Effects Of PI3K/Akt Signaling Pathway On PrPc Induced Drug Resistance In Gastric Cancer

【Background】In our previous work, PrPc was identified as one MDR related molecule and played an important role in drug resistance of gastric cancer. However, little is known about the molecular mechanism involved in this process. PI3K/Akt signaling pathway is one of the important signal transduction pathways that can regulate cell growth, cell proliferation and cell survival through phosphorylating its downstream molecules.Recently accumulated evidence indicates that PI3K/Akt activity plays a significant role in tumor MDR. Previous studies show that PrPc plays a positive role in neuronal survival in cell culture of primary neurons through activation of PI3K/Akt pathway.To investigate whether PI3K/Akt pathway contributed to PrPc induced MDR in gastric cancer cell , we observed the expression of PrPc and p-Akt in precancerous tissues and gastric cancer tissues, and established the gastric cancer cell line stably transfected with PrP gene, then analyzed the effects of PI3K selective inhibitors on PrPc induced MDR in gastric cancer cells. It enriches our knowledge of gastric cancer MDR induced by PrPc and provides a new sight into the mechanisms of gastric malignancy transformation and its reversal.【Objectives】1. To examine the expression and relationship between PrPc and p-Akt in gastric cancer tissues and pericancerous tissues. 2. To detect the expression of Akt in PrP stably transfected gastric cancer cells. 3. To analyze the effects and mechanisms of PI3K selective inhibitors on MDR of PrPc stably transfected cells.【Methods】1.Immunohistochemistry was perfomed to examine the expression of PrPc and p-Akt in human gastric cancer tissues and precancerous tissues. 2. pcDNA3.1-PrP was transfected into SGC7901 by using Lipofectamine 2000, and multiple clones were isolated after screened by G418 and then verified by western blot. 3. The expression of t-Akt and p-Akt were detected byWestern blot at the protein level in PrP tranfected cells and control ones. 4. MTT assay was performed to determine the drug sensitivity of the transfectants with or without the inhibitor of PI3K-LY294002, and the IC50 values were calculated. 5. Apoptosis of the PrP transfected cells was examined by Annexin V/PI staining; The accumulation and retention of adriamycin in tranfected cells were determined by flow cytometric analysis. 6. RT-PCR and Western blot were used to detect the MDR related molecules P-gp and Bcl-2 in transfected cells with or without LY294002.7. The luciferase reporter vector containing MDRl promoter gene was constructed and Luciferase assay was performed to detect the fluoresence in the transfected cells to investigate the transcriptional regulation function of PrPc and Akt on MDR1 promoter.【Results】1.In cancer tissues, PrP was detected by a positive rate of 82.4%, and p-Akt 88.2%. PrP and p-Akt were exclusively cytoplasmic and membrance in cancer cells. The correlation coefficient of PrP and p-Akt expression in cancer tissues was evaluated by spearman correlation analysis according to the total score of immunohistochemistry from each case that combines staining intensity with positive cell percentage. Correlation coefficient between PrP and p-Akt was 0.514(p<0.01). When analyzed according to differentiation grade of each case of cancer sample, both PrP and p-Akt were found to have significant correlation with gastric cancer differentiation status (rs= 0.548, rs= 0.69, P<0.01 respectively), that's to say, the poorer differentiation, the higher expression of PrPc and p-Akt. 2. pcDNA-PrP was stably transfected into SGC7901. Western blot analysis confirmed that p-Akt was highly expressed in PrP transfected cells (SGC7901/PrP) compared to cells transfected with empty vector (SGC7901/3.1B) and its parent cells(SGC7901). 3. MTT assay showed LY294002 obviously increased the effects of chemotherapeutic agents on transfeceted cells in a concentration-dependent manner. When treated with 30μM LY294002, the IC50 value of SGC7901/PrP for adriamycin or vincristine was similar to its control cells. 4. Flow cytometric analysis showed LY294002 not only significantly increased vincristine-induced apoptosis but also increased adriamycin accumulation and retention in transfeceted cells in a concentration-dependent manner.5. Western blot and RT-PCR showed that LY294002 down-regulated the expressions of P-gp and Bcl-2 at both mRNA and protein level in SGC7901/PrP cells .6. The luciferase reporter gene vector containing MDRl promoter was successfully constructed. The luc activity in SGC7901/PrP cells was higher than those in control ones. LY294002, however, significantly inhibited the luc activity of SGC7901/PrP cells. 【Conclusions】1.PrPc and p-Akt play important roles in gasric cancer: PrPc and p-Akt were closely correlated with each other in tumor and adjacent precancerous tissues and both of them correlated with the differentiation degree of gastric cancer. Western blot analysis confirmed that p-Akt was highly expressed in PrP transfected cells, suggesting that PrPc and p-Akt play important roles in gasric cancer and PrPc might activate PI3K/Akt signaling pathway.2. Inhibiting PI3K/Akt pathway activation can reverse PrPc induced MDR: We found that Inhibition of PI3K/Akt signaling pathway by LY2940002 could lead to the inhibition of PrPc induced drug resistance in gastric cancer cells, suggesting PrPc induced drug resistance correlated with activation of PI3K/Akt pathway.3. Inhibiting PI3K/Akt pathway activation can down-regulated the expression of P-gp and Bcl-2:LY294002 could decrease the expression of P-gp and Bcl-2 at both protein and mRNA level in SGC7901/PrP, while had no effect on control cells. We had successfully constructed the luciferase reporter gene vector containing MDRl promoter and found LY294002 could significantly reduce the luc activity in SGC7901/PrP compared with control ones. All these data indicated that PrPc might increase the expression of P-gp and Bcl-2 by activating PI3K/Akt pathway.