Study On Radiosensitizing Effect Of Inhibition Of Glucose Metabolism Enzyme PDK Activity On Gastric Cancer

Objective:(1)To explore the expression and function analysis of pyruvate dehydrogenase kinase(PDK)molecules in gastric cancer;(2)To investigate the effect of inhibition of glucose metabolism enzyme PDK activity on the biological behavior of gastric cancer cells after irradiation.Methods:(1)Sanger Box online portal,Xiantao academic website,GEO and other databases were used to analyze the expression of PDK molecules in gastric cancer.The GEPIA2 portal and Xiantao academic website were used to analyze the influence of PDK family on the prognosis of gastric cancer patients;(2)The Linked Omics database was employed to analyze PDK1 co-expressed genes and their enrichment,and the STRING database was used to research the protein interaction network of PDK1;(3)TIMER 2.0 database was applied to analyze the connection between immune cells in gastric cancer immune microenvironment and the PDK family,and TISIDB portal was employed to analyze the correlation between the expression of PDK family and immune subtypes and molecular subtypes;(4)RNA from gastric cancer cells HGC-27,AGS,MKN-45 and gastric mucosal epithelial cells GES-1 were extracted for reverse transcription and fluorescence quantitative analysis,and the expression of PDK1 in gastric cancer cells was analyzed;(5)The effect of PDK inhibitor dichloroacetate(DCA)on the proliferation of gastric cancer cells was detected by CCK8,and the inhibition rate and half maximal inhibitory concentration(IC50)of cell proliferation were calculated;(6)The effect of PDK inhibitors on the clonal formation ability of irradiated gastric cancer cells was investigated by cell cloning formation experiment;(7)Western blot was used to detect the abundance of gamma-H2 Ax,a marker of DNA double-strand break,and analyze the effects of PDK inhibitors on DNA integrity of irradiated gastric cancer cells;(8)The effects of PDK inhibitors on ROS production in irradiated gastric cancer cells were investigated by using fluorescent enzyme labeling instrument and fluorescence microscope;(9)The effects of PDK inhibitors and radiation on lactic acid production in gastric cancer cells were investigated by chemiluminescence method.Results:(1)The expression of PDK1,PDK2 and PDK3 in gastric cancer tissues was higher than that in normal tissues,while the expression of PDK4 in gastric cancer tissues was lower than that in normal tissues.There was no substantial relation between PDK1 expressions and overall survival of gastric cancer patients.Subgroup analysis demonstrated that high expression of PDK1 was an independent prognostic factor in gastric cancer patients older than 65 years(P<0.001);(2)Enrichment analysis results showed that PDK1 expression may play an important role in the occurrence and development of gastric cancer by regulating nucleotide formation,aminoacyl-t RNA biosynthesis,cell cycle regulation and DNA double-strand break repair.In addition,PDK1 was closely related to hypoxia inducible factor-1α(HIF-1α)in gastric cancer in the protein-protein interaction network;(3)The expression of PDK1 was correlated with immune cell infiltration,and there were different among different immune subtypes of gastric cancer;(4)The expression of PDK1 in gastric cancer cells was measured by quantitative fluorescence PCR technology,among which MKN-45 and AGS had high expression.These two cell lines were selected as subsequent experimental studies;(5)The anti-proliferative effect of the PDK inhibitor DCA was measured against gastric cancer cells using the CCK8 kit.With increased dose and time of DCA,the activity of gastric cancer cells decreased.DCA inhibited gastric cancer cell proliferation in a dose-and time-dependent manner.The IC50 of DCA on AGS and MKN-45 cells were 47.94 m M and 44.19 m M,respectively;(6)By cell cloning and formation experiment,the SER of 20 m M DCA combined with radiotherapy was 1.38 in AGS cells,and 1.45 in MKN-45 cells.The results showed that inhibiting PDK activity had a radio-had sensitization effect on irradiated gastric cancer cells;(7)The abundance of γ-H2 AX,a DNA double-strand break marker,was significantly increased in DCA and radiation combined intervention group(P < 0.001),indicating that the PDK inhibitor could increase the DNA double-strand breaks and enhance the radiosensitivity of gastric cancer cells;(8)The effects of PDK inhibitors on ROS content of irradiated gastric cancer cells were detected by fluorescent enzyme labeling instrument and fluorescence microscope.The results demonstrated that the ROS content in the 40 m M DCA combined with the radiation group was significantly higher than in the other groups(P<0.001);(9)Lactic acid content in gastric cancer cells was detected by chemiluminescence method.The results showed that PDK inhibitor inhibited lactic acid production in gastric cancer cells.Conclusion:(1)PDK1 is an important target for the treatment of gastric cancer,which has promising research prospects.High expression of PDK1 was a prognostic factor for overall survival in gastric cancer patients older than 65 years;(2)The expression PDK1 can play an important role in gastric cancer by regulating nucleotide formation,energy metabolism and DNA repair,and is associated with the infiltration of immune cells among gastric cancer tissue;(3)PDK inhibitors inhibited gastric cancer cell proliferation in a dose-and timedependent manner;(4)PDK inhibitors can improve the ROS level,inhibit the cloning formation ability against irradiated gastric cancer cells.Thus,inhibiting PDK activity may promote the radiotherapy sensitivity of gastric cancer cells.