Colorectal cancer (CRC) is one of the most representative high-risk cancers, ranking third in terms of cancer incidence and second in terms of mortality, according to 2020 GLOBOCAN statistics (Sung et al., 2021). CRC risk factors vary by country according to age and gender, with males showing higher incidence and mortality rates than females (Khil et al., 2021). Hormones can be considered as the primary factor contributing to gender sensitivity, and several previous studies have revealed the protective effect of estrogen against CRC (Di Leo et al., 2001).

Therefore, this study focused on the G protein-coupled estrogen receptor (GPER), which has previously been identified as an additional receptor for estrogen. GPER is one of the seven transmembrane receptors and acts as a mediator of estrogen in the nervous, immune, cardiovascular, and reproductive systems, as well as in bone metabolism and cancer. Considering that GPERs are not located inside the nucleus like typical estrogen receptors (ERs), such as ERα and ERβ, but are rather located within the cell membrane, they have different mechanisms of action, which have not yet been fully clarified (Liu et al., 2017; Jung, 2019; Luo and Liu, 2020).

To understand the action of GPER, several ligands including estrogen have been investigated (Qiu et al., 2021). G1 Tomoxifen, as a selective ER modulator, and ICI 182,780, as an ER antagonist, are known as GPER agonists, while G15 is known as a GPER antagonist (Hsu et al., 2019). Various flavonoids known as phytoestrogen are also considered to act via GPER (D’Arrigo et al., 2021). Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a member of the natural flavonoid family including various fruits and vegetables, has various physiological activities such as anti-inflammatory and antioxidant effects (Arai et al., 2000; Xia et al., 2017; Meng et al., 2021). Therefore, it is a common ingredient in healthy functional foods and known for its minimal adverse effects (Khan et al., 2013). Several previous studies have reported that resveratrol shows anticancer effects, although its molecular mechanism remains unclear (Ko et al., 2017; Lucas et al., 2018; Ren et al., 2021).

In this study, resveratrol as a GPER agonist was investigated and compared with G1 and G15 as negative control (Fig. 1). To elucidate whether GPER could play a role as a target on the chemotherapeutic effect of CRC, we investigated whether resveratrol showed an anticancer effect depending on GPER expression.

Figure 1. Structural formulas of compounds used in the experiments.

Cell lines and culture

Human colorectal cancer RKO cells (CRL-2577, ATCC, Manassas, VA, USA) and WiDr cells (CCL-218, ATCC) were grown in an Eagle’s minimum essential medium (GenDEPOT, Barker, TX, USA) containing 10% fetal bovine serum (GW Vitek, Seoul, Korea) and 1% penicillin/streptomycin (GenDEPOT) at 37°C in a humidified 5% CO₂ incubator.

Organoid culture

This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Korea Cancer Center Hospital (approval no. KIRAMS-2017-07-001). Informed consent was not required for this study. Human CRC patient-derived organoids (30T and 33T) were obtained from the KIRAMS. The culture medium contained 1×B27 (Gibco, Grand Island, NY, USA), 1.25 mM N-acetyl cysteine (United States Pharmacopeia, Rockville, MD, USA), 50 ng/mL human epidermal growth factor (BioVision Inc., Milpitas, CA, USA), 50 ng/mL human Noggin (Peprotech, Rocky Hill, NJ, USA), 10 nM gastrin (Sigma-Aldrich, Merck KGaA, St. Louis, MO, USA), 500 nM A83-01 (BioVision Inc.), and 100 mg/mL primocin (InvivoGen, San Diego, CA, USA). For the first 2-3 d, organoids were cultivated in a culture medium also containing 10 M Y-27632 to prevent anoikis.

Cell viability assay

The viability of RKO and WiDr cells was evaluated via MTT assay. The cells (3-5×10³ cells/well) were seeded into 96-well plates before being incubated for 24 h. Resveratrol (CAS No. 501-36-0, Aladdin, Shanghai, China), G1 (Cayman, MI, USA), and G15 (Cayman) were dissolved in 0.2 % dimethyl sulfoxide (DMSO) (Sigma-Aldrich). The cells were then treated with resveratrol (25, 50, 60, and 70 μM), G1 (0.5, 0.75, 1, 1.25 μM), and G15 (0.34, 0.67, 1.35, 2.7 μM) for 48 h. A 10 μL solution of MTT (5 mg/mL, BioVision, Inc.) was applied to each well before incubating for 3 h at 37°C. After removing the medium, 100 μL of DMSO (Sigma-Aldrich) was added to each well before being incubated for 30 min.

