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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.45 No.1 pp.11-20
DOI : https://doi.org/10.17779/KAOMP.2021.45.1.002

The Effects of Luzindole on Cells Migration and Invasion and Epithelial-mesenchymal Transition in YD15 and HSC5 Head and Neck Cancer Cells

MinA Jang1), Uk-Kyu Kim2), Seung-hwa Jeong3), Hye-Yeon Han4), Mi Heon Ryu5)*
1)Department of Oral Pathology, School of Dentistry, Dental and Life Science Institute, Pusan National University
2)Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University
3)Department of Preventive & Community Dentistry, School of Dentistry, Pusan National University
4)Department of Dental Hygeine, Jinju Health Collge
5)Department of Oral Pathology, Education and Research Team for Life Science on Dentistry, School of Dentistry, Dental and Life Science Institute, Pusan National University
* Correspondence: Mi Heon Ryu, Department of Oral Pathology, School of Dentistry, Pusan National University, 49, Busandaehak-ro, Mulgeum-eup, Yangsan, 50612, Republic of Korea Tel: +82-51-510-8251, Fax: +82-51-510-8249 E-mail: apollon@pusan.ac.kr
January 9, 2021 January 22, 2021 February 5, 2021

Abstract


Human melatonin receptors consist of melatonin receptor 1A (MT1) and melatonin receptor 1B (MT2), and possess various biological activations, which include the control of circadian rhythm and immune regulation. Recently, it have been found that melatonin receptors inhibit cell proliferation and have oncostatic properties, which is being researched in the treatment strategies of breast cancer, prostate cancer, and Non-Small Cell Lung Cancer. Also, interest in the effect of melatonin receptor’s correlation to head and neck carcinogenesis and application possibilities on head and neck cancer has been found. However, in head and neck cancer, how melatonin receptor relates and functions with epithelial-mesenchymal transition (EMT), which plays a major role in human carcinogenesis, is yet unknown. In this research, in HSC5 cell and YD15 cell, the head and neck cancer cell lines, a selective melatonin receptor antagonist, Luzindole, was utilized to examine the effect of melatonin receptors on EMT. After treating Luzindole on HSC5 cells and YD15 cells, the authors evaluated cell viability rate with CCK 8 assay, and performing colony forming assay, invasion assay and western blot analysis, to confirm melatonin receptor’s effect on EMT. When Luzindole was treated on HSC5 cells and YD15 cells in low concentration of 100nM, no significant difference in cell viability was found, whereas Luzindole-treated cells had a significantly increase in the invasion assay. As a result of colony forming assay, in YD15 cells, the number of colony formation decreased slightly, whereas in HSC3 cells, the number of colony formation increased. According to the western blotting, no difference in E-cadherin, Slug, and vimentin protein expression was shown. This result of research indicates the possibility of melatonin receptor being related to EMT and new chemotherapeutic target in the carcinogenesis of head and neck cancer.


Melatonin receptor , Luzindole , Epithelial-mesenchymal transition , Head and neck cancer

두경부 암종세포에서 Luzindole을 이용한 YD15 세포와 HSC5 세포의 상피간엽이행 발현 변화 분석

장 민아1), 김 욱규2), 정 승화3), 한 혜연4), 유 미현5)*
1)부산대학교 치의학전문대학원 구강병리학 교실, 치의생명과학연구소
2)부산대학교 치의학전문대학원 구강악안면외과학 교실
3)부산대학교 치의학전문대학원 예방과 사회치의학 교실
4)진주보건대학교 치위생과
5)부산대학교 치의학전문대학원 구강병리학 교실, 부산대학교 치의생명과학 교육연구팀, 치의생명과학연구소

초록

    Pusan National University(PNU)

    Ⅰ. INTRODUCTION

    Head and neck cancer (HNC), carcinoma occurring in a wide range of human body parts such as skin, oral cavity, oropharynx, and salivary gland, shows a variety of clinical manifestations and prognosis1). About 650,000 people are newly diagnosed with HNC every year around the world, leading to high mortality rate2). In U.S., Since 66% of patients are diagnosed with an advanced stage of HNC (stage 3 or more), the cure rate is lower and the prognosis tends to be worse3). Squamous cell carcinoma accounts for more than 90% of HNC and if it is early detected and treated, excellent prognosis would be expected4).

