Experimental Study Of The Key Glycolytic Enzyme PKM2and Drug Resistance Of Gastric Cancer

Background and purpose:multidrug resistance becomes a stumbling block to gastric cancer, but the mechanism of multidrug resistance is still not clear. MDRl/P-gp is one of the important resistance protein of multidrug resistance and is highly expressed in gastric cancer cell lines. The key glycolytic enzyme PKM2is over-expressed in gastric cancer and is correlated with the differentiation of gastric cancer cells. PK.M2is closely associated with drug resistancein in lung cancer and ovarian cancer. This study assessed whether PKM2is associated with multidrug resistance in gastric cancer and whether PKM2change the drug sensitivity by regulating the MDR1/P-gp expression.Methods:The protein level of PKM2and P-gp in MKN28, MKN45, SGC7901, BGC823was detected by Western Blot, which was used to estimate the relationship between PKM2and P-gp. shRNA lentiviral vector for PKM2interference was constructed, enzyme digested and sequenced correctly. Using three plasmid systems packaging virus in293T cells, transfecing target cells and selecting stable transfected lines with puromycin. The effect of silencing PKM2gene was detected by Western Blot. MTT was used to detect the ability of cell proliferation and the response to antitumor drugs. At last, the change of related molecules protein level such as FoxO3a and P-gp was detected by western blot after silencing PKM2, preliminary studing on the relationship between PKM2and drug resistance.Results:MDR1was expressed higher in high differentiated gastric cancer cell line MKN28, but PKM2expression is relatively lower, on the contrary, P-gp was expressed lower in poor differentiated gastric cancer cell line MKN45, SGC7901and BGC823, but PKM2expressed relatively higher. shPKM2lentiviral vector was successfully constructed and transfected into MKN45cells, the protein level of PKM2after silencing PKM2was assayed by western blot and the results showed that PKM2expression was significantly inhibited. MTT methods displayed that the growth of PKM2was significantly inhibited after silencing PKM2in MKN45. MTT was used to detect the drug sensitivity of5-Fu, DDP and EP1, the results showed that compared with LeshNC group, LeshPKM2group for5-Fu, DDP, EPI showed certain resistance, but there was no significant difference in5-Fu group. At least20μM in DDP group (inhibition rate60%vs68.58%, P=0.0002) and30μM in EPI group (inhibition rate73.90%vs80.13%, P<0.0001), there was statistical significance (P<0.05) and the inhibition of MKN45-PKM2-shRNA significantly decreased. The protein level of Fox03a and P-gp was up-regulated after silencing PKM2.Conclusion:PKM2was associated with multidrug resistance in gastric cancer. PKM2regulated FoxO3a and P-gp leading to the resistance to cisplatin and epirubicin in gastric cancer cells.