不同p53状态的肝癌细胞系DNA损伤修复能力的比较

目的 观察不同p53状态的肝癌细胞在DNA损伤因素存在下，探讨p53在肝细胞癌发生过程中的作用。方法 选用内源性表达野生型p53、突变型p53、p53全基因缺失的HepG2、PLC／PRF／5、Hep3B肝癌细胞系，将含有p53结合序列的CAT报告基因质粒分别转染各细胞，通过ELISA方法检测报告基因CAT活化情况，观察p53功能状态；用细胞生长曲线比较不同p53状态的细胞生长速度的差异；给予一定剂量的紫外线照射，检测各细胞程序外DNA损伤修复(UDS)能力；观察紫外线照射后细胞克隆形成情况，反映不同细胞系在紫外线照射后细胞存活率的不同。结果 报告基因CAT在HepG2细胞表达最强，而在PLC／PRF／5、Hep3B肝癌细胞均处于较低水平，说明HepG2细胞具有功能性的p53表达；HepG2细胞生长速度明显慢于其他两种细胞；紫外线照射后，HepG2具有较好的DNA损伤修复能力以及较高的细胞生存率。结论 野生型p53具有抑制肝癌细胞生长、保持细胞良好的DNA损伤修复的功能。     

非增殖型腺病毒和肿瘤特异性增殖型腺病毒携带IL-12基因治疗肝癌的体外实验研究

目的 比较携带小鼠IL-12基因的增殖型腺病毒(CNHK200-mIL12)和非增殖型腺病毒(Adv-mIL12)对IL-12基因的表达以及对肝癌细胞的杀伤能力。方法 通过MTT以及病毒增殖实验．评估E1B-55000缺陷的增殖型腺病毒CNHK200-mIL12和ONYX-015(dl1520),以及非增殖型腺病毒Adv-mIL12对人正常肝细胞株LO2、人肝癌细胞株HepG2和Hep3B的杀伤能力。采用蛋白质印迹分析和ELISA法,检测CNHK200-mIL12和Adv-mill2感染HepG2和Hep3B细胞后,小鼠IL-12基因的表达情况。结果 CNHK200-mIL12感染HepG2和Hep3B细胞后大量增殖,在感染后96h时检测,分别增殖3160倍和630倍,在极低的MOI(空斑形成单位／细胞)值和极短的时间内(HepG2细胞：MOI=0．2,第4天;Hep3B细胞：MOI=0．005,第2天),可大量杀伤肿瘤细胞,而对LO2细胞无明显杀伤。CNHK200-miLl2和Adv-mIL12感染HepG2细胞后,其IL-12基因表达量,前者是后者的101倍;感染Hep3B细胞后,前者是后者的20倍。结论 增殖型腺病毒载体对肿瘤细胞的杀伤能力和目的基因的表达,明显优于传统的非增殖型腺病毒载体,应用前景广阔。     

装载mIFN-γ基因的溶瘤腺病毒CNHK200的抗肝癌活性

目的:研究鼠源γ-干扰素(mIFN-γ)基因插入溶瘤腺病毒CNHK200后,该病毒对不同的肝癌细胞系及其裸鼠移植瘤的增殖力及特异性杀伤作用。方法:用MTT法检测CNHK200-mIFN-γ在体外对人正常肝细胞L02、人肝癌细胞Hep3B、HepGⅡ的特异性杀伤作用;裸鼠体内试验观察CNHK200-mIFN-γ对肝癌Hep3B模型的抗肿瘤疗效。结果:CNHK200-mIFN-γ对正常人肝细胞L02无杀伤作用,但能特异性杀伤肝癌细胞,mIFN-γ的插入使病毒对肿瘤细胞的杀伤能力提高;动物体内,CNHK200-mIFN-γ具有明显的肿瘤生长抑制作用。结论:CNHK200-mIFN-γ可以高效率地杀死肝癌肿瘤细胞,而不杀伤正常细胞,可能具有良好的临床应用前景。     

