Sugiol suppresses the growth, migration, and invasion of human endometrial cancer cells via induction of apoptosis and autophagy

Abstract

Recently, diterpenoids have been shown to exhibit several health benefits including cancer prevention. In the present study, we examined the anticancer effects of sugiol diterpene against the endometrial carcinoma and attempted to explore the underlying mechanisms. The results showed that sugiol significantly (P < 0.05) inhibited the proliferation of the endometrial carcinoma cell lines (HEC-1-A, HEC-1-B, and KLE) as compared to the normal THESCs cells. The IC₅₀ of sugiol against all the three endometrial carcinoma cell lines ranged between 14 and 18 µM as against an IC₅₀ of 110 μM against the normal THESCs cells. Sugiol caused several changes in the morphology of the HEC-1-B cells characteristic of apoptosis. The DAPI and annexin PI assays confirmed the induction of apoptosis in HEC-1-B cells. Sugiol also triggered increase in Bax and decrease in Bcl-2 expression. The acridine orange staining revealed that the formation of autolysosomes in HEC-1-B cells upon treatment with sugiol suggestive of autophagy. The autophagy was further confirmed by increase in the expression of LC3B-II, Beclin-1, Atg5, and Atg12 and decrease in the expression of P62. The transwell assay showed that relative to the untreated HEC-1-B cells, the migration and invasion of the sugiol-treated HEC-1-B cells was significantly (P < 0.05) inhibited. Collectively, the finding of the present study revealed that sugiol suppresses the growth of human endometrial cells via induction of apoptosis and autophagy. Consistently, sugiol may prove to be an important lead molecule in the development of chemotherapy for endometrial carcinoma.

Introduction

Endometrial carcinoma is a pathological condition characterized by the unrestrained or abnormal growth of precursor endometrium (uterus lining) cells (Piulats et al. 2017). This is a lethal gynecological syndrome predominant in women, globally (Travaglino et al. 2020). Currently, endometrial carcinoma is ranked as the 6th most prevalent type of cancer in women. Approximately 0.38 million new cases and 0.08 million endometrial carcinoma related deaths were reported across the globe in the year 2018 (Coll-de la Rubia et al. 2020). Unfortunately, the incidence of endometrial carcinoma is continuously increasing, the prognosis is poor and survival rate has declined from 88 to 84% since 1997 (Leslie et al. 2012). As such, there is urgent need to look for efficient treatment strategies for the management of endometrial carcinoma. Natural products are regarded as an important source of chemopreventive drugs since decades (Xu et al. 2010). Several epidemiological studies have reported an inverse relationship between the high intake of plant secondary metabolites (including flavonoids, terpenoids and alkaloids in the form of beverages, fruits, and vegetables) and lower risk of chronic and acute diseases including cancer (Knekt et al. 2002). Moreover, 72.9% of the anticancer drugs are natural products or drugs based on them. Out of all the natural products, plants show a remarkable diversity of chemicals that have been approved or are in clinical trials for chemotherapy. Several drugs including vinblastine, vincristine, and taxol have plants as their origin (Newman and Cragg 2012). Diterpenoids represent a diverse class of natural products with remarkable pharmacological properties. Moreover, the anticancer effects of diterpenoids are well reported in literature (Núñez et al. 2011). Sugiol is a natural diterpene mostly isolated from various species of Salvia genus (Ulubelen et al. 2000). It has also been isolated from Juniperus formosana (Kou and Yu 1996), Calocedrus formosana (Chao et al. 2005) and Metasequoia glyptostroboides (Bajpai et al. 2014a, b). Sugiol has been reported to exhibit a wide array of bioactivities such as antioxidant, antimicrobial, anticancer, and anti-inflammatory activities, to name a few (Bajpai et al. 2014a, b). Sugiol has been reported to inhibit the proliferation of human ovarian cancer cells via induction of apoptosis (Wang 2020). Similarly, it has been reported to suppress the growth of pancreatic cancer cells via G₂/M cell cycle arrest (Hao et al. 2018). In yet another study, sugiol inhibits STAT3 activity via regulation of transketolase and ROS-mediated ERK activation in DU145 prostate (Jung et al. 2015). However, there is no report on the anticancer effects of sugiol against the human endometrial cancer cells. Additionally, to the best of our knowledge, there is not a single study which reports the autophagy inducing potential of sugiol. Against this backdrop, the present study was designed to evaluate the anticancer effects of sugiol against the human endometrial carcinoma cells and to attempt to unveil the underlying molecular mechanisms. Additionally, this study also investigated the effects of sugiol on the migration and invasion of the human endometrial carcinoma cells.

