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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.43 No.1 pp.1-10
DOI : https://doi.org/10.17779/KAOMP.2018.43.1.001

Methanol Extract Saururus chinensis Inhibits the Cell Proliferation of HSC3 Oral Squamous Cell Carcinoma Cell Line through Autophagy

Mihyang Do1), Hye-Yeon Han1), Sung-Hee Jeong2), Jiyeon Kim3), Mi Heon Ryu4)*
1)Research Institute for Oral Biotechnology, School of Dentistry, Pusan National University, Yangsan, Republic of Korea
2)Department of Oral Medicine, School of Dentistry, Dental Research Institute, Pusan National University, Yangsan, Republic of Korea
3)Department of Pediatric Dentistry, School of Dentistry, Dental Research Institute, Pusan National University, South Korea
4)Department of Oral Pathology, BK21 Plus project, School of Dentistry, Pusan National University, Yangsan, Republic of Korea
Correspondence: Mi Heon Ryu, Department of Oral Pathology, BK21 PLUS project, School of Dentistry, Pusan National University, 49 Busandaehak-ro, Yangsan, Gyeongnam, 50612, Republic of Korea Tel: +82-51-510-8251, Fax: +82-51-510-8249 E-mail: apollon@pusan.ac.kr
December 6, 2018 January 4, 2019 February 22, 2019

Abstract


Autophagy is recently receiving the spotlight as the development strategy for promising anticancer drugs. In particular, the majority of anticancer drugs originating from natural products are known to induce autophagy. Saururus chinensis has been used for treating various inflammatory diseases. Recent research has revealed that the extract of Saururus chinensis possess cytotoxicity for various types of human cancer cells. However, the exact action mechanism of Saururus chinensis extract for oral squamous cell carcinoma (OSCC) has not been studied yet. Therefore, the authors of this research aim to study the effect of methanol extract of S. chinensis (MESC) on OSCC cells. To observe the cell proliferation inhibitory effect of MESC on HSC3 cells, the authors conducted the trypan blue exclusion assay. Also, the action mechanism of MESC was studied by conducting the cell cycle analysis, acidic vesicular organelle (AVO) staining and flow cytometry analysis, monodansylcadaverine (MDC) staining, propidium iodide staining, and Western blotting on MESC-treated HSC3 cells. When HSC3 cells were treated in MESC, the cell proliferation was suppressed in time-dependent and dose-dependent manners. Also, the number of sub-G1 arrested cells increased in a dose-dependent manner. MDC punctate and AVO puncta significantly increased respectively. Western blot analysis demonstrated the expression of autophagy-related proteins increased, but apoptotic proteins were not observed. Also, the pAkt protein was reduced, while the p-p38 protein and pERK protein increased. According to our results, MESC induced autophagy and accompanied changes in the cell cycle in HSC3 cells. Also, the alteration in Akt, ERK, and p38 pathways were confirmed. This result suggested the possibility of MESC as the new promising adjuvant for treating OSCC patients.



Saururus chinensis 추출물에 의한 자가포식 유도를 통한 HSC3 구강편평세포암종 세포주에서의 세포 증식 억제 기전

도 미향1), 한 혜연1), 정 성희2), 김 지연3), 유 미현4)*
1)부산대학교 치의학전문대학원 구강생물공학연구소
2)부산대학교 치의학전문대학원 구강내과학 교실
3)부산대학교 치의학전문대학원 소아치과학 교실
4)부산대학교 치의학전문대학원 구강병리학 교실, BK21 플러스 사업단

초록


    Ⅰ. INTRODUCTION

    Autophagy is the mechanism of a cell, which lacks nourishment and is in disadvantageous growth environments, increasing its chance of survival by decomposing cell organelle and useless structures inside of the cell1). This is an important mechanism for the survival and maintenance of homeostasis of the cell2). When there are abnormalities in the autophagy regulation such as abnormal decreases or increases in autophagy compared to the normal condition, nerve diseases, inflammation, degenerative diseases and cancer may develop3). The action mechanisms of autophagy and its roles in cancer are recently being studied, but the exact mechanism has yet been identified. Also, its interactive relationship with apoptosis, which is known as the main cell death mechanism, remains unclear.

