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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.42 No.6 pp.167-174
DOI : https://doi.org/10.17779/KAOMP.2018.42.6.002

Apoptosis and Autophagy Induced by Methanol Extract of Kochia scoparia in Human Mucoepidermoid Carcinoma Cell Line

Mihyang Do1), Mi Heon Ryu2), Uk-Kyu Kim3)*
1)Research Institute for Oral Biotechnology, School of Dentistry, Pusan National University, Yangsan, Republic of Korea
2)Department of Oral Pathology, BK21 Plus project, School of Dentistry, Pusan National University, Yangsan, Republic of Korea
3)Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University, Yangsan, Republic of Korea
Correspondence: Uk-Kyu Kim, Department of Oral Maxillofacial Surgery, School of Dentistry, Pusan National University, 49 Busandaehak-ro, Yangsan, Gyeongnam, 50612, Republic of Korea Tel: +82-55-360-5112, Fax: 82-55-360-5004 E-mail: kuksjs@pusan.ac.kr
November 29, 2018 December 7, 2018 December 14, 2018

Abstract


Natural products are vastly utilized as a source of chemotherapeutic agents for human cancers. Kochia scopraia is traditionally used for the cure of urological and dermatological diseases. Recently, methanol extract of Kochia scoparia (MEKS) has been shown to have anti-cancer activity to various human cancers. However, there is no report demonstrating the anti-cancer activity of MEKS in human mucoepidermoid carcinoma (MEC) cells. In this study, the authors studied the effects of MEKS on the cell proliferation and underlying mechanism in YD15 human MEC cells. MEKS decreased YD15 cell proliferation proven by trypan blue exclusion assay and induced apoptosis, evidenced by cell cycle analysis and western blotting. Autophagy induction by MEKS was verified by western blotting. In addition, MEKS regulated the expression of phosphorylated Akt, phosphorylated p38 and Nrf2 protein. This results can imply that MEKS might be a potential candidate for the treatment of human MEC cells.


Mucoepidermoid carcinoma , Kochia scoparia , Apoptosis , Autophagy , Chemotherapeutic agent

점액표피양암종 세포주에서 Kochia scoparia 추출물의 세포자멸과 자가포식 유도 효과

도 미향1), 유 미현2), 김 욱규3)*
1)부산대학교 구강생물공학연구소
2)부산대학교 치의학전문대학원 구강병리학 교실, BK21 플러스 사업단
3)부산대학교 치의학전문대학원 구강악안면외과학 교실

초록

    Pusan National University

    Ⅰ. INTRODUCTION

    Mucoepidermoid carcinoma (MEC) is one of the most common malignant tumors occurring in the salivary glands1). This tumor accounts for 15% of tumors occurring in the head and neck region and approximately 30% of all malignant salivary gland tumors2,3). In the past, it was considered a benign tumor and was called a mucoepidermoid tumor. However, it has been classified as a malignant tumor (mucoepidermoid carcinoma) from the revised WHO classification in 19904). MEC has a lot of phenotypes and a wide variety of biologic behaviors, which is associated with a histologic grade5). High-grade MEC is highly aggressive and shows a very low 5-year survival rate6). In addition, MEC has a low susceptibility to chemotherapy and radiotherapy. Therefore, the treatments of MEC mainly depend on surgical procedures. It is urgent to develop appropriate chemotherapeutic drugs because the surgical procedures cause aesthetic and functional defects to patients with MEC7).

    Target therapy has been applied to treat cancers recently and it gains therapeutic effects by specifically targeting a substance that plays an important role in the development and progression of cancer8-10). The Mitogen-activated protein kinase (MAPK) pathway plays an important role in the survival and cancer growth of various cancer cell subtypes and has drawn attention as one of the important chemotherapeutic targets11). The p38 MAPK pathway acts as a tumor suppressor and it is also involved in regulating the cell cycle progression negatively and inducing apoptosis12,13). The Akt pathway is a type of the serine/threonine protein kinases. It is known to be involved in cell proliferation, differentiation, and growth and is also associated with apoptosis14-16). Moreover, the Akt pathway may regulate a signaling molecule that can suppress the abnormal proliferation of cancer cells.

