Cancer Letters

Cancer Letters

Volume 332, Issue 1, 10 May 2013, Pages 11-18
Cancer Letters

Gastrin acting on the cholecystokinin2 receptor induces cyclooxygenase-2 expression through JAK2/STAT3/PI3K/Akt pathway in human gastric cancer cells

https://doi.org/10.1016/j.canlet.2012.12.030Get rights and content

Abstract

Gastrin, cholecystokinin2 receptor (CCK2R), and cyclooxygenase-2 (COX-2) have been implicated in the carcinogenesis and progression of gastric cancer. Our study demonstrated that antagonist or siRNA against CCK2R blocked amidated gastrin (G17)-induced activation of STAT3 and Akt in gastric cancer cell lines. G17-increased COX-2 expression and cell proliferation were effectively blocked by CCK2R antagonist and inhibitors of JAK2 and PI3K. In addition, knockdown of STAT3 expression significantly attenuated G17-induced PI3K/Akt activation, COX-2 expression, and cell proliferation. These results suggest that CCK2R-mediated COX-2 up-regulation via JAK2/STAT3/PI3K/Akt pathway is involved in the proliferative effect of G17 on human gastric cancer cells.

Highlights

► Antagonist or siRNA against CCK2R blocked G17-induced activation of STAT3 and Akt in gastric cancer cell lines. ► G17-increased COX-2 expression and cell proliferation were blocked by CCK2R antago-31 nist and inhibitors of JAK2 and PI3K. ► Knockdown of STAT3 expression attenuated G17-induced PI3K/Akt activation, COX-2 expression, and cell proliferation.

Introduction

Gastric cancer remains the second most common cause of cancer-related death worldwide and is a major cause of cancer-related mortality in China [1]. Among the multiple risk factors leading to the occurrence of gastric cancer, infection with Helicobacter pylori (H. pylori) is the major predisposing factor for this malignant disease [2], [3]. Recent studies have demonstrated that hypergastrinemia induced by H. pylori infection is often associated with increased cyclooxygenase-2 (COX-2) expression in gastric and colorectal malignancies [4], [5].

Gastrin is a polypeptide hormone that is synthesized in the gastric G cells and plays an important role in modulating various functions in the gastrointestinal tract, including acid secretion, motility, and cell proliferation. The trophic effect of gastrin on a number of established gastric cancer cells has been well documented in vivo and in vitro [6], [7]. Mature amidated gastrin (G17) acts through the G-protein coupled cholecystokinin2 receptor (CCK2R), formerly named CCKB/gastrin receptor, which also plays a crucial role in gastrointestinal malignancy [8], [9].

Cyclooxygenase (COX), a key enzyme in the conversion of arachidonic acid to prostaglandins (PGs) and other eicosanoids, exists as two isoforms: constitutive COX-1 and mitogen-inducible COX-2. COX-2 is also constitutively expressed in gastric cancer and is related to cell proliferation and apoptosis, tumor invasiveness and metastasis [10], [11], [12]. Some studies have shown that COX-2 is co-expressed with gastrin in stomach and colon tumor cells [4], [5], [13]. On the other hand, in vitro study indicates that gastrin stimulates COX-2 gene and protein expression in human gastric cancer cells [14], [15]. Our recent study has shown that blockade of CCK2R and COX-2 exerts synergistic anti-tumour effect on human gastric cancer [16]. In view of above data, we speculate that COX-2 may be one of the important downstream targets of gastrin. However, the signaling pathways linking CCK2R to the expression of COX-2 in gastric cancer cells remain unclear.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is the principal signaling mechanism for a wide array of cytokines and growth factors [17]. The activation of STAT3 has been linked with the proliferation, survival, invasion, and angiogenesis of a variety of human cancer cells, including gastric cancer [17], [18]. In addition, another attractive candidate is the phosphatidyl inositol 3′-kinase (PI3K)/Akt pathway, which is involved in the regulation of many cell processes including proliferation and survival [19]. PI3K exists as a heterodimer that consists of a regulatory subunit (p85) and a catalytic subunit (p110). Previous studies have shown that the induced expressions of COX-2 mRNA and protein can be mediated by a wide variety of signaling pathways, such as the JAK2/STAT3, MAPK/ERK, and PI3K/Akt pathways [20], [21], [22]. However, the exact molecular mechanism by which G17 acting on the CCK2R regulates COX-2 expression in gastric cancer cells is not well understood. In the present study, two human gastric cancer cell lines SGC-7901 and MKN-45, in which the CCK2R, as well as COX-2 were found to be expressed [16], [23], [24], were applied to examine whether the activation of the CCK2R by G17 leads to up-regulation of COX-2 expression in gastric cancer cells and, if so, to determine the signaling pathway(s) linking CCK2R to COX-2 expression.

Section snippets

Reagents

Human amidated gastrin (G17), the JAK2 inhibitor AG490, dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CCK2R antagonist YM022 was purchased from Tocris Bioscience (Bristol, UK). The PI3K inhibitor LY294002 was purchased from Cell Signaling Technology (Danvers, MA, USA). Selective COX-2 inhibitor NS-398 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions of YM022, AG490,

G17 induces COX-2 expression in gastric cancer cells

COX-2 expression in gastric cancer cells was detected by western blot analyses. As shown in Fig. 1A, G17 dose-dependently increased COX-2 expression in SGC-7901 and MKN-45 cells. For the time sequence study, the increase of COX-2 expression was evident after 6 h of incubation, reached a peak at 24 h and declined at 48 h (Fig. 1B).

G17-induced COX-2 expression is CCK2R dependent

CCK2R antagonist YM022 significantly reduced the constitutive and G17-induced COX-2 expression in SGC-7901 and MKN-45 cells (Fig. 2A). To confirm that CCK2R mediates

Discussion

Gastrin has been shown to be a growth factor for gastric cancers in both established cell lines as well as cells derived from human gastric cancer specimens [6], [7]. This tumor promoting effect of gastrin has been proven to be mediated by CCK2R, which has also been reported to be present in both gastric cancer cell lines [6], [8] and human gastric cancer tissues [28]. The present study clearly shows that G17 significantly increases the proliferation of SGC-7901 and MKN-45 cells in a

Acknowledgments

The present study was supported by Grants from the Nature Science Foundation of China (No. 81072030) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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