR-30 downregulation induced by the SDF-1/CXCR4 system in oral cancer cells

Nobuyuki Kuribayashi; Daisuke Uchida; Makoto Kinouchi; Chikako Koshiji; Sayaka Izumi; Kembun Hakata; Yuske Komiyama; Shuji Tsuchida; Tomonori Hasegawa; Hitoshi Kawamata

We have previously demonstrated that stromal cell-derived factor (SDF)-1/CXCR4 system enhances the metastases of oral cancer cells via induction of metabotropic glutamate receptor (mGluR) 5. However, the mechanism(s) of mGluR5 expression induced by the SDF-1/CXCR4 system are largely unknown. Recently, small non-coding RNAs, microRNAs (miRNAs) are involved in the metastatic process of several types of cancers. In this study, we examined the miRNA association involved in the mGluR5 expression using oral cancer cells, B88, which express functional CXCR4 and exhibit highly metastatic potentials. We examined the metastasis-related miRNAs in SDF-1 stimulated B88 cells by use of a miRNA microarray analysis. Consequently, we isolated miR-30 family which has predictive binding sites in 3’-UTR of mGluR5 mRNA in silico analysis. Among these miRNAs, we confirmed the downregulation of all miR-30 family in SDF-1 stimulated B88 cells by the real-time PCR analysis. Next, we transfected these miR-30 families in SDF-1 stimulated B88 cells. We confirmed the downregulation of mGluR5 by the miR-30a-5p and miR-30c-5p overexpression. These results indicated that SDF-1/CXCR4 system might regulate the metastases of oral cancer by the upregulation of mGluR5 via downregulation of miR-30 family. Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Chikako Koshiji, Sayaka Izumi, Kembun Hakata, Yuske Komiyama, Shuji Tsuchida, Tomonori Hasegawa, Hitoshi Kawamata. Regulation of metabotropic glutamate receptor 5 expression by the miR-30 downregulation induced by the SDF-1/CXCR4 system in oral cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1925.


Cancer Research | 2016

Abstract 4117: AMD070, a novel orally bioavailable CXCR4 inhibitor, inhibits the metastases of oral cancer via SDF-1/CXCR4 system

Makoto Kinouchi; Daisuke Uchida; Nobuyuki Kuribayashi; Takahiro Wakui; Kyoko Kuribayashi; Maki Okubo; Masahiro Saito; Eri Masuyama; Hitoshi Kawamata

We have demonstrated that the stromal cell-derived factor (SDF)-1/CXCR4 system is involved in metastatic processes in oral cancer. Moreover, we have also reported that blocking CXCR4 with AMD3100, a CXCR4 antagonist, may be a potent anti-metastatic therapy for CXCR4-related head and neck cancer. Recently, AMD070 has been described as a novel orally bioavailable inhibitor of CXCR4, being minimally invasive than AMD3100. In this study, we examined the effect of AMD070 on the SDF-1/CXCR4 dependent-metastases in oral cancer cells, B88-SDF-1, which express high levels of SDF-1 and CXCR4. We examined the effect of CXCR4-specific antagonists, AMD3100 and AMD070 on the cell growth, cell migration and cell invasion of B88-SDF-1 cells. Both antagonists did not affect the anchorage-dependent growth of B88-SDF-1 cells, but did inhibit the SDF-1/CXCR4 dependent-migration of the cells. Moreover, AMD070 significantly suppressed anchorage-independent growth and invasion of B88-SDF-1 cells. Next we examined the effect of AMD070 to distant metastasis in vivo. Daily administration of AMD070 per os significantly inhibited the metastasis of B88-SDF-1 cells to the lungs of nude mice. These results indicated that AMD070 might be a novel orally bioavailable inhibitor on the metastases of oral cancer by the SDF-1/CXCR4 system. Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Takahiro Wakui, Kyoko Kuribayashi, Maki Okubo, Masahiro Saito, Eri Masuyama, Hitoshi Kawamata. AMD070, a novel orally bioavailable CXCR4 inhibitor, inhibits the metastases of oral cancer via SDF-1/CXCR4 system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4117.


Cancer Research | 2015

Abstract 3059: miR-518c-5p promotes the metastasis of oral cancer cells in a CXCR4-dependent and -independent manner

Makoto Kinouchi; Daisuke Uchida; Nobuyuki Kuribayashi; Tetsuya Tamatani; Hirokazu Nagai; Hitoshi Kawamata; Youji Miyamoto

We have demonstrated that CXCR4 system is involved in the metastatic process of oral cancer. Recently, we identified the miR-518c-5p as a downstream target of the CXCR4 system, which was involved in the metastasis of B88, a CXCR4-expressing oral cancer cell line (2014 AACR annual meeting). In this study, we examined the function of miR-518c-5p in an oral cancer cell line, CAL27, which did not express CXCR4 and did not exhibit metastatic potential in vivo. We transfected a miR-518c expression vector into parental CAL27 cells and isolated a stable transfectant, CAL27-518c. We could detect the upregulation of miR-518c-5p in the miR-518c transfectants compared with mock cells, which was completely inhibited by treatment with the miR-518c-5p LNA inhibitor. The growth and migration of CAL27-518c cells were significantly enhanced compared with those of mock cells. The miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of CAL27-518c cells, indicating that these phenomena were mainly dependent on the expression of miR-518c-5p. Next, we examined the function of miR-518c-5p in vivo. miR-518c or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with miR-518c transfectants. These results indicate miR-518c-5p promotes the metastasis of oral cancer in a CXCR4-dependent and -independent manner. Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Tetsuya Tamatani, Hirokazu Nagai, Hitoshi Kawamata, Youji Miyamoto. miR-518c-5p promotes the metastasis of oral cancer cells in a CXCR4-dependent and -independent manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3059. doi:10.1158/1538-7445.AM2015-3059

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Daisuke Uchida

Dokkyo Medical University

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Nobuyuki Kuribayashi

Translational Research Institute

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Go Ohe

University of Tokushima

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Kanae Hara

University of Tokushima

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