    Kochia scoparia is an annual plant which is native to Korea, Japan and China and has been used for treating urological and dermatological diseases from old times17). The active ingredients of this plant include momordin Ic and triterpenoid saponin18,19). The representative medical actions of this plant are antiallergic actions, anti-inflammatory actions, and the reduction of pruritus18). Recent studies have reported that this plant has anti-cancer effects to cancer cells in the human body. Particularly, it has been reported that it is effective in treating breast cancer, hepatocellular carcinoma, and oral cancer. However, studies on the anti-cancer activities of Kochia scoparia are still at an early stage. The objective of this study was to provide baseline data for developing chemotherapeutic agents that can be used to treat MEC by evaluating the anti-cancer effects of methanol extract of Kochia scoparia (MEKS) on the MEC cell line.

    Ⅱ. MATERIALS and METHODS

    1. Chemicals

    The MEKS used in this experiment was purchased from Korea Plant Extract Bank (Cheongju, Chungbuk) and dissolved in dimethyl sulfoxide (DMSO) to make a 20 mg/ml stock solution for further use.

    2. Cell culture and MEKS treatment

    YD15 cells (human MEC cell line) were purchased from Korean Cell Line Bank (Seoul, Korea). The cells were cultured using the RPMI media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) and incubated at 37°C in a 5% CO2 incubator.

    For the MEKS treatment, the same number of cells was seeded in each cell culture dish, stabilized over night, and treated with MEKS after reaching 50-60% confluency. The control group was treated with DMSO and the MEKS-treataed experimental group was treated with appropriately diluted MEKS.

    3. Reagents and antibodies

    Trypan blue was purchased from Gibco (Paisley, UK). Propidium iodide (PI), and the antibody for LC3II were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for phosphorylated p38, p38, phosphorylated Akt, Akt, cleaved PARP, and cleaved caspase 3 were obtained from Cell Signaling Technology (Beverly, MA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for the Nrf2 was obtained from Abcam Biologicals (Cambridge, UK).

    4. trypan blue exclusion assay

    After YD15 cells were seeded in 24 well-culture plate for cell cycle analysis, the cells were stabilized and the confluency of the cells was confirmed. The control group was treated with DMSO and the MEKS-treataed experimental group was treated with MEKS at 40, 60, 80, 100, or 120ug/ml for 24 hours or 48 hours. Afterward, the cells were washed with Phosphate Buffered Saline (PBS) and detached from the cell culture plate. The detached cells were dyed with trypan blue and the number of living cells was measured using a haemocytometer.

    5. cell cycle analysis

    YD15 cells were treated with MEKS at 0, 40, 60, and 80ug/ml for 24 hours, and cells were detached from the culture plate using trypsin. The isolated cells were centrifuged and fixed in ethanol solution at -20°C for one day. Afterward, it was dyed with propidium iodide (PI) solution (0.02mg/ml) and RNAase A (0.05mg/ml), and the cell cycle was evaluated using the FACScan flow cytometry analysis.

    6. Western blotting analysis

    The quantity of protein was measured after dissolving the YD15 cells treated with DMSO or MEKS using lysis buffer. Twenty grams of proteins were electrrophoresed by size by using SDS-PAGE and transferred to nitrocellulose membrane. After treating the membrane with blocking solution at room temperature for 1 hour, it was reacted with primary antibody overnight at 4 °C. Next, it was treated with secondary antibody attached with horseradish peroxidase (HRP) at room temperature for 2 hours. The samples were washed with PBS twice for each step. The protein bound to the antibody was detected and the bands were observed.

