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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.45 No.3 pp.87-98
DOI : https://doi.org/10.17779/KAOMP.2021.45.3.002

Anti-cancer Effects of the Mixture of Spirodelae Herba and Perilla Frutescens Extracts on Head and Neck Cancer Cell Line

Risu Na1), Ok-Su Kim2), Byunggook Kim3), Ying Yang1), Jianan Song1), Danyang Liu1), Sangmi Jeon1), Ok Joon Kim1)*
1)Department of Oral Pathology, School of Dentistry, Chonnam National University
2)Department of Periodontology, School of Dentistry, Chonnam National University
3)Department of Oral Medicine, School of Dentistry, Chonnam National University
†

These authors contributed equally to this work.


* Correspondence: Ok Joon Kim, D.,D.,S., Ph,.D., Department of Oral Pathology, School of Dentistry, Chonnam National University, Buk-Gu, Gwangju, 61186, Korea Tel: +82-62-530-4832, Fax: +82-62-530-4839 Email: js3894@jnu.ac.kr
May 17, 2021 May 21, 2021 June 4, 2021

Abstract


Spirodelae Herba (SH) and Perilla Frutescens (PF) extracts have been widely used in clinical practice with various disorders for thousands of years. There are some reports regarding the anticancer effects of SH and PF each by each, but their mixture have not been investigated and their mechanisms also have not been clear. The purpose of the present study was to investigate the anticancer mechanisms and their effects of the mixture of SH and PF extracts on head and neck cancer cell line.



Head and neck carcinoma KB cells were treated with SH, PF and their mixture. Anticancer effects were investigated by searching cancer cell death pathway; apoptosis and autophagy, which have been regarded to be effective and safe methods. Apoptosis, which is termed a programmed cell death, was observed by TUNNEL assay. Autophagy, which is termed a type II programmed cell death, was observed by acridine orange red staining. Additionally, the protein expressions associated with apoptosis and autophagy were detected for their mechanism by western blots.



The mixture of SH and PF extracts induced autophagic and apoptotic cell death simultaneously in cancer cells. And 0.4 mg/ml of the mixture with SH and PF extracts down-regulated the expression of mTOR, however, the expressions of ATG5 and LC3-II, which induced autophagy, up-regulated. The mixture of SH and PF extracts also down-regulated the expressions of Bcl-2, but up-regulate the expressions of PARP-1 cleavage, Caspase-9 cleavage, Caspase-3 cleavage and BAX, which induced apoptosis.



Taken together, these results suggested that the mixture of SH and PF extracts induce autophagic and apoptotic cell death simultaneously in head and neck cancer cells and it could be used as an alternative for anti-cancer drugs.


Spirodelae Herba (SH) and Perilla Frutescens (PF) extracts , Anti-cancer , Apoptosis , Autophagy

Spirodelae Herba와 Perilla Frutescens의 혼합물이 두경부암에 미치는 영향

Risu Na1), 김 옥수2), 김 병국3), Ying Yang1), Jianan Song1), Danyang Liu1), 전 상미1), 김 옥준1)*
1)Department of Oral Pathology, School of Dentistry, Chonnam National University
2)Department of Periodontology, School of Dentistry, Chonnam National University
3)Department of Oral Medicine, School of Dentistry, Chonnam National University

초록

    Ⅰ. INTRODUCTION

    Every year, more than 350,000 patients loss their lives due to Head-neck cancer in the world wide1). The serious point is that almost 650,000 new patient have been diagnosed as head and neck cancer every year1). In most countries, head-neck malignant tumor is more likely to occur in men compare to women, and age of patient is higher than 50 years old2). At present, due to the limitation of medical conditions in undeveloped counties, most patients were always diagnosed in advanced period and the result was unsatisfactory after related treatments3). At the same time, significant nerves, vessels and vital organs were distributed in the head and neck area, and anatomical relations is fairly complex which is difficult to approach cancerous area, so multidisciplinary medical trial is needed to achieve satisfactory consequence4). Traditionally, principal treatment of head-neck cancer including surgical excision, radiation therapy and chemotherapy that cause various complications; such as pain, scar, movement disorder, deformity, nausea, pancytopenia, which leads to reduce the patient's quality of life in varying degrees5). Therefore, it is necessary to find out less toxic, more effective and safe conservative treatment to head-neck cancer. Recently, due to the anti-tumor character and obtaining from the nature, the herbal medicine get more and more people's attention all over the world. Herbal drug is a relatively safe auxiliary modality6). However, the only drawback of herbal extracts for cancer suppressing performance is frequently limited.

