2.1 Effects of PPIs on the expression of PI3K/AKT/mTOR/HIF-1α signaling pathway and downstream factor PKM2 in gastric adenocarcinoma cell lines with different degrees of differentiation
Protein expression of PKM2 in different gastric adenocarcinoma cell lines with different degrees of differentiation (MKN28, SGC7901, and BGC823) was analyzed by western blotting analysis, while the HELA cells were taken as a control. The results showed that the expression of PKM2 increased in human gastric adenocarcinoma cells with high, moderate and poor differentiation (Fig.1A), suggesting that there was a higher level of aerobic glycolysis in the gastric adenocarcinoma cell with higher malignancy.
After PPIs treatment with the concentrations of 0 and 100 μg/ml (pH6.5)，the expressions of V-ATPases and PKM2 were measured, respectively. The results showed that omeprazole, pantoprazole and esomeprazole could respectively inhibit the expression of V-ATPases at a concentration of 10 μg/ml in MKN28 whereas it had no significantly inhibitory effects on the expression of PKM2. However, after omeprazole or esomeprazole pretreatment with the concentration of 100 μg/ml, the expression of PKM2 was obviously inhibited, consistent with those in the SGC7901 and the BGC823 cells (Fig. 1B).
2.2 PPIs and rapamycin could affect protein expression of PI3K/AKT/mTOR/HIF-1α/PKM2 signaling pathway
SGC7901 cells were treated with PPIs (0, 20, 40, 80, 160 μg/ml) for 24 hr with absence or presence of rapamycin (20 nmol) (Fig.2-1). It was shown that esomeprazole could enhance the inhibitory effects of rapamycin on p-mTOR at a concentration of 20 μg/ml (P<0.05), and could enhance the inhibitory effects of rapamycin on HIF-1α at 10 μg/ml (P<0.05). Nevertheless, at a concentration of 20 or 40 μg/ml, esomeprazole showed the synergistic inhibitory effects with rapamycin on p-mTOR and HIF-1α，whereas it showed that inhibition of downstream factor PKM2 was not statistically different from using rapamycin alone (P＞0.05). Only when the concentration of esomeprazole was enhanced to 80 μg/ml could the combination be superior to using rapamycin alone. Dramatically, it seemed that esomeprazole did not inhibit p-AKT caused by rapamycin, but promote the expression of p-AKT conversely (P<0.05). We speculated that esomeprazole was similar to rapamycin, inhibiting p-mTOR, but had no effects on the total mTOR expression. In addition, it was indicated in Fig.2-2 (A-D) that both PPIs and rapamycin could change protein localization of PKM2, and when the two drugs combined together, the effects became more significant coefficiently.
2.3 PPIs could inhibit expressions and change the intracellular localization of V-ATPases, PI3K， AKT， mTOR， HIF-1α and PKM2 in the gastric cancer cell lines
It was showed in Fig. 3A that in the condition of pH 6.5, when compared with that in the control group (0 μg/ml), the mRNA expression of V-ATPases, PI3K, AKT, HIF-1α and PKM2 was inhibited in the SGC7901 cells pretreated previously with esomeprazole (20 μg/ml) for 24 hr (P<0.05). The mRNA expression of V-ATPases, PI3K, AKT, HIF-1α and PKM2 decreased conversely as the esomeprazole concentration increased (40, 80, 100 μg/ml).
Correspondingly, in the condition of pH 6.5, compared with those in the control group (0 μg/ml), esomeprazole at relatively lower concentration could not inhibit protein expressions, while the protein expressions of V-ATPases, PI3K, AKT, HIF-1α and PKM2 were significantly inhibited in the SGC7901 cell line pretreated by esomeprazole (40 μg/ml) for 24 hr (P<0.05) concentration-dependently. As the concentration increased, the protein expression decreased conversely (Fig. 3B).
Besides, after pretreating SGC7901 with esomeprazole (0, 10, 40, 100 μg/ml) for 24 hr, we detected V-ATPases, PI3K, AKT, mTOR, HIF-1α and PKM2 expression by the immunofluorescence imaging. The fluorescent images showed that esomeprazole (40 μg/ml) could reduce intracellular expressions of V-ATPases, PI3K, AKT, mTOR, HIF-1α and PKM2 (Fig. 3C).