Materials and methods

Cell lines, cultural conditions, reagents, and antibodies

Sugiol (> 98% purity through HPLC, CAS# 511-05-7) was purchased from BioCrick Biotech Co. Ltd Sichuan, China. The RMPI-1640 media and all reagents, unless otherwise mentioned, were purchased from Invitrogen, Inc. (Carlsbad, CA, United States). The 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), Acridine Orange (AO), Annexin-V/Propidium Iodide (Annexin-V/PI) and 4, 6-diamino-2-phenylindole (DAPI) were purchased from Serva (Heidelberg, Germany). All the primary and secondary antibodies involved in this study were purchased from Santa-Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). The normal THESCs cells (ATCC; CRL-4003) and cancerous HEC-1-B (ATCC; HTB-113), HEC-1-A (ATCC, HTB-112), and KLE (ATCC; CRL-1622) endometrial cell lines were procured from Type Culture Collection of Chinese Academy of Science (Shanghai, China). The cells were cultured in penicillin–streptomycin (100 U/mL and 100 µg/mL, respectively) and 10% fetal bovine serum maintaining RMPI-1640 medium. Cells were placed in a humidified environment having 5% CO₂ and 37 °C of temperature.

Cell viability assay

The HEC-1B and THESCs cells were plated in complete RMPI-1640 medium within 96-well plates (Costar, Cambridge, MA, United States) at a concentration of 4 × 10³ cells/well. Post culture for 24 h, the media were substituted by fresh one suspended with different sugiol doses (0–640 µM). The untreated HEC-1B and THESCs cells were kept as control. After 48 h of treatment, cells were subjected to incubation with 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 5 mg/ml) for 4 h at 37 °C. The precipitate then developed was dissolved using dimethyl sulphoxide. Consequently, microplate reader (Bio-Rad Laboratories, Hercules, CA, United States) was set at 570 nm to record optical density. The proliferation percentage was finally calculated using the following formula:

$$\mathrm{Inhibition}(\%)=100-\frac{\mathrm{OD}\mathrm{of}\mathrm{treated}\mathrm{cells}}{\mathrm{OD}\mathrm{of}\mathrm{untreated}\mathrm{control}\mathrm{cells}}\times 100$$

Phase-contrast microscopy

The HEC-1-B cells at 2 × 10⁴ cells/well of density were plated into 24-well plates (Corning Inc., Corning, NY, United States) administrated with altering doses of sugiol viz 7, 14 and 28 µM for 48 h. The untreated HEC-1-B cells were kept as control. Sugiol-induced changes in morphology of HEC-1-B were examined under a phase-contrast microscope (Olympus, Tokyo, Japan).

The 4′,6-diamidino-2-phenylindole (DAPI) staining assay

The HEC-1-B cells at 2 × 10⁴ cells/well of density were plated into 6-well plates administrated with altering doses of sugiol viz 7, 14, and 28 µM for 24 h. The untreated HEC-1-B cells kept as control. After sugiol treatment, DAPI staining was conceded by incubating the sugiol-treated cells for 20 min in 6-well plates in dark. After DAPI staining was accomplished, cells were washed in phosphate buffered saline (PBS). Then HEC-1-B cells were fixed in 10% formaldehyde and rewashed again in PBS. Consequently, DAPI stained HEC-1-B cells were loaded to glass slides and probed under fluorescence microscope.

