Experimental Study About The Antitumor Effects Of Tumor-targeted IFN-α2a-NGR On Cellular Level

Interferon-α(IFN-α) are pleiotropic cytokines, extensively used in the treatment of patients with some types of cancer and viral infectious diseases. However, some kinds of cancer are not sensitive to IFN-αand significant toxicity of IFN-αtherapy led by high doses were recorded, which have impacted their clinical application. NGR is an oligopeptide which has the ability of tumor angiogenesis specific binding through the surface expression of aminopeptidase N (CD13) in tumor angiogenesis. To increase the anti-tumor activity of IFN-α2a, promote its application in solid tumor and lower its side-effects, the fusion protein IFN-α2a-NGR has been constructed, which was confirmed to possess significant antiangiogenesis activity in vivo and in vitro. After nude mice bearing tumors were treated with IFN-α2a-NGR, the previous results showed that, IFN-α2a-NGR can bind tumor vessels whereas IFN-α2a can not. IFN-α2a-NGR has a more significant inhibition effect on invasion, migration and tube formation of tumors than IFN-α2a. When achieved the similar inhibiting effect of IFN-α2a, the dose of IFN-α2a-NGR would be one-third of the former. Furthermore administration of IFN-α2a-NGR was extremely well tolerated with very high doses in animals, and the use of IFN-α2a-NGR is considered to be safe just as IFN-α2a.However, some kinds of cancer are still not sensitive to IFN-α2a-NGR. In the previous study, we have demonstrated that Interferon-αreceptor (IFNAR) is an important determinant of tumor sensitivity to IFN-α2a therapy. To further investigate the molecular mechanisms of the anti-tumor effect of targeted IFN-α2a-NGR and find the key molecules of this signal pathway and the way to reverse drug sensitivity, we chose two cellular models in the further research. One is interferon-sensitive cell line A549 which expresses high level of IFNAR,the other is MKN-45 which is not sensitive to interferon and its IFNAR level is much lower than A549. We determined the anti-tumor effect of IFN-α2a-NGR on cellular and molecular level, the results should be helpful for the further development of IFN-α2a-NGR.In the present research MTT method was used to determine the growth inhibitory effects of IFN-α2a-NGR; Flow cytometry and Western Blot were employed to detect the expression of STAT1,p-STAT1,p53,OAS and SOCS1; SOCS1 gene knock down was carried out by synthesized siRNA interference. We have demonstrated that both IFN-α2a-NGR and IFN-α2a can activate the JAK/STAT signal pathway and there were no differences on antitumor effects between IFN-α2a-NGR and IFN-α2a on cellular or molecular level. However, there were many differences between MKN-45 and A549. When stimulated with IFN-α2a-NGR or IFN-α2a , a notable increased expression of STAT1,p-STAT1,p53 and OAS were observed in A549 cells while SOCS1 expresssion was significantly increased in MKN-45 cells. After knocking down the expression of SOCS1, cell proliferation was increased in MKN-45 cells but not in A549 cells.In conclusion, our study indicated that the different sensitivity of IFN-α2a-NGR in different cells may be partly due to the expression of IFNR, SOCS1, P53 and p-STAT1. These findings are important for further development of IFN-α2a-NGR and will be helpful for the sensitivity evaluation of its application.