    There are many articles regarding herbal medicine for remedying carcinoma, dealing with apoptosis and autophagy in vitro and in vivo, such as Curcumin7) by inducing apoptosis via PI3K/Akt-signalling pathways in SKOV3 cells, Glycyrrhiza glabra8) by inducing apoptosis in the HT-29 Colon Cancer Cell Line, Shikonin9) by inducing apoptosis via the proteasome, mitochondria, and caspase cascades in SCC25 cells, 3-β-Εrythrodiol10) by inducing apoptosis and reduces tumor weight and volume in mouse xenograft model, Tetrandrine11) by inducing beclin-1-mediated cell autophagy in human oral cancer CAL 27 cells, Platycodin D12) by triggering autophagy through activation of extracellular signal- regulated kinase in hepatocellular carcinoma HepG2 cells, Tetrandrine13, 14) by inducing autophagy through activating ROS and Notch1 signaling, Diallyl Disulfide15) by inducing apoptosis and autophagy via the mTOR pathway in myeloid leukemic cell line, Tanshinones16) by inducing apoptosis and autophagy via reactive oxygen species in lung cancer 95D cells, Hedyotis diffusa plus Scutellaria barbata17) by inducing apoptosis via inhibiting Akt signaling pathway through by down-regulating miR-155 expression in bladder cancer cells.

    Apoptosis, the physiological progressive cell death happened under genetic control, is the response to physiological signal, the change of environmental conditions or slight injuries and cause the orderly cell death process18). Morphological alteration contain cell volume contraction, connection between cells weakened, density of cytoplasmic enhanced, nuclear concentration, nucleoli and nuclear membrane broken, membrane form to several small bubble with intact structure which is called apoptotic body. In this process inexistence of contents overflow, so there is no induce any kinds of inflammation, and this apoptotic body will be devoured by phagocytes rapidly.

    In cells, if the organelles or some protein molecules are damaged, autophagy may be happened and these defective particles are packaged by double membrane structure of autophagic vesicles and transferred to the lysosome to degrade 19). Autophagy, also known as type II cell death, is that cell under the control of autophagy related genes using the lysosome to degrade its own damaged organelles and macromolecule material.

    Spirodelae Herba (Fig. 1A), an aquatic perennial plant, has been used in herbal medicine for long time for treating skin disease, urticaria, and inflammation and it distributed in most regions of oriental countries, such as China, Japan and Korea20). Kim et al. concluded that vitexin, orientin and other flavonoids from Spirodela polyrhiza can inhibit adipogenesis in 3T3-L1 cells21). Mou et al. concluded that alcohol- extracts of Herba Spirodelae and Radix Salviae Miltiorrhizae can promote growth and proliferation of cultured melanocytes22). Peng et al. concluded that Spirodela polyrrhiza Schleid extract have the protective effect on ECV-304 injury induced by hydrogen peroxide23).

    Perilla frutescens (Fig. 1B) is a traditional Chinese medicinal herb and have long been used in the Oriental countries for treating different kind of disease including tumor, depression, infection, inflammation, and allergies24). Hiroshi et al. concluded that Perilla frutescens can inhibits inflammation, allergic response, and tumor necrosis factor-a production in mice25). Norihiro et al. concluded that Perilla frutescens have anti-inflammatory and antitumor-promoting effects26). Naomi et al. concluded that Perilla frutescens extract have the anti-carcinogenic effects via two independent mechanisms: inhibition of the inflammatory response and scavenging of reactive oxygen radicals27). These several literature mention about Spirodelae Herba and Perilla frutescens have Shown in the Table 1.