The viability of organoids was assessed by MTS assay according to a previous study (Park et al., 2020). Suspended organoids (5,000 cells) in the gel were then divided into a 96-well plate including 10 µL/well of matrigel, before 100 µL of culture medium was added. After 24 h, organoids were treated with resveratrol (25 and 50 μM in 0.2% DMSO) for 7 d. MTS solution (20 µL in each well, G3580, Promega, Madison, WI, USA) was added to each well before being incubated at 37°C for 3 h.

Subsequently, 560 nm (MTT assay) or 490 nm (MTS assay) of absorbance was measured using a microplate reader (Infinite M200 PRO, TECAN, Männedorf, Switzerland). All assays were repeated thrice. Additionally, for all assays, data are presented as mean ± standard deviation (SD) (n=6).

Clonogenic assay

RKO and WiDr cells were both seeded in 6-well plates at densities of 100 cells/well (control cells) and 500 cells/well (resveratrol- or G1-treated cells) (n=6 per group), respectively. After treatment with resveratrol (25 μM) and G1 (0.5 μM), cells were incubated for 10 d to form colonies of 50 cells or more. Subsequently, 0.25% crystal violet (Sigma-Aldrich) in 25% methanol (DUKSAN, Seoul, Korea) was applied to stain the colonies. The plating efficiency (PE) was used to standardize the variation in the number of seeded cells. The survival fraction (SF) was calculated using each of the following equations:

Animals

The animal study was approved by the Duksung Women’s University Institutional Animal Care and Use Committee (2022-001-012) according to the regulations for the care and use of laboratory animals. BALB/c-Foxn1nu/Arc (Balb/c-nude) mice (male, 5-weeks old) were purchased from JA BIO (Suwon, Korea). The mice were acclimated to the animal laboratory environment for a week, under the following conditions: 28°C, 50% humidity, a 12 h light/dark cycle, and unlimited food and water.

Tumor growth curve

To establish the CRC cells-derived xenograft models, 100 µL/mice of RKO (5×10⁷ cells/mL with 10% matrigel) and WiDr (3×10⁷ cells/mL with 10% matrigel) cells were both subcutaneously transplanted into Balb/c-nude. Once the volume of tumors reached approximately 50 mm³, the mice were randomly divided into two groups (control and resveratrol-treated groups, n=5/group). Resveratrol (200 mg/kg) was intraperitoneally injected 3 d per week for four weeks, as previously described (Chávez et al., 2008; Ji et al., 2015). Tumor size was also measured thrice per week using calipers, with tumor volume being calculated using the following equation:

Western blot analysis

CRC cells (RKO and WiDr), patient-derived organoids (30T and 33T), and tumor tissues were all lysed with radioimmunoprecipitation assay (RIPA) buffer (GenDEPOT) containing protease (P3100, GenDEPOT) and phosphatase (Roche, Basel, Switzerland) inhibitors. Proteins (20 g) were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Sigma-Aldrich). Primary antibodies were incubated on the membranes overnight at 4°C. Antibodies included extracellular signal-regulated kinase (ERK, Invitrogen, Carlsbad, CA, USA, 1:1,000), p-ERK (Invitrogen, 1:1,000), Akt (Santa Cruz Biotechnology, Dallas, TX, USA, 1:1,000), p-Akt, poly (ADP-ribose) polymerase (PARP, Cell signaling technology, 1:1,000), Bax (BD Biosciences, Franklin Lakes, USA, 1:1,000), GPER (ab137479, Abcam, Eugene, OR, USA, 1:1,000), and GAPDH (Sigma-Aldrich, 1:5,000). Subsequently, the washed membranes were incubated with secondary anti-mouse and anti-rabbit antibodies (1:3,000) for 3 h at 25°C. Proteins were then visualized using a chemiluminescent solution (Thermo Fisher Scientific, Waltham, MA, USA), and images were obtained using a Chemi-doc (FluorChem E system, San Jose, CA, USA).

