The Effects Of Testes Specific Protease 50 On Cellular Glucose Metabolism And Its Correlation With Tumorigenesis

Malignant tumor is a disease harmful to human health.Its occurrence and development are closely related to the activation of oncogene and the inactivation of tumor suppressor gene.Testis-specific protease 50(TSP50)is a proto-oncogene found in recent years.The gene is specifically expressed in spermatocytes of the testis and is rarely expressed in mature spermatozoa and normal tissues,but is highly expressed in various cancerous tissues such as human lung cancer,liver cancer,prostate cancer and colon cancer,etc.TSP50 can induce tumorigenesis by activating NF-κB signaling pathway and promote tumor metastasis.Abnormal energy metabolism is one of the ten characteristics of cancer cells,and glucose metabolism,which is the main source of energy,becomes the main focus of energy metabolism research.In the 1920 s,German biologist Otta Warburg found that malignant tumor cells,even in the case of adequate oxygen supply,are still supply energy by glycolytic rather then by oxidative phosphorylation,and this phenomenon of tumor cells is also known as the“Warburg effect”.Previous studies on TSP50 are mainly focused on its effect on cell proliferation,metastasis and immune function regulation,but its effect on tumor glucose metabolism is unclear.Therefore,this study is aimed to explore the effect of TSP50 on cellular glucose metabolism and its correlation with tumorigenesis,which can provide a theoretical basis for tumor therapy targeting TSP50.In order to explore the effect of TSP50 on cellular glucose metabolism,we first detected the expression of TSP50 in mammary cell lines by immunoblotting assay,and then we found the normal mammary epithelial cells MCF-10 A with low expression of TSP50 and two breast cancer cells MDA-MB-231 and ER7530 with high expressiong of TSP50.Then we detected the changes of glucose metabolism after overexpressing in MCF-10 A or knockdowning TSP50 in MDA-MB-231 and ER7530.Following results were obtained.1.Effect of overexpressing TSP50 on cellular glucose metabolism.In order to investigate the effect of TSP50 on cellular glucose metabolism,we first overexpressed TSP50 in normal breast epithelial cell line MCF-10 A,which was low expression of TSP50,and then examined the changes of the glucose consumption,lactate production,the activity of LDH,a key enzyme in glycolysis pathway and the expression of glucose metabolism related protein(GLUT1,HK2,PKM2 and LDHA)and the content of ATP in cells,for measuring the effect of TSP50 on cellular glucose metabolism.The results showed that the amount of glucose consumption and lactate producton were increased in MCF-10 A cells after overexpressing TSP50,and the activity of lactate dehydrogenase is also increased.At the same time,the expression level of glucose transporter GLUT1,the key enzyme hexokinase HK2,pyruvate kinase PKM2 and lactate dehydrogenase LDHA were also increased.Meanwhile,the content of ATP is increased significantly,and there were significant differences statistically.The above results suggest that overexpression of TSP50 can promote cell aerobic glycolysis.2.Effect of knocking down TSP50 on cellular glucose metabolism.To further confirm the effect of TSP50 on cellular glucose metabolism,we transfected the TSP50 siRNA to knock down the expression of TSP50 in breast cancer cells MDA-MB-231 and ER7530,and then examined the glucose metabolism-related indexes.The results showed that comparing to the control cells,the glucose consumption and lactate production were significantly decreased,and the activity of lactate dehydrogenase is also decreased after knocking down TSP50 in MDA-MB-231 and ER7530 cells.At the same time,the expression level of the glucose transporter GLUT1,the key enzyme hexokinase HK2,the pyruvate kinase PKM2 and lactate dehydrogenase LDHA are also decreased.Meanwhile,the content of ATP is decreased significantly,and there were significant differences statistically in all of these results.The above results suggest that knockdown of TSP50 expression can significantly inhibit cell aerobic glycolysis.Above results suggest that TSP50 can significantly promote cell aerobic glycolysis.3.Correlation between the effect of TSP50 on glucose metabolism and its effect on tumorigenesisPrevious studies have shown that TSP50 has an important role in tumorigenesis.In order to clarify the relationship between the cellular glycolysis of TSP50 and its effect on tumorigenesis,we selected a small molecule glycolytic inhibitor 2-Deoxy-D-glucose(2-DG)to block glycolysis in TSP50-overexpressing MCF-10 A cells,and then detect the changes in cell proliferation and migration capacity.The results showed that the proliferation ability and migration ability of MCF-10 A cells were significantly attenuated and the degree of weakening was significantly stronger than that of control cells.The results suggest that there is a significant correlation between the effect of TSP50 on glycolytic activity and its role in tumorigenesis,and its effect on tumorigenesis is at least partially dependent on the energy provided by cell glycolysis.In summary,this study indicate that TSP50 can promote the aerobic glycolysis in cells,and preliminarily elucidated that there was a correlation between the effect of TSP50 on glucose metabolism and its effect on tumorigenesis.These results provided an experimental basis for elucidating the mechanism of TSP50 in terms of energy metabolism,and also provides a novel theoretical basis for tumor therapy targeting TSP50.