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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.47 No.2 pp.27-34
DOI : https://doi.org/10.17779/KAOMP.2023.47.2.001

The Effect of Methanol Extract of Taxillus Yadoriki (Siebold) Dancer on MC3 and YD15 Mucoepidermoid Carcinoma Cells

Hye-Yeon Han1), MinA Jang2), Mi Heon Ryu3)*
1)Department of Dental Hygeine, Jinju Health College
2)Department of Oral Pathology, School of Dentistry, Dental and Life Science Institute, Pusan National University
3)Department of Oral Pathology, School of Dentistry, Education and Research Team for Life Science on 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 Email: apollon@pusan.ac.kr
March 16, 2023 March 20, 2023 April 14, 2023

Abstract


Taxillus yadoriki (Siebold) Dancer is a parasitic plant that grows on camellia trees and is common on Jeju Island. The branches of T. yadoriki have long been used to treat various diseases, including hypertension, diabetes mellitus, viral infections, and arthritis. Although recent studies reported that T. yadoriki has anticancer effects in various human cancer cell lines, including lung cancer, the exact molecular mechanisms supporting its anticancer effects are not well understood. This study aims to assess the anticancer effect of the methanol extract of T. yadoriki branches (METY) on mucoepidermoid carcinoma (MEC) cell lines (MC3 cells and YD15 cells) and explore its mechanism of action. Inhibitory activity of MEC cell proliferation was assessed using the CCK-8 assay. The mechanism of the anticancer effect on METY-treated MC3 cells and YD15 cells was evaluated with Hoechst 33342 stain and Western blot. After treating MC3 cells and YD15 cells with METY for 48 hours, the cytotoxicity of MC3 and YD15 cells increased, and nuclear fragmentation increased in both METY-treated MEC cells. Caspase-3 and cleaved PARP activation demonstrated apoptosis of METY-treated MEC cells. Cell proliferation inhibition with METY was alleviated in METY-treated MEC cells pretreated with zVAD-FMK, supporting the cell proliferation inhibition effect by apoptosis. METY-induced apoptosis in MEC cells occurs through MAP kinase pathways such as p38 and pAkt. MEC cell. METY-induced apoptosis of MEC cells occurs via the p38 and pAkt MAPK pathways. Therefore, METY may be a promising anticancer candidate for the MEC therapeutic strategy.


Taxillus yadoriki , Mucoepidermoid carcinoma , Apoptosis , Akt , MAPK

점액표피양암종세포에서 겨우살이 가지 추출물을 이용한 MC3 세포주와 YD15 세포주의 세포사 기전 분석

한혜연1), 장민아2), 유미현3)*
1)진주보건대학교 치위생과
2)부산대학교 치의학전문대학원 구강병리학교실, 치의생명과학연구소
3)부산대학교 치의학전문대학원 구강병리학교실, 부산대학교 치의생명과학 교육연구팀, 치의생명과학연구소

초록

    Ⅰ. INTRODUCTION

    Mucoepidermoid carcinoma (MEC) is a malignancy of the salivary gland in humans and the most common tumor of malignant salivary gland neoplasm, accounting for approximately one-third of all salivary gland malignancies1, 2). Histologically, it is composed of mucin-producing cells, intermediate cells, and epidermoid cells and is classified as low grade, intermediate grade, and high grade based on the composition ratio of these cells3). The main treatment for the tumor is surgical excision, and radiation therapy or chemotherapy can be administered depending on tumor stage4). Chemotherapeutic drugs for MEC include cisplatin plus vinorelbine, paclitaxel, trastuzumab, sorafenib, and so forth, but these show limited effectiveness5); thus, there is an urgent need to develop a new chemotherapeutic agent that can be effectively used for salivary gland tumors.

    Taxillus yadoriki is a parasitic plant that grows on trees such as camellia and oak in Korea (Jeju Island) and Japan. The branch of T. yadoriki has long been used to treat various diseases such as hypertension, diabetes mellitus, viral infection, and arthritis. Recently, extracts of T. yadoriki have been reported to have anti-inflammatory effects6), but they also have anticancer effects in cells lines such as the pancreatic cancer cell line AsPC-1, colorectal cancer cell line, breast cancer cell line MDA-MB-231, prostate cancer cell line PC-37), and lung cancer cell line A5498). However, prior studies were not conducted on various types of tumor cell lines and were carried out only by certain research groups. Furthermore, it is not known what effects the extract of T. yadoriki has on head and neck tumors, especially MEC, and only a limited number of studies have been reported. In addition, the reported mechanism of action is limited to cyclin D1, and further research is therefore needed.