    The treatment of HNC is varied depending on the legions and often includes surgical resection, radiotherapy and chemotherapy5). Among them, the surgical resection which is most primarily conducted as the treatment of it may result in difficulty in speaking and breathing, dysphasia and damage to the head and neck region of patients, and moreover, bring about functional, esthetic and psychological problems in patients with it3).

    Epithelial-mesenchymal transition (EMT), the process in which epithelial cells are transformed into motile cells, mesenchymal cells, recently provides a new model for studies on more differentiated and malignant carcinoma. EMT is involved with many proteins such as receptor tyrosine kinase (RTK) and TGF-β, and the E-cadherin line of genes and proteins in the downstream of signal transduction are inactivated, leading to the elimination of the epithelial phenotype6).

    The mechanism involved with EMT is found to be very similar with the embryonic development, in the process in which carcinoma is initiated and progressed. In the process of EMT, cells are remodeled and epithelial cells are transformed into mesenchymal cells with abilities of migration and invasion7). Recent research on EMT emerges as a new approach for discovering important targets in studies on the initiation and progression of cancers. Understanding of the core mechanism related to EMT in the initiation and progression of HNC and developing of chemotherapeutic agent targeting it may provide the foundation of efficient chemotherapy, except for the traditional radiotherapy or surgical techniques, as the treatments for HNC.

    Luzindole (N‐Acetyl‐2‐benzyltryptamine) is the medicine acting as a selective melatonin receptor antagonist8)(Fig. 1). Melatonin (N-acetyl-5-methoxytryptamine) as a neurohormone primarily released from the pineal glands in mammals controls the cardiac rhythm and has several functions of innate and acquired immunity, such as immunoregulation, antioxidant activity and neuroendocrine, in playing a differentiated role and exerting its effect by combining with melatonin receptor9-13). As human melatonin receptors, melatonin receptor 1A (MT1) and melatonin receptor 1B (MT2) exist. Each of MT1 and MT2 is often discovered in all kinds of organs including skin and in retina, respectively. These receptors are differently controlled, depending on their in vivo concentrations. Luzindole has 11-25 times higher affinity to MT2 than that to MT114,15), and is observed to not only has an anti-depressant effect but also disrupt the circadian rhythm14,16).

    Since the inhibitory effect of melatonin receptors on cancer cells and their oncostatic properties are recently are proved, so they are studied to treat breast, prostate cancer and non-small cell lung cancer. In addition, the association between melatonin receptors and HNC has increasingly attracted more attention, as the effect of melatonin on head and neck regions and the applicability of it to HNC have been verified. However, substantial parts of melatonin receptors' expressions and underlying mechanisms in HNC are not yet clarified.

    By examining the head and neck carcinogenesis of the YD 15 cell line and HSC5 cell line, the cell strains of HNC, this study attempts to provide an understanding of the initiation and progression mechanism of HNC and basic data for cancer treatment, by observing the effects of Luzindole, a melatonin receptor antagonist, on EMT, and investigating related mechanisms in HNC cell lines.

    Ⅱ. SUBJECTS AND METHODS

    1. Reagents and antibodies

    Luzindole was supplied from Tocris Bioscience (Ellisville, MO, USA). Cell Counting Kit 8 (CCK-8) was purchased from Dojindo Laboratories (Dojindo Laboratories, Kumamoto, Japan). Cell culture reagents such as Iscove's Modified Dulbecco's Medium (IMDM) and Penicillin/streptomycin (p/s) were purchased from GibcoBRL (Grand Island, NY, USA). Roswell Park Memorial Institute 1640 (RPMI1640) medium was obtained from Hyclone (UT, USA). Matrigel-coated transwell cell culture chambers (8μM pore size) was obtained from Corning Incorporated (Corning, NY, USA).

    Antibodies against E-cadherin, Slug, vimentin and MNTR1A were supplied by Abcam Trading (Abcam, Cambridge, MA, UK). Antibody against beta-actin antibody was obtained from Santa Cruz Biotechnology, Inc (Dallas, TX, USA).

    2. Cell culture

    The authors used two cell lines in this experiment, HSC5 cells and YD15 cells. As human skin squamous cell carcinoma cell line, HSC5 cells were obtained from Japanese Collection of Research Bioresources Cell Bank (JCRB cell Bank, Osaka, Japan). HSC5 cells were cultured in culture medium with F12, 10% FBS, 1% P/S, NaHCO3, Insulin, T3 (triiodothyronine), apo-tranferrin, choleratoxin and hydrocortisone.