    Chemotherapeutic agents for cancer treatments are very toxic for both cancer cells and normal cells, which can reduce the efficiency of anticancer treatment by causing side effects such as bone marrow suppression, nausea and vomiting, severe anemia, stomatitis, alopecia and so on4). To overcome these side effects, the developments of drugs from natural products are increasing rapidly. Medical drugs that use natural products are the mixtures of diverse components, and have the advantages of working simultaneously on many targets, effectively regulating complex causes and symptoms, and being relatively less toxic and advantageous for long-term use. Saururus chinensis is herbaceous perennial plant that grows naturally in Korea, Japan and Southeast Asia, and possesses phytochemicals such as Sauchinone and neoglignans5). Methanol extract of Saururus chinensis (MESC) is known to have anticancer effects on various cancer cells such as stomach and lung cancer cell lines6), but there are few studies on the effect of MESC in oral squamous cell carcinoma (OSCC) cells.

    OSCC is the most common type of oral cancer, accounting for more than 90%.7) Due to anatomical characteristics, oral cancer in the oral cavity can be discovered with the naked eye, which makes it convenient for early detection, treatment and prevention of oral cancer. Therefore, it is considered to be the most appropriate model for cancer prevention. However, the prognosis of oral cancer patients is poor since oral cancer progresses fast, and lymph node metastasis and recurrences are frequent. Also, when oral cancer is surgically removed, aesthetic and functional defects such as masticatory and pronunciation issues arise, causing problems with the rehabilitation treatment of oral cancer patients. Therefore, the development of effective oral cancer drugs that are less toxic is in demand.

    The purpose of the present study is to explore natural product substances that have anticancer effects, and to reveal the selective anticancer effects of OSCC cells by investigating the effect of MESC on OSCC cells and its underlying mechanism.

    Ⅱ. MATERIALS and METHODS

    1. Chemicals

    MESC used in this experiment was purchased from the Korea Plant Extract Bank (Cheongwon-gu, Chungbuk, Korea). MESC was dissolved in dimethyl sulfoxide(DMSO) and stored at the temperature of –20℃ refrigerator prior to usage.

    2. HSC3 cell culture

    HSC3 OSCC cells were used in this experiment, which was purchased from Japanese Collection of Research Bioresources Cell Bank (JCRB)(Tokyo, Japan), Cell culture was conducted using Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics, and cultured in the 37℃, 5% CO2 incubator with consistent humidity.

    3. Reagents and antibiotics

    Trypan blue was obtained from Gibco (Paisley, UK). Acidic vesicular organelle (AVO) stain, monodansylcadaverine (MDC), propidium iodide (PI), and the antibody for LC3II were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for phosphorylated ERK, ERK, phosphorylated p38, p38, phosphorylated Akt, Akt, cleaved PARP, and cleaved caspase 3 were purchased from Cell Signaling Technology (Beverly, MA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-rabbit IgG antibody, and rabbit anti-mouse IgG antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for the autophagy-related gene (Atg) 5 and Beclin 1 were obtained from Abcam Biologicals (Cambridge, UK). RNase A solution (DNase free) was purchased from Biosesang (Seong-nam, Korea).

    4. Analysis of cell viability

    In order to examine the effect of MESC on cell viability, the trypan blue exclusion assay was carried out. 1×105 HSC3 cells were seeded on the 24-well cell culture plate, kept overnight, and after being MESC-treated for 24 and 48 hours respectively, the number of living cells was estimated by staining with the trypan blue. For the control group, DMSO was treated and for the MESC-treated experimental group, 20, 40, 80, and 160 ug/ml of MESC were treated for 24 and 48 hours respectively. The average value of three repeated experiments was set as the estimated value.

    5. Analysis of cell cycle distribution

    In order to reveal the cell proliferation inhibitory mechanism induced by MESC on HSC3 cells, the cell cycle analysis was conducted. HSC3 cell was treated with MESC for 24 hours, then the cell was detached from the culture dish using the trypsin. Then, centrifugation was conducted for the detached cell, and fixed for a day in the –20℃ ethanol solution. Next, the cell cycle was evaluated after dyeing with 0.02mg/ml P) solution and 0.05mg/ml RNase A and conducting the FACScan flow cytometry analysis.