    In this article, we use experiments such as the Cell Counting Kit 8 (CCK-8) assay, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) stain, and Western blot to study the effects and mode of action of T. yadoriki extract on MEC cell lines.

    Ⅱ. MATERIALS AND METHODS

    1. Reagents and antibodies

    We obtained CCK-8 from Dojindo Laboratories (Kumamoto, Japan). DAPI was purchased from Sigma-Aldrich Chemical Co (St, Louis, MO, USA).

    Antibodies against cleaved caspase 3, cleaved PARP, Bcl-2, Bax, ERK, p-ERK, Akt, p-Akt, p38, and p-p38 were acquired from Cell Signaling Technology, Inc. (Danvers, MA, USA). The beta-actin antibody was provided by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

    zVAD-FMK was obtained from R&D Systems (Minneapolis, MN, USA), and LY294002 (an AKT inhibitor) was obtained from Abcam (Cambridge, UK). SB203580 (a p38 inhibitor) was obtained from Invitrogen (San Diego, CA, USA).

    2. Cell cultures

    In this experiment, wethe authors used two human MEC cell lines, the MC3 cell line and the YD15 cell line. The MC3 cells were a kind gift from Professor Wu Junzheng at the Fourth Military Medical University (Xi’an, China), and the YD-15 cell line was purchased from the Korean Cell Line Bank (Seoul, Korea). Both of these cell lines were cultured in RPMI1640 medium with the addition of 10% fetal bovine serum and maintained in a 5% CO2 incubator at 37°C.

    3. Treatment of methanol extract of T. yadoriki (Siebold) Dancer (METY)

    The plant extract used in this experiment was purchased from The Korea Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea; distribution No. KPM040-077). Branches from T. yadoriki (Siebold) Dancer, which is parasitic to Korean oak, were extracted with methanol (METY) and dissolved in DMSO for use in the experiment. After treating the MC3 and YD15 cells with METY at concentrations of 0, 40, 80, and 120 μg/mL for 48 hours, the cells were submitted for the next experiment.

    4. Cell viability assessment

    We seeded 5 × 103 MC3 and YD15 cells into a 96-well plate. The cells were stabilized overnight and then treated with METY at a concentration of 0, 40, 80, and 120 μg/mL when they reached approximately 60% confluency. After 48 hours of METY treatment, we used a CCK-8 kit to measure the optical density value of the cells for both MC3 and YD15 cells at each concentration.

    5. DAPI stain

    To evaluate the induction of apoptosis by METY treatment, MC3 and YD15 cells were seeded at 50%–60% confluency in a 24-well plate and stabilized overnight. The cells were treated with METY at concentrations of 0, 40, 80, and 120 μg/mL for 48 hours. After treatment, the cells were stained with DAPI and observed under a fluorescence microscope to count the number of cells showing nuclear fragmentation. We performed a statistical analysis of the results9).

    6. Western blot

    For the experiment, cells were seeded in 60-mm dishes and stabilized overnight to achieve a cell confluency of 50% –60% for the experiment. After treating MC3 cells and YD15 cells with METY for 48 hours at the indicated concentrations, the cells were collected and proteins extracted. We measured the concentration of the isolated proteins and separated them by molecular weight on a sodium dodecyl– sulfate polyacrylamide gel electrophoresis gel. The gel was subsequently transferred onto nitrocellulose membranes, blocked with bovine serum albumin, and subjected to antigen– antibody reactions using primary antibodies such as cleaved caspase 3, cleaved PARP, Bax, Bcl-2, ERK, phosphorylated- ERK, p38, phosphorylated p38, Akt, and phosphorylated Akt. Then, we incubated the membranes with secondary antibody. Finally, we used the illumination kit to detect the band.

    7. Statistical analysis

    Data derived from both the control group and the experimental group were evaluated for statistical significance using the chi-squared test in the SPSS 27.0 program (SPSS, Chicago, IL, USA). A p value of less than 0.05 was considered statistically significant.

    Ⅲ. RESULTS

    1. Effect of METY on MEC cell lines

    WeThe authors first treated 0, 40, 80, and 120 μg/mL of METY on MC3 cells and YD15 cells for 24 hours and 48 hours and then measured cell viability using the CCK-8 assay kit.

