ABSTRACT
ObjectiveTo study the possible molecular mechanism of baicalein （BAI）-mediated focal adhesion kinase （FAK） in the regulation of phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway to inhibit the proliferation and migration of gastric cancer HGC-27 cells. MethodThe gastric epithelial GES-1 cells and gastric cancer HGC-27 cells were respectively treated with BAI （0， 5， 15， 25， and 50 μmol·L-1） for 48 h， and then methyl thiazolyl tetrazolium （MTT） assay was adopted to detect effect of BAI on cell proliferation. Western blot （WB） was employed to detect the expression of FAK and the proteins related to epithelial-mesenchymal transition （EMT） and PI3K signaling pathway after intervention with different concentrations of BAI. The HGC-27 cells stably overexpressing FAK were constructed with lentivirus-mediated transfection technique， and the transfection of FAK was detected through WB and green fluorescent protein （GFP）. The cells were divided into empty vector （NC） group， BAI group， FAK overexpression group， and BAI-treated FAK overexpression group， and cell proliferation activity was detected by MTT assay. The colony formation and cell migration were observed via colony formation assay and Transwell migration assay， respectively. The expression of proteins involved in EMT and PI3K signaling pathways were detected by Western blot. ResultCompared with the NC group， BAI （15， 25 and 50 μmol·L-1） inhibited the proliferation of HGC-27 cells in a dose-dependent manner （P<0.05， P<0.01） while did not affect that of GES-1 cells. BAI （5， 15 and 25 μmol·L-1） down-regulated the expression level of p-FAK （P<0.05， P<0.01）. Compared with NC group， FAK overexpression group showed up-regulated expression level of FAK in HGC-27 cells. The HGC-27 cells in both NC group and FAK overexpression group had green fluorescence. Compared with NC group， BAI inhibited the growth， colony formation， and migration， while FAK overexpression promoted those of HGC-27 cells. The treatment of FAK overexpression group with BAI inhibited the enhancement of cell proliferation and migration （P<0.05）. WB showed that compared with NC group， BAI （15， 25 μmol·L-1） significantly up-regulated the expression of E-cadherin protein and down-regulated that of Vimentin， Snail， p-PI3K， and p-Akt protein in HGC-27 cells （P<0.05， P<0.01）. Compared with NC group， FAK overexpression group showed down-regulated expression of E-cadherin， up-regulated expression of p-FAK， Vimentin， and Snail， and increased ratios of p-FAK/FAK， p-PI3K/PI3K and p-Akt/Akt （P<0.05）. This phenomenon would be reversed after BAI treatment. ConclusionBAI can affect the proliferation and migration of gastric cancer HGC-27 cells by mediating FAK to regulate PI3K/Akt signaling pathway.
ABSTRACT
The aim of the present study was to investigate the effects of cyclic adenosine monophosphate (cAMP) on rat gastric antral circular smooth muscle function. Forskolin, a direct activator of adenylyl cyclase (AC), was used to observe the influences of cAMP. Multi-channel physiological recorder was used to record spontaneous contraction activity of gastric antral circular muscle from Wistar rats. And ELISA method was used to detect the change of cAMP production in perfusate. The results showed that forskolin concentration-dependently suppressed the amplitude and frequency of the spontaneous contraction of the gastric antral muscle, and lowered the baseline of contraction movement significantly. Forskolin concentration-dependently increased the production of cAMP in the perfusate, which showed a significant negative correlation with the contraction amplitude of gastric antral ring muscle. The inhibitory effect of forskolin on spontaneous contraction activity of rat gastric antral circular muscle could be blocked by cAMP-dependent protein kinase (PKA) inhibitor H-89. These results suggest forskolin increases cAMP production and then activates PKA pathway, resulting in the inhibition of the spontaneous contraction activity of rat gastric antral circular smooth muscle.