    As the human mucoepidermoid carcinoma cell line, YD15 cells were supplied from Korean Cell Line Bank (KCLB, Seoul, Korea; cat.nos. 60504) and were culture in RPMI1640 medium containing 10% FBS and 1% P/S. Both cell lines were maintained to be monolayers in each culture dish, with medium replaced once per two days. The cells were cultured in an incubator with 5% CO2 at 37℃ and humidified environment.

    3. Luzindole treatment

    After seeding 2,500 HSC5 and YD15 cells in each dish with the diameter of 35mm, they were cultured overnight, to stabilize them. Then, they were cultured in fresh medium replacing the medium, which contained 100nM of Luzindole (vehicle: 0.2% dimethyl sulfoxide (DMSO)) for 24 hours in HSC5 cells and 10nM of Luzindole for 24 hours in YD15 cells. As the control group, Both HSC5 and YD15 cells were also cultured in the medium with 0.2 % (the final concentration) DMSO for the same period (24 hours). After the Luzindole treatment, the cells were used in CCK 8 assay, colony forming assay, invasion assay and western blot assay.

    4. CCK-8 assay

    The CCK-8 assay was conducted to examine whether Luzindole treatment influences the cell viability in HSC5 and YD15 cells. The cells were Luzindole -treated for 24 hours, after they were cultured in 48 well plates. After the Luzindole treatment, CCK-8 solution was cultured in an incubator for one hour, by putting it into each well of 48 well plates, according to the manufacturer's instructions. Then, the OD value was measured at the absorbance of 450nm by using a microplate reader.

    5. Colony forming assay

    At first, 500 uL of cell culture fluid with 0.9% soft agar, 10% FBS and 1% PS was adequately mixed and put into a cell culture dish, and then, was hardened at 4 ℃ for 30 minutes and put into a cell incubator at 37 ℃, to create the base layer. Next, the cells which were Luzindole-treated for 24 hours and the same amount of DMSO-treated cells were trypsinized. From each group, 50, 100 and 200 cells were counted and the resuspension of them to the medium with 0.4% soft agar was conducted. After the solution was adequately mixed and sprayed on the base layer, it was hardened at 4 ℃ for 10 minutes. Finally, the formation of colony was observed for 14 days, after 250 μL of cell culture fluid was put into each agar plate. After 14 days of the culture, all colonies dyed with 2% crystal violet in 2% ethanol were counted.

    6. Invasion assay

    For the invasion assay, matrigel-coated transwell cell culture chambers with the pore size of 8μM were used. The membranes of transwells were coated with 40㎕ of the mixture created by mixing serum free media with matrigel in the ratio of 3:1, and air-dried for 3 hours. After putting serum-free medium into the upper chamber of matrigel-coated transwell cell culture chambers and 600㎕of media with 10% FBS into the lower chamber of them, the researcher cultured 3 ×104 cells by putting them into the upper chamber. Matrigel-coated transwell cell culture chambers containing cells were cultured at 37℃ under 5% CO2 atmosphere for 24 and 48 hours. Then, the cells that could not penetrate through the membrane of the upper chamber were eliminated with cotton swaps. The other cells on the lower surface, which penetrated through matrigel and membranes, were fixed with 4% formaldehyde and dyed with 2% crystal violet in 2% ethanol. From each membrane, three zones were randomly selected, and the number of cells was counted by photographing them at 40 ☓ magnification, with a light microscope.

    7. Western blot analysis

    After both vehicle- and Luzindole-treated cells were harvested, proteins were extracted by using RIPA buffer and lysis buffer. The proteins were quantified and loaded to gel according to the conventional method, and electrophoresis was conducted. Then, several steps were taken: transfer, membrane blocking and primary antibody incubation. After washed with Tris-buffered saline with 0.1% Tween® 20 Detergent (TBST), the secondary antibody was diluted and treated at room temperature for 2 hours, and then, bands were detected.

    8. Statistical Analysis

    The Student's t-test was conducted to examine differences in the number of HSC5 and YD15 cells, colonies formed and invaded cells between the vehicle-treated group and the Luzindole-treated group. In addition, the numbers were converted the ratios of experimental values of the control group to those of the Luzindole-treated group. Statistical analyses of them were conducted by using SPSS 25.0 (SPSS Inc., NY, USA). In all statistical analyses, the statistical significance was regarded to be at p<0.05.

    Ⅲ. RESULTS

    1. CCK-8 assay in HSC5 and YD15 cells

    After both HSC5 and YD15 cells were Luzindole-treated for 24 hours, the cell viability was measured by using the CCK-8 assay kit. From the cell viability test, it was found that there were no differences in the cell viability between them, regardless of Luzindole treatment (Fig. 2).