    In the past, only one kind of herbal drug was commonly used to treating cancer. In order to achieve an effective results, cancer patient have to be taken high doses of drug that may lead to the obvious side effects. At present, doctors usually use cocktail herbal drug to treat different kinds of cancer and gain satisfying effects28). Lee et al. concluded in his report that an oriental herbal cocktail, ka-mi-kae-kyuk-tang, exerts anti-cancer activities by targeting angiogenesis, apoptosis and metastasis29). Inweon et al. also concluded that Ka-mi-kae-kyuk-tang oriental herbal cocktail might be a safe and effective herbal complementary and alternative medicine modality to alleviate cancer drug-induced hematological side effects in addition to its anticancer activities 30). Chia et al. concluded in his article that Chinese herbal cocktail Tien-Hsien Liquid had broad-spectra anti-cancer activities by inhibiting metastasis, angiogenesis, and tumor growth31).

    In this research, the mixture of Spirodelae Herba and Perilla frutescens was applied to the KB cells, belongs to head-neck cancer line. In pilot study, Spirodelae Herba possess anti-tumor function via autophagic mechanism and Perilla frutescens contain cancer inhibitory ability through apoptotic pathway and a large amount of literatures also testified their tumor suppressing properties32-34). Hypothesis was carry out that the mixture of these two kinds of herbal medicine can reduce the number of cancer cells more effectively with the mechanisms both autoghagy and apoptosis simultaneously.

    Ⅱ. Materials and Methods

    1. Plant material extraction and sample preparation

    Dried herbs were obtained from Dong Kyung PHARM (Boun, Korea). As shown in Fig. 2, the dried herb (1,000 g) was grinded into powder. The grinded materials weighed 300 g. It was then extracted with 1,500 ml 70% EtOH at room temperature for 48 h by a dynamoelectric stirrer (MTOPS, Korea). After filtration with the 70% EtOH extraction was evaporated by a rotary vacuum evaporator (EYELA, Kubota, Japan).

    2. Cell viability assay

    Cell viability was measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5- -diphenyltetrazolium bro- mide (MTT) to insoluble formazan. The cells were cultured (1X105/ml) in 96-well plates (Corning Inc. USA). The extraction of SH and PF, and the mixture of these two herb extracts (MIX) were added to the culture medium, and the cells were incubated for 24 h. After 24 h, incubated with 100 μl of PBS containing MTT at 37 ℃ for 3 h. The formazan product was solubilized by the addition of 50 μl of dimethyl sulfoxide (Calbiochem, USA). Optical density was measured at 570 nm using an ELx800uv ELISA reader (Bio Tek Instruments, Winooski, VT, USA).

    3. Diff-Quick® staining

    To observed the cell morphology, KB cells were seeded in 4-well plates on cover slips at 1X105 per well with 1 mL of medium in each well. After 24 h, the cover slips were then treated with Diff-Quick® solution (International Reagent Co., Kobe, Japan) and mounted on a slide for visualization by light microscopy.

    4. Acridine orange red staining

    The cells were treated with the extracts of SH, PF and MIX for 24 h, briefly washed with PBS, and incubated with acridine orange hydrochloride solution (0.5 μg/ml in DDW) for 20 min. The cell images were collected using a confocal laser scanning microscope (Beckman Coulter, USA).

