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。     

E1B55kDa缺陷型腺病毒对人肝癌细胞的杀伤研究

目的 探讨E1B55kDa缺陷型腺病毒dl1520对肝癌细胞的体内外杀伤作用。方法 用dl1520分别感染p53基因型不同的人肝癌细胞,感染后第4天用染色的方法检测存活细胞。用RT－PCR检测细胞内p53和p21^Waf-1基因表达的改变,通过检测腺病毒衣壳蛋白hexon基因的表达证实腺病毒的感染。在SCID裸鼠瘤体内注射dl1520,观察dl1520对肝癌细胞的体内杀伤作用。结果 p53基因缺失的肝癌细胞Hep3B对dl1520诱导的细胞毒性作用最敏感,超过60％的细菌被杀伤,而不足20％的PLC／PRF／5（p53基因突变型）和HepG2(p53基因野生型）肝癌细胞被杀伤。腺病毒感染后,HepG2细胞内p53和p21^Waf-1基因表达水平均明显升高。瘤体内注射dl1520,可显著抑制Hep3B裸鼠移植瘤的生长,而对PLC／PRF／5和HepG2的裸鼠移植瘤则无明显的生长抑制作用。结论 E1B55kDa缺失的腺病毒可以选择性地杀伤p53基因缺失的肝癌细胞,是一种潜在的肿瘤治疗手段。     

重组复制缺陷型腺病毒Ad p53AIP1的抗肿瘤效应及其作用机制

目的:探讨外源性p53AIP1（p53regulated apoptosisinducing protein 1）基因抗肿瘤效应及其作用机制，以评估p53AIP1基因在肿瘤基因治疗中应用的可行性。方法：构建携带p53AIP1基因的重组复制缺陷型腺病毒Adp53AIP1，感染人肝癌细胞HepG2，应用MTT比色法、Western blotting、流式细胞术、罗丹明染色以及电镜等观察外源性p53AIP1基因表达对肿瘤细胞的作用及其相关机制。小鼠皮下接种感染Adp53AIP1的小鼠乳腺癌4T1细胞，观察Adp53AIP1对肿瘤细胞体内成瘤性的影响;建立4T1细胞皮下移植瘤小鼠模型, 直接瘤内多点注射重组腺病毒，观察Adp53AIP1的抗肿瘤疗效。结果：感染Adp53AIP1的HepG2细胞能够高效表达P53AIP1蛋白。MTT检测显示Adp53AIP1对人肝癌细胞HepG2的生长抑制率达50％以上；流式细胞术分析证实p53AIP1使肿瘤细胞周期阻滞于G2/M期；罗丹明染色、电镜观察以及凋亡相关蛋白PARP表达的检测均证实p53AIP1 能诱导肿瘤细胞发生明显凋亡。感染Adp53AIP1的肿瘤细胞体内成瘤受到非常明显的抑制（P<001）, Adp53AIP1瘤体注射对移植瘤生长也有明显的抑制作用（P<0.05）。Western blotting以及RTPCR检测证实Adp53AIP1对p53mRNA表达无影响，能下调mdm2 基因的表达；p53AIP1能上调P53蛋白的表达、下调MDM2蛋白的表达水平，同时还影响P21等细胞周期相关蛋白和凋亡相关蛋白的表达。结论：p53AIP1有明显的体内外抗肿瘤作用，其作用机制与其反向调控P53蛋白、调控多种细胞周期和细胞凋亡相关蛋白、诱导细胞周期阻滞和细胞凋亡有关，该基因在肿瘤基因治疗中具有潜在的应用前景。     

