Cloning, Expression And Anticancer Activity Of Human Granulin A

Peptides play crucial roles in many physiological processes and a lot of peptides have been used clinically to treat human disorders. For many years, researchers are trying to search for the anticancer peptides from human body, since peptides from human display little immunogenicity. Several peptides from human resources are successfully used clinically for the treatment of human cancers, such as interferons and interleukins. However, these kinds of peptides usually do not inhibit cancer cell growth directly; they affect the growth of tumor via immune effect. In the present study, we found that the human granulin A (hGRN A) possesses the ability to inhibit the proliferation of cancer cells directly. This result provides primary evidence that hGRN A have the potential to be developed as a novel kind of anticancer agent.Granulins (GRN), also known as epithelins, consists of a family of cysteine-rich peptides with diverse functions. All members of the GRN family contain 12 cysteine residues arranged in highly conserved positions, and the cysteine residues form intramolecular disulfide bridges, resulting in the characteristic of tightly packed structures. GRN A is an acid- and heat-stable polypeptide with low molecular mass. The peptide is found in a wide range of organisms, from the eubacteria to mammals. Studies have shown that GRN A plays a critical role in epithelial homeostasis, tumorigenesis, and in reproductive, immunological, and neuronal functions.In this study, we constructed the expression vector of hGRN A, pGAPZaA-hGRN A, and the plasmid was transformed into the yeast, SMD1168H. Recombinant hGRN A was purificated using the Ni+2 affinity chromotograghy column. The condition for fermentation of hGRN A was optimized. The inhibition rate of several human tumor cells was determined by MTT method. DAPI staining and flow cytometry were performed to analyze the proapoptotic effects of hGRN A on human tumor cells. The main research results were as follows:1. Construction of hGRN A expression vector. The hGRN A gene was engineered into the vector pGAPZaA, and the expression vector, pGAPZaA-hGRN A, was constructed successfully.2. Tansformation of pGAPZaA-hGRN A into SMD1168H cells. The linearized plasmid was transformed into a yeast expression strain, SMD1168H. By using the resistance zeocin gene carried by the expression vector, the recombinant clones were selected with different concentrations of zeocin. Finally, high yield clones were found when the concentration of zeocin was 100μg/mL.3. Optimization of fermentation condations. The fermentation conditions, including medium composition, inoculation amount, fermentation temperature and culture time, were optimized. The composition of culture medium was defined as yeast powder 1％, peptone 2％, glucose 2％, KH2PO40.4％, (NH4)2SO40.2％, MgSO4 0.05％in a final concentration of 0.1 mol/L phosphate buffer, and the optimal fermentation conditions were determined as:inoculation amount of 7%, fermentation temperature 25℃, culture time 96 h. Using the optimized fermentation conditions, the hGRN A yield increased from 5 mg/L to 9.8 mg/L.4. Purification of. hGRN A was purified by affinity chromatography using a nickel column. After applying on the column, the column was washed with 20 mmol/L imidazole and the hGRN A was eluted with 250 mmol/L imidazole. Purified hGRN A was obtained.5. Cytotoxicity of on human tumor cells. The inhibitory rate of recombinant hGRN A on human tumor cells was determined by MTT method, the ICsos of hGRN A for PANC-28, HCT-116 and BEL-7402 cancer cells were 85.6,100.4 and 90.9 μg/mL respectively. The results showed that the recombinant hGRN A could inhibit the proliferation of tumor cells in a dose-dependent manner.6. Cell apoptosis inducing activity of hGRN A. (1) Nuclear morphology was observed by DAPI staining. Compared with the control group, the nucleus cytoplasm pycnosis, condensation and half moon appeared in the hGRN A group. With the increase of hGRN A concentration, the number of apoptotic cells increased significantly. (2) The inhibition of recombinant hGRN A on tumor cells was verified with flow cytometry. At the concentrations of 50,100 and 150 μg/mL, tumor cell apoptosis rates were 18.52%,44.13% and 69.5% respectively, significantly higher than that of the control group (9.64%) (p< 0.05).In conclusion, the results of present study confirmed that hGRN A displays cytotoxicity to several human cancer cells, and induced cell death via apoptotic pathway in human pancreatic cells. This study provides primary evidence that hGRN A possesses the potential as a novel anticancer agent, and also provides basis for the further study of the function of hGRN A both in vitro and in vivo.