    5. TUNEL assay

    To investigate the DNA strand breaks in the nuclei at the cellular level, a terminal deoxynucleotidyl transferase- mediated dUTP nick end-labeling (TUNEL) assay was performed using a TUNEL Assay System (R&D Systems), as previously described (Kim and Soh, 2009). Briefly, the cells were fixed with 4% paraformaldehyde, and then washed with an immersing solution (potassium buffered saline, PBS) at room temperature. The cells were permeabilized with a 0.2% TritonX-100 solution, and washed twice with PBS. The cells were incubated with 100 μl of a reaction mix (biotinylated nucleotide mix and recombinant terminal deoxynucleotidyl transferase, rTdT in equilibration buffer) for 60 min. To remove the unincorporated biotinylated nucleotides, the cells were incubated again in 2xSSC (1xSSC is 0.15 M NaCl plus 0.015 M sodium citrate) solution at room temperature. The endogenous peroxidases were blocked by immersing the slides in 0.3% hydrogen peroxide at room temperature. After washing four times with PBS, horseradish peroxidase-labeled streptavidin (HRP-streptavidin) diluted in PBS was added to the slides for 30 min at room temperature. 3, 3’-diaminobenzidine (DAB) was used to stain the apoptotic cells. The DNase I-treated tissue was used as a positive control. The reaction without the rTdT enzyme was used as a negative control.

    6. Western blot

    The proteins from the cell culture were extracted with a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP.40, 1 mM PMSF and 1% protease inhibitor cocktail. The concentration of the protein extract was determined using the BCA reagent method (Promega, USA). A 500 μg aliquot of the protein was prepared in a reducing sample buffer and run through 10% SDS polyacrylamide gel. The nonspecific binding sites were blocked by incubating the membranes in 5% nonfat milk in Tris-buffered saline (TBS) for 1 h at room temperature. The membranes were incubated with the primary antibodies in a blocking solution for 12 h at 4 ℃. After washing with 1XTBST, the membranes were incubated with anti-mouse or anti-rabbit antibodies conjugated with HRP for 3 h at room temperature, and washed three times with TBST for 20 min. The immunoreactive proteins were visualized using an ECL Kit (Santa Cruz, CA, USA) according to the manufacturer’s instructions. The levels were determined by densitometric analysis using Scion Image software (Scion Corp, Frederick, MD, USA).

    7. Statistical analysis

    Data was expressed as the means±standard deviation. All experiments were carried out three times, and the data was calculated using Microsoft Excel. For significance testing, Student’s t-test was used (P<0.05)

    Ⅲ. Results

    1. Effects of the extracts on cell viability and cell morphology in KB cells.

    The KB cells were treated with a range concentrations of the extracts of SH, PF and MIX for 24 h to determine the cell viability. As shown in Fig.3A, the viability of the KB cells decreased in a dose-dependent manner in SH, PF and especially in MIX group. At 0.4 mg/ml, the viability of the KB cells decreased to around 53%, 52% and 23%, respectively.

    The KB cells were treated with 0.4 mg/ml concentrations of the extracts of SH, PF and MIX for 24 h and checked the histomorphologic changes by Diff-Quick® staining. As shown in low magnification pictures in Fig.3B, the KB cells were closely packed with each other in CON group, whereas the KB cells were detached from each other in experimental group. Another point is that the number of KB cells, comparing to CON group, was decreased in SH and PF, especially in MIX group, which is consistent with the pilot study. In high magnification, the vacuoles in cytoplasm indicating autophagic cells can be observed in SH group and cell membrane blebbing which indicated apoptotic cells can be observed in PF and MIX group respectively.

    2. The mode of cell death induced by the extracts of SH, PF and MIX.

    To confirm the autophagy, the KB cells were treated with 0.4 mg/ml dose of SH, PF and MIX, followed by incubating 24 h. Autophagic cells were checked by acridine orange red staining. As shown in Fig.4A, the autophagic cells stained with orange red color were detected in SH and MIX group, but not in CON and PF group.

    To determine the apoptosis, apoptotic cells were detected by TUNEL assays after incubating 24 h of 0.4 mg/ml the extracts of SH, PF and MIX. As shown in Fig.4B, apoptotic cells showing green fluorescence were detected in PF and MIX group, but not in CON and SH group.

    3. Autophagic protein expression by SH, PF and MIX

    To analyze the expression levels of proteins related to the autophagy, the representative proteins of mTOR signal pathway were detected using protein antibody of mTOR, ATG5 and LC3. As shown in Fig.5, mTOR expression was down-regulated in SH and MIX group, whereas ATG5 and LC3II/LC3I protein expression was up-regulated. These data suggested that herbal extracts of SH and MIX induce autophagy through mTOR signaling pathways.

