Human IL-24Recombinant Protein Inhibits The Proliferation Of Esophageal Carcinoma Cells By Rceptors Mediated Pathway

Esophageal cancer, which its morbidity and mortality is the fourth in China, is one of the eight most common malignant neoplasm, and a serious threat to the human life and health in the world. Patients with esophageal cancer with5-year survival rates of10%-25%in China accounted for more than50%of the world. The majority of patients were initially treated with stage II or III disease and display poor prognosis. Unfortunately, despite advances in initial debulking surgery and chemotherapy of esophageal cancer, many patients will have recurrence frequently, which is not responsive to further chemotherapy and carries a negative prognosis. Many studies shown that some anti-tumor drugs, such as5-FU, DDP, MTX or Taxol were unable to inhibit esophageal cancer cell proliferation and prevent angiogenesis and had side effect on normal tissue or cells with same frequency. These facts emphasize the need for development of new effective therapies for esophageal cancer.One candidate is melanoma differentiation associated gene-7(mda-7/IL-24), which have demonstrated that delivery of IL-24by a replication-incompetent adenovirus induces growth suppression and promotes cancer cell-specific apoptosis in a wide variety of human cancers in vitro and in vivo, without adverse effects toward normal cells or tissues. The protein drugs generated by genetic engineering are safer than protein drugs that were expressed by adenovirus in clinic treatment and the biological activity of IL-24recombinant proteins expressing from bacteria or E. coli is far lower compared with mammalian cell. Secreted soluble IL-24recombinant protein (sIL-24) derived from mammalian cell supernatant has the ability to anti-tumor activity, but its secreted expression is lower relatively. At the same time, there is no report whether IL-24can play similar role in growth-suppression and induction apoptosis on esophageal cancer cells. At the present study, we explored the effect of IL-24recombinant protein secreted and expressed on esophageal cancer cell (Eca-109) proliferation and apoptosis and preliminary researched its mechanism of growth-suppression and induction apoptosis on Eca-109cells through biology and cell biology methods. What we had achieved is as follows:1. IL-24gene was integrated into Flp-In CHO cell genome by recombinase mediated using Flp-In site-specific integration system. Western blot analysis shown His6-IL-24was detected in cell supernatants and lysates. Therefore, we have successfully established a stable engineering cell lines expressing and secreting IL-24recombinant protein.2. His6-IL-24recombinant protein was purified from FCHO-IL-24cell culture supernatant using affinity chromatography and ultrafiltration, and subsequent HPLC indicated its purity is94.23%and concentration is500ng/mL as a result of ELISA.3. The A375and Eca-109cell express IL-24receptors, the proliferation of which was significantly inhibited by IL-24recombinant protein and their inhibition rate are34.69%and36.21%respectively when both tumor cells were treated with50ng/mL IL-24recombinant protein. These results didn’t find in A549cell expressing imperfect IL-24receptors and normal HEL cell expressing IL-24receptors under the same condtions. Besides, IL-24recombinant protein could significantly inhibit A375and Eca cell clone formation, with no effect on A549and HEL cell and the same as MTT.4. AnnexinV-FITC stained and flow cytometry demonstrated that IL-24recombinant protein could induce A375and Eca cell apoptosis, and their apoptosis rate were64.5%and34.5%when A375and Eca cells were treated with40ng/mL IL-24recombinant protein respectively.5. Antibody neutralize results indicated that A375and Eca-109cell growth-suppression by IL-24recombinant protein-mediated was remarkably neutralized by IL-24antibody (AF1965, MAB1965),1L-20R1,1L-20R2, IL-22R, IL-20R1plus IL-20R2or IL-22R plus IL-20R2, respectively, which suggested that secreted IL-24recombinant protein bound to its functional receptor leading to inhibit A375and Eca-109cell proliferation.6. STAT3phosphorylation was activated in A375and Eca cells treated with20ng/mL IL-24recombinant protein for4h, and without activation for1h, while this effect was attenuate at the present of STAT3phosphorylation inhibitor (500ng/mL), which suggested that IL-24activatd STAT3signaling pathway by binding to its receptors, and inhibited cell proliferation and induced apoptosis in A375and Eca cells.Growth-suppression of tumor cell is the major target of tumorbio therapy. It is an urgent task of fundamental research to clarify the mechanism of the functional molecule and carcinogenesis. In our study, the IL-24engineering cell line was successfully established and received the high purity IL-24recombinant protein, which can not only inhibit Eca-109and A375cell proliferation and clone formation, but also induce appoptsis. IL-24recombinant protein might bind to its functional receptors leading to active STAT3molecule culminate in suppressing cell proliferation and inducing appoptsis. This study has provided experimental basis for biological activity of the purified IL-24recombinant protein in the form of protein drugs used in esophageal cancer treatment.