    2. Luzindole treatment inhibits invasive capacity in HSC5 and YD15 cell lines

    The matrigel invasion assay was conducted to evaluate the invasive capacity influencing the metastasis of cancer. The number of invasive HSC5 cells for 24 and 48 hours in the group which was Luzindole-treated, slightly increased, compared to that in the vehicle-treated HSC5 cells (Fig. 3a). In addition, the number of YD15 cells which penetrated through a matrigel-coated filter for 48 hours was higher in the Luzindole-treated control group than in the vehicle-treated group (Fig. 3b). Luzindole treatment was thus found to enhance the invasive potential in HSC5 and YD15 cells.

    3. Luzindole treatment is involved in colony forming potential in HSC5 and YD15 cell lines

    To acquire more specific understanding of the oncogenic potential of melatonin receptors in HSC5 and YD15 cells, the colony forming assay was conducted, after Luzindole was treated for 24 hours. The results of the experiment showed that the number of colony formation in Luzindole-treated HSC5 cells slightly increased. However, Luzindole treatment resulted in the reduction of colony formation in YD15 cells. Although the number of cells seeded for this experiment amounted to 50, 100 and 200, the distinctive reduction in colony formation was observed (Fig. 4).

    4. Luzindole treatment did not involved in the expression of E-cadherin, Slug and vimentin proteins in HSC5 and YD15 cell lines

    The western blot analysis was conducted to observe changes in the expression of proteins related to Epithelial-Mesenchymal Transition (EMT) in Luzindole-treated HSC5 and YD15 cells. The results of western blotting demonstrated that the expression of E-cardherin protein, the epithelial marker, slug and vimentin proteins, mesenchymal markers, did not show any difference (Fig. 5).

    Ⅳ. DISCUSSION

    The results of our experiment showed that when low concentration Luzindole is administered to HSC5 and YD15 cells, it could not inhibit the cell proliferation and had no cytotoxic activity. When Luzindole treatment was conducted respectively for 24 hours, to examine the invasive activity of HNC's cell lines, the invasiveness of both HSC5 and YD15 cells was increased. The results from a colony forming assay to examine the oncogenic potential showed that the number of colonies was increased in HSC5 cells, while it was slightly decreased in YD15 cells. There were no changes in the expression of E-cadherin, slug and vimentin, when a western blot analysis was conducted to investigate the changes in the EMT signal.

    Although many parts of melatonin's role have not yet been elucidated, some studies using cancer cell lines or in vivo research have reported that it is involved in the proliferation of cancer cells. According to Kanishi et al., the proliferation of cells are inhibited, if the endometrial cancer cell line is treated with melatonin17). In the oral cavity, the concentration of it in saliva released from the salivary gland reaches its peak overnight, and also has anti-inflammatory and anti-oxiditive activity. Such actions lead to the inhibition of oral cancer and other diseases which may be caused by free radical components18). Drazen et al. demonstrated that melatonin promotes the proliferation of splenocyte but such an effect is reduced by the Luzindole treatment19).

    Although there have not been many studies on what roles melatonin receptors play in human cancers, it is known to have an effect on the initiation, promotion and progression of a tumor20). Dillon et al. demonstrated that MT1 is increasingly expressed in malignant human breast tissues21)and prostate cancer, by analyzing the MT1 immunoreactivity of both normal and malignant human breast tissues. Melatonin engages in the altered expression of both E-cadherin and vimentin protein22). The melatonin's control is thought to have an effect on the carcinogenesis of breast cancer. In addition, a study on the correlation between the distribution of MT1 and MT2 and the prognosis of patients demonstrated that both MT1 and MT2 increase in lung cancer, and that favorable prognosis appears when the immunoexpression of MT2 is increased23).

    Osanai et al. reported that the proliferation and invasiveness of the endometrial cancer cell line would be inhibited if Ramelton, a MT1/MT2 receptor agonist, is administered to the cell line24). In addition, both MMP-2 and MMP-9 are involved in the process. Such a mechanism is associated with the inhibition of calmodulin or the changed expression of ERK pathways. Moreover, it is also known to engage in the inhibition of cyclic adenosin monophosphate (cAMP), linoleic acid metabolism pathways and cell proliferation. MT2 receptors are reported to have an inhibitory effect on the proliferation of ER positive endometrial cancer cells by combining with estrogen receptors and to be associated with telomerase activity25,26). Our experiment also examined whether the concentration at which Luzindole has no effect on cell proliferation is associated with EMT, by administering Luzindole whose concentration is as low as 10nM or 100nM to the cell lines of HNC. By using Luzindole at such low concentration, the experiment acquired significant outcomes from the invasiveness and colony forming assay of the cell lines of HNC. The melatonin receptor treatment is determined to have a potential adjuvant therapeutic effect on HNC.