重组复制缺陷型腺病毒Ad-p53AIP1的抗肿瘤效应及其作用机制

目的：探讨外源性p53AIP1（p53-regulated apoptosis—inducing protein1）基因抗肿瘤效应及其作用机制，以评估声3A，H基因在肿瘤基因治疗中应用的可行性。方法：构建携带p53AIP1基因的重组复制缺陷型腺病毒Ad—p53AIP1，感染人肝癌细胞HepG2，应用MTT比色法、Westernblotting、流式细胞术、罗丹明染色以及电镜等观察外源性p53AIP1基因表达对肿瘤细胞的作用及其相关机制。小鼠皮下接种感染Ad—p53AIPl的小鼠乳腺癌4T1细胞，观察Ad—p53AIP1对肿瘤细胞体内成瘤性的影响；建立4T1细胞皮下移植瘤小鼠模型，直接瘤内多点注射重组腺病毒，观察Ad—p53AIP1的抗肿瘤疗效。结果：感染Ad—p53AIP1的HepG2细胞能够高效表达p53AIP1蛋白。MTT检测显示Ad—p53AIP1对人肝癌细胞HepG2的生长抑制率达50％以上；流式细胞术分析证实p53AIP1使肿瘤细胞周期阻滞于G2／M期；罗丹明染色、电镜观察以及凋亡相关蛋白PARP表达的检测均证实p53AIP1能诱导肿瘤细胞发生明显凋亡。感染Ad—p53AIPl的肿瘤细胞体内成瘤受到非常明显的抑制（P〈0．01），Ad—p53AIP1瘤体注射对移植瘤生长也有明显的抑制作用（P〈0．05）。Western blotting以及RT—PCR检测证实Ad—p53AIP1对声3mRNA表达无影响，能下调mdm2基因的表达；p53AIP1能上调P53蛋白的表达、下调MDM2蛋白的表达水平，同时还影响P21等细胞周期相关蛋白和凋亡相关蛋白的表达。结论：p53AIP1有明显的体内外抗肿瘤作用，其作用机制与其反向调控P53蛋白、调控多种细胞周期和细胞凋亡相关蛋白、诱导细胞周期阻滞和细胞凋亡有关，该基因在肿瘤基因治疗中具有潜在的应用前景。     

p16基因治疗对肝癌细胞生物学行为的影响

背景与目的：p16基因是原发性肝癌（hepatocellular carcinoma,HCC）中失活频率很高的抑癌基因之一,应是治疗HCC的理想靶基因。本研究的目的是探讨转入野生型p16cDNA对肝细胞性肝癌细胞生物学行为的影响。方法：用我们构建的p16基因的逆转录病毒表达载体pcLXSN-p16,分别感染p16蛋白表达阴性与表达阳性的肝癌细胞株SNU-449与HepG2.2.15,筛选出稳定的表达株,对转基因后肿瘤细胞的生物学行为进行观察。结果：p16蛋白表达阴性的SNU-449细胞转入野生型p16基因后,细胞生长速度明显减慢,G0-G1期细胞明显多于转基因前,其裸鼠首次接种成瘤率（可在一定程度上反映瘤细胞对组织的侵袭能力）、在裸鼠体内的生长速度以及对裸鼠的致死性均低于未转基因者。而p16蛋白表达阳性的HepG2.2.15细胞转入外源p16cDNA后,其生长状况及细胞周期则未发现有明显改变。就SNU-449与HepG2.2.15细胞株而言,转入p16cDNA均未能诱导细胞凋亡。结论：转入外源野生型p16基因可抑制p16蛋白表达阴性的肝癌细胞的生长并降低其侵袭能力,但对p16蛋白表达阳性的肝癌细胞的生长则无影响。     

