Isoliquiritigenin Inhibits Gastric Cancer Stemness, Modulates Tumor Microenvironment, and Suppresses Tumor Growth through Glucose-Regulated Protein 78 Downregulation

Abstract

Chemotherapy is the treatment of choice for gastric cancer; however, the currently available therapeutic drugs for treatment have limited efficacy. Cancer stemness and the tumor microenvironment may play crucial roles in tumor growth and chemoresistance. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum chaperone facilitating protein folding and cell homeostasis during stress and may participate in chemoresistance. Isoliquiritigenin (ISL) is a bioactive flavonoid found in licorice. In this study, we demonstrated the role of GRP78 in gastric cancer stemness and evaluated GRP78-mediated stemness inhibition, tumor microenvironment regulation, and chemosensitivity promotion by ISL. ISL not only suppressed GRP78-mediated gastric cancer stem cell-like characteristics, stemness-related protein expression, and cancer-associated fibroblast activation but also gastric tumor growth in xenograft animal studies. The findings indicated that ISL is a promising candidate for clinical use in combination chemotherapy.

Keywords: GRP78; cancer stemness; chemosensitivity; gastric cancer; isoliquiritigenin; tumor microenvironment.

Figure 1

Glucose-regulated protein 78 (GRP78) was overexpressed in patients with gastric cancer. (A) Expression of GRP78 in tumor and normal tissues in patients with stage I cancer. (B) Quantification of GRP78 expression in the tissue samples of patients with gastric cancer. (C) GRP78 expression was analyzed by performing immunohistochemistry (IHC) analysis in tumor and normal tissue samples of patients with stage I to stage IV cancer. (D) Quantification of GRP78 expression in the tumor tissue samples of patients with stage I gastric cancer compared with their corresponding normal tissues through IHC staining. (E) Kaplan–Meier curves for overall survival rates associated with GRP78 expression in gastric cancer. (F) Quantification of GRP78 expression analyzed through IHC staining in the tumor tissues of patients with stage I to stage IV cancer. Data are expressed as the mean ± standard error of mean; n ≥ 3 independent experiments, two-tailed Student’s t test: ** p < 0.01, *** p < 0.005.

Figure 2

Glucose-regulated protein 78 (GRP78) was overexpressed in spheroid gastric cancer cells. (A) A representative picture demonstrating the MKN45 cells with the spheroid-forming ability. (B) Protein expression of GRP78 in parental and spheroid body-forming MKN45 cells. (C) Quantification of GRP78 expression in parental and spheroid body–forming MKN45 cells. Data are expressed as the mean ± standard error of mean; n ≥ 3 independent experiments, two-tailed Student’s t test: ** p < 0.01.

Figure 3

Glucose-regulated protein 78 (GRP78) promoted stemness in MKN45 cells. (A) Representative pictures showing spheroid body-forming and (B) colony-forming MKN45/ctrl and MKN45/GRP78⁺ cells. (C) Stemness-related surface makers (LGR5, CD24, and CD44) were analyzed through flow cytometry in MKN45/ctrl and MKN45/GRP78⁺ cells. (D) Quantification of the expression of surface makers (LGR5, CD24, and CD44). (E) Aldehyde dehydrogenase 1-positive cells were analyzed through flow cytometry among MKN45/ctrl and MKN45/GRP78⁺ cells. (F) Protein expression of stemness-related transcription factors (SOX2 and Nanog) and GRP78 in MKN45/ctrl and MKN45/GRP78⁺ cells. (G) Quantification of SOX2, Nanog, and GRP78 expression in MKN45/ctrl and MKN45/GRP78⁺ cells. Data are expressed as the mean ± standard error of mean; n ≥ 3 independent experiments, two-tailed Student’s t test: * p < 0.05, ** p < 0.01.

Figure 4

The stemness characteristic was reduced by isoliquiritigenin (ISL) treatment. (A) Structural formula of ISL. (B) Glucose-regulated protein 78 (GRP78) expression in MKN45 cells was inhibited after treatment with 15 or 25 μg/mL ISL for 72 h. (C) Quantification of GRP78 expression after ISL treatment. (D) The protein expression of the transcription factor CREB3L1 was downregulated in MKN45 cells after treatment with 15 or 25 μg/mL ISL for 72 h. (E) Quantification of CREB3L1. (F) Representative pictures demonstrating that the colony-forming MKN45 cells were decreased after treatment with 25 μg/mL ISL. (G) Representative pictures demonstrating that the spheroid body-forming MKN45 cells were decreased after treatment with 25 μg/mL ISL. (H,I) The protein expression of stemness-related transcription factors (SOX2 and Nanog) was downregulated in MKN45 cells after treatment with 15 or 25 μg/mL ISL for 72 h. Data are presented as the mean ± standard error of mean; n ≥ 3 independent experiments, two-tailed Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 5

