穿膜肽11R在携带P53基因的溶瘤腺病毒中抗肿瘤作用的研究
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摘要
本文旨在探讨携带有11R-P53基因的溶瘤腺病毒是否能够有效的抑制肝癌细胞的生长,同时对我们的基因病毒治疗系统SG7605-11R-P53的靶向性、有效性和安全性作以评价。
背景:P53是一个关键的肿瘤抑制因子,对于它的研究已经有30多年的历史,但是目前人们对它还没有一个较全面的认识。它是一个分子量为53kDa的核转录因子,在G1期检查点扮演着重要角色,应答于DNA损伤或致癌基因的激活,从而以确保基因组的完整性。一旦发生DNA损伤或致癌基因激活时,它就会被激活,从而使得细胞周期停滞或诱导其发生凋亡。事实上由于基因突变使得P53很容易失活,从而失去对突变细胞的诱导凋亡作用。在发现的人类肿瘤中有一半以上会发生P53基因的突变,并且其碱基的突变率是非常高的。大量数据表明,突变的P53基因不仅失去了抑制肿瘤的功能,而且也获得了新的促进肿瘤发生的能力[1]。.我们实验室前期工作已经证明加入P53基因的溶瘤腺病毒能够更有效地抑制那些P53基因突变的癌细胞的生长[2-3]。
细胞穿膜肽(Cell penetrating peptides, CPPs)又可称为蛋白转导域(Protein transduction domain)是一种能携带大分子物质穿过细胞膜进入细胞的短肽。细胞穿膜肽多聚精氨酸11R是一个能够转导生物活性分子进入真核细胞内的短肽,Kazuhito Tomizawa研究小组表明11R-P53融合蛋白能够增强对肿瘤细胞的诱导凋亡率[4-5],从而增强了抗肿瘤的作用。本课题利用穿膜肽11R的特殊穿膜能力,以期增强携带有P53基因的溶瘤腺病毒的再次感染肿瘤细胞的机会,以达到彻底杀灭肿瘤的目的。
方法与结果:利用本实验室构建的携带有细胞穿膜肽11R的溶瘤腺病毒SG7605-11R-P53以及对照病毒SG7605-P53分别感染肝癌细胞株HepGII、SMMC-7721、Hep3B、Huh7和成纤维细胞株BJ,用western blot方法检测目的蛋白P53的表达情况,结果显示SG7605-11R-P53和SG7605-P53均能在肝癌细胞株中和正常细胞内正常表达。而增殖实验表明,SG7605-11R-P53能在肝癌细胞HepGII、SMMC-7721、Hep3B、Huh7中大量增殖,而在正常肝细胞LO2内和成纤维细胞株BJ内复制能力较弱,而且SG7605-11R-P53的增殖倍数要高于SG7605-P53的;同样,MTT实验结果显示SG7605-11R-P53对肝癌细胞株HepGII、SMMC-7721、Hep3B、Huh7有较强的杀伤效果,而对正常成纤维细胞株BJ的杀伤力较弱,并且SG7605-11R-P53具有更强的杀伤效果。
分别将携带细胞穿膜肽11R的溶瘤腺病毒SG7605-11R-EGFP以及不含细胞穿膜肽11R的溶瘤腺病毒SG7605-EGFP感染肝癌细胞株HepGII、Hep3B、Huh7和正常肝细胞LO2,荧光显微镜下观察并拍照发现,在肝癌细胞HepGII、Hep3B、Huh7中SG7605-11R-EGFP的荧光在亮度和密度上都强于相同MOI条件下的SG7605-EGFP的作用效果,同时我们也观察了不同时间点SG7605-11R-EGFP在肝癌细胞内和正常细胞内的荧光变化情况,结果显示,随着时间的增加,肝癌细胞株HepGII、Hep3B中的荧光亮度和密度是逐渐增强的,而在正常细胞中,荧光亮度和密度是几乎不变的。将SG7605-11R-EGFP、SG7605-EGFP以及带有标记的自由肽11R与SG7605-EGFP的组合分别感染肝癌细胞,我们发现相同条件下SG7605-11R-EGFP的感染效率最高,其次分别是SG7605-EGFP+11R和SG7605-EGFP。
在BALB/c裸鼠皮下制备肝癌Huh7的肿瘤模型,在肿瘤生长至7～9mm时进行随机分3组治疗,分别进行瘤内注射SG7605-11R-P53和SG7605-P53以及对照组的PBS,自第一次治疗开始,定期测量肿瘤体积。结果显示经过SG7605-11R-P53和SG7605-P53治疗的裸鼠的肿瘤体积明显的小于空白对照组,并且SG7605-11R-P53对肿瘤生长的抑制率明显的高于SG7605-P53的。
由上述可知,我们的携带细胞穿膜肽11R的溶瘤腺病毒SG7605-11R-P53能在肝癌细胞中大量增殖,并对肝癌细胞具有高效的杀伤效果,而对正常细胞具有较弱的侵染能力和杀伤力,充分表明了SG7605-11R-P53是一种安全并且有效的溶瘤腺病毒。通过本课题的研究,我们联合细胞穿膜肽11R的特殊穿膜能力和P53基因对肿瘤的诱导凋亡的能力以及对溶瘤腺病毒的靶向性调控,用于对肿瘤有效地治疗,进一步完善了我们基因-病毒治疗系统,为以溶瘤腺病毒载体系统为主的靶向治疗癌症建立了基础。
Anti-cancer effect of membrane penetrating peptide 11R in Oncolytic Adenoviruses carrying P53
Abstract 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.