2.4 PPIs could inhibit cell viability and induce early apoptosis of human gastric adenocarcinoma cell line SGC7901.
Effects of PPIs pretreatment with different concentrations for 24 hr or 48 hr on the cell viability of SGC7901 were then evaluated by CCK-8 cell proliferation assay. The results showed that pretreatment of SGC7901 cells with different concentrations of esomeprazole for 24 hr or 48 hr could inhibit the viability of SGC7901 cells to a certain extent. The viability of SGC7901 cells with esomeprazole (20 μg/ml) pretreatment for 24 hr was (88.97±3.39)%, which was significantly lower than that in the control group simultaneously (P<0.05) (Figure. 4A). With the increasing drug concentration and the prolongation of treating time, cell viability inhibition became more evident, indicating that the inhibitory effects of esomeprazole on the viability of SGC7901 cells was in a concentration- and time-dependent manner.
Furthermore, the SGC7901 cells were treated with esomeprazole at the concentration of 0, 10, 20, 40, 80 and 160 μg/ml for 48 hr. Flowing cytometry analysis showed that the early apoptotic rate in the 20 μg/ml group was significantly higher than in the control group (Figure.4B and 4C) (P<0.05), indicating that esomeprazole could induce early apoptosis of the SGC7901 cells.
2.5 Knockdown V-ATPases could inhibit the protein expressions of intracellular PI3K/AKT/mTOR/HIF-1α/PKM2 signaling pathway
By using plasmid transduction of V-ATPases shRNA, we successfully performed stable transfected SGC7901 cells targeted knockdown of V-ATPases (named pshRNA-V-ATPases) and the negative control cells (named pshRNA-Con). As shown in Fig.5A, the protein expression of V-ATPases significantly decreased by suppressing V-ATPases. Compared with pshRNA-Con group and negative control group, the protein expression of PI3K, AKT, mTOR, HIF-1α and PKM2 was significantly inhibited in the pshRNA-V-ATPases group (Fig.5B) (P<0.05).
2.6 Effects of LY294002, a specific phosphatidylinositol‑3‑kinase inhibitor, on protein expressions of intracellular PI3K/AKT/mTOR/HIF-1α signaling pathway and PKM2
LY294002 is a specific phosphatidylinositol‑3‑kinase inhibitor. The BGC823 cells were pretreated with LY294002 (0, 10, 20, 50, 100 μmol/L) or DMSO (0.2%) as a control. As shown in Fig. 6A and 6B, compared with the untreated group and DMSO group, LY294002 with 10 μmol concentration could significantly inhibit protein expression of p-AKT and p-mTOR in the BGC823 cells after 48 hr treatment (P<0.05). When the concentration was elevated to 20 umol/L, the expression of HIF-1α was significantly inhibited (P<0.05), while LY294002 with the concentration of 50 μmol/L could significantly inhibit the expression of PKM2 (P<0.05). The inhibition differed obviouly as the concentration increased. The inhibitory effects were on a concentration-dependent manner (P<0.05). It was indicated that LY294002 could inhibit the activation of PI3K/AKT/mTOR/HIF-1α signaling pathway at a lower concentration, but only at a higher concentration could it exert the inhibitory effects on the expression of PKM2.
2.7 Knockdown of HIF-1α had some effects on upstream and downstream molecules.
After the expression of HIF-1α was knocked down by si-RNA in the BGC823 cell line, the expression of HIF-1 significantly decreased, and the expression of PKM2 decreased in the downstream, while the expression of p-mTOR in the upstream also simultaneously increased (Fig.6C). It could be speculated that it might be related to the existence of a feedback mechanism. Over-suppressing on HIF-1α might lead to feedback up-regulation of p-mTOR and thus slowed down PKM2 reduction in the downstream.
2.8 Effects of knocking down PKM2 on human gastric adenocarcinoma cells.
The BGC823 and SGC7901 cells were treated with PKM2 plasmid or empty plasmid for 48 hr (named pShRNA-PKM2/pShRNA-Con), and then the messenger RNA (mRNA) and protein expressions of PKM2 were detected by RT‐PCR and Western blotting analysis. And the protein localization also was detected by immunofluorescence imaging (Fig.7A). The results showed that PKM2 mRNA and protein expressions significantly decreased in the PKM2 transfection group when compared with the control group (P<0.05) (Fig.7B and 7C).
