1.Background

Medical diagnostic technologies and therapeutic drugs are being actively developed to treat and prevent heart diseases, cancer, and diabetes, which increase as the human lifespan increases. Approximately 50% of the drugs cur-rently used to treat diseases are derived from natural pro-ducts¹⁾. Attempts have been made to secure various plant resources distributed worldwide to research and develop new natural products and separate and produce functional drug components. In Korea, Stillen (a stomach mucosa protection agent, Dong-A Pharmaceutical) and Joins (an arthritis treatment agent, SK Pharmaceutical) are represen-tative of new natural medicines approved by the Korea Food and Drug Administration. These drugs contain com-ponents derived from multiple natural products²⁾. Clinical trials are underway to develop new natural products to treat many human diseases²⁾.

Smilax glabra, a lily family plant, is a widely used traditional herbal medicine, and approximately 200 com-pounds, such as flavonoids, flavonoid glycosides, phenolic acids, and steroids, have been isolated from the root³⁾. Various pharmacological activities such as cytotoxicity, anti-inflammatory and immune control, antioxidant, liver protection, antiviral, antibacterial, and cardiovascular pro-tection of Smilax glabra have been reported through in vivo and in vitro studies³⁾. Smilax glabra has also been observed to reduce serum uric acid levels⁴⁾, kidney disease⁵⁾, has anti-tumor effects in various cancers^6-9), and nerve cell induction¹⁰⁾. Additionally, tumor growth and lung metas-tasis in mice were inhibited by the ethyl acetate extract of Smilax glabra Roxb. Anti-tumor activity of the ethyl ace-tate extract of Smilax glabra Roxb. increased the convert-sion of tumor-related macrophages to the M1 phenotype and promotes the recruitment of CD4^＋ and CD8^＋ T cells to the tumor microenvironment¹¹⁾.

2.Objectives

Oral cancer accounts for 2% to 5% of all malignant tumors occurring in the human body, and the mortality rate due to oral cancer is 2% to 3% of all cancer deaths¹²⁾. The most common type of oral cancer is squamous cell carci-noma, which accounts for approximately 80% to 90%¹²⁾. Surgical therapy is the preferred treatment; however, if cervical lymph node metastasis is suspected, cervical clea-ning is performed simultaneously with cancer removal¹³⁾. Patients with early-stage carcinomas without cervical metastasis undergo radiation therapy to reduce facial defor-mation and dysfunction complications caused by surgery¹³⁾. For advanced carcinomas, a composite treatment with che-motherapy is performed to increase the survival rate¹⁴⁾. The survival rate increases when appropriate treatment is administered after early diagnosis¹²⁾. However, oral cancer is often diagnosed after the stage has progressed consi-derably, therefore, the survival rate decreases significantly even if surgery is performed. In addition, serious func-tional and aesthetic problems caused by complications and reconstruction reduce the quality of patient life¹³⁾. Because the oral cavity is composed of tissues with various charac-teristics, there are many limitations to the development of effective anticancer drugs. Although the anticancer effect of Smilax glabra has been reported in various carcinomas, its effect and mechanism of action in oral cancer have not yet been reported. In this study, the anticancer effects of the ethanol extract of Smilax glabra (EESG) and its mechanism in YD10B oral squamous cell carcinoma cells were investigated.

1.Ethic statement

The materials and methods used in this study do not require IRB review.

2.Materials

Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12 nutrient mixture, fetal bovine serum (FBS), phosphate- buffered saline (PBS), and 0.05% trypsin–EDTA were purchased from Gibco BRL (Rockville, MD, USA). 3- (4,5-Dimethylthiazol-2-yl)2,4-diphenyl tetrazolium bromide (MTT), RNase A, dimethyl disulfide (DMSO), propidium iodide (PI), and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were purchased from their res-pective sources: phosphorylated histone H2AX (gH2AX) at Ser139 (Upstate Biotechnology, Charlottesville, CA, USA); poly(ADP-ribose) polymerase (PARP), procaspase-3, and procaspase-9 (Cell Signaling Technology, Denver, MA, USA); b-actin and horseradish peroxidase-conju-gated secondary antibodies (Amersham Life Science, Little Chalfont, UK).