    Ⅲ. RESULTS

    1. Cell proliferation inhibition in MEKS-treated YD15 cells

    The YD15 cells, which belong to the MEC cell line, was treated with MEKS (40, 60, 80, 100, or 120 ug/ml) for 24 or 48 hours and trypan blue exclusion assay was conducted to confirm the cell proliferation inhibition effects of MEKS treatment. The results of this experiment confirmed that the number of living cells in the MEKS-treated experimental group was significantly lower than that in the control group (Fig. 1B, C). The number of live cells reduced in a MEKS-concentration dependent and a time-dependent manner. Moreover, when YD15 cells were observed with phase contrast microscope, those in the control group proliferated densely and normally, but those treated with MEKS had fewer number of cells in a condensed and irregular shape (Fig. 1A).

    2. Alteration of cell cycle by in MEKS-treated YD15 cells

    When the cell cycle of MEKS-treated YD15 cells was analyzed using flow cytometry, the number of cells in the sub-G1 phase (indicating apoptotic cells) and G0/G1 phase increased (Fig. 2). It was also confirmed that it significantly increased in the MEKS dose-dependent manner.

    3. Expression of apoptotic protein and autophagy related proteins

    The expression of cleaved PARP protein, indicating that apoptosis is induced, and that of LC3II protein, a hallmark of autophagy induction, were confirmed using western blotting. GAPDH was used as an internal control. The results of the experiment showed that the expression of the cleaved PARP protein increased when it was treated with MEKS at 40, 60, or 80ug/ml for 24 hours in a MEKS dose dependent manner. Moreover, the expression of the LC3II protein increased in a MEKS dose-dependent manner, too (Fig. 3).

    4. Involvement of p38 and Akt pathway and Nrf2 protein in MEKS-induced cell death

    The changes in the expression of the p38 and Akt pathways were examined using western blotting in order to confirm the underlying mechanism of cell proliferation inhibition by MEKS treatment. The results showed that the expression of phosphorylated Akt increased at 60 and 80ug/ml MEKS. Although the level of phosphorylated p38 protein increased concentration at 40ug/ml of MEKS, the that of phosphorylated p38 protein decreased again at 60 and 80ug/ml of MEKS (Fig. 4). It was observed that the expression of the Nrf2 protein, which is closely related to the MAPK pathway, significantly increased at 60 and 80ug/ml of MEKS (Fig. 4).

    Ⅳ. DISCUSSION

    Our results of this study indicated that MEKS had cell proliferation inhibition effects in the YD15 cells (MEC cell line) and showed anti-cancer activity. When the morphological changes of YD15 cells were observed, the cells treated by MEKS (the MEKS-treated experimental group) illustrated more distinct condensation and morphological changes of the cells than those of the control group. It was also confirmed that the number of cells in the sub G1 phase increased using flow cytometry analysis. It was observed that pAkt, p38 pathway, and Nrf2 protein expression were changed.

    MEKS treatment has performed to YD15 cells for 24 and 48 hours at 0, 40, 60, or 80ug/ml and then trypan blue exclusion assay was carried out in order to examine the cell proliferation inhibitory effects of MEKS treatment on YD15 cells. The results of the present study showed that the number of live cells in the MEKS-treated experimental group significantly decreased than that in the control group. Moreover, it was confirmed that the number of live cells decreased in a dose-dependent and time-dependent manner. The results suggested that MEKS had the potential to inhibit the cell proliferation of the MEC cell line.

    Cell cycle analysis was conducted using FACScan flow cytometry to understand the subtype of MEKS-induced cell death in YD15 cells. The results revealed that the number of cells undergoing sub-G1 arrest increased more in the MEKStreated experimental group than in the control group and the difference between the two groups increased with a higher MEKS concentration. The results implied that sub-G1 arrest is essential for inhibiting the cell proliferation of YD15 cells.

    In order to evaluate the cell death pattern of MEKS-induced cell death, the authors observed the expression of proteins that are induced by apoptosis and autophagy using western blotting. The cleaved PARP protein expressed when apoptosiswas induced20), demonstrated a MEKS concentration-dependent increase when MEKS concentrations were 40, 60, and 80ug/ml. Additionally, the expression of LC3II protein, which is induced by autophagy21), revealed a MEKS concentration-dependent increase within the same MEKS concentration range.