    4. Apoptotic protein expression by SH, PF and MIX

    To analyze the expression levels of proteins related to the apoptosis, the representative protein expression of PARP-1, BAX, Bcl-2, Caspase-9, Caspase-8 and Caspase-3 were investigated. As shown in Fig.6, PARP-1 cleavage, BAX, caspase-9 cleavage and caspase-3 cleavage were increased whereas Bcl-2 expression was down-regulated in PF and MIX group. Caspase-8 cleavage was not detected in present study. These data suggested that herbal extracts of PF and MIX induce apoptosis through intrinsic mitochondria- dependent pathway.

    Ⅳ. Discussion

    Spirodelae Herba, an aquatic perennial plant, has been used in herbal medicine for long time for treating skin disease, urticaria, and inflammation and it distributed in most regions of oriental countries, such as China, Japan and Korea20). Kim et al. concluded that vitexin, orientin and other flavonoids from Spirodela polyrhiza can inhibit adipogenesis in 3T3-L1 cells21). It was reported that alcohol-extracts of Herba Spirodelae and Radix Salviae Miltiorrhizae can promote growth and proliferation of cultured melanocytes 22). Peng et al. reported that Spirodela polyrrhiza Schleid extract have the protective effect on ECV-304 injury induced by hydrogen peroxide23). Although there were numerous literatures regarding various application of SH extracts, but no literature reported its anti-cancer effects. In the present study, herbal extracts of SH have been elucidated the anti- cancer effects by inducing autophagic pathway.

    Perilla frutescens is a traditional Chinese medicinal herb and have long been used in the Oriental countries for treating tumor, depression, infection, inflammation, and allergies 24). Kwon et al. concluded that herbal extracts of Perilla frutescens inhibits growth and induces apoptosis in melanoma cells via activation of ROS-mediated caspase-dependent and -independent pathways35). It was reported that herbal extracts of Perilla frutescens induces cell cycle arrest and apoptosis in HT-29 human colon cancer cells36). It was concluded that herbal extracts of Perilla frutescens inhibits growth and induces apoptosis on human hepatoma HepG2 cells37). Kwak et al. reported that herbal extracts of Perilla frutescens induces apoptosis through the combinations of mitochondrial, death receptor-mediated, and endoplasmic reticulum pathways and suppressed the cell proliferation via p21-mediated G1 phase arrest in HL-60 cells38). In the present study, herbal extracts of PF could induce apoptosis through intrinsic mitochondria-dependent pathway in head and neck cancer cell lines. The different mechanism of anti- cancer effects of PF extracts between previous literatures and present study maybe due to the possible reason that is a different experimental system; such as dose, cell line, incubation time, and etc.

    In the present study, SH and PF extracts were mixed by 1:1 ratio to obtain MIX extracts. Although total concentration of herbal extracts were same in 3 experimental groups, but mathematically in MIX treatment group, half concentration of effective components were coming from herb SH and another half concentration of effective components were coming from herb PF. In other words, although half concentration of original herb was applied, the MIX extracts at 0.4 mg/ml can kill KB cancer cells more effectively comparing to single SH or PF extracts treatment. So it was predicted that this result may be due to possible reasons of unknown components from SH and PF extracts, which might promote anti-cancer effects synergistically.

    There are three kinds of cell death: necrosis, apoptosis and autophagy. Apoptosis and autophagy, which are programmed cell death, are valuable for cancer treatment. If two kinds of herbal extracts, which induce apoptosis and autophagy each by each, were mixed and treat cancer KB cells, there were several hypothesis as follow: 1) none of apoptosis and autophagy was happened; 2) only apoptosis was happened 3) only autophagy was happened 4) both apoptosis and autophagy were happened. According to the results of acridine orange red staining, TUNEL assay and western blot analysis in this study, herbal extracts of MIX can kill the cancer KB cells more effectively comparing to single SH or PF extracts by inducing autophagy and apoptosis simultaneously. Although it is not known what kind of chemical reaction happen after mixing herbal extracts of SH and PF, but anyway, anti-cancer character of SH extracts did not disappear, and also anti-cancer character of PF extracts. So it was predicted that some unknown chemical compo nents from SH and PF extracts induce chemical reaction and promote total anti-cancer characters synergistically. According to this prediction, it can be made a strategy to develop a new drug for cancer in future.