3.Ethanol extract of Smilax glabra

The EESG was obtained after classification and extra-ction by COSMAX Inc. R&I Center (Seongnam, Korea). Smilax glabra grown in Uiryeong (Gyeongsangnam-do, Korea) was used for the extract. Smilax glabra roots washed with distilled water, ground (HMF-4070TG; Hanil Electric., Bucheon, Korea), and treated with 70% ethanol for 72 hours at room temperature. The extraction solution was filtered through Whatman filter paper (Merck, St. Louis, MO, USA) and concentrated under reduced pre-ssure using a rotary evaporator. Extracts were dried and stored in −70° for analysis. 10 mg/ml stock solution of the extract powder dissolved in DMSO was used for analysis and stored in −70°C until the experiment.

4.Cell culture

YD10B cells and normal gingival fibroblasts (NGF) were cultured in DMEM/F12 complete medium supplemented with 10% FBS at 37°C in 5% CO₂ incubator. YD10B is a squamous cell carcinoma cell line and originated from the tongue of a 67-year-old Mongolian male. YD10B cells were purchased from Korea Cell Line Bank (Korean Cell Line Research Foundation, Seoul, Korea), and NGF was kindly provided by Xianglan Zhang in Yonsei University College of Dentistry (Seoul, Korea).

5.MTT assay

Cells (YD10B, 5×10³ cells/well; NGF, 4×10³ cells/well) were plated into a 96-well culture plate and left to adhere overnight. After washing with fresh complete media, cells were treated with 0∼40 mg/ml of EESG for 24 hours. DMSO was treated as a reagent solution control. Cells media were changed with fresh complete medium with 5 mg/ml MTT solution and further cultured for 1 hour at 37°C. The cell media were then removed, and 100 ml DMSO solution was added. Absorbance was measured at 570 nm using Synergy^TM HTX Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA).

6.DAPI staining

Cells (2×10³ cells/well) were cultured in 4 well-chamber slides overnight to adhere. Fresh complete media with EESG was added for 24 hours. Cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes. After washing with PBS solution (pH 7.4) and stained with a 1 mg/ml DAPI solution (Sigma Chemical, St. Louis, MO, USA) in PBS for 10 minutes. Apoptotic cells with condensed or fragmented nuclei were observed under an EVOS FL monochrome fluorescence microscopy (Thermo Fisher Scientific, Pittsburgh, PA, USA).

7.TUNEL assay

The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using the In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Cells (1×10³ cells/well) were cultured in 4 well-chamber slides overnight to adhere. Fresh complete media with EESG was treated for 24 hours. Cells were washed with PBS solution and fixed with 4% parafor-maldehyde in PBS (pH 7.4) for 1 hour. Cells were then permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate and quenched in 3% H₂O₂ solution. Cells were stained with TUNEL reaction mixture contained terminal transferase for 1 hour in 37°C incubator. The stained cells were observed under a fluorescence microscopy. To assess apoptotic cells, TUNEL-positive fluorescent cells were quantified by counting approximately 300 cells in four to five separate random fields.

8.Annexin V-FITC and PI staining

Annexin V-FITC apoptosis detection kit was used to detect apoptotic cells according to the manufacturer’s instructions. EESG-treated cells were collected by centri-fugation. The cells were suspended in a binding buffer contained Annexin V-FITC and PI for 5 minutes in the dark at room temperature. Cells were analyzed Annexin V-FITC via flow cytometry (Ex=488 nm, Em=350 nm; BD, Franklin Lakes, NJ, USA).

9.Cell cycle phase distribution

The cells (4×10⁵ cells/well) were treated with EESG in the complete culture medium for 24 hours. The cells were harvested by trypsinization and washed in cold PBS. Cells were then fixed in 70% ethanol and stored at 4°C for 2 hours. For DNA content analysis, the cells were treated with 0.25 mg/ml RNase A for 30 minutes and stained with 50 mg/ml PI in 1.12% sodium citrate at room temperature. DNA content was analyzed via flow cytometry using a FACSCalibur using WinMDI 2.8 software (BD, Franklin Lakes, NJ, USA).