    The changes in the Akt pathway and p38 pathway were evaluated using western blotting in order to examine the cell proliferation inhibition mechanism of MEKS treatment. The results of this study showed that the expression of phosphorylated Akt protein increased at 60 and 80ug/ml of MEKS. Phosphorylated p38 protein increased at 40ug/ml of MEKS but it decreased again at 60 and 80ug/ml of MEKS. It was observed that the expression of the Nrf2 protein, which is closely related to the MAPK pathway, significantly increased at 60 and 80ug/ml. This implies the inhibition of cell proliferation and anti-cancer activity of MEKS in YD15 cells can function through the regulation of p38 pathway and Nrf2 protein.

    Phytochemicals, derived from natural products often act through the increased expression of phase 2 detoxifying antioxidant enzyme, which is regulated by the Nrf2 protein22,23). This process is affected by the higher signal transduction mechanisms such as the p38 pathway and the PI3K/Akt pathway. It is reported that these actions play an important role in the treatment of cancer and chemoprevention. Keum et al. reported that Nrf2 protein regulation in HepG2 cells was associated with p38 pathway24). In addition, Kocanova et al. also reported that the p38 and PI3K pathways induced apoptotic cell death through Nrf2 protein regulation in the treatment using phytochemical25). Previous studies have shown that most of the phytochemical originated from natural products functions by inducing the expression of the Nrf2 protein, which is considered as a potential molecular target for cancer chemoprevention26). The results suggest that MEKS has inhibited cell proliferation and chemoprevention functions by regulating Nrf2 protein activity in YD15 cells.

    Surgical resection and eventual adjuvant radiotherapy are main treatment methods for MEC. MEC shows resistance to conventional chemotherapies for a variety of reasons27). MEC also often recurs or metastasized to other distant organs. However, the overall response rate for the treatment is frequently unsatisfactory or the effect of treatment is shortlived 28,29). Therefore, it is urgent to develop MEC-specific chemotherapeutic agents. The development of anti-cancer drugs that take into account advanced salivary gland neoplasm, and development of effective anti-cancer drugs that are specific to a specific target is urgently needed.

    The results of this study confirmed that MEKS induced apoptosis and autophagy through the Akt and p38 pathways. The results of this study suggested the possibility of using MEKS as an anti-cancer drug for the treatment of MEC, along with the existing study achievements showing that MEKS has anti-cancer effects on various human cancer cell lines. Authors believe that the results of the present study will provide an important basis for understanding the anticancer mechanism of head and neck cancer and developing anti-cancer drugs. Furthermore, the results will be able to present an important element to various topics encompassing therapeutic method development and studies on head and neck cancer.

    ACKNOWLEDGEMENT

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

    Figure

    KOAMP-42-167_F1.gif

    The inhibition of cell proliferation by MEKS treatment in YD15 cells. The cells were treated with MEKS at 0, 40, 60, 80, 100, or 120 ug/ml for 24 hours or 48 hours. (a) Morphologic changes of MEKS-treated YD15 cells. The number of viable cells were counted after 24 hours (b) and 48 hours (c) using trypan blue exclusion assays.

    KOAMP-42-167_F2.gif

    MEKS-treated YD15 cells showed the accumulation of sub-G1 phase and G0/G1 phase. The YD15 cells were treated with indicated MEKS concentrations and then were stained with PI. The distribution of cell cycle was assessed using flow cytometry; (a) a histogram of cell cycle analysis, Graphs (b) and table showing the cell cycle distribution of MEKS-treated YD15 cells.

    KOAMP-42-167_F3.gif

    MEKS triggers cleavage of PARP, and LC3II induction in YD15 cells. The cells were treated with 0, 40, 60, or 80 ug/ml of MEKS for 24 hours and the expression of cleaved PARP and LC3II proteins were examined using western blotting assay.

    KOAMP-42-167_F4.gif

    Activation of the Akt and p38 pathways in the MEKS-treated YD15 cells; The YD15 cells were treated with 0, 40, 60, or 80 ug/ml of MEKS for 24 hours

    Table

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