    As compared with the control group, cell viability decreased statistically significantly over time when METY was treated for 24 hours and 48 hours in both MC3 cells and YD15 cells. Furthermore, the number of cells decreased to a statistically significant degree as the concentration of METY increased (Figure 1).

    2. Assessment of apoptotic cells in METY-treated MEC cells

    To understand the mode of action in METY-treated MEC cells, we applied DAPI staining on the cells treated with METY concentrations of 0, 40, 80, and 120 μg/mL. As a result, we observed an increase in nuclear fragmentation under a fluorescence microscope, in proportion to the METY concentration (p < 0.05).

    3. Evaluation of apoptosis-related proteins and Bax/Bcl-2 ratio

    To analyze the effect of METY on the expression of apoptosis- related proteins of MEC cells, we used Western blotting to evaluate the level of the expression of cleaved caspase 3, cleaved PARP protein, and the change in the Bax/Bcl-2 ratio in MC3 and YD15 cells treated with different concentrations of METY (0, 40, 80, and 120 μg/mL). The results showed changes in the expression levels of cleaved caspase 3, cleaved PARP, and the Bax/Bcl-2 ratio in both cell lines. Figure 3A shows the results for cleaved caspase 3, Figure 3B shows the results for cleaved PARP, and Figure 3C shows the results for the Bax/Bcl-2 ratio. As a result of the experiment, the expression of cleaved caspase 3 protein and cleaved PARP protein increased in both MC3 and YD15 cells treated with METY, and the Bax/Bcl-2 ratio was also statistically significantly increased in MC3 cells.

    4. Study of the cellular mechanism of cell death in METY-treated MEC cells

    To investigate the mechanism of cell death in the METY-treated cells, we investigated the expression of phosphorylated- ERK, ERK, phosphorylated p38, p38, and phosphorylated Akt and Akt proteins using Western blot. We found that as the concentration of METY increases, the degree of expression of phosphorylation of ERK, p38, and Akt proteins decreases.

    5. Verification of the cell death mechanism of METY-treated MEC cells by Z-VAD, LY, and SB treatment

    To verify cell death in the cells treated with METY, we evaluated cell viability by 1 hour pretreatment of zVAD-FMK, LY294002 (10 μg/mL), and SB 239063 (10 μg/mL) pretreating METY-treated MC3 and YD15 cells. The results showed a significant difference in cell viability between the two groups (p < 0.05).

    Ⅳ. DISCUSSION

    The results of the present study indicate that METY inhibits the proliferation of MEC cells by inducing apoptosis. We further confirmed the alteration of cell viability by observing changes in cell morphology and number under a phase contrast microscope. The apoptotic effects of METY were supported by DAPI staining and Western blotting. The cell death induced by METY treatment likely occurs through the inactivation of the Akt/mTOR-mediated pathway and the p38 and ERK signaling pathways.

    In recent years, phytochemicals of plant origin have been widely used in the treatment and prevention of malignant tumors in humans. Most of these phytochemicals are believed to exhibit anticarcinogenic activity by inhibiting the tumor development or progression. Among the natural product–based substances used as chemotherapeutic agents are tea polyphenol, EGCG, curcumin, and resveratrol10). However, drugs used in chemotherapy for salivary gland tumors, such as cetuximab, imatinib, dasatinib, lapatinib, and paclitaxel, are not significantly different from those used for other tumors11, 12). Furthermore, there are relatively few studies on natural product–based substances that can be used in the chemotherapy for salivary gland tumors, such as research on Convallaria keiskei extract13), curcumin14, 15), and combination therapy with filipin or verapamil16). Therefore, the goal of this study was to discover substances that could be used as potential chemotherapeutic agents in MEC, the most common malignant tumor in salivary gland tumors. The METY treatment used in this study effectively induced cell death in MEC cells in a concentration-dependent and time-dependent manner, even at relatively low concentrations. Therefore, the results of this study demonstrate the potential of METY as a candidate chemotherapeutic agent for MEC cells.