Annexin-V/PI staining

To determine the percentage of the apoptotic cells in sugiol-treated and control-untreated HEC-1-B cells, FITC annexin-V/PI apoptosis detection kit (BD Pharmingen™) was employed in accordance with the guidelines of manufacturer. Concisely, the HEC-1-B cells were placed onto 6-well plates at a density of 3 × 10⁴ cells/well and exposed to different sugiol concentrations viz 7, 14, and 28 µM. Post 24 h of incubation period, cells were harvested followed by washing two times in cold PBS. Cell suspension was placed for centrifugation then resuspended in 1X annexin V binding buffer. Thereafter, annexin V/PI staining was performed by adding FITC annexin V (5 µL) and PI (5 µL) to each well of 6-well plates for 15 min at room temperature. Post-incubation, 1X annexin V binding buffer (400 µL) was added, and apoptotic cell percentage was determined by flow cytometry (FACScan, Dection Dickinson, United States).

Acridine-Orange (AO) staining

The HEC-1-B cells at 2 × 10⁴ cells/well of density were plated into 6-well plates. After preculturing was accomplished, each well plate was administrated with different sugiol doses (7, 14, and 28 µM) and incubated for 24 h. The untreated HEC-1-B cells were taken as control. Post-sugiol treatment, HEC-1-B cells were subjected to washing in PBS followed by staining at 37 °C by 1 µg/ml of AO (Sigma) for 20 min. Consequently, sugiol-treated and AO-stained HEC-1-B cells were investigated under confocal microscopy.

Transwell assay

Assaying of migration and invasion of endometrial HEC-1-B carcinoma cells was done using transwell chambers (BD Biosciences, United States) fitted with PET track-etched membranes. Briefly, HEC-1-B cells were cultured for 24 h and serum-starved 5 × 10³ cells were transferred into upper chambers of transwell maintaining RMPI-1640 serum-free medium and different sugiol doses viz 7, 14, and 28 µM. Untreated HEC-1-B cells were kept as control. In contrast to upper chambers, lower chambers were added of medium alone. For invasion assay, upper chambers were coated with 630 µL of Matrigel diluted to 1:3 ratio in serum-free medium prior its supplementation to serum-starved HEC-1-B cells. The HEC-1-B cells were left overnight with sugiol at incubation. When the incubation period was completed, non-migrated/non-invasive cells left over on upper membranes were swabbed and the cells on lower membranes were fixed in 100% pure alcohol and stained for 2 h with 0.5% crystal violet. Consequently, the migrated/invaded HEC-1-B cells on lower membranes were numbered and pictured under 200 × of magnification of a microscope (Nikon, Japan).

Western blotting

The primary and secondary antibodies involved in the assessment of apoptosis and autophagy allied proteins in sugiol-treated HEC-1-B cells were bought from QIAGEN Biotechnology Malaysia Sdn Bhd (Cell signalling Technology, United States). HEC-1-B cells were cultured with changing sugiol concentrations (control-28 µM) within 24-well plates at a concentration of 1 × 10⁵ cells/well for 24 h. Thereafter, protein content was extracted by lysing cells with RIPA lysis buffer comprising of protease inhibitor. Lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (12.5%) for the purpose of separation. Proteins separated were then loaded to nitrocellulose membranes which were blocked using Bowene Serum Albumin (BSA, 5%) in TBS. Blocked membranes were subjected to probing with primary antibodies overnight at 4 °C. Post primary antibodies exposure membranes were washed in tris-buffered saline, with Tween (TBST) three times. Thereafter, membranes were treated with 1:1000 dilutions of soothing HRP-conjugated secondary antibodies. Consequently, these membranes were subjected to incubation in 1X Red Alert before visualization of bands using ECL kit (GE healthcare, Uppsala, Sweden).

Statistical significance

The experiments were performed in triplicate and the final values are presented as mean ± standard deviation (SD). SPSS 15.0 software was used to carry student’s t test and one-way ANOVA. P values < 0.05 were taken as the measure of statistically significant difference.