The cell viability was then evaluated by the CCK‐8 assay. It was indicated in Fig.8A that compared with those in the pshRNA-Con group and negative control group, knockdown of PKM2 inhibited cell viability in the SGC7901and BGC823 cells. The transwell assay revealed that the number of migrated cells significantly decreased in the PKM2 transfection group compared with the control group in the BGC823 cells (P<0.05), while there was no statistical difference between the PKM2 transfection group and the control group in SGC7901 cells (Fig. 8B).
We further detected the effects of the transfection of PKM2 plasmids on cell aerobic glycolysis. SGC7901 cells were transfected with PKM2 siRNA (PKM2 siRNA group) or empty plasmids (con siRNA group) and we used SGC7901 cells with no treatment as the control group. SGC7901 cells in the control group and the transfection group were cultured for 24 hr, and the total protein was extracted, as determined by Western blotting analysis. The protein expressions of GLUT‐1 and LDHA in the transfected group were lower than those in the control group (Fig.8C). Furthermore, the cells of the control group and the transfection group were cultured for 24 hr, and the extracellular glucose, as well as lactic acid contents, were detected respectively. The results showed that the contents of glucose and lactate were lower in the transfection group than that in the control group (Fig. 8D-F). These results indicated that knockdown of PKM2 had negative effects on aerobic glycolysis of gastric adenocarcinoma cells.
2.9 In vivo experiments corresponded with experiments in vitro
The athymic nude mice were randomly divided into five groups, accordingly. In the MOCK group SGC7901 cell suspension was inoculated subcutaneously into the back of the mouse and the mouse were then untreated. The NS group: SGC7901 cell suspension was inoculated subcutaneously into the back of the mouse and the mouse were treated by gavage with 5 ml NS every two days. In the PPI group: SGC7901 cell suspension was inoculated subcutaneously into the back of the mouse and the mouse were treated by gavage with pantoprazole (75 mg/kg) in 50 ml NS every two days. The PKM2 knocked down group: PKM2 knocked down SGC7901 cell suspension was inoculated subcutaneously into the back of the mouse and the mice were then untreated. The NC group: empty plasmids injected SGC7901 cell suspension was inoculated subcutaneously into the back of the mouse and the mouse were then untreated. Weight of the mouse was measured and food or water intake was then recorded. After 5 weeks treatment, the mice were sacrificed and the tumors were resected for weighing and western blotting assay.
The results suggested that tumor growth was suppressed and weight loss of mouse was alleviated in the PPI group and the PKM2 knocked down group. It can be seen that the PKM2 knocked down group and the PPI group have the smallest tumor size compared with the MOCK group and the NC group (P<0.05, as shown in Fig. 9A and Fig. 9B). In the pantoprazole group, the tumor weight was significantly lower than that in the NS group (P<0.05, as shown in Fig.9C). The tumor weight was also significantly lower in the PKM2 interference group than that in the NC group (P< 0.05, as shown in Fig. 9D).
The body weight of the mouse in the pantoprazole group was significantly higher than that in the NS group and in the MOCK group (P<0.05, as shown in Fig. 9E). The body weight of the mouse in the PKM2 knocked down group was significantly higher than that in the NC group and the MOCK group, (P<0.05, as shown in Fig. 9F).
The average daily food intake of mouse in the PPIs group was lower than that in the MOCK and the NS group, but the difference was not statistically significant (P>0.05) (as shown in Fig.9G). The average daily intake of mouse in the PPIs group was lower than that in the MOCK group and the NS group, but the difference also was not statistically significant (P>0.05) (as shown in Fig. 9H).
As shown in Fig.10, PPZ treatment might inhibit the expression of PI3K, p-AKT, p-mTOR, HIF-1α and PKM2 of tissues of tumor-bearing mice accordingly.
2.10 MAPK signaling pathway is the most relevant downstream of PKM2.
To further verify the downstream of PKM2, PKM2 were knocked down in GC cells and untreated cells played the role of the control. Then we performed gene chip experiment to find out downstream of PKM2. Total RNA was extracted from the cells and RNA samples that passed the quality test were used for gene chip experiment.
Gene set enrichment analysis (GSEA) was performed based on the selected differential genes. According to the genetic information of all pathways in KEGG and BIOCARTA, the differential genes were analyzed by GSEA (Fig. 11A and 11B). After sorting according to the P value, we selected the top ten to display (Fig.11B). We can see from Fig.11A and 13B that MAPK pathway is the downstream of PKM2.