背景:P53是一个关键的肿瘤抑制因子,对于它的研究已经有30多年的历史,但是目前人们对它还没有一个较全面的认识。它是一个分子量为53kDa的核转录因子,在G1期检查点扮演着重要角色,应答于DNA损伤或致癌基因的激活,从而以确保基因组的完整性。一旦发生DNA损伤或致癌基因激活时,它就会被激活,从而使得细胞周期停滞或诱导其发生凋亡。事实上由于基因突变使得P53很容易失活,从而失去对突变细胞的诱导凋亡作用。在发现的人类肿瘤中有一半以上会发生P53基因的突变,并且其碱基的突变率是非常高的。大量数据表明,突变的P53基因不仅失去了抑制肿瘤的功能,而且也获得了新的促进肿瘤发生的能力[1]。.我们实验室前期工作已经证明加入P53基因的溶瘤腺病毒能够更有效地抑制那些P53基因突变的癌细胞的生长[2-3]。
细胞穿膜肽(Cell penetrating peptides, CPPs)又可称为蛋白转导域(Protein transduction domain)是一种能携带大分子物质穿过细胞膜进入细胞的短肽。细胞穿膜肽多聚精氨酸11R是一个能够转导生物活性分子进入真核细胞内的短肽,Kazuhito Tomizawa研究小组表明11R-P53融合蛋白能够增强对肿瘤细胞的诱导凋亡率[4-5],从而增强了抗肿瘤的作用。本课题利用穿膜肽11R的特殊穿膜能力,以期增强携带有P53基因的溶瘤腺病毒的再次感染肿瘤细胞的机会,以达到彻底杀灭肿瘤的目的。
方法与结果:利用本实验室构建的携带有细胞穿膜肽11R的溶瘤腺病毒SG7605-11R-P53以及对照病毒SG7605-P53分别感染肝癌细胞株HepGII、SMMC-7721、Hep3B、Huh7和成纤维细胞株BJ,用western blot方法检测目的蛋白P53的表达情况,结果显示SG7605-11R-P53和SG7605-P53均能在肝癌细胞株中和正常细胞内正常表达。而增殖实验表明,SG7605-11R-P53能在肝癌细胞HepGII、SMMC-7721、Hep3B、Huh7中大量增殖,而在正常肝细胞LO2内和成纤维细胞株BJ内复制能力较弱,而且SG7605-11R-P53的增殖倍数要高于SG7605-P53的;同样,MTT实验结果显示SG7605-11R-P53对肝癌细胞株HepGII、SMMC-7721、Hep3B、Huh7有较强的杀伤效果,而对正常成纤维细胞株BJ的杀伤力较弱,并且SG7605-11R-P53具有更强的杀伤效果。
分别将携带细胞穿膜肽11R的溶瘤腺病毒SG7605-11R-EGFP以及不含细胞穿膜肽11R的溶瘤腺病毒SG7605-EGFP感染肝癌细胞株HepGII、Hep3B、Huh7和正常肝细胞LO2,荧光显微镜下观察并拍照发现,在肝癌细胞HepGII、Hep3B、Huh7中SG7605-11R-EGFP的荧光在亮度和密度上都强于相同MOI条件下的SG7605-EGFP的作用效果,同时我们也观察了不同时间点SG7605-11R-EGFP在肝癌细胞内和正常细胞内的荧光变化情况,结果显示,随着时间的增加,肝癌细胞株HepGII、Hep3B中的荧光亮度和密度是逐渐增强的,而在正常细胞中,荧光亮度和密度是几乎不变的。将SG7605-11R-EGFP、SG7605-EGFP以及带有标记的自由肽11R与SG7605-EGFP的组合分别感染肝癌细胞,我们发现相同条件下SG7605-11R-EGFP的感染效率最高,其次分别是SG7605-EGFP+11R和SG7605-EGFP。
在BALB/c裸鼠皮下制备肝癌Huh7的肿瘤模型,在肿瘤生长至7～9mm时进行随机分3组治疗,分别进行瘤内注射SG7605-11R-P53和SG7605-P53以及对照组的PBS,自第一次治疗开始,定期测量肿瘤体积。结果显示经过SG7605-11R-P53和SG7605-P53治疗的裸鼠的肿瘤体积明显的小于空白对照组,并且SG7605-11R-P53对肿瘤生长的抑制率明显的高于SG7605-P53的。
由上述可知,我们的携带细胞穿膜肽11R的溶瘤腺病毒SG7605-11R-P53能在肝癌细胞中大量增殖,并对肝癌细胞具有高效的杀伤效果,而对正常细胞具有较弱的侵染能力和杀伤力,充分表明了SG7605-11R-P53是一种安全并且有效的溶瘤腺病毒。通过本课题的研究,我们联合细胞穿膜肽11R的特殊穿膜能力和P53基因对肿瘤的诱导凋亡的能力以及对溶瘤腺病毒的靶向性调控,用于对肿瘤有效地治疗,进一步完善了我们基因-病毒治疗系统,为以溶瘤腺病毒载体系统为主的靶向治疗癌症建立了基础。
Anti-cancer effect of membrane penetrating peptide 11R in Oncolytic Adenoviruses carrying P53
Abstract 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.
引文
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