Results

Sugiol inhibits the proliferation of endometrial carcinoma cells

The effects of sugiol molecule (Fig. 1a) on the viability of endometrial carcinoma cell lines (HEC-1-A, HEC-1-B and KLE) and the normal cell line (THESCs) was determined by MTT assay. The results showed that sugiol inhibited the viability of all the endometrial carcinoma in a dose-dependent manner. The IC₅₀ of sugiol against three endometrial carcinoma cell lines tanged between 14 and 18 µM (Table 1). The antiproliferative effects of sugiol were and significantly (P < 0.05) higher against the endometrial carcinoma cells as compared to normal THESCs cells (Fig. 1a). Sugiol exhibited an IC₅₀ value of 110 µM against the THESCs cells (Fig. 1b). Further, morphological assessment of the sugiol-treated HEC-1-B cells was performed by phase contrast microscopy. It was found that that untreated HEC-1-B control cells maintained their cellular integrity and original morphology while sugiol-treated groups showed distorted morphology including loss of cellular integrity, original shape, nucleus dislocation, membrane integrity, and rupture (Fig. 1c). When the sugiol dosage was increased, the number of dead HEC-1-B cells increased dose-dependently.

Fig. 1.

Sugiol inhibits the proliferation of the human endometrial carcinoma cells a Chemical structure of sugiol molecule. b Cell proliferation determination via MTT assay. The endometrial HEC-1-B carcinoma and THESCs normal cells were exposed to sugiol at altering concentrations as indicated for 48 h and then stained using MTT. The results denoted that sugiol inhibits the proliferation of HEC-1-B cells with higher potential (IC₅₀ of 14 µM) than THESCs cells (IC₅₀ of 110 µM). c Phase-contrast microscopy was used to analyze the morphology of sugiol-treated HEC-1-B cells. Results illustrated that sugiol-induced changes in the normal cellular morphology including loss in cellular and membrane integrity, nucleus dislocation and membrane rupture. The experiments were performed in triplicate and the values are presented as mean ± SD (P < 0.05)

Table 1.

The IC₅₀ of sugiol against different endometrial carcinoma cell lines and the normal cells as determined by the MTT cell viability assay

S. No	Cell lines	IC50 (μM)
1	HEC-1-A	14
2	HEC-1-B	14
3	KLE	16
4	THESCs	110

Sugiol induces apoptosis in endometrial carcinoma cells

Apoptosis being the primary target in chemoprevention, the proapoptotic effects of sugiol in HEC-1-B cells were evaluated using DAPI staining, annexin-V/PI staining, and Western blotting. The results revealed in Fig. 2a showed that sugiol-treated HEC-1-B cells demonstrated membrane blebbing, DNA fragmentation, and formation of apoptotic crops as compared to the untreated control cells. Further, annexin-V/PI assay results showed enhanced numbers of apoptotic (early and late) and necrotic cells in comparison to control group. The percentage apoptotic HEC-1-B cells increased in a dosage-dependent manner. The results of annexin-V/PI staining were demonstrated in dot-plot graphs representing upper left quadrant (necrotic cells), upper right quadrant (late-apoptotic cells), lower right quadrant (early apoptotic cells), and lower left quadrant (viable cells). The total percentage apoptotic cells were found to be 21.78% at IC₅₀ and 33.28% at 28 µM of sugiol dose in comparison to 3.04% apoptotic cells in control (Fig. 2b). Moreover, Western blotting showed that sugiol caused considerable increase in Bax and decrease in the expression of Bcl-2 proteins in HEC-1-B cells (Fig. 2c). Therefore, our findings indicate that sugiol-induced antiproliferative effects on HEC-1-B could be due to the induction of apoptosis.

Fig. 2.

