Removing a Cystein Group On Interferon Alpha 2b at Position 2 and 99 does Not Diminish Antitumor Activity of the Protein, Even Better

Heni RACHMAWATI

doi:10.3797/scipharm.isp.2015.04

Abstract

Interferon alpha 2b is the only standard therapeutic protein for hepatitis virus infections. Further study demonstrated that this protein also posseses antitumor activity in several cancerous organs. One main pathway of this antitumor activity is mediated through antiproliferation as well as proapoptotic effects. Previously, we have successfully developed recombinant human interferon alpha 2b (rhIFNα2b) by using a synthetic gene. In addition, two mutein forms of rhIFNα2b were generated to improve the characteristics of this protein. Two point mutations showed better pharmacokinetic profiles than one point mutation as well as the native form. In the present study, this mutein form was studied for ist antitumor effect in vitro using HepG2 cells. As a comparison, the native form as well as a commercial rIFNα2b were used. Several parameters were investigated including the MTT assay, cell viability test, cell cycle using flow cytometric analysis, and the genes and protein expressions involved in cell growth. The latest was observed to study the mechanism of rhIFNα2b. There was no significant difference in the MTT assay and cell viability after cells were treated with both forms of rhIFNα2b. However, the mutein rhIFNα2b tended to show better proapoptotic activity reflected by flow cytometric data, protein expression of pSTAT1, and DNA expression of caspase 3.

Highlights

Interferons are a group of multi-functional cytokines that were originally identified as the proteins responsible for the formation of cellular resistance to bacterial lipopolysaccharides (LPS) and viral infections [1]
As reported by Asano et al, IFN-α affected cell cycle arrest of mouse macrophages showed that the Cdk inhibitors p19 and p21 were strongly up-regulated after treatment with IFN-α, and that the binding of these inhibitors to the G1 cyclin/Cdk complex leads to reduction of its kinase activities and results in G1 arrest in the early phases of IFN treatment [4]
Since STAT1 has been shown to promote apoptosis and carry tumor suppressor functions in different types of cancers, the correlation among parameters we reported here will help to explain the mechanism of our developed interferon alpha 2b in the cancer treatment

Summary

Introduction

Interferons are a group of multi-functional cytokines that were originally identified as the proteins responsible for the formation of cellular resistance to bacterial lipopolysaccharides (LPS) and viral infections [1]. Interferons play a role in the control of cell growth, differentiation and regulation of the immune system. Their used in anticancer therapy had been well documented for a long time and often possess relatively high antitumor activity. Interferon affects different phases of the mitotic cycle in different cell systems with the most common effect is G1 arrest [3]. Treatment with interferon induces Cdk inhibitors p15 and p27 [5,6,7] as well, resulting in cell-cycle arrest at the G1 phase

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