    6. Analysis of MESC's cell death types and its mode of action

    In order to reveal MESC's cell death types and cell proliferation inhibitory mechanism, the Western blotting analysis was conducted. Protein was extracted from the MESC-treated HSC3 cell, and the protein concentration was determined using the Bradford assay. Electrophoresis was conducted on approximately 40ug of protein with 10% SDS-PAGE, blotted to the nitrocellulose membrane, kept for overnight at 4℃ then hybridized with the primary antibody. The membrane was washed with Phosphate-buffered saline (PBS), then the secondary antibody with the horseradish peroxidase (HRP) tag was treated at the room temperature for 2 hours. Then, the protein expression was analyzed using the chemilluminescence detection system.

    7. AVO and MDC stain

    To measure MESC's autophagy induction, AVO and MDC stain, which are generated from the autophagy induction steps, were carried out. HSC3 cells were seeded with the number of 5×104 cells on the 6-well cell culture plate, treated MESC for 24 hours. As for the vehicle control, DMSO was treated, which has the same concentration as the MESC-treated experimental group. After AVO/MDC stain, the fluorescence microscope (excitation = 330-385 nm, emission = 420nm wavelength filter used) was used for the observation. Among 100 cells, the number of cells that show positive dots in cytoplasm due to AVO/MDC stains was counted, and the average value was set as the final value after conducting each experiment three times. Also, the result of AVO/MDC stain was evaluated using the flow cytometry analysis.

    8. Statistical analysis

    SPSS 23.0 software was used for the statistical analysis of the experimental data, with p-value less than 0.05 (p<0.05) considered to be statistically significant.

    Ⅲ. RESULTS

    1. Analysis of the changes in the cell viability for HSC3 cells

    In order to investigate MESC's cell proliferation inhibitory effect, 0, 20, 40, 80, and 160 ug/ml MESC was treated in HSC3 cells for 24 and 48 hours respectively, then the trypan blue exclusion assay was conducted. When compared with the control group, the number of live HSC3 cells was significantly reduced for the MESC-treated experimental group. After MESC treatment for 24 hours, the number of cells significantly decreased starting from the concentration of 40 ug/ml, and when treated for 48 hours, the number of cells decreased significantly starting from the concentration of 20 ug/ml (Fig. 1A). The reduction of cells was shown to be dependent on the time and concentration of MESC.

    2. Analysis of the cell cycle for HSC3 cells

    When the cell cycle was analyzed using the flow cytometry, the number of HSC3 cells in the sub-G1 phase increased. This was also confirmed to be dependent on the concentration of MESC (Fig. 1B, 1C, 1D).

    3. Analysis of the expression of apoptotic and autophagy related proteins

    To determine if MESC's cell proliferation inhibitory mechanism is apoptosis or autophagy, expressions of apoptotic proteins such as cleaved PARP, cleaved caspase 3, Bcl2, and Bax protein and autophagy related proteins such as LC3II, Beclin1, and Atg 4B were examined using the western blotting. As for the internal control, GAPDH was used. Experimental results showed that the expression of LC3II and Beclin1 proteins increased when treated with 30, 60 and 90 ug/ml of MESC. However, the expression of cleaved PARP and cleaved caspase 3 was not observed(as for the positive control, the protein from taxol-treated HSC3 cell was used). Also, the expression of Bcl2 protein was reduced, and the expression of Bax protein did not change (Fig. 2).

    4. Analysis of MESC's autophagy inducement ability

    To confirm that the cell proliferation inhibitory mechanism in MESC-treated HSC3 cells is autophagy, AVO stain and MDC stain were conducted. Our results revealed that compared to the control group, the number of cells that possesses AVO puncta increased significantly in the MESC-treated experimental group. When the FACScan flow cytometry was used to measure AVO puncta cells, significant increase compared to the control group was again confirmed. Also, the result of MDC stain revealed that the number of cells which have MDC-positive dots in cytoplasm increased significantly in MESC-treated HSC3 cells (Fig. 3).

