Effect And Mechanism Of A Scorpion Serine Protease Inhibitor Sj7170Promting The Growth And Metastasis Of Glioblastoma

Glioblastoma is the most frequent primary malignant brain tumor in adults. Currently approximately half of the newly diagnosed glioblastoma patients are in the late stages. The average life expectancy for patients with glioblastoma remains14-18months. Thus glioblastoma is one of the most aggressive malignancies. Current scientific research indicates that the specific molecular mechanisms underlying the invasive behavior of glioblastomas remain largely unclear. Therefore, the development of effective modulators (activators or inhibitors) against glioblastoma as molecular tools will not only help to reveal the molecular mechanism of glioblastoma tumorigenesis, but also contribute to find drugs and methods for glioblastoma diagnosis and therapy.Serine protease inhibitors (SPIs) are widely found in a variety of organisms and their organizations, such as parasites, hematophagous invertebrates, amphibian skins, and the venom glands of poisonous animals. SPIs have very fundamental physiological/pathological functions. Recent studies demonstrate that the overexpression of a number of SPIs from the Serpin and Kunitz families results in the enhancement of cancer cell malignancy. However, all of these SPIs are secreted by endogenous human cells, and none of exogenous SPIs of tunor promoting effect are reported. Furthermore, the molecular mechanism of promoting tumor of endogenous SPIs remains unclear. And more importantly, the research of SPIs with promoting tumor research has never been involved in glioblastoma. Therefore, the development of the mechanism of promoting glioblastoma effect of novel SPIs has an important scientific significance and application value.This study screened and identified a cDNA sequence from the venomous gland cDNA library of Euscorpiops puerensis, named Sj7170.The full length of Sj7170cDNA sequence is459nt sequence, of which261nt sequence of open reading frame encodes86residues containing a signal peptide of24residues and a mature peptide of62residues with five disulfide bonds. The sequence alignment indicated that Sj7170was of typical Ascaris family features with potential serine protease inhibitor activity. Based on the cDNA sequence of Sj7170, we designed the specific primers and amplified the mature peptide sequence by PCR and constructed the recombinant expression plasmid pET-28a-Sj7170for transforming into E. coli/Rosetta. Chromatographically pure recombinant Sj7170peptide was obtained. Serine protease inhibitory activity of recombinant Sj7170peptide in vivo and in vitro were detected. The results showed that rSj7170specifically inhibited the activity of α-chymotrypsin with3.9×10-7M of Ki value in vitro, while it had no effects on trypsin and elastse.Further studies found and clarified that the scorpion serine protease inhibitor Sj7170promoted the proliferation of glioblastoma cells. The results of cell number assay showed that rSj7170significantly promoted U87and U251cells proliferation. Colony formation assays indicated that rSj7170also enhanced the ability of single U87cell to form a clone. Cell cycle analysis by flow cytometric showed that rSj7170accelerated the transition of U87cells from G1phase to S phase, speeded up the process of the cell cycle. The results of western blot showed that rSj7170upregulated the expression of cyclinD1and cycle transcriptional activator E2F1, enhanced the phosphorylation of Rb2/p130and inhibited the expression of cycle transcriptional inhibitor E2F5. The construction of cyclinD1knockdown cell lines further comfirmed the important role of cyclinD1on promoting cell proliferation by Sj7170. Furthermore, animal experiment in vivo comfirmed that rSj7170promoted subcutaneous tumor formation and growth of U87cells in nude mice and upregulated the expression of cyclinD1in tumor tissues.This study also found that the scorpion serine protease inhibitor Sj7170promoted glioblastoma cells migration and invasion. Transwell assays showed that rSj7170significantly promoted cell migration and invasion of U87and U251cells, change mutiples are more than10times. The evaluation of EMT marker proteins showed that rSj7170upregulated the expression of Vimentin and Snail in U87and U251cells, supressed the expression of E-cadherin and induced the epithelial-mesenchymal transition process of U87and U251cells. The construction of Snail knockdown cell lines indicated rSj7170lost the ability of promoting cell migration and invasion when Snail was scilenced. Therefore, upregulation of Snail and occurrence of EMT progress played an essential role in promoting migration and invasion function of Sj7170. However, rSj7170still promoted cell migration and invasion when cyclinDl was scilenced. Thus cyclinDl did not play a role in such process. Immunofluorescence results showed that rSj7170induced cytoskeletal rearrangement via Rho GTPase pathway, so that the spherical morphology of U87glioma cells was changed to a more adherent.Finally, this study further explored the relation betweeen protease inhibitory activity and the effect of Sj7170on promoting the growth and metastasis of glioblastoma cells. According to the key sites of Sj7170for the chymotrypsin inhibitory activity, we designed ten Sj7170mutants. We obtained the DNAs of these ten Sj7170mutants using overlapping PCR. Then we expressed and purified these ten mutants using the same system for wild-type Sj7170. We measured their protease inhibitory activity and their abilities to promote the proliferation and metastasis of glioblastoma cells. Chymotrypsin inhibitory activity assay revealed that mutant Sj7170-G35A had signigicant changes on its secondary structure and protease inhibitory activity. The Ki value is2.1X10-9M, the inhibition activity increased185.71times. But glioblastoma cells proliferation assay indicated that mutant Sj7170-G35A could not promote the proliferation and growth of U87cells. Meanwhile the protease inhibitory activity of other nine mutant peptides was not changed and they also lost the ability of promoting cell prliferation. Cell migration assay indicated that there was no correlation between the migration ability and the protease inhibitory activity of ten Sj7170mutants. These results suggested that there was no direct relation between two functions of Sj7170. The function of promoting glioblastoma cell proliferation is independent of anti-protease activity. These two functions may have different biological effects in vivo.In summary, Sj7170is the first isolated and characterized dual-function peptide which has serine protease inhibitory activity and glioblastoma activating function. This study not only provides a new understanding of the natural peptide library of scorpion venom, but also supports new findings of tumorigenesis/metastasis modulators from serine protease inhibitors. All results of this study strengthens the functional links between serine protease inhibitors and tumorigenesis/metastasis activators, which help to further reveal the molecular mechanism of tumor development and provide the possibility of potential value.