Crocin reverses 1-methyl-3-nitroso-1-nitroguanidine (MNNG)-induced malignant transformation in GES-1 cells through the Nrf2/Hippo signaling pathway

Abstract

Background: Crocin, an active constituent of saffron, has anticancer activity. In this study, we investigated the relationship of Crocin with human gastric epithelial cells induced by 1-methyl-3-nitroso-1-nitroguanidine (MNNG), and explored the underlying mechanism.

Methods: In vivo, the animal growth and atypical hyperplasia were observed in Sprague-Dawley rats. A cell model was established by treating the human gastric mucosa epithelial cell line GES-1 with MNNG. The effects of Crocin on proliferation, cell cycle, apoptosis, and epithelial-mesenchymal transition (EMT) in GES-1 cells were analyzed using Cell Counting Kit-8, colony formation, flow cytometry, and Transwell assay, respectively. Western blot was used to explore the potential mechanism..

Results: The gastric mucosa of animal model deteriorated obviously, the weight growth rate slowed down, and the atypical hyperplasia of gastric mucosa increased. The GES-1 cells had characteristics of malignant cells such as proliferation, apoptosis, and metastasis ability. It was found that Crocin suppressed the cell proliferation, increased apoptosis, and blocked the cycle arrest in G0/G1 phase simultaneously. Furthermore, Crocin negatively regulated the invasion ability of MNNG-treated GES-1 cells and EMT process. Crocin also increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), decreased TAZ in MNNG-treated GES-1 cells. Interestingly, Crocin regulated the expression of TAZ and yes-associated protein (YAP) by increasing Nrf2 level, as well as their upstream targets, mercaptopyruvate sulfurtransferase (MST) and large tumor suppressor (LATS).

Conclusions: Crocin protected against MNNG-induced malignant transformation through the Nrf2/Hippo signaling pathway, which might be a drug candidate for clinical gastric cancer management.

Keywords: Crocin; GES-1; MNNG; gastric cancer.

Figure 1

Effect of Crocin on animal growth and cell viability. (A) The weight and (B) atypical hyperplasia of animals were evaluated. (C) The cell viability of GES-1, BGC-823, and MC cells was detected by CCK-8 assay. Cell viability (%) = (OD490 value of samples/OD490 value of control) ×100%. (D) Colony formation was analyzed in GES-1, BGC-823, and MC cells using plate clone assay. (E) The protein levels of Ki67 and proliferating cell nuclear antigen PCNA were detected by western blot. Data are presented as the mean ± SD. *P<0.05; **P<0.01. All experiments were conducted in triplicate. MC, malignant cell; CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen; SD, standard deviation.

Figure 2

Effect of Crocin on cell cycle. (A) The cell cycle of GES-1, BGC-823, and MC cells was analyzed by flow cytometry. (B) The protein levels of cyclin A, cyclin B, cyclin D1, and P21 were detected by western blot. Data are presented as the mean ± SD. *P<0.05; **P<0.01. All experiments were conducted in triplicate. MC, malignant cell; SD, standard deviation.

Figure 3

Effect of Crocin on cell apoptosis. (A,B) Apoptosis was analyzed in GES-1, BGC-823, and MC cells using flow cytometry. (C) The protein levels of Bcl-2 and Bax were detected by western blot. Data are presented as the mean ± SD. *P<0.05; **P<0.01. All experiments were conducted in triplicate. MC, malignant cell; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated x; SD, standard deviation.

Figure 4

Effect of Crocin on cell mobility. (A) The migration capability of the cells was determined by Transwell assay, magnification at 100×. (B) The protein levels of CDX2, Claudin-4, and E-cadherin were detected by western blot. (C) The protein levels of Vimentin, Fibronectin, and Transgelin were detected by western blot. Data are presented as the mean ± SD. *P<0.05; **P<0.01. All experiments were conducted in triplicate. MC, malignant cell; SD, standard deviation.

Figure 5

Effect of Crocin on the Nrf2/Hippo signaling pathway. (A,B,C,D) The protein levels of Nrf2, HO-1, and TAZ were detected by western blot. (E,F) After the addition of the Nrf2 inhibitor ML385, the protein levels of Nrf2, TAZ, YAP, p-YAP, MST, p-MST, LATS, and p-LATS were detected by western blot. Data are presented as the mean ± SD. *P<0.05; **P<0.01. All experiments were conducted in triplicate. MC, malignant cell; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; TAZ, tafazzin; YAP, yes-associated protein; MST, mercaptopyruvate sulfurtransferase; LATS, large tumor suppressor; SD, standard deviation.