Glucose-regulated protein 78 (GRP78) silencing reduced the stemness capacity of MKN45 cells. (A) Representative pictures showing the spheroid-forming capacity between MKN45/ctrl and MKN45/sh-GRP78 cells. (B) Representative pictures showing the colony-forming capacity between MKN45/ctrl and MKN45/sh-GRP78 cells. (C) Expression of surface makers (LGR5, CD24, and CD44) was analyzed through flow cytometry in MKN45/ctrl and MKN45/sh-GRP78 cells. (D) Quantification of surface makers. (E) Aldehyde dehydrogenase 1-positive cells were analyzed through flow cytometry in MKN45/ctrl and MKN45/sh-GRP78 cells. (F) Protein expression of stemness-related transcription factors (SOX2 and Nanog) and GRP78 was analyzed through Western blot in MKN45/ctrl and MKN45/sh-GRP78 cells. (G) Quantification of the expression of stemness markers (SOX2 and Nano) and GRP78. Data are presented as the mean ± standard error of the mean; n ≥ 3 independent experiments, two-tailed Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 6

Knockdown of glucose-regulated protein 78 (GRP78) reduced tumor growth in gastric cancer xenografts. (A) Schematic of gastric cancer xenografts between MKN45/ctrl and MKN45/shGRP78 groups. (B) The curves of tumor growth in mice between MKN45/ctrl and MKN45/shGRP78 groups. (C) Representative images of hematoxylin and eosin and immunohistochemical (IHC) staining of ki-67 between MKN45/ctrl and MKN45/shGRP78 groups. (D) Representative IHC analysis of GRP78 in MKN45/ctrl and MKN45/shGRP78 gastric cancer xenografts and relative quantification per intensity of staining scoring. (E) Representative IHC analysis of ki-67 staining in MKN45/ctrl and MKN45/shGRP78 gastric cancer xenografts and relative quantification per intensity of staining scoring. (F) Curves of body weight of mice between MKN45/ctrl and MKN45/shGRP78 groups. (G) Tumor sizes of each group. Data are presented as the mean ± standard error of mean; n ≥ 4 independent experiments, two-tailed Student’s t test: ** p < 0.01, *** p < 0.005.

Figure 7

Glucose-regulated protein 78 (GRP78) expression in gastric cancer cells induces cancer-associated fibroblast activity. (A) Levels of tumor growth factor (TGF)-β1 in the condition medium of MKN45/ctrl, MKN45/GRP78⁺, and MKN45/sh-GRP78 cells. (B) Levels of TGF-β1 in the condition medium in MKN45 cells with or without ISL treatment. (C) Protein expression of α-SMA and matrix metalloproteinase (MMP-9) was analyzed through a Western blot after treatment with 15 and 25 μg/mL isoliquiritigenin (ISL) for 48 h in h-GCA N3 cells. (D,E) Quantification of α-SMA and MMP-9 expression was analyzed through a Western blot after treatment with 15 and 25 μg/mL ISL for 48 h. (F) Levels of IL-6 in the conditioned medium in h-GCA N3 cells with or without ISL treatment. (G) Levels of TGF-β1 in the conditioned medium in h-GCA N3 cells with or without ISL treatment. Data are presented as the mean ± standard error of mean; n ≥ 3 independent experiments, two-tailed Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 8

Isoliquiritigenin (ISL) inhibited 5-fluorouracil (5-FU)-induced glucose-regulated protein 78 (GRP78)-mediated stemness. (A) GRP78 expression was analyzed through Western blot after treatment with 0.5 μM 5-FU for 72 h. (B) Quantification of GRP78 expression. (C) Expression of surface markers (LGR5, CD24, and CD44) was analyzed through flow cytometry in MKN45 cells after treatment with ISL or ISL combined with 5-FU. (D) Quantification of surface makers. (E,F) Representative pictures showing the spheroid-forming capacity of MKN45 cells treated with or without ISL combined with 5-FU. (G) The viability of the MKN45, ISL-treated MKN45, MKN45/sh-GRP78, 5-FU-treated MKN45, 5-FU+ISL-treated MKN45, and 5-Fu-treated MKN45/sh-GRP78 cells was evaluated. Data are presented as the mean ± standard error of mean; n ≥ 3 independent experiments, two-tailed Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 9

Pretreatment of isoliquiritigenin (ISL) reduced tumor growth and promoted chemosensitivity to 5-fluorouracil (5-FU). (A) Schematic of gastric cancer xenografts in each group. (B) Tumor growth curves of the Ctrl, ISL, 5-FU, and ISL+5-FU groups. (C) Representative images of hematoxylin and eosin and immunohistochemical (IHC) staining of GRP78 and α-SMA in each group. (D) Representative IHC analysis of GRP78 in gastric tumor xenografts (E) Representative IHC analysis of α-SMA in gastric tumor xenografts. (F) Curves of body weight of mice in each group. (G) Gastric tumors harvested from each group. Data are presented as the mean ± standard error of mean; n ≥ 7 independent experiments, two-tailed Student’s t test: ^# p < 0.05, ^### p < 0.005, *** p < 0.005.

Figure 10

Graphical scheme depicting that isoliquiritigenin (ISL) inhibited gastric cancer stemness and tumor growth and regulated the tumor microenvironment by downregulating GRP78.