Anti-cancer Effect Of Membrane Penetrating Peptide 11R In Oncolytic Adenoviruses Carrying P53

Anti-cancer effect of membrane penetrating peptide 11R in Oncolytic Adenoviruses carrying P53Abstract Here we try to study whether the oncolytic adenovirus carrying 11R-P53 can efficiently inhibit the growth of liver cancer cell. At the same time we also evaluate the targeting and safety of our gene-viral therapeutic system SG7605-11R-P53.Background P53 is a key tumor suppressor, we have been studying it over 30 years, however it is still not fully understood. P53 is a nuclear transcription factor molecularly weighed 53kDa, and plays an important role in checking DNA damage or oncogene activation in G1 stage, ensuring the total genomic integrity. Once DNA damage or oncogene activation occur, P53 would be induced activated, and then lead to cell cycle arrest or apoptosis. In fact, P53 can be easily inactivated by natural mutation and lose its function inducing mutant cell apoptosis. It is up to 50% of human cancers that we had known are developed by P53 mutation, and the mutation rate of its amino acids is extremely high. Many datas indicate that P53 mutation does not only lead to the loss of its tumor suppressing function, but also the gain of tumor promoting function [1]. Initial work of our lab have proved that the oncolytic adenovirus carried P53 gene could inhibit the growth of cancer cells which caused by P53 mutation [2-3].Cell penetrating peptides (CPPs) , also called protein transduction domain, is a kind of short peptide that can penetrate into cell membrane when carrying large molecular substance. Polyarginine 11R is a short peptide which can be used to transduce bioactive molecular into eukaryotic cell. The group led by Kazuhito Tomizawa have showed that 11R-P53 fusion protein can effectively improve the inducing rate of tumor cell apoptosis, thus enhance the effect of cancer therapy[4-5]. In this article we used the special ability of 11R to enhance the infectious chance of the oncolytic adenovirus carrying P53 gene again, aiming at eradicating tumor cells. Methods and results We used the oncolytic adenovirus carrying 11R SG7605-11R-P53 and the control virus carrying SG7605-P53 to infect the liver cancer cell HepGII,SMMC-7721,Hep3B,Huh7 and normal fibroblast BJ, then we used western blot assay to evaluate the expression of P53 protein. The result showed that SG7605-11R-P53 and SG7605-P53 can be expressed in both hepatoma carcinoma cell and normal cell. Multiplication experiment manifested that SG7605-11R-P53 and SG7605-P53 could effectively reduplicate in cancer cells, but in normal cells they had poor reduplication capacity. Meanwhile SG7605-11R-P53 had a higher reduplication rate than SG7605-P53 in the same cells. In the MTT experiment, we had the same results that SG7605-11R-P53 had a stronger effect in liver cancer cells than it in normal cells, and it also had a stronger killing effect than viral SG7605-P53.Fluorescence photograph showed that in HepGII,Hep3B,Huh7 and LO2 cells, SG7605-11R-EGFP had brighter flurescence than SG7605-EGFP in the same condition. We found that in the same condition SG7605-11R-EGFP in the liver cancer cells had stronger fluorescence, both in the brightness and density. In the same method, we also observed the change of the fluorescence in different times and in different cells. Results showed that the brightness and density of the fluorescence became stronger with the increasing time in liver cancer cells, but in normal cells almost had no change. We used different combinations of virals and free petides with maker to infect liver cancer cells. We found that SG7605-11R-EGFP had a highest infection rate, followed by SG7605-EGFP+11R and SG7605-EGFP.Preparation the tumor model of liver cancer Huh7 in the subcutaneously of BALB/c nude mouse and randomly separated the nude mouse into 3 groups when the tumor had a size about 7-9mm. Then treated them with SG7605-11R-P53,SG7605-P53 and PBS as control. After treatment, periodical measurement of the gross tumor volume was taken. Results showed that after treated with SG7605-11R-P53 and SG7605-P53, tumors were obviously smaller than the control group. Moreover, the group treated by SG7605-11R-P53 was more effective than the group treated by SG7605-P53.From above mention, the oncolytic adenovirus SG7605-11R-P53 could bulk reduplicate in liver cancer cells and haved a higher inhibiting effect over the growth of liver cancer cells, but had a poor effect in normal cells. This sufficiently manifest that SG7605-11R-P53 is a kind of safe and effective oncolytic adenovirus. In this study, we combined a short peptide 11R which has special penetrating membrane ability, and P53 which can induce the apoptosis of cancer cells and regulate the targeting of the oncolytic adenovirus in order to cure the tumor effectively. This may improve our gene-viral therapeutic system and can be used to set up the basic for using the oncolytic adenovirus vector system as major tool for tumor therapy.