Kazuhito Izukuri

Reactive oxygen species (ROS) reduce the expression of BRAK/CXCL14 in human head and neck squamous cell carcinoma cells.

Abstract The present study investigated the effects of oxidative stress induced by reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and hydroxyl radical (HO•), on the expression of both BRAK , which is also known as non-ELR motif angiostatic CXC chemokine ligand 14 (CXCL14), in head and neck squamous cell carcinoma (HNSCC) cells. When HNSCC cells were cultured in the presence of ROS, the expression of BRAK was significantly decreased whereas that of IL-8 was increased. Interestingly, the effects on the expression of both genes in HNSCC cells were much greater with HO▪ than with H2O2. The effects of ROS on both BRAK and IL-8 expression were attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK) inhibitors. These results indicate that oxidative stress induced by H2O2 or HO• stimulates angiogenesis and tumuor progression by altering the gene expression of BRAK and IL-8 via the EGFR/MEK/ERK pathway in human HNSCC cells.

Suppressed rate of carcinogenesis and decreases in tumour volume and lung metastasis in CXCL14/BRAK transgenic mice

Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention.

Expression of tumour‐suppressing chemokine BRAK/CXCL14 reduces cell migration rate of HSC‐3 tongue carcinoma cells and stimulates attachment to collagen and formation of elongated focal adhesions in vitro

BRAK/CXCL14 (breast‐ and kidney‐expressed chemokine/CXC chemokine ligand 14) is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues, including HNSCC (head and neck squamous cell carcinoma). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC (HSC‐3 BRAK) cells decreased the rate of tumour formation and size of tumour xenografts compared with mock‐vector‐introduced (HSC‐3 Mock) cells in athymic nude mice, even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that high‐level expression of the gene is important for the suppression of tumour establishment in vivo. For the first step to study the mechanisms of BRAK‐dependent tumour suppression, we compared characteristics between HSC‐3 BRAK and HSC‐3 Mock cells under in vitro culture conditions. The cell migration rate was lower in HSC‐3 BRAK cells than in HSC‐3 Mock cells. Also, HSC‐3 BRAK cells showed more rapid adhesion than HSC‐3 Mock cells when cultured on type I collagen‐coated dishes but not on fibronectin or laminin 1‐coated ones. This adhesion was mediated by α2β1 integrin. Immunofluorescent analysis of the cells cultured on type I collagen showed that HSC‐3 BRAK cells formed much more elongated focal adhesions co‐localized with paxillin and actin stress fibres than did HSC‐3 Mock cells. Treatment of parental HSC‐3 cells with recombinant BRAK stimulated the activation of Rap1, which is a ras family small GTPase, and formation of elongated focal adhesions, indicating that the difference in cell character observed between HSC‐3 Mock and HSC‐3 BRAK was not due to selection of clones of different character but due to expression of BRAK in the cells. The characteristic morphology of focal adhesions in HSC‐3 BRAK cells was perturbed by the introduction of an expression vector of the Rap‐binding domain of the Ral guanine nucleotide dissociation stimulator, a target of Rap1, into HSC‐3 BRAK cells, suggesting that Rap1 regulated the formation of the morphology of the focal adhesions. These data indicate that the expression of BRAK stimulated the formation of elongated focal adhesions of the HSC‐3 cells in an autocrine or paracrine fashion, in which stimulation may be responsible for the reduced migration of the cells.

Determination of Serum BRAK/CXCL14 Levels in Healthy Volunteers

Background: BRAK/CXCL14 is a chemokine expressed in many normal cells and tissues. Several papers have reported tissue distribution, but no study has examined the serum protein level of this chemokine. Methods: We evaluated serum levels of BRAK/ CXCL14 by enzyme-linked immunosorbent assay, after determining the conditions necessary to increase the sensitivity and reproducibility of the method. Results: The average level of males was significantly higher than that of females. Two percent of the volunteers showed a value 2 S.D. higher than the average value without any obvious abnormality, but no person was lower than 1 S.D. of the average value. The serum BRAK/CXCL14 levels of individual subjects were constant. Conclusion: These data indicate that the serum BRAK/CXCL14 level is highly conserved and suggest that the levels are determined by the genetic background and/or lifestyle of the individual.