TIP30基因腺病毒载体的构建及体内外抑癌作用

目的 利用缺陷型腺病毒载体表达TIP30基因,观察其体内外抑瘤作用。方法 用Ad-Easy^TM系统,在大肠杆菌同源重组,构建Ad-TIP30腺病毒载体,经在293细胞内成功包装并鉴定后,感染p53基因型不同的肝癌细胞株HepG2(p53-wt)、PLC／PRL／5(p53-mut)和骨肉瘤细胞株Saos-2(p53-null)。用台盼蓝拒染法检测存活细胞,观察TIP30的体外抑瘤作用;用RT-PCR方法检测HepG2细胞p53基因的表达水平;通过裸鼠皮下肝癌移植瘤模型观察Ad-TIP30的体内抑瘤效果。结果 Ad-TIP30对3种肿瘤细胞的增殖均有抑制作用,但对p53基因野生型的肝癌细胞株HepG2抑制作用最明显。经Ad-TIP30感染后,HepG2细胞p53基因表达水平显著升高。Ad-TIP30可显著抑制裸鼠皮下肿瘤的生长,其抑瘤率达62．8％。结论 腺病毒载体表达TIP30基因可通过p53基因依赖性或非依赖性途径抑制肿瘤细胞的增殖,是一种潜在的肿瘤生物治疗手段。     

溶瘤腺病毒SG7605-11R-P53对肝癌细胞的体外杀伤作用

目的:研究携带细胞穿膜肽11R和P53的溶瘤腺病毒SG7605-11R-P53对肝癌细胞的体外杀伤作用。方法:以本课题组前期实验构建的携细胞穿膜肽11R和P53的溶瘤腺病毒SG7605-11R-P53和不携11R的溶瘤腺病毒SG7605-P53感染肝癌细胞HepG2、SMMC-7721、Hep3B、Huh7和正常成纤维细胞株BJ,Western blotting检测感染后细胞P53和11R-P53的表达情况,TCID50法检测SG7605-11R-P53和SG7605-P53在肝癌细胞中的增殖能力,MTT法检测SG7605-11R-P53对肝癌细胞及正常细胞的杀伤作用。结果:SG7605-11R-P53和SG7605-P53能在肝癌细胞中高表达P53和11R-P53蛋白。SG7605-11R-P53可在HepG2、SMMC-7721、Hep3B和Huh7细胞中大量增殖,其增殖倍数是SG7605-P53的10~100倍,但在正常BJ细胞内几乎不增殖。SG7605-11R-P53在MOI=0.1时对Hep3B细胞的杀伤率达90%,对于正常BJ细胞只有当MOI=50时才有很弱的抑制作用;SG7605-11R-P53对4种肝癌细胞杀伤作用的大小依次为Hep3B、HepG2、Huh7和SMMC-7721细胞。结论:携带细胞穿膜肽11R和P53的SG7605-11R-P53溶瘤腺病毒体外对4种肝癌细胞株均有较好的靶向杀伤作用,尤其对Hep3B细胞的杀伤作用最强。     

复制缺陷型腺病毒Ad-p14ARF治疗肝细胞癌的实验研究及作用机制的探讨

目的 研究复制缺陷型重组腺病毒Ad—p14ARF用于肿瘤基因治疗的可行性及其作用机制。方法 利用Ad—Easy系统构建并扩增了Ad—p14ARF复制缺陷型腺病毒;应用台盼蓝细胞计数法和倒置相差显微镜观察Ad-p14ARF对肝癌细胞的生长抑制作用;AnnexinV／PI双染法结合流式细胞仪检测不同滴度的Ad-p14ARF作用后肝癌细胞的凋亡情况;Western blot法检测相关蛋白的表达情况,构建皮下肝癌移植瘤裸鼠模型,研究Ad—p14ARF在体内的抑瘤活性。结果 Ad—p14ARF可以明显抑制野牛型p53和突变型p53表型的肝癌细胞增殖、生长。AnnexinV／PI双染色法显示,Ad—p14ARF能促进肝癌细胞株HepG2和BEL7402的细胞凋亡,凋亡细胞比例与腺病毒滴度呈现一定的剂量相关性。凋亡相关蛋白检测提示,Ad—p14ARF能上调p53下游基因Bax、p21的表达。体内抑瘤试验显示,Ad—ECRG2可以明显抑制肝癌细胞BEL7402裸鼠移植瘤的生长。结论 p14ARF基因的导入可以通过p53依赖和非依赖途径抑制肿瘤细胞增殖,促进肿瘤细胞凋亡,有望在恶性肿瘤的基因治疗中发挥重要作用。     