Statistical analysis

All data were evaluated by Student’s t-test or analysis of variance (ANOVA) using GraphPad Prism 7 (GraphPad Software Inc., CA, USA). Dunnett’s test and Sidak’s test were also used for multiple comparisons via the one-way and two-way ANOVA post hoc analyses, respectively. Data are presented as the mean ± SD, with statistical significance being set at p<0.05.

Comparison of GPER expression levels in CRC cell lines and patient-derived organoids

To investigate whether the effect of resveratrol was affected by GPER expression level, the expression levels of GPER were compared among cell lines and patient-derived organoids. As shown in Fig. 2A, RKO was selected as a CRC cell line with high GPER expression, whereas WiDr was selected as a CRC cell line with low expression. Additionally, among the eight patient-derived CRC organoids, 30T was selected as an organoid with high GPER expression, whereas 33T was selected as an organoid with low GPER expression (Fig. 2B).

Figure 2. Protein expression level of GPER in (A) CRC cells and (B) human CRC organoids. GPER protein levels were examined in (A) three types of CRC cells and (B) each CRC organoids derived from eight patients.

Anticancer effect of resveratrol is more sensitive in RKO cells than in WiDr cells

Resveratrol inhibited the cell viability more sensitively in RKO cells than in WiDr cells (Fig. 3A). The IC50 values of resveratrol were 58.7 μM for RKO cells and 181.9 μM for WiDr cells. G1 as a GPER agonist also showed higher cytotoxicity in RKO cells than in WiDr cells, whereas G15 as a GPER antagonist showed no cytotoxicity in either cells. In addition, resveratrol significantly suppressed cell proliferation in RKO cells than in WiDr cells (Fig. 3B). To investigate the mechanism of resveratrol, several protein levels were compared in these cells treated with resveratrol. As shown in Fig. 3C, the expression levels of p-ERK, Bax, and cleaved-PARP proteins were increased in RKO cells but remained unchanged in WiDr cells. However, resveratrol decreased the expression level of p-Akt only in WiDr cells.

Figure 3. Anticancer effect of resveratrol in CRC cells. (A) Inhibition of cell viability by resveratrol (Res). Res, G1, and G15 were treated in RKO and WiDr cells for 48 h, while cell viability was measured by MTT assay as described in the Materials and Methods. Data represent the mean ± SD (n=6). *p<0.05; ***p<0.001 (vs. control, one-way ANOVA with Dunnett’s post hoc test). (B) Suppression of cell proliferation by Res. As the clonogenic assay, the survival fraction (SF) was calculated. Data represent the mean ± SD (n=3). *p<0.05; **p<0.001; ***p<0.0001 (One-way ANOVA with Dunnett’s post hoc test). (C) Change in protein expression levels by Res. Cells were treated with Res (100 μM) and G1 (2 μM) for 3 h. All data represent the mean ± SD (n=3). *p<0.05, **p<0.0001 (Two-way ANOVA with Sidak’s post hoc test).

Inhibition of tumor growth by resveratrol in CRC cell-derived xenograft models

Resveratrol significantly inhibited tumor growth in the RKO cell-derived xenograft model but not in the WiDr cell-derived xenograft model (Fig. 4A). No change was observed in the body weight for these models (Fig. 4B). However, according to the ethical guidelines for experimental animals, a tumor volume exceeding 2,000 mm³ was considered as an indicator of death; therefore, survival rate was measured (Fig. 4C). The final survival rate of the control group was 16.7%, whereas that of resveratrol-treated group was 50%. Furthermore, resveratrol increased p-ERK, Bax, and cleaved-PARP protein expression only in the tumor tissues of the RKO cell-derived xenograft model but not in those of the WiDr cell-derived xenograft model (Fig. 4D).

Figure 4. Suppression of tumor growth by resveratrol in xenograft models. Res (200 mg/kg, i.p., 3 times/week) was treated into RKO cells- and WiDr cells-derived xenograft models. (A) Tumor growth delay. (B) Change in body weight in xenograft models. (C) Survival rate of Res in RKO cells-derived xenograft model. All data represent the mean ± SD (n=5). *p<0.05 (Two-way ANOVA with Sidak’s post hoc test). (D) Protein expression levels of tumor tissues derived from xenograft models. Data represent the mean ± SD (n=6). *p<0.05, **p<0.0001 (Two-way ANOVA with Sidak’s post hoc test).

