Berberine Overcomes Gemcitabine-Associated Chemoresistance through Regulation of Rap1/PI3K-Akt Signaling in Pancreatic Ductal Adenocarcinoma

Abstract

Gemcitabine (Gem)-based chemotherapy is one of the first-line treatments for pancreatic ductal adenocarcinoma (PDAC). However, its clinical effect is limited due to development of chemoresistance. Various naturally occurring compounds, including Berberine (BBR), provide an anti-cancer efficacy with time-tested safety, individually and in combination with chemotherapeutic drugs. Accordingly, we hypothesized that BBR might enhance the chemosensitivity to Gem in PDAC. In this study, cell culture studies using MIA PaCa-2 and BxPC-3 cells, followed by analysis in patient-derived organoids were performed to evaluate the anti-cancer effects of BBR in PDAC. Considering that cancer is a significant manifestation of increased chronic inflammatory stress, systems biology approaches are prudent for the identification of molecular pathways and networks responsible for phytochemical-induced anti-cancer activity, we used these approaches for BBR-mediated chemosensitization to Gem. Firstly, Gem-resistant (Gem-R) PDAC cells were established, and the combination of BBR and Gem revealed superior anti-cancer efficacy in Gem-R cells. Furthermore, the combination treatment induced cell cycle arrest and apoptosis in Gem-R PDAC cells. Transcriptomic profiling investigated the Rap1 and PI3K-Akt signaling pathway as a key regulator of Gem-resistance and was a key mediator for BBR-mediated chemosensitization in PDAC cells. All cell culture-based findings were successfully validated in patient-derived organoids. In conclusion, we demonstrate that BBR-mediated reversal of chemoresistance to Gem manifests through Rap1/PI3K-Akt signaling in PDAC.

Keywords: Berberine; Gemcitabine; Rap1/PI3K-Akt signaling pathway; chemoresistance; pancreatic ductal adenocarcinoma.

Figure 1

Berberine enhances the chemosensitivity to Gemcitabine-resistant PDAC cells in inhibiting cell proliferation. (A) Drug dose–response curves comparing cell viability following treatment with Gem in parental and Gem-R PDAC cells. Error bars are the mean ± SD; (B) Drug dose–response curves comparing cell viability following treatment with BBR in Gem-R PDAC cells. Error bars are the mean ± SD; (C) Drug dose–response curves comparing cell viability following treatment with combination of BBR and Gem in Gem-R PDAC cells. Error bars are the mean ± SD; (D) Cell proliferation assays in Gem-R PDAC cells with BBR, Gem, and their combination. Total viable cells were measured by CCK-8 assays on the indicated days. Error bars are the mean ± SD (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). Gem-R, Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; CCK-8, Cell Counting Kit-8; SD, standard deviation.

Figure 2

Berberine enhances the chemosensitivity of Gemcitabine-resistant PDAC cells by inhibiting colony formation, migration, and invasion. (A) Colony formation assays of Gem-R PDAC cells following treatment. The average (column) ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem); (B) Wound healing assays following in Gem-R PDAC cells. Images show representative areas (marked by red lines). Scale bar = 250 μm. The average (column) ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem); (C) Invasion assays following treatment in Gem-R PDAC cells. Scale bar = 100 μm. The number of cells was randomly counted at four fields, and then relative invasion ratios were calculated. The average (column) ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). Images show representative fields on the membrane (magnification x100). Gem-R, Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; SD, standard deviation.

Figure 3

Berberine and Gemcitabine induce the G0/G1 phase cell cycle arrest and enhance cell apoptosis in Gemcitabine-resistant PDAC cells. (A) Representative images of cell cycle assays following treatment in Gem-R PDAC cells. The graph indicated the average ratio (column) ± SD of cells at each stage of cell cycle (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). (B) Representative images of apoptotic cells that stained for annexin V assays following treatment in Gem-R PDAC cells. The average ratio (column) ± SD of cells undergoing apoptosis is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). Gem-R, Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; SD, standard deviation.

Figure 4

Rap1/PI3K-Akt signaling pathway correlates with Gemcitabine-resistance in PDAC cells. (A) Schematic of the differentially expressed gene discovery in Gem-R PDAC cells using GSE148200 and GSE140077. (B) Venn-diagram of up- and down-regulated expression (log₂FC > ±1.0 and p < 0.01) of the genes in Gem-R PDAC cells. (C,D) Scatter plot of KEGG pathway enrichment analysis of up- (C) and down-regulated (D) genes in Gem-R PDAC cells. The number of differentially expressed genes in the pathway is indicated by the circle area, and the circle color represents the range of p value. (E) qRT-PCR analysis of key differentially expressed genes of Rap1/PI3K-Akt signaling pathway in parental and Gem-R PDAC cells. β-Actin mRNA expression was used as an internal control. The average (column) ± SD is indicated (* p < 0.05 vs. parental). Gem-R, Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; FC, fold change; SD, standard deviation.

Figure 5

The combination treatment of Berberine and Gemcitabine regulates the activity of Rap1/PI3K-Akt signaling pathway in Gemcitabine-resistant PDAC cells. (A,B) Western immunoblotting of Rap1, PI3K, Akt, and phospho-Akt (ser473) (p-Akt) expression in Gem-R MIA PaCa-2 (A) and BxPC-3 (B) following treatment. The protein of β-Actin was used as an internal control. The average (column) ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). Gem-R, Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; SD, standard deviation.

Figure 6

The combination of Berberine and Gemcitabine suppressed spheroid formation and growth of human PDAC tumor-derived organoids. (A) Representative images of sphere forming Gem-R PDAC cells following treatment. Scale bar = 250 μm (Magnification ×40). The average (column) ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). (B) Representative images of tumor organoids following treatment. Scale bar = 100 μm (Magnification ×100). The average (column) ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). Gem-R, Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; SD, standard deviation.