    5. Analysis of MESC-induced cell death mechanism

    In order to confirm the cell proliferation inhibitory mechanism caused by MESC, the altered expressions of Akt pathway, ERK pathway and p38 pathway were verified using the western blotting. Experimental results showed that compared to the control group, the expressions of p38 and ERK proteins in MESC-treated cells increased in a concentrationdependent manner, However, the expression of pAkt proteins gradually decreased (Fig. 4). Therefore, it was confirmed that Akt, ERK, and p38 pathway are involved with HSC3 cell proliferation inhibitory actions induced by MESC treatment.

    Ⅳ. DISCUSSION

    To assess MESC's cell proliferation inhibitory effects in the HSC3 cell, which is the human OSCC cell line, MESC was treated for 24 and 48 hours respectively in concentrations of 0, 20, 40, 80 and 160 ug/ml, and the trypan blue exclusion assay was carried out. Experimental results showed that compared to the control group, MESC-treated experimental group's number of live cells decreased significantly. Also, the reduction of live cells was confirmed to be dose-dependent and time-dependent. It is believed that MESC possesses the cell proliferation inhibitory potential in OSCC cells.

    In order to determine the types of cell death that occur in MESC-treated HSC3 cells, the western blotting was used to observe the expression of proteins that are induced when apoptosis and autophagy are induced respectively. Experimental results showed that the expression of LC3II and Beclin 1 proteins, which are expressed when autophagy occurs8,9), increased as the MESC concentration increased. However, the expression of cleaved PARP and cleaved caspase 3 proteins, which are expressed when apoptosis is induced, was not detected. To confirm that the type of MESC-induced cell death is autophagy, the authors conducted AVO stain and MDC stain. When autophagy is induced, AVO is created and continues to increase. The estimation and observation of AVOs can be conducted by staining the cells using the acridine orange reagent. When the cells have been stained with the acridine orange reagent, cytoplasm glow a fluorescent green, but when it is acidified through the formation of AVO, it glows a fluorescent red. Also, MDC stain can evaluate the autophagy activation by dyeing the autophagic vacuole. According to the experimental results, the number of cells that show positive reactions to AVO stain and MDC stain increased significantly, and in case of AVO stain, significant increase was also observed in the detection reaction using the flow cytometry, Therefore, MESC-induced cell death is believed to be autophagy.

    As for the initiation pathway of apoptosis, there is the extrinsic pathway which activates caspase 8 by stimulating the death receptor, and the intrinsic pathway which forms a complex with caspase 9 by mitochondria's cytochrome c as the cytoplasm10). The intrinsic pathway is regulated by the relative proportions of anti-apoptotic proteins such as Bcl-2, and pro-apoptotic proteins such as Bax11, 12). As for the progression of apoptosis, caspase 3 is ultimately activated by cleaved caspase 8 and caspase 9, and PAPR protein, which is its substrate of cleaved caspase 3, is cleaved13). In this study, cleaved caspase 3 and cleaved PARP were not detected. Also, expression changes in Bax protein was not observed, which led to the belief that apoptosis did not occur in MESC induced cell death.

    To examine MESC's cell proliferation inhibitory mechanism, altered expression in the Akt pathway and p38, ERK pathway were observed using the western blotting. According to our results, the expression of pAkt protein was reduced in MESC-treated HSC3 cells, and p38 pathway and ERK pathway increased as MESC's concentration increased. It is known that in autophagy's development mechanism, Akt pathway is reduced and ERK pathway is activated14). Shinojima et al. reported that in autophagy inducement using curcumin, Akt pathway is reduced and ERK pathway is activated15). p38 pathway plays the role of inducing autophagy by reacting to chemotherapeutic agents, and carries out dual regulations for the positive or negative regulators when it comes to the induction of autophagy16). Also, it is known to play the role of adjusting the balance between apoptosis and autophagy by reacting to the genotoxic stress17). Since such regulation mechanism of the p38 pathway are not widely known yet, it is believed that further studies are necessary.