Abstract 424: Chemokine CXCL14 is a multistep tumor suppressor

PURPOSE: The multistep nature of tumor formation has been well established and each step depends on the mutation or abnormal regulation of various genes. In order to elucidate the in vivo function of CXCL14, we used CXCL14 transgenic (Tg) mice and investigated the effects of this chemokine at multiple stages during cancer development, in addition to the effects on the overall survival rate. Furthermore, we also sought to determine the role of CXCL14 in the action of natural killer (NK) and natural killer T (NKT) cells. EXPERIMENTAL PROCEDURES: Carcinogenic rate was determined in mice undergoing AOM/DSS-induced colorectal carcinogenesis. The growth rate of implanted tumor cells was determined by injecting Lewis lung carcinoma (LLC) cells or B16 melanoma cells subcutaneously into the dorsolateral region of Tg or isogenic wild type C57BL/6 (Wt) mice. Tumor cells were also injected intravenously via a tail vein into Wt, T-cell and B-cell deficient SCID mice or T-cell, B-cell and NK cell-deficient NOG mice to investigate colonization to the lungs. Tg or Wt mice were also pre-injected with NK cell function inhibitory anti-asialo GM1 antibodies to see the role of NK cells in CXCL14-dependent tumor suppression in vivo. Melanoma cells that had been engineered to express the CXCL14 gene under the control of doxycycline (B16-LMT3-Tet/OnBRAK) were also used. RESULTS: The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that in Wt mice. When tumor cells were injected into these mice, the size of the tumors that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumor cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity plays an important role during CXCL14-mediated suppression of tumor growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed on the B16-LMT3-Tet/OnBRAK melanoma cells. The injection of alpha-galactosylceramide, an activator of NKT cells appeared to decrease the degree of melanoma cell metastasis in both the Wt and Tg mice, but the effect was much stronger in the Tg mice than in the Wt mice, indicating that the presence of both CXCL14 and alpha-galactosylceramide resulted in a synergistic effect and even greater tumor suppression. Finally, the survival rates after tumor cell injection were significantly increased for the Tg mice. CONCLUSION: High expression of the CXCL14 gene either in host cells, or tumor cells resulted in suppression of growth and metastasis of tumor cells. As these Tg mice showed no obvious signs of abnormality, we propose CXCL14 to be a promising molecular target for cancer suppression/prevention. Citation Format: Ryu-Ichiro Hata, Kazuhito Izukuri, Yasumasa Kato, Soichiro Sasaki, Chihiro Miyamoto, Tetsu Akasaka, Xiaoyan Yang, Yojiro Maehata, Yoji Nagashima, Kazuyoshi Takeda, Tohru Kiyono, Naofumi Mukaida, Masaru Taniguchi. Chemokine CXCL14 is a multistep tumor suppressor. [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 424. doi:10.1158/1538-7445.AM2015-424

Abstract 1541: NK cells are indispensable for suppression of tumor growth and metastasis in transgenic mice overexpressing chemokine CXCL14/BRAK