10.Western blot analysis

Cells were collected by scrapping and lysed in RIPA lysis buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM EDTA [pH 8.0]; Thermo Fisher Scientific) with protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Equal amount of cell extracts was separated to SDS-PAGE and transferred to nitrocellulose membrane in Tris-Glycine transfer buffer at 100 V for 1 hour at constant current. The membrane was blocked in 5% skim milk in PBS and incubated with each primary antibody (dilution 1:1,000 in 5% skim milk in PBS) overnight at 4°C. The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (dilution 1:3,000 in 5% skim milk in PBS) for 2 hours at room temperature. The targeted proteins were visualized using ECL reagents (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

11.Caspase-3 activity

Caspase-3 activity assay was performed according to the protocols recommended by the manufacturers (Caspase-3 colo-rimetric assay kit; GenScript Corp., Piscataway, NJ, USA). Briefly, cells (3×10⁶/well) were treated with EESG for 24 hours and lysed in 50 ml of chilled lysis buffer containing 10 mM dithiothreitol on ice for 60 minutes. The lysate was centrifuged at 10,000 rpm for 10 minutes at 4°C, and super-natant was added to 50 ml of 2× reaction buffer containing 10 mM dithiothreitol. About 5 ml of reconstituted caspase-3 substrate (final 200 mM, DEVE-pNA) was added to each reaction mixture and allowed to incubate for 8 hours at 37°C. The absorbance of pNA was quantified using a spectrophotometer at 405 nm, and the results of the induced group’s caspase-3 activity were obtained by com-puting OD_inducer/OD_{negative control}.

12.Statistical analysis

GraphPad Prism ver. 5.02 statistical software was used for statistical analyses. Data were expressed as the means± standard deviation of three independent experiments. The Kruskal–Wallis ANOVA with Dunn’s post hoc analysis was used for the multiple comparisons. p-values of less than 0.05 were considered statistically significant.

1.Effect of EESG on cell viability

The cytotoxic effect of EESG on the growth of NGF and YD10B cells was determined using the MTT assay. As shown in Fig. 1, treatment with 20 mg/ml EESG decreased the viability of YD10B cells at 24 hours. NGF viability was not significantly affected by the EESG treatment.

Fig. 1. Effects of ethanol extract of Smilax glabra (EESG) on cell viability of normal gingival fibroblast (NGF) and YD10B oral squamous cell carcinoma cells. Cells were treated with the indicated concentrations of EESG for 24 hours, and cytotoxicity were determined by MTT assay. Dimethyl disulfide (DMSO) was treated as control. The percentage of viable cells was calculated as a ratio of treated to control cells (treated with DMSO). Data are mean±standard deviation of three independent experiment. Statistically significant difference compared with the control group (*p<0.05, **p<0.01) versus DMSO control.

2.Apoptotic effect of EESG

To determine the reason for cancer cell growth inhibition by EESG, YD10B cells were treated with 20 mg/ml EESG, and the cell cycle phase distribution were monitored by flow cytometry. As shown in Fig. 2A, subdiploid DNA content was increased by EESG treatment. Apoptotic morphological change, including chromatin condensation and formation of apoptotic bodies, were detected by DAPI staining in YD10B cells treated with EESG for 24 hours (Fig. 2B). The TUNEL assay showed that EESG treatment increased the number of cells with DNA strand breaks. In addition, flow cytometric analysis of EESG-treated YD10B cells stained with Annexin V and PI showed that 20 mg/ml EESG induced early apoptosis, with high annexin and low PI staining in 49.6% of the YD10B cells (Fig. 2C). These results suggest that EESG induces apoptosis in YD10B cells. The EESG-treated YD10B cells were stained with antibodies against gH2AX, a DNA damage marker. Eight hours of treatment with 20 mg/ml EESG induced significant nuclear foci of gH2AX (Fig. 2D). Induction of gH2AX foci-positive cells occurred in a dose-dependent manner. Additionally, decreased levels of procaspase-3 and procas-pase-9, and PARP cleavage were detected in EESG-treated YD10B cells (Fig. 2E). The activation of procaspase-3 in EESG-treated YD10B cells were supported by a dose- dependently increase in caspase-3 activity (Fig. 2F). Col-lectively, these results indicate that EESG induces apo-ptosis through caspase activation in YD10B cells.