    Melatonin receptors are G protein-coupled receptors, and signaling pathways are varied depending on whether they are MT1 or MT2 monomeric receptors or MT1/MT2 heterodimer receptors. When melatonin receptors are MT1 or MT2 monomeric receptors, they inhibit cAMP, protein kinase A signaling, resulting in the inhibition of CREB phosphorylation27). When melatonin receptors are MT1/MT2 heterodimer receptors, they form homodimer or heterodimer. Heterodimer acts via the heterodimerspecific phospholipase C or the protein kinase C pathway28). Such a mechanism influences the downregulation of genes associated with cell proliferation and the phosphorylation level of transcription factors, resulting in the anti-proliferation effect23).

    Recently, the chemotherapy for breast cancer has been conducted by using melatonin receptors. Lissoni et al. reported that the outcomes from patients with metastatic breast cancer, who were treated with tamoxifen combined with melatonin therapy, were better than those from the same patients who were treated with only tamoxifen29). The result is thought to be resulted from the fact that melatonin receptors inhibit the insulin growth factor-1 or the prolactin level by responding to the melatonin treatment, suggesting the potential of melatonin as an adjuvant therapy. In addition, the lower the melatonin level, the higher the cancer risk, indicating the potential of it as a cancer preventive agent20). The effects of melatonin also have positive relations with cytotoxic effect of the existing chemotherapeutic agents: the chemoprotective effect and the myelostimulatory effect30). This may be due to the detoxification ability within melatonin cells, suggesting the potential of it as a clinical adjuvant therapy.

    Minimizing the side-effects of the existing chemotherapeutic agents and enhancing therapeutic effects are very important in increasing patients' quality of life and prognosis, when strategies for treating cancer are selected. The findings show the potential of melatonin receptors as a chemotherapeutic agent even for HNC cell lines, given their roles such as oncostatic effect and anti-proliferative activity on cancer cells. Another tumor-specific mechanism that has not yet been discovered is implicated, in that it showed slightly different responses to different cell lines used in this study. A further basic study for more deeply understanding specific mechanisms of melatonin receptors, which can differently appear in various types of head and neck cancer. Moreover, a follow-up study on the establishment of animal experimental models using melatonin receptors and the possibility of the experiment is thought to be required.

    Ⅴ. CONCLUSION

    The study examined the effect of Luzindole on the invasiveness and colony forming ability of head and neck cancer cell lines. Even relatively low concentration Luzindole had enhanced on the invasiveness and colony forming ability of HNC cells. The treatment against melatonin receptors is, therefore, determined to has an adjuvant therapeutic potential for HNC, and the result of this research is thought to be used as basic data for developing new therapeutic strategies for patients with head and neck cancer. A further basic study is thought to be necessary for more deeply understanding melanin receptors' specific functions and mechanism for treating head and neck cancer.

    ACKNOWLEDGMENTS

    This work was supported by a 2-Year Research Grant of Pusan National University

    Figure

    KAOMP-45-1-11_F1.gif

    The chemical structure of Luzindole.

    KAOMP-45-1-11_F2.gif

    Luzindole treatment has no effect on HSC5 cells (A) and YD15 cells (B). DMSO, dimethyl sulfoxide.

    KAOMP-45-1-11_F3.gif

    Invasion assay after Luzindole treatment on HSC5 and YD15 cells. After the Luzindole treatment, the number of invasive HSC5 and YD15 cells decreased, compared with Control (Student's t-test).

    KAOMP-45-1-11_F4.gif

    Colony forming assay after Luzindole treatment on HSC5 and YD15 cells. The number of colonies in Luzindole-treated HSC5 cells were slightly increased, whereas the number of colonies in YD15 cells has gradually decreased, compared with Control.

    KAOMP-45-1-11_F5.gif

    Western blot analysis after Luzindole treatment on HSC5 and YD15 cells. In the expressions of E-cadherin, Slug and vimentin of Luzindol-treated cells, no difference was observed.

    Table

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