Sugiol induces apoptosis in endometrial carcinoma cells a DAPI-staining assay was carried out to monitor apoptosis in sugiol-treated HEC-1-B cells. It demonstrated membrane blebbing and apoptotic crops in treated groups in comparison to controls. b Annexin-V/PI staining assay was used to quantify the apoptotic effects of sugiol in HEC-1-B cells. The results were presented in dot-plot graphs indicated increase in the number of apoptotic and necrotic cells in treated groups. c Western blotting was used to examine the expressions of Bax and Bcl-2. In sugiol-treated groups, the expressions of Bax were higher and that of Bcl-2 was lower. The experiments were performed in triplicate

Sugiol induces autophagy in endometrial carcinoma cells

To ascertain whether autophagy has a role in antiproliferative effects induced by sugiol, we performed AO staining of the HEC-1-B cells to monitor the formation of autophagic vesicles. AO is being extensively used as an indicator for acid vesicular organelles as it switches from green to red inside acidic compartments like autophagosomes and lysosomes. We noticed enhanced red fluorescence in sugiol-treated groups while control groups showed only green fluorescence (Fig. 3a). Meanwhile, we observed that sugiol caused accumulation of LC3B-1, LC3B-II, Beclin-1, Atg5, and Atg12 in HEC-1-B cells and decrease of p62 in comparison to control group (Fig. 3b). These proteins are considered as autophagosomal markers in multicellular organisms. Therefore, our findings suggest the involvement of autophagy together with apoptosis in sugiol triggered antiproliferative effects against the endometrial carcinoma cells.

Fig. 3.

Sugiol induces autophagy in endometrial carcinoma cells a AO staining was performed to assess the formation of autophagosomes. Results showed higher transformation of green fluorescence to red fluorescence in sugiol-treated groups than in untreated controls. This indicated the formation of acidic vesicles or autophagosomes in HEC-1-B cells. b Western blotting was performed to monitor the activity of autophagy marker proteins. Results demonstrated higher expression of LC3B-1, LC3B-2, Beclin-1, Atg5, and Atg12 and lower activity of p62 in sugiol-treated groups. The experiments were performed in triplicate

Sugiol inhibits migration and invasion of endometrial cancer cells

To examine the effects of sugiol on cellular migration and invasion of HEC-1-B cells, we performed transwell assays. Results showed that sugiol significantly (P < 0.05) migration and invasion of HEC-1-B cells in a concentration-dependent manner (Fig. 4a, b). The percentage migration cells reduced from 100 to 21% on increasing sugiol dosage ranging from 0 to 28 µM and at IC₅₀ of sugiol the percentage migration was observed to be 52%. The percentage invasion cells reduced from 100 to 16% on increasing sugiol dosage from 0 to 28 µM and at IC₅₀, the percentage migration was observed to be 43%. Therefore, our results suggest that sugiol retards the migration and invasion of endometrial carcinoma cells.

Fig. 4.

Sugiol inhibits migration and invasion of human endometrial carcinoma cells. a Transwell assay showing migration of HEC-1B was inhibited by sugiol in a dose-dependent manner. b Transwell assay showing invasion of HEC-1B was inhibited by sugiol in a dose-dependent manner. The experiments were performed in triplicate and the values are presented as mean ± SD (P < 0.05)

Discussion

Endometrial carcinomas are the most common primary gynecological tumors found in women and standard treatment methodologies include abdominal hysterectomy, hormone therapy, radiation therapy, and chemotherapy (Li et al. 2018). Unfortunately, despite the recent advancements in cancer research, the overall survival rates for the advanced stages endometrial carcinoma are still far from descent (Gibson et al. 2016). Therefore, there is an emergency to develop new potential treatment methodologies for endometrial carcinoma in general and for advanced stages of the disease in particular. In the present investigational study, we examined the anticancer effects of sugiol diterpene against the human endometrial carcinoma and also evaluated its possible mechanism of action. The results revealed that sugiol inhibits the proliferation of endometrial carcinoma cells with comparatively lower antiproliferative effects against the normal endometrial carcinoma cells. These results suggest that sugiol selectively targets the cancer cells and points toward the applicability of the as an anticancer agent. The high-antiproliferative activity against the endometrial carcinoma cells and low-antiproliferative effects on the normal cells may allow the use of sugiol at lower doses to specifically kill cancer cells without affecting the normal cells. Several signaling cascades are aberrantly activated or deactivated in cancer cells (Wang et al. 2020) and the potential of sugiol to modulate these pathways may be responsible for the specific anticancer activity of sugiol. Nonetheless, the understating of exact underlying mechanism for cancer-cell-specific antiproliferative effects of sugiol requires further research endeavors and needs to be elucidated in future studies. Our findings are consistent with previous studies wherein sugiol has been shown to inhibit the proliferation of human cancer cells (Córdova et al. 2006; Shin et al. 2007).