    The mitogen-activated protein kinases (MAPK) pathway plays a role in various aspects of cell activity, such as cell proliferation, differentiation, cell death, and morphological changes17). It also plays an important role in the development of oral cancer by encouraging tumor cell proliferation and inhibiting tumor cell apoptosis18). The ERK pathway responds to genotoxic, osmotic, hypoxic, and oxidative stress19). In this study, wethe authors found that the proteins in the MAPK pathway were inhibited during METY-induced apoptosis (Figure 4). In addition, cell viability was significantly recovered when the p38 pathway inhibitor SB203580 was treated with METY (Figure 5). These results indicate that METY-induced apoptosis occurs in MC3 and YD15 cells through the regulation of the ERK and p38 pathways. Moreover, the Akt-mTOR pathway is closely related to the MAPK pathway and plays an important role in determining the response to various anticancer agents20). Yue et al. reported that a ferulic acid derivative inhibited cell growth and migration in A549 lung cancer cells by inhibiting the ERK/p38 and AKT/mTOR pathways21). Similarly, it is thought that METY inhibits apoptosis in MEC cell lines by passing through the ERK/p38 and AKT/mTOR pathways.

    Furthermore, it appears that METY is involved in apoptosis through alteration of the expression of proapoptotic and antiapoptotic proteins, and changes in the ratio of various Bcl2 family members, such as Bcl-2 and bax, play an important role in determining whether cells will undergo apoptosis in response to external stimuli22). In MEC cell lines, METY treatment was found to induce apoptosis by altering the bcl-2/bax ratio, which is thought to occur through the ERK pathway, p38 pathway, and Akt-mTOR pathway. To verify the involvement of caspases in METY-induced cell death, we performed a CCK-8 assay after zVAD-fmk treatment 1 hour before METY treatment on MEC cells. However, the results showed that METY-induced cell death was not prevented by zVAD-fmk treatment. Although Western blotting showed an increased expression in cleaved caspase 3 and cleaved PARP protein, the results using zVAD-fmk were not significantly significant, suggesting the possibility of caspase-independent cell death in METY-induced cell death. In cancer cells, genes related to apoptosis can mutate or there may be no caspase activity, and resistance to traditional chemotherapy drugs can occur23, 24). Therefore, caspase-independent cell death may be an alternative cancer therapeutic strategy. Further research that provides a more detailed investigation is needed.

    In this study, wethe authors demonstrated that METY can suppress the proliferation of MEC MC3 and YD15 cells by inducing apoptosis through the downregulation of the ERK/p38 and AKT/mTOR signaling pathways. These results may offer a scientific basis for the development of new anticancer agents using METY. Therefore, METY is considered to be a promising candidate for anticancer therapy.

    ACKNOWLEDGMENTS

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

    Figure

    KAOMP-47-2-27_F1.gif

    Effects of METY on cell viability in MC3 and YD15 cells. (A) The effects of METY on cell viability were analyzed by CCK-8 assay. The graph represents the mean ± standard deviation of triplicate experiments. (B) Representative photographs of MC3 and YD15 cells treated with various concentrations of METY for 24 hours and 48 hours (concentration: 0 μg/mL, 40 μg/mL, 80 μg/mL, and 120 μg/mL), respectively. Scale bars, 100 μm.

    KAOMP-47-2-27_F2.gif

    Assessment of apoptosis induction in MC3 and YD15 cells after 48-hour treatment with vehicle, 40 μg/mL, 80 μg/mL, and 120 μg/mL of METY. (A) Representative photographs of DAPI-stained cells. (B) Quantification of nuclear fragmentation detected by a fluorescence microscope with DAPI staining of the MC3 cells after 48 hours of METY treatment. (C) Quantification of nuclear fragmentation detected by fluorescence microscope with DAPI staining in the YD15 cells after 48-hour METY treatment. (*:p < 0.05, **:p<0.01, ***:p<0.001)

    KAOMP-47-2-27_F3.gif

    Western blot analysis of the level of apoptosis-related proteins in MC3 and YD15 cells after treatment with 0, 40, 80, and 120 μg/mL of METY. (A) Cleaved caspase 3. (B) Cleaved PARP. (C) Bax and Bcl-2 protein (Bax/Bcl2 ratio) *p < 0.05.

    KAOMP-47-2-27_F4.gif

    Altered expression of the MAPK pathway and Akt in MC3 and YD15 cells treated with vehicle and with 40, 80, or 120 μg/mL METY solutions after 48 hours have elapsed. (A) Expression of ERK and p38 and phosphorylated proteins. (B) Expression of Akt and phosphorylated Akt proteins. *p < 0.05.

    KAOMP-47-2-27_F5.gif

    Assessment of cell viability using LY294002 (10 μg/mL) and SB 239063 (10 μg/mL) pretreatment in METY-treated MC3 and YD15 cells. (A) MC3 cells. (B) YD15 cells. *p < 0.05.

    Table

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