Fig. 2. Apoptotic effects of ethanol extract of Smilax glabra (EESG) on YD10B oral squamous cell carcinoma cells. (A) Flow cytometry analysis of EESG-treated YD10B cells. Cells were cultured for 24 hours in the presence of 20 mg/ml EESG and stained with propidium iodide (PI) for flow cytometric analysis. (B) EESG-treated YD10B cells were stained with a 1 mg/ml DAPI and the condensed or fragmented nuclei in apoptotic cells were observed under a fluorescence microscope (×40 original magnification). The TUNEL assay of EESG-treated cancer cells were also performed using an In Situ Cell Death Detection Kit (×10 original magnification). (C) EESG-treated YD10B cells were stained using a FITC-Annexin V kit, then analyzed via flow cytometry. (D) EESG-treated YD10B cells with gH2AX foci were stained with antibodies against gH2AX. gH2AX foci were counted. Nuclei with more than 10 foci were counted as gH2AX-positive. More than 200 nuclei were counted in each group and the results represent the mean±standard deviation of three independent experiments (×40 original magnification). (E) The expression of apoptosis-related proteins was analyzed via western blotting using PARP, procaspase-3, and procaspase-9 primary antibodies. (F) Caspase-3 activity was evaluated in the lysates of EESG-treated cancer cells using a caspase ELISA kit. *p<0.001 versus non control.

1.Interpretation

Despite the availability of precise diagnostic equipment, such as MRI and PET-CT, there is a limit to diagnosing early cancer without symptoms. Unlike other carcinomas, oral cancer has a low incidence; however, its 5-year sur-vival rate is as low as 50%^12-14). Most oral cancers are squamous epithelial cell carcinomas, which occur in various tissues such as lips, tongue, cheek, hard palate, and gums; however, they also occur in salivary gland cancer, sarcoma, lymphoma, and melanoma^12-14). The tissue characteristics of oral cancer vary depending on the site of occurrence; therefore, chemotherapy has many side effects, and the cancer treatment strategy and prognosis are very different. Therefore, unlike other types of carcinomas, oral cancer has many restrictions on the development of effective anticancer treatments. To reduce these therapeutic side effects, many studies are being conducted on various plant resources to develop anticancer functional drug compo-nents from natural products that have traditionally been used²⁾. Effective anticancer activity has been observed in functional drug components developed from natural pro-ducts alone; however, research on complex compositions to increase pharmacological action while minimizing drug side effects is also ongoing.

2.Comparison with previous studies

Smilax glabra is a deciduous vine of the lily family and is distributed in Korea, Japan, China, the Philippines, and Indochina, and has numerous pharmacological effects on the roots containing flavonoids and flavonoid glycosides (taxifolin, naringenin, astibin, engeletin, etc.), phenolic and phenolic acids (smiglabrone A, smiglabrone B, vanillin, acetovanillone, glucopyranoside, etc.), stilbene and orga-nic acids (piceatannol, syringic acid, palmitic acid, etc.), phenylpropanoids and lignans (trans-caffeic acid, juncusyl ester B, kompasinol A, smiglaside, smiglabranol, etc.), steroids and steroid glycosides (daucosterol, diosgenin, stigmasterol, etc.), and volatile oil (heptanoic acid, longi-pinanes, hexadecane, eicosane, etc.)³⁾. Most of them are components that can be extracted by organic solvents. In this study, the effect of the EESG were observed in oral squamous cell carcinoma cells. 40 mg/ml EESG had little cytotoxicity in NGF (Fig. 1). While 20 mg/ml and 40 mg/ml EESG significantly inhibited cell proliferation of YD10B cells. Flow cytometry analysis confirmed an increase in subdiploid DNA content in YD10B treated with 20 mg/ml EESG (Fig. 2A). This indicates that the inhibition of cell proliferation of YD10B cells by EESG is related to apoptotic pathways. According to other reports, the water extract of Smilax glabra increased the G0/G1 and G2/M phases and changed the expression of genes related to proliferative and apoptotic pathways (MKI67, HER2, EGFR, MDM2, TNFa, PI3KCA, KRAS, BAX, and CASP8) to prevent breast cancer proliferation (MCF7, T47D, MDA- MB-231, and MDA-MB-468), resulting in the inhibition of cell proliferation¹⁵⁾. The water extract of Smilax glabra also disrupted the intracellular reduced glutathione/ oxidized glutathione (GSH/GSSG) balance to activate the redox-dependent ERK1/2 pathway, which also inhibited gastric cancer (AGS), colon cancer (HT-29), and acidosis (GSSG)¹⁶⁾. Several previously reported results, including our data, indicate that Smilax glabra is an effective func-tional natural product with anticancer activity through cancer cell death.