Apoptosis is a primary cell killing mechanism in higher mammals often termed as type-1 programmed cell death (Redza-Dutordoir and Averill-Bates 2016). Apoptosis is regulated by a number of intrinsic and extrinsic factors and several pathways are involved in its onset. The members of Bcl-2 family proteins are regarded as key apoptosis regulatory proteins including proapoptotic Bax and antiapoptotic Bcl-2 (Burlacu 2003). Apoptosis is a first line target of chemopreventives and the compounds that can induce apoptosis are considered essential for the development of chemotherapy (Burlacu 2003). The morphological changes in sugiol-treated HEC-1-B cells indicated apoptosis as a possible mechanism for antiproliferative effects of sugiol in HEC-1-B cells. Based on this, we performed DAPI and annexin-V/PI staining in HEC-1-B cells to monitor proapoptotic effects of sugiol. Interestingly, we found that sugiol-induced membrane blebbing, DNA fragmentation, and formation of apoptotic crops, which indicated apoptosis. Further, the expressions of Bax and Bcl-2 were determined to ensure the apoptotic cell death in sugiol-treated HEC-1-B cells. We found enhanced Bax and reduced Bcl-2 levels which further strengthened the proapoptotic potential of sugiol. Our results are parallel to previous studies wherein sugiol-induced apoptosis against a number of human cancer cells including colon cancer and ovarian cancer (Souren et al. 2013; Wang et al. 2020).

Autophagy is a self-renewal process which plays housekeeping role including elimination of damaged/malfunctioning cells, peroxisomes, endoplasmic reticulum and mitochondria, and aggregated/misfolded proteins (Glick et al. 2010). In the present study, it was found that sugiol-induced autophagy-mediated antiproliferative effects in HEC-1-B cells as indicated by the results of AO staining. Further, the activities of marker proteins LC3B-1, LC3B-2, Beclin-1, Atg5, and Atg12 were elevated in sugiol-treated groups. The activity of autophagy suppresser gene p62 was downregulated by sugiol in HEC-1-B cells in comparison to controls. Therefore, our results were in consistent with previous study by Scariot et al. wherein sugiol triggered autophagy in leishmania infantum (Scariot et al. 2019). Cellular migration and cellular invasion are the vital features of cancer metastasis and progression (Takano et al. 1994). Herein, we found that sugiol suppresses the migration and invasion of the of HEC-1-B cells via transwell assay via inhibition of MM-2/9 expressions. These results suggest that sugiol might exhibit anti-metastatic effects on human cancer cells.

Taken together, the findings of the present study revealed that sugiol selectively inhibits the growth of human endometrial carcinoma cells via induction of apoptosis and autophagy. These results point toward the applicability of sugiol as a lead molecule for the development of chemotherapy for endometrial carcinoma. However, the evaluation of sugiol against more cell lines and under in vivo conditions is required for further validation of the present findings. Additionally, research studies may be directed to semi-synthesize sugiol derivatives with comparatively higher efficacy against human endometrial carcinoma.

Author contributions

Conceptualization: HZ and XZ; methodology: HZ and XZ; formal analysis and investigation: HZ and XZ; writing—original draft preparation: HZ and XZ; writing—review and editing critically for important intellectual content: HZ and XZ; supervision: XZ.

Funding

This research did not obtain any specific Grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interest

All the authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.