[PURPOSE] We reported previously that the forced expression of the chemokine CXCL14/BRAK (BRAK) in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor cell transplants compared with those of mock vector-treated cells in athymic nude mice or in severe combined immunodeficiency (SCID) mice, even though the growth rates of these cells were the same under in vitro culture conditions. The aim of this study was to determine whether BRAK transgenic mice (Tg) would show resistance to tumor cell transplant and tumor metastasis or not, and if so, to find the mechanism of tumor suppression. [EXPERIMENTAL PROCEDURES] Lewis lung carcinoma (LLC) cells or B16 melanoma cells were injected subcutaneously into dorso-lateral region of Tg or wild type (Wt) C57BL/6J mice. Tumor cells were also injected intravenously via a tail vein into Wt, SCID mice or NK cell-deficient NOG mice to investigate colonization to the lungs. Tg or Wt mice were also pre-injected with NK cell function inhibitory anti-NK 1.1 antibody or anti-asialo GM1 antibody to see the role of NK cells in BRAK-dependent tumor suppression in vivo. Melanoma cells that had been engineered to express the BRAK gene under the control of doxycycline (B16-luc-2-LMT3-Tet/OnBRAK) were also used. [RESULTS] Sizes of LLC or B16 melanoma cell tumors in the Tg mice were significantly smaller than those in the control Wt mice, indicating that BRAK, first found as a suppressor of tumor growth of head and neck squamous cell carcinomas, also suppressed the growth of carcinomas of other tissue origins. This suppression was attenuated by the injection of anti-asialo-GM1 antibody. Colonization of LLC cells or B16 melanoma cells to the lungs was also suppressed in the Tg mice, and this suppression was attenuated by the treatment with anti-asialo-GM1 antibody or anti-NK 1.1 antibody. When Wt mice were fed doxycycline-containing sucrose solution after the injection of B16-luc-2-LMT3-Tet/OnBRAK cells, the number of metastatic nodules in the lungs was significantly lower than that for the mice fed control sucrose solution. In the case of SCID mice, the number of metastatic nodules was 4 times higher that for the Wt, but still the number was significantly lower than that for the BRAK-expressing melanoma cells. On the other hand, when the B16-luc-2-LMT3-Tet/OnBRAK cells were injected into NK-cell-deficient NOG mice, the number of metastatic nodules was ten times higher than that in the Wt mice; and also no difference was observed between BRAK-expressing and non-expressing melanoma cells, indicating NK cell activity to be indispensable for suppression of tumor-cell metastasis to the lung. [CONCLUSION] High expression of the BRAK gene either in host cells, or tumor cells resulted in suppression of growth and metastasis of tumor cells, and NK cells were indispensable for either suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1541. doi:1538-7445.AM2012-1541

Inhibitory effects by a submandibular gland extract on luteinizing hormone‐stimulated testosterone production by testicular cells

In order to evaluate the role of the submandibular gland (SMG) on testosterone (T) production by the testis, primary cultured testicular cells were prepared from rats that had the submandibular gland surgically ablated (G‐) and control (sham operated) rats (S.O.) respectively. The cells were incubated with or without 100 ng/ml luteinizing hormone (LH) and/or SMG extract. The same linear increase in T secretion was shown by both S.O.G‐ cells on multi‐stimulation with LH for up to 96 hrs. However, while an equivalent response was shown for S.O. cells after a single LH stimulation at 96 hrs, T secretion by the G‐ cells reached a plateau after 24 hrs. The level at 96 hrs was thus approximate 30% and 33% of those of S.O. cells with and without multi‐stimulus by LH for 96 hrs, respectively. When S.O. cells were cultured with SMG extract, LH‐stimulated T secretion was dose‐dependently inhibited and there was no effect on basal T secretion. The inhibitory effect was abolished by treatment at 95 °C for 5 min. Ultra‐filtration indicated that the molecular size of the inhibitory agent was greater than 30,000. It is proposed that SMG may contain a high molecular weight, heat labile soluble factor(s) which affects T secretion by inhibiting LH action in testicular cells.

Inhibition of estradiol‐17β secretion in ovarian granulosa cells by an extract from the submandibular gland of the rat

The submandibular salivary gland (SMG) in the rodent is a rich source of growth factors. The estradiol‐17β (E2) secretions were studied in the primary cultures of ovarian granulosa cells from sialoadenectomized (surgical removal of SMG, SDX) immature rats. The secretions of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) were also studied in the primary cultures of anterior pituitary cells from SDX rats. E2 secretion from the ovarian granulosa cells of SDX rats was not affected by male and female SMG extracts. FSH‐stimulated E2 secretion, however, was inhibited by the SMG extracts. Substances in the SMG extract, which inhibited FSH‐stimulated E2 secretion in the cells, were heat and urea stable, and were not stripped by charcoal. SMG extracts did not affect the secretions of LH and FSH in the anterior pituitary cells, with or without luteinizing hormone‐releasing hormone. The present findings suggest that male and female rat SMG contain the E2 modulating factor which affect ovarian granulosa cells.