Chemotherapeutic effect of resveratrol in CRC patient-derived organoids with high GPER expression

The chemotherapeutic effect of resveratrol was further investigated in two CRC patient-derived organoids, as shown in Fig. 2B. Resveratrol significantly inhibited cell proliferation only in 30 T with high-GEPR expression but not in 33 T with low GPER expression (Fig. 5A). The morphology of two organoids was shown in Fig. 5B. The expression levels of p-ERK, Bax, and cleaved-PARP proteins were increased by resveratrol via GPER in organoids (Fig. 5C). However, resveratrol decreased the expression level of p-Akt in 33T as in WiDr cells.

Figure 5. Anticancer effect of resveratrol in CRC patient-derived organoids. (A) Suppression of organoid viability. Res was treated into 30T and 33T organoids for 7 d, and viability was measured by MTS assay as described in the Materials and Methods. Data represent the mean ± SD (n=6). ***p<0.001 (vs. control, one-way ANOVA with Dunnett’s post hoc test). (B) Morphology of CRC patient-derived organoids (30T and 33T) treated with Res. (C) Change in protein expression levels by Res.

Our results suggested that resveratrol stimulated an anticancer effect via GPER activation, which is a target for the therapeutic strategy of CRC. To elucidate the anticancer effect of resveratrol via GPER, we compared them between two CRC cell lines and two xenograft models, depending on GPER expression level (Fig. 3, 4). Notably, our hypothesis was also demonstrated in CRC patient-derived organoids (Fig. 5), which have the patient’s physiological and genetic characteristics (Broutier et al., 2017; Kondo and Inoue, 2019; Kwon et al., 2021; Sakalem et al., 2021).

Resveratrol reportedly increases the expression of antioxidant enzymes (Xia et al., 2017) and regulates molecular signaling pathways such as AMPK, ROS, and Wnt (Vernousfaderani et al., 2021). Resveratrol also possesses anti-inflammatory and antioxidant activities, which can potentially serve as chemo-adjuvant (Arai et al., 2000; Xia et al., 2017; Meng et al., 2021). The cytotoxicity of resveratrol in WiDr cells expressed a low level of GPER, which may have been shown by these physiological activities (Fig. 3). As its mechanism, resveratrol inhibits CRC epithelial-to-mesenchymal metastasis through the expression of Snail/E-cadherin via the TGF-β1/Smads signaling pathway (Ji et al., 2015) and induces apoptosis by increasing reactive oxygen species through mitochondrial pathway (Fu et al., 2021). Our results demonstrated that resveratrol significantly delayed tumor growth, while inducing apoptosis through the ERK and Akt pathways in CRC. The ERK (Zhou et al., 2019), Bax (Bahadori et al., 2016), and PARP pathways (Cruz-Nova et al., 2018) have all been reported to induce apoptosis and inhibit cell proliferation.

The role of GPER in the progression of CRC has not yet been elucidated and thus remains a topic of debate. Steroid sulfatase activates the estrogen pathway via GPER activation and increased the proliferation of CRC (Gilligan et al., 2017). However, GPER activation increased ER stress in breast cancer MCF-7 cells (Vo et al., 2019) and blocked Wnt-induced-JUN upregulation in CRC H29 cells (Abancens et al., 2022), leading to apoptosis. Our results showed that resveratrol stimulated the anticancer effect on high GPER-expressed cancer cells. D’Arrigo et al. (2021) previously reported that resveratrol showed direct docking to GPER binding pocket. Our results also suggested that resveratrol induced the phosphorylation of ERK and increased Bax, while cleaving PARP via GPER in vitro and in vivo. This indicated that the activity of GPER-mediated signaling by resveratrol inhibited CRC progression (Fig. 6). In particular, the anticancer effect of resveratrol on CRC patient-derived organoids suggests its potential for clinical applications in CRC treatment.

Figure 6. Anticancer effect of resveratrol depending on GPER expression in CRC.

This research was supported by an NRF grant funded by MSIT (NRF-2021R1A2C2004535), the Priority Research Centers Program through NRF funded by the Ministry of Education (2016R1A6A1A03007648) (J.J.), and a grant of the KIRAMS funded by MSIT, the Republic of Korea (No. 50542-2022) (U.S.).

The authors have no conflicts of interest relevant to this study to disclose.