br Materials and methods br Results br Discussion Ghrelin

Materials and methods
Results
Discussion Ghrelin is reported to regulate a number of processes related to cancer progression including tumor cell proliferation, apoptosis and metastasis [3,7,[15], [16], [17], [18], [19], [20], [21], [22]]. In the present study, we examined the potential effects of ghrelin on A549 cells proliferation and investigated its underlying molecular mechanism. Our findings show that ghrelin-induced A549 cells proliferation is accompanied by GHSR-dependent activation of PI3K/Akt/mTOR/P70S6K and ERK signaling. The ghrelin gene (GHRL) encodes a 28-amino Gemfibrozil peptide hormone. GHSR has two isoforms, GHSR1a and GHSR1b. The expression of GHSR has been observed in many tumors including breast cancer, lung cancer, renal cell carcinoma, gastric cancer, pancreatic cancer, thyroid cancer, colorectal cancer, prostate cancer, ovarian cancer, oral cancer [[23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33]]. GHSR1a is the functional receptor responsible for many of the physiological effects mediated by ghrelin. Many studies used [D-Lys3]-GHRP-6 (the GHSR antagonist) or ghrelin neutralizing antibody to block ghrelin's biological effects [5,34]. There is evidence showing that ghrelin-induced colon cancer cell proliferation was attenuated by [D-Lys3]-GHRP-6 [5]. Our previous study has showed that [D-Lys3]-GHRP-6 reversed the anti-apoptosis effect induced by ghrelin in HUVECs [35]. In the present study, we found that ghrelin promotes A549 cells proliferation, to investigate whether GHSR is involved in ghrelin-induced cell proliferation, we down-regulated GHSR expression by GHSR siRNA in A549 cells and found that GHSR siRNA completely abolishes ghrelin-induced A549 cell proliferation. This result indicates that GHSR mediated the tumor cell proliferation stimulated by ghrelin. However, another study has demonstrated that ghrelin's proliferative effects was GHSR independent in rat intestine cell line [13]. Further study is needed to examine which GHSR sub-type mediates ghrelin-induced cell proliferation in A549 cells. There is evidence suggesting that desacyl ghrelin inhibited colon cancer cell lines proliferation [36]. In our study, we found that ghrelin promoted proliferation of A549 cells, with a maximal response after treatment with 5 nM ghrelin, whereas when the concentration of ghrelin was 10 nM, the proliferation of A549 cells decreased. It may be the number of GHSR in A549 cells is limited, when all the GHSR bound to ghrelin, the remaining ghrelin inhibited cell proliferation instead of promoting tumor cell proliferation. Recently, a study reported that ghrelin stimulated colon cancer cell proliferation via the GHSR/Ras/PI3K/Akt/mTOR signaling pathway [28]. A previous study showed that ghrelin promoted the proliferation of IEC-6 cells through ERK1/2 phosphorylation [13]. The PI3K/Akt signaling was reported to participate in promoting tumor cell proliferation and PI3K was shown to be essential for Akt activation. In the present study, we found that ghrelin-induced proliferation was partially inhibited by treatment with LY294002 or PD98059. Whereas the combination of PD98059 and LY294002 almost completely inhibited ghrelin-induced A549 cells proliferation. Moreover, ghrelin induced PI3K/Akt and ERK phosphorylation, PI3K inhibitor LY294002 blocked Akt phosphorylation without affecting ERK phosphorylation. PD98059 had no effect on Akt phosphorylation. These results suggest that PI3K/Akt and ERK are two mutually independent signal pathways in A549 cells activated by ghrelin. mTOR is reported to play important roles in cancer development. Activation of the mTOR is involved in tumor cell proliferation by various stimulation in different cell types. Our previous study showed that ghrelin activated mTOR/P70S6K signaling in HUVECs [35]. In this study, we found that ghrelin stimulated mTOR/P70S6K phosphorylation and GHSR siRNA reversed ghrelin's effects. Given that ghrelin induce phosphorylation of PI3K/Akt and mTOR/P70S6K in A549 cells, we further to investigate the relationship between PI3K/Akt or ERK and mTOR/P70S6K signaling involved in ghrelin-induced proliferation. Our results showed that LY294002 inhibited mTOR/P70S6K phosphorylation, whereas PD98059 did not affect mTOR/P70S6K phosphorylation. These results further confirmed that PI3K/Akt and ERK signaling are two parallel pathways activated by ghrelin in A549 cells. Furthermore, rapamycin abolished the proliferation of A549 cells and P70S6K phosphorylation induced by ghrelin. These data provided strong evidence that mTOR/P70S6K activation participates in ghrelin-caused A549 cells proliferation.