人肝癌组织及肝癌细胞系中TIP30基因与VEGF的关系

目的:观察人肝癌组织中抑癌基因TIP30的表达与VEGF(vascular endothelial growth factor)表达的关系;并在高转移性人肝癌细胞系中外源性导入TIP30基因后检测VEGF的表达,探讨TIP30基因抑制肿瘤转移的机理.方法:免疫组织化学染色法检测肝癌组织中TIP30及VEGF的表达;腺病毒感染将TIP30基因导入HCC-LM3细胞,ELISA检测感染病毒后不同时间培养上清中VEGF的分泌;同时利用RT-PCR检测导入TIP30基因后HCC-LM3细胞中VEGF的转录.结果:1)24例肝癌组织中,与癌旁组织相比11例TIP30蛋白表达降低,其中VEGF表达相对升高的有8例;13例TIP30蛋白表达水平相对无变化者中有4例VEGF表达升高;TIP30蛋白与VEGF的表达相关(P＜0.05).2)感染病毒后12h,三组中VEGF的表达水平无差别;与对照组相比,感染Ad-TIP30病毒后24h,HCC-LM3细胞培养上清中VEGF的水平开始下降,且差别显著(P＜0.05);48h后,VEGF表达水平Ad-TIP30组与两对照组比统计学意义极显著(P＜0.01).RT-PCR检测也表明Ad-TIP30病毒感染48h后HCC-LM3细胞中VEGF的转录水平降低.结论:肝癌组织中TIP30蛋白与VEGF蛋白的表达存在正相关;高转移性的肝癌细胞中外源性TIP30基因可以抑制VEGF的转录;TIP30基因抑制肿瘤细胞的转移可能部分是由于通过抑制VEGF的转录或表达从而抑制肿瘤血管形成来完成的.为今后临床上克服肿瘤转移的抗肿瘤血管治疗研究提供一个新的靶点.     

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。     

Ad-ING4对人前列腺癌细胞PC-3体内外抑癌效应的研究

背景与目的：腺病毒作为载体已被广泛用于肿瘤的基因治疗。ING4是生长抑制因子家族中的一个成员,是一种潜在的抑癌基因。本研究旨在探讨腺病毒介导的人ING4基因（Ad-ING4）对人前列腺癌细胞PC-3的体内外抑癌效应及其分子机制。方法：将扩增的Ad-ING4重组腺病毒感染PC-3细胞,用RT-PCR法检测ING4在PC-3细胞中的转录;MTT法检测ING4基因对PC-3细胞增殖的影响：流式细胞术和Hoechst33258染色法检测细胞凋亡的变化;半定量RT-PCR法检测ING4基因的表达对PC．3细胞中的bcl-2、bax、p53和caspase-3凋亡相关基因表达的影响。用Ad-ING4重组腺病毒在裸鼠PC-3移植瘤的瘤体内注射治疗,观察肿瘤生长变化,15d后处死裸鼠,摘除瘤体,称瘤重;用免疫组化法检测瘤组织中Bcl-2、Bax、Caspase-3和CD34蛋白的表达。结果：腺病毒介导的人ING4基因在PC-3细胞中成功转录,明显抑制PC-3细胞增殖,上调p53、bax、caspase．3基因表达和下调bcI-2基因表达,并诱导细胞凋亡。Ad-ING4重组腺病毒能显著抑制裸鼠PC-3移植瘤的生长,瘤重的抑制率达37％,与空病毒载体Ad-GFP组和细胞对照PBS组比较差异有统计学意义（P〈0．05）;免疫组化结果显示Ad-ING4重组腺病毒能明显上调Bax和Caspase-3蛋白的表达水平,下调Bcl-2和CD34蛋白的表达水平。结论：腺病毒介导的ING4基因在体内外均可明显抑制人前列腺癌细胞PC-3的生长,诱导其凋亡,其机制可能是上调P53、Bax、Caspase-3蛋白和下调Bcl-2蛋白表达水平。     

肝癌靶向性腺病毒载体的构建及其作用的研究

背景与目的:抑癌基因p16是肿瘤基因治疗最常用的治疗基因之一,通过腺病毒载体介导p16基因表达引发肿瘤细胞凋亡在多种p16基因失活的肿瘤细胞系中都得到了证实。研究表明,p16在人原发性肝细胞癌中存在高频率失活。因此,本研究通过观察外源性p16基因经AFP启动子驱动表达后对AFP阳性肝癌细胞的影响,以探讨p16基因在肝癌靶向基因治疗中应用的可行性。方法:将外源性p16基因置于人AFP核心启动子AF0.3下游,构建了携带p16基因的重组复制缺陷型腺病毒Ad-AFP-p16,感染肝癌细胞后,应用MTT比色法、Westernblot、DNA片段化分析、流式细胞术分析等方法在体外研究外源性p16基因表达对细胞生长的影响。小鼠皮下接种感染重组腺病毒的鼠肝癌细胞H22,观察病毒对肝癌细胞体内成瘤性的影响。结果:感染Ad-AFP-p16的肝癌细胞高效表达P