To confirm the mechanism of cancer cell death by EESG in more detail, we first observed DNA using TUNEL and gH2AX assays (Fig. 2B∼2D). DNA fragmentation is an important feature in the late stages of apoptosis. The exposed 3’-OH of the fragmented DNA can bind to fluorochrome-labeled dUTP as a catalyst for terminal deo-xynucleotidyl transferase (TdT), which can detect apoptotic cells by fluorescence microscopy or flow cytometry¹⁷⁾. Phosphorylation of H2AX (gH2AX) is a key reaction that occurs early in DNA double-strand breaks in cells¹⁸⁾. DNA double-strand breaks can destabilize genes or cause genetic mutations. DNA double-strand breaks are caused by intra-cellular and external stimuli, stress during DNA replication, intracellular reactive oxygen species, ionizing radiation, and genotoxic substances. To repair the DNA double- strand break, the Ser139 residue at the C-terminus of the histone protein H2AX is phosphorylated, and DNA damage response proteins MDC1, 53BP1, and MRN (MRE11- RAD50-NBS1) complex repair DNA damage¹⁸⁾. When DNA repair is complete, PP2A (protein phosphatase 2A) dephosphorylates gH2AX and reduces it to H2AX. A sustained gH2AX state indicates that the repair process of the cleaved double-strand DNA proceeds very slowly or that permanent repair is impossible, leading to cell senes-cence or cell death. Significantly increased levels of TUNEL and gH2AX were observed in YD10B treated with 20 mg/ml EESG compared to the control group. This indicated that the DNA was cleaved by EESG.

Unlike cell death (necrosis), apoptosis is a process in which cells die by themselves through a suicide signaling mechanism that occurs within cells¹⁹⁾. Apoptosis is a pro-cess in which cells die on their own by suicide signaling mechanisms that rarely induce an immune response, and the cell remnants are removed by immune cells such as macrophages¹⁹⁾. Apoptosis is induced by the activation of intracellular proteolytic enzymes called caspases²⁰⁾. In the early stage of apoptosis, the inactive form of caspase-3 is converted into its active form by cleavage, and it triggers the apoptotic process by degrading various apoptosis- related proteins. PARP is an enzyme that helps repair da-maged DNA; however, during apoptosis, PARP is cleaved by activated caspase-3 and loses its function²¹⁾. Compared to the control group, procaspase-3 and procaspase-9 levels were reduced, and PARP cleavage was observed in YD10B cells treated with EESG, and this response was dependent on the EESG concentration (Fig. 2E). This result indicates that the inhibition of YD10B cell proliferation by EESG involved caspase-mediated apoptosis. Consistent with our results, the EESG also inhibited cell proliferation in a dose-dependent manner in human breast cancer cell line MCF7, colon carcinoma cell line HT-29, and gastric cancer cell line BGC-823²²⁾. In an in vivo mouse model, the anticancer effect of inhibiting cancer growth of HT-29 and murine hepatoma H22 cells by the EESG was also observed²²⁾. In addition, the proliferation of MCF-7 breast cancer cells was inhibited by the fetuin-binding glycoprotein of Smilax glabra²³⁾.

3.Limitations

For pharmacological ingredients with proven anticancer activities to be clinically applicable, a process to separate and purify functional ingredients from natural materials is required. This study investigated the anticancer activity of the EESG against oral squamous cell carcinoma cell lines. Further research is needed to isolate and identify the components with anticancer activity among the EESG and to verify their functionality in vitro and in vivo. In addi-tion, it is necessary to compare the anticancer activity of Smilax glabra through comparative analysis with drugs currently used as chemotherapy for oral cancer.

4.Conclusions

In this study, the anticancer effects of the EESG in YD10B cells were determined. The EESG inhibited cancer cell growth by inducing apoptotic cell death through G0/G1 phase arrest in YD10B cells. Through this, the pharmacological function of the EESG in controlling oral cancer growth was verified in vitro.

None.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Ethical Approval

This study does not require IRB review because it is an experimental paper using commercially available cells.

Funding

This research was supported by Eulji University in 2023 and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2022R1F1A1063204).

Data availability

The data and materials of this article are included within the article. The data supporting the findings of this study are available from the corresponding author upon reaso-nable request.