Interaction Of Granulin A And Enolase 1 Attenuates The Migration And Invasion Of Human Hepatoma Cells

HCC(Hepatocellular carcinoma,HCC)is one of the most common malignant tumors in the world for its high incidence,recurrence and metastasis rate as well as its mortality.China is one of the major countries of liver cancer occurrence in the world.The incidence of HCC in China has become the Third in males,while ranked fifth in females;it's mortality rate is second in males,while forth in females.At present,surgery is still the main approach for the treatment of HCC.However,due to the high recurrence and metastasis rate after surgery,its efficacy is not ideal.Therefore,it is promising for developing novel strategies to interfere with the cancer metastasis to increase the survival rate of HCC patients.Peptides play a crucial role in many physiological processes.However,a small number of peptides have been commercialized as drugs because of their limited in low yield and poor bioavailability.It is promising to develop human peptides as drugs due to its low immunogenicity as well as its safety.Granulin A(GRN A)is a peptide with a molecular 6 kDa derived from proteolysis of progranulin(PGRN).Previous study in our laboratory has shown that GRN A is able to inhibit cancer cell growth significantly,suggesting that the polypeptide possesses potential to be developed as a novel anticancer agent.However,its molecular mechanism is unknown.To identify the molecular target of GRN A,we initially performed confocal experiment to determine the distribution of the polypeptide.Our results showed that GRN A is distributed in both the cytoplasm as well as cell membrane.We then performed SDS-PAGE,Pull-down and LC-MS/MS analysis to identify the prey protein that interacted with GRN A.Our results revealed that a clear band appeared with molecular weight of around 50 kDa.LC-MS/MS analysis confirmed that the prey protein displayed high similarity with ENO1.To further confirm the targeted protein of GRN A,Western blotting analysis was performed.The results showed that ENO1 is able to interact with the ENO1-monoclonal antibody specifically.SPR analysis was further applied to determine the interaction of GRN A and ENO1.Our results show that the Kd value(affinity constant)between ENO1 and GRN A is 56.04 ?M.The results provide solid evidence that there is interaction between ENO1 and GRN A.ENO1 plays a critical role in glycometabolism,catalyzing the transformation of 2-phosphate-d-glycerate(2-PG)to phosphoric acid-pyruvate(PEP)during glycolysis.To confirm the effect of interaction between GRN A and ENO1 on glycometabolism,intracellular glucose uptake levels were determined.Recent study identified an ENO1 inhibitor,AP-III-a4(Cas no.1177827-73-4),called enoblock,which can bind with ENO1 and inhibit glycometabolism.In the present study,paralleled experiments were performed to compare the effect of GRN A and enoblock on glucose uptake.The results showed that treatment of the HepG-2 cells with GRN A increased the glucose uptake significantly,similar with that of the cells treated with enoblock.Further study suggested that GRN A can increase the gluconeogenesis via inhibiting the expression of phosphoenolpyruvate carboxykinase 1(PCK1)and phosphoenolpyruvate carboxykinase 2(PCK2).Previous studies have shown that ENO1 is closely related with cancer cell metastasis;high expression of ENO1 increases the cell migration and invasion.To study if the interaction between GRN A and ENO1 affects cancer cell migration and invasion,Scratch wound healing assay and Transwell experiments were performed.Scratch wound healing assay showed that the migration was remarkably inhibited in cells treated with GRN A;the relative migration rate were 29.60 ± 2.33% and 40.97 ± 1.23%,when treated the cells with GRN A at a concentration of 2 ?M for 12 or 24 h.Transwell assay also indicated that treatment of the cancer cells with GRN A inhibited cell migration;the number of cells migrated to the opposite side were 378 ± 28,297 ± 35,210 ± 13,146 ± 17,when treated the cells with GRN A at a concentration of 0,0.5,1.0,2.0 ?M respectively.Treatment of the cancer cells with enoblock(from 0 to 5 ?M)also displayed similar results.Moreover,GRN A treatment also inhibited cancer cell invasion in a dose-dependent manner,and the number of cells invaded to the opposite side were 208 ± 16,167 ± 16,115 ± 3,62 ± 9,when treated the cells with GRN A at a concentration of 0,0.5,1.0,2.0 ?M respectively,similar as the results of enoblock.These results confirmed that GRN A is able to interfere with the metastasis and inhibits migration and invasion of cancer cells.To study if the impact of GRN A on cancer cells is associated with ENO1,we studied the effect of GRN A in cells overexpressing ENO1.The results showed that the migration cells number in HepG-2 cells transfected with ENO1 increased from 227 ± 15 to 427 ± 49,suggesting that ENO1 is capable of enhancing the ability of cell migration.Additionally,treatment with GRN A inhibited the cell migration significantly;the migration cells number decreased from 227 ± 15 to 144 ± 21 in cells transfected with control constructs.In contrast,overexpression of ENO1 reversed the effect of GRN A on migration of cancer cells;the number of migrated cells was increased to 346 ± 46 in ENO1 transfected cancer cells treated with GRN A,compared with that 144 ± 21 in cells transfected with the control plasmid.Similarly,ENO1 overexpression attenuated the GRN A associated inhibitory effect of cell invasion;the invaded cells number transfected with ENO1 increased from 123 ± 7 to 165 ± 10,indicating that ENO1 is able to promote the ability of cell invasion.In addition,treatment with GRN A inhibited the cell invasion significantly;the invasion cells number decreased from 123 ± 7 to 67 ± 10.However,overexpression of ENO1 reversed the effect of GRN A on migration of cancer cells significantly;the number of migrated cells was increased to 112 ± 16 in cells transfected with ENO1,compared with that in cells transfected with the control constructs.The study provides solid evidence that there is the interaction between GRN A and ENO1 and the interaction is responsible for the effects of GRN A on cancer cell migration and invasion.We then studied if over-expression of ENO1 affects the expression of apoptosis proteins using Western blotting analysis.The results showed that the expression of antiapoptosis proteins,displayed opposite effects between normal HepG-2 cells and the cells over-expression of ENO1 when treated with GRN A;the expression of Bcl-x L and c-Myc are diminished significantly in cells treated with GRN A.However,the expression of these genes was elevated significantly in cells over-expressing ENO1.The results indicated that GRN A induced cell apoptosis is associated with ENO1.Similar results were also found in gluconeogenesis proteins;GRN A treatment results in inhibition of the expression of PCK1 and PCK2 significantly.However,these genes expression was enhanced obviously in cells overexpressing ENO1;suggesting that the effect of GRN A on glucose uptake is also associated with ENO1.In summary,our findings suggest that GRN A inhibits migration and invasion of cancer cells.Treatment of hepatoma cancer HepG-2 cells with GRN A is able to increase glucose uptake.The interacts with ENO1 and the interaction is responsible for inhibition of PCK1 and PCK2 expression,as well as the suppression of cancer cell migration and invasion.The study of provides evidence that GRN A possesses potential to be developed as a novel anticancer agent.This study is also important for dissecting the function of ENO1 for its possibility as a novel anticancer target.