    Autophagy is known for playing very various roles in the development and progression of cancer18-20). In the initial stage of cancer development, autophagy plays the role of suppressing cancer development21, 22). ATG6/BECN1, which is the essential gene for the induction of autophagy, is reportedly deleted in human prostate cancer, breast cancer and ovarian cancer23). Also, the growth of cancer cells that are deficient autophagy is stimulated, and the development of hepatocellular carcinoma, lung cancer and lymphoma increases in a mouse that possesses BECN1 heterozygous mutation24,25). However, autophagy is known to stimulate the progression of cancer at the stage where cancer has been progressed to a certain extent. In carcinoma with Ras mutation, autophagy stimulates the progression of cancer26-28). In particular, autophagy reportedly occurs frequently in the tumor's hypoxia region, and the cause is believed to be autophagy stimulating the survival of cancer cells in the hypoxic status. Also, the development of autophagy is supposedly different depending on the types of carcinoma29,30). In such ways, the roles of autophagy are different depending on various conditions, such as the stage of cancer progression, types of carcinoma and mutations.

    Autophagy is emerging as the promising alternative to anticancer treatments, in case of cancer that has resistance against anticancer treatments due to the damaged apoptosis mechanism. Therefore, studies that provoke cell death by using the autophagy induction agent are being actively conducted. Autophagy induction agent causes autophagic cell death by inducing autophagy through targeting autophagy pathways such as BNIP3, Beclin1 and mTOR, and suppressing apoptosis by using caspase inhibitors such as zVAD31, 32). Compounds such as Rapamycin, bortezomib, arsenic trioxide and Zinc are known to be effective for breast cancer, hematologic malignancy and colorectal cancer33-38). However, the exact mechanism behind autophagy switching from cell protecting mechanism to cell destruction mechanism has not been verified yet, which leads to the need for further studies.

    According to the findings of our study, MESC showed cell proliferation inhibitory effects in HSC3 cells, and this effect was shown to come from autophagy. This cell proliferation inhibitory effect is related to the altered expressions in p38, Akt and ERK proteins. Such findings imply the possibility as the anticancer drug for the treatment of OSCC. However, it is believed that additional studies on the confirmation of anticancer effects in various cancer cell lines and in vivo, as well as detailed verification of the mechanism are necessary.

    V. ACKNOWLEDGEMENT

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

    Figure

    KAOMP-43-1-1_F1.gif

    Effect of MESC on cell viability and sub-G1 arrest in human OSCC cell lines. HSC3 cells were treated with DMSO or indicated concentrations of MESC for 24 hours or 48 hours. (A) The effect of MESC on HSC3 cell viability was determined using a trypan blue exclusion assay. (B) The analysis of cell cylcle in MESC-treated HSC3 cells. (C) Qualitative assessments of cell cycle distribution of MESC-treated HSC3 cells. The graphs represent the mean ± S.D. (D) Qualitative assessments of cells undergoing sub-G1 phase. The graphs represent the mean ± S.D.

    KAOMP-43-1-1_F2.gif

    The expression of apoptotic proteins and autophagy-related proteins by MESC treatment in HSC3 cell lines. Expression of the proteins was determined by Western blotting. (A) Apoptotic proteins including Cleaved PARP, Cleaved caspase 3, Bcl2 and Bax. (B) Autophagy related proteins including LC3II, Beclin1, Atg 4B.

    KAOMP-43-1-1_F3.gif

    MESC induces the level of AVO/MDC stain in HSC3 cells. (A) The level of AVO puncta was confirmed by immunofluorescence using florescence microscopy. The graphs represent the mean ± S.D. (B) The level of AVO puncta was confirmed by immunofluorescence using florescence microscopy. (C) Qualitative assessments of cells with MDC puncta. The graphs represent the mean ± S.D.

    KAOMP-43-1-1_F4.gif

    MESC regulates the expression levels of pAkt, pERK and p-p38 in HSC3 cell lines. The expression of these proteins was determined by Western blotting in (A) Protein levels of p-Akt and Akt (B) Protein levels of pERK and ERK (C) Protein levels of p-p38 and p38.

    Table

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