    The present study demonstrates that the mixture of Spirodelae Herba and Perilla frutescens extract induces cell death by apoptosis and autophagy in KB cell line. Furthermore, it is expected that the mixture of Spirodelae Herba and Perilla frutescens extract will be clinically useful for the treatment of patients with head and neck cancers. The findings support that the mixture of Spirodelae Herba and Perilla frutescens extract can be used as an anti-cancer drug.

    ACKNOWLEDGMENTS

    This work was supported by National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIP) (No. 2019R1F1A1044073, 2019R1F1A1056887 and 2019R1F1A1 059492)

    Figure

    KAOMP-45-3-87_F1.gif

    Spirodelae Herba (A) and Perilla Frutescens (B).

    KAOMP-45-3-87_F2.gif

    A schematic diagram of sample preparation. The dried herb (1,000 g) was grinded into powder and extracted with 1,500 ml 70% EtOH at room temperature for 48 h by a dynamoelectric stirrer. After filtration with the 70% EtOH, the extraction was evaporated by a rotary vacuum evaporator to obtain the freeze dry herb.

    KAOMP-45-3-87_F3.gif

    Effects of the extracts on KB Cell Viability(Fig. A) and histomorphologic evaluation in KB cells(Fig. B). Lower panel presents (x40) and upper panel presents (x200). Data are presented as mean±SD (n=3). *p<0.05, **p<0.01.

    KAOMP-45-3-87_F4.gif

    The mode of cell death induced by the extracts of SH, PF and MIX. Autophagosome detection using (A) Acridine orange red staining, the red color indicated autophagic cells (B) Apoptotic cells were detected with the TUNEL assay, the green color indicated apoptotic cells. Bars indicate 20 μm.

    KAOMP-45-3-87_F5.gif

    Autophagic protein expression by SH, PF and MIX. (A) mTOR, ATG5 and LC3II/LC3I protein expression and (B) mTOR, (C) ATG5, (D) LC3II/LC3I protein levels was graphed. GAPDH was used to ensure equal protein loading. Data are presented as mean±SD (n=3). *p<0.05, **p<0.01.

    KAOMP-45-3-87_F6.gif

    The protein expression related to apoptosis by SH, PF and MIX. (A) PARP-1, BAX, Bcl-2, Caspase-9, Caspase-8 and Caspase-3 protein expression (B) PARP-1 & its cleavage, (C) BAX/Bcl-2, (D) Caspase-9 & its cleavage, (E) Caspase-8 & its cleavage, (F) Caspase-3 & its cleavage protein levels was graphed. GAPDH was used to ensure equal protein loading. Data are presented as mean±SD (n=3). *p<0.05, **P<0.01.

    Table

    Literature review of Spirodelae Herba and Perilla frutescens

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    35. Kwon SJ, Lee JH, Moon KD, Jeong IY, Ahn DU, Lee MK, Seo KI: Induction of apoptosis by isoegomaketone from Perilla frutescens L. in B16 melanoma cells is mediated through ROS generation and mitochondrial-dependent, -independent pathway. Food Chem Toxicol 2014;65:97-104.
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    38. Kwak CS, Yeo EJ, Moon SC, Kim YW, Ahn HJ, Park SC: Perilla Leaf, Perilla frutescens, Induces Apoptosis and G1 Phase Arrest in Human Leukemia HL-60 Cells Through the Combinations of Death Receptor-Mediated, Mitochondrial, and Endoplasmic Reticulum Stress-Induced Pathways. J Med Food 2009;12:508-517.
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