腺病毒介导HSV—tk自杀基因联合野生型p53基因对直肠癌细胞的杀伤作用

目的 观察重组腺病毒介导单纯疱疹病毒胸苷激酶（HSV－tk)基因联合人野生型p53基因共转染,对直肠癌细胞杀伤作用。方法 构建重组腺病毒质粒pAdCMV-Link1(tk/p53)、pAdCMV-Link(tik),pAdCMV-Link(p53),分别感染p53炎变的人直肠癌细胞SW837。进行细胞集落形成实验、细胞存活率的测定和裸鼠移植瘤治疗实验,观察HSV－tk/GCV系统与野生p53基因联合对肿瘤细胞的杀伤作用。结果 应用pAdCMV-Link1(tk/p53),pAdCMV-Link(-),pAdCMV-Link(tk),pAdCMV-Link(p53)重组腺病毒感染SW837细胞,加入GCV,各组细胞集落数分别为8、95、40、70。pAdCMV-Link(CD/53)组肿瘤细胞集落形成减少、细胞存活率显著下降（P＜0．01）。裸鼠移植肿瘤生长抑制率分别为76．5％、0．8％、55．8％、23．2％,pACMV-Link(tk/p53)重组腺病毒对肿瘤的抑制作用最强。结论 HSV－tk自杀基因与野生型p53基因共转染,对肿瘤细胞有更强的杀伤作用。     

53基因及CAR受体对ONYX-015肿瘤杀伤作用的影响

目的:利用肿瘤特异性增殖型腺病毒ONYX-015分别感染具有柯萨奇病毒和腺病毒联合受体(CAR)水平正常、p53正常或突变的,以及CAR水平低下、p53突变的肿瘤细胞株,研究ONYX 015对这些肿瘤细胞的特异性增殖及杀伤能力。方法:以正常的肝细胞株L02作为对照,用细胞病变效应(CPE)实验观察ONYX-015对细胞的选择性杀伤效应;病毒增殖实验检测野生型腺病毒(Ad5)、ONYX 015在多种肿瘤细胞中的增殖能力。结果:ONYX 015对正常的肝细胞L02无杀伤性,但能够有效地杀伤p53突变的肝癌细胞Hep3B、p53正常的肝癌细胞HepG2及肺癌细胞A549,不能杀伤p53突变的人乳腺癌细胞株MDA-MB 231。在CAR受体水平正常的癌细胞株Hep3B、HepG2和A549中,Ad5和ONYX-015均可增殖。在CAR受体水平低下、p53突变的人乳腺癌细胞株MDA-MB 231中,两种病毒均不增殖。结论:CAR受体对ONYX-015的增殖力起着至关重要的作用。在CAR受体水平正常的前提下,无论肿瘤细胞的p53基因正常与否,ONYX-015均可以有效增殖并杀伤细胞;相反,如果CAR受体水平低下,即使该种肿瘤细胞p53基因突变,ONYX-015在该细胞中的增殖力也会受到限制。ONYX-015不杀伤CAR受体及p53基因均正常的正常肝细胞。     

p53基因及CAR受体对ONYX-015肿瘤杀伤作用的影响

目的：利用肿瘤特异性增殖型腺病毒ONYX-015分别感染具有柯萨奇病毒和腺病毒联合受体（CAR）水平正常、p53正常或突变的,以及CAR水平低下、p53突变的肿瘤细胞株,研究ONYX-015对这些肿瘤细胞的特异性增殖及杀伤能力。方法：以正常的肝细胞株L02作为对照,用细胞病变效应（CPE）实验观察ONYX-015对细胞的选择性杀伤效应;病毒增殖实验检测野生型腺病毒（Ad5）、ONYX-015在多种肿瘤细胞中的增殖能力。结果：ONYX-015对正常的肝细胞L02无杀伤性,但能够有效地杀伤p53突变的肝癌细胞Hep3B、p53正常的肝癌细胞HepG2及肺癌细胞A549,不能杀伤p53突变的人乳腺癌细胞株MDA—MB-231。在CAR受体水平正常的癌细胞株Hep3B、HepG2和A549中,Ad5和ONYX-015均可增殖。在CAR受体水平低下、p53突变的人乳腺癌细胞株MDA—MB-231中,两种病毒均不增殖。结论：CAR受体对ONYX-015的增殖力起着至关重要的作用。在CAR受体水平正常的前提下,无论肿瘤细胞的p53基因正常与否,ONYX-015均可以有效增殖并杀伤细胞;相反,如果CAR受体水平低下,即使该种肿瘤细胞p53基因突变,ONYX-015在该细胞中的增殖力也会受到限制。ONYX-015不杀伤CAR受体及p53基因均正常的正常肝细胞。     

肝癌选择性溶瘤腺病毒的构建及其体外抑瘤作用

目的: 构建由AFP基因增强子启动子调控,并携带自杀基因TK的新型条件复制型腺病毒载体,观察该载体的选择复制能力、溶瘤作用及其与前药GCV联合处理对肝癌细胞的杀伤作用。方法:以HepG2基因组DNA为模板,PCR扩增AFP基因启动子（AFPp）和增强子(AFPe),构建表达质粒pAFPpEGFPluc和pAFPepEGFPluc,再构建由AFPep调控E1A表达、并携带TK基因的穿梭质粒pDC311AFPepE1A/CMVTK,利用AdMax系统包装腺病毒Ad.AFPepE1A/CMVTK;利用Western blotting、病毒增殖测试、细胞病变效应实验、病毒联合前药GCV对肝癌细胞的杀伤实验等鉴定病毒的复制能力、溶瘤作用和对肝癌细胞的杀伤作用。结果：成功构建的腺病毒载体 Ad.AFPepE1A/CMVTK在AFP阳性细胞中选择性复制,病毒自身具有一定的溶瘤作用;该病毒载体联合GCV前药系统处理肝癌细胞后,AFP阳性肝癌HepG2细胞和Hep3B细胞的存活率分别为(10.35±1.07)%、(15.49±5.80)%,AFP阴性的张氏肝细胞和人肺癌NCIH460细胞的存活率分别为(73.55±436)%、(74.54±9.89)% (P<0.01)。结论:构建的新型溶瘤腺病毒载体具有选择性杀伤肝癌细胞的能力,在肝癌治疗方面有良好的应用前景。     

p53 selective and nonselective replication of an E1B-deleted adenovirus in hepatocellular carcinoma.

An E1B gene-attenuated adenovirus (dl1520) has been proposed to have a selective cytolytic activity in cancer cells with a mutation or deletion in the p53 tumor suppressor gene (p53-null), a defect present in almost half of human hepatocellular carcinomas (HCCs). In this study, the in vitro and in vivo antitumor activity of dl1520 was investigated focusing on two human HCC cell lines, a p53-wild type (p53-wt) cell line and a p53-null cell line. dl1520 was tested for in vitro cytopathic effects and viral replication in the human HCC cell lines Hep3B (p53-null) and HepG2 (p53-wt). The in vivo antitumor effects of dl1520 were investigated in tumors grown s.c. in a severe combined immunodeficient mouse model. In addition, the combination of dl1520 infection with systemic chemotherapy was assessed in these tumor xenografts. At low multiplicities of infection, dl1520 had an apparent p53-dependent in vitro viral growth in HCC cell lines. At higher multiplicities of infection, dl1520 viral replication was independent of the p53 status of the target cells. In vivo, dl1520 significantly retarded the growth of the p53-null Hep3B xenografts, an effect augmented by the addition of cisplatin. However, complete tumor regressions were rare, and most tumors eventually grew progressively. dl1520 had no effect on the in vivo growth of the p53-wt HepG2 cells, with or without cisplatin treatment. The E1B-deleted adenoviral vector dl1520 has an apparent p53-dependent effect in HCC cell lines. However, this effect is lost at higher viral doses and only induces partial tumor regressions without tumor cures in a human HCC xenograft model.     

Radiation-induced intercellular signaling mediated by cytochrome-c via a p53-dependent pathway in hepatoma cells

The tumor suppressor p53 has a crucial role in cellular response to DNA damage caused by ionizing radiation, but it is still unclear whether p53 can modulate radiation-induced bystander effects (RIBE). In the present work, three different hepatoma cell lines, namely HepG2 (wild p53), PLC/PRF/5 (mutation p53) and Hep3B (p53 null), were irradiated with γ-rays and then co-cultured with normal Chang liver cell (wild p53) in order to elucidate the mechanisms of RIBE. Results showed that the radiosensitivity of HepG2 cells was higher than that of PLC/PRF/5 and Hep3B cells. Only irradiated HepG2 cells, rather than irradiated PLC/PRF/5 or Hep3B cells, could induce bystander effect of micronuclei (MN) formation in the neighboring Chang liver cells. When HepG2 cells were treated with 20?μM pifithrin-α, an inhibitor of p53 function, or 5?μM cyclosporin A (CsA), an inhibitor of cytochrome-c release from mitochondria, the MN induction in bystander Chang liver cells was diminished. In fact, it was found that after irradiation, cytochrome-c was released from mitochondria into the cytoplasm only in HepG2 cells in a p53-dependent manner, but not in PLC/PRF/5 and Hep3B cells. Interestingly, when 50?μg/ml exogenous cytochrome-c was added into cell co-culture medium, RIBE was significantly triggered by irradiated PLC/PRF/5 and Hep3B cells, which previously failed to provoke a bystander effect. In addition, this exogenous cytochrome-c also partly recovered the RIBE induced by irradiated HepG2 cells even with CsA treatment. Our results provide new evidence that the RIBE can be modulated by the p53 status of irradiated hepatoma cells and that a p53-dependent release of cytochrome-c may be involved in the RIBE.     

Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma

PR-104 is a clinical stage bioreductive prodrug that is converted in vivo to its cognate alcohol, PR-104A. This dinitrobenzamide mustard is reduced to activated DNA cross-linking metabolites (hydroxylamine PR-104H and amine PR-104M) under hypoxia by one-electron reductases and independently of hypoxia by the 2-electron reductase aldo-keto reductase 1C3 (AKR1C3). High expression of AKR1C3, along with extensive hypoxia, suggested the potential of PR-104 for treatment of hepatocellular carcinoma (HCC). However, a phase IB trial with sorafenib demonstrated significant toxicity that was ascribed in part to reduced PR-104A clearance, likely reflecting compromised glucuronidation in patients with advanced HCC. Here, we evaluate the activity of PR-104 in HCC xenografts (HepG2, PLC/PRF/5, SNU-398, Hep3B) in mice, which do not significantly glucuronidate PR-104A. Cell line differences in sensitivity to PR-104A in vitro under aerobic conditions could be accounted for by differences in both expression of AKR1C3 (high in HepG2 and PLC/PRF/5) and sensitivity to the major active metabolite PR-104H, to which PLC/PRF/5 was relatively resistant, while hypoxic selectivity of PR-104A cytotoxicity and reductive metabolism was greatest in the low-AKR1C3 SNU-398 and Hep3B lines. Expression of AKR1C3 in HepG2 and PLC/PRF/5 xenografts was in the range seen in 21 human HCC specimens. PR-104 monotherapy elicited significant reductions in growth of Hep3B and HepG2 xenografts, and the combination with sorafenib was significantly active in all 4 xenograft models. The results suggest that better-tolerated analogs of PR-104, without a glucuronidation liability, may have the potential to exploit AKR1C3 and/or hypoxia in HCC in humans.     

Adenovirus-mediated tBid overexpression results in therapeutic effects on p53-resistant hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, gene therapy may represent an alternative for HCC management. Our earlier study has shown that Bid plays a role in the development of HCC. The aim of our study is to evaluate the possibility of using truncated Bid (tBid) as a novel therapy for HCC treatment. Two HCC cell lines, Hep3B and PLC/PRF/5, were used in the experiment. Hep3B was a p53-resistant while PLC/PRF/5 a p53-sensitive. A recombinant adenovirus-Ad/AFPtBid, which contained a tBid gene driven by an alpha-fetoprotein (AFP) promoter, was constructed. Both Hep3B and PLC/PRF/5 cells infected with Ad/AFPtBid showed a significant decrease in cell viability. The decrease in cell viability by Ad/AFPtBid resulted from apoptosis of HCC cells, evident by enhanced activity of caspases and increased release of cytochrome c. In vivo experiment was performed by the intratumor injection of Ad/AFPtBid in nude mice inoculated with Hep3B. Ad/AFPtBid injection significantly inhibited tumor growth, and tumor tissues showed a marked increase in TUNEL-positive cells. Our experiment also demonstrated that Ad/AFPtBid only targeted AFP-producing cells but not those non-AFP producing cells. In conclusion, these results indicate that the introduction of Ad/AFPtBid can not only significantly but specifically kill HCC cells that produce AFP. The cell death induced by Ad/AFPtBid in HCC cells is via an apoptotic pathway that can be independent of p53 status.     

Assessment of growth inhibition and morphological changes in in vitro and in vivo hepatocellular carcinoma models post treatment with dl1520 adenovirus

BACKGROUND AND AIMS: E1B-deleted virus dl1520 (ONYX-015) has been previously used in clinical trials mainly for treatment of head and neck tumors, and has been shown to have beneficial effects independent of p53 status. The main aim of this investigation was to carry out a preclinical study for assessment of the use of dl1520 in in vitro and in vivo hepatocellular carcinoma (HCC) models with various p53 status (deleted, mutant, and wild type), and study the ultrastructural changes in the carcinoma cells during and following treatment with dl1520. METHODS: dl1520 (ONYX-015) virus was used for treatment of three HCC cell lines in culture, then for treatment of developed xenografts in SCID mice. The effects of dl1520 on HCC cell growth and accompanied morphological changes were assessed by various techniques including transmission electron microscopy. dl1520 infection was confirmed using polymerase chain reaction and immunolabeling at transmission electron microscopy level. RESULTS: dl1520 was effective in killing cells and inhibiting HCC cell growth both in vitro and in vivo. The cell killing was at higher levels in cells possessing abnormal p53. Survival rates in SCID mice treated with dl1520 were statistically significantly higher in HCC tumors with deleted and mutant p53, than in tumors with wild-type p53. CONCLUSIONS: The findings in this study suggest that dl1520 could be safely and effectively used for treatment of HCC dependent on the p53 status of the cells in vivo. Characteristic morphological changes that took place in the dl1520-treated HCC cells/tumors were distinct at transmission electron microscopy level and are the first of their kind to be reported.     

Growth arrest induced by C75, A fatty acid synthase inhibitor, was partially modulated by p38 MAPK but not by p53 in human hepatocellular carcinoma

C75, a well-known fatty acid synthase (FAS) inhibitor, has been shown to possess potent anti-cancer activity in vitro and in vivo. In this study, we reveal that C75 is a cell cycle arrest inducer and explore the potential mechanisms for this effect in hepatocellular carcinoma (HCC) cell lines with abundant FAS expression: HepG2 and SMMC7721 cells with wt-p53, and Hep3B cells with null p53. The results showed FAS protein expression and basal activity levels were higher in HepG2 cells than in the other two HCC cell lines. Treatment with C75 inhibited FAS activity within 30 min of administration and induced G(2) phase arrest accompanied by p53 overexpression in HepG2 and SMMC7721 cells. By contrast, C75 triggered G(1) phase arrest in Hep3B cells, and RNA interference targeting p53 did not attenuate C75-induced G(2) arrest in HepG2 cells. Similarly, p53 overexpression via p53 plasmid transfection did not affect C75-induced G(1) phase arrest in Hep3B cells. However, we observed a clear correlation between p38 MAPK activation triggered by C75 and the induction of cell cycle arrest in all three HCC cells. Furthermore, treatment with the p38 MAPK inhibitor SB203580 reduced p38 MAPK activity and cell cycle arrest, and also partially restored cyclin A, cyclin B1, cyclin D1 and p21 protein levels. Collectively, it was p38 MAPK but not p53 involved in C75-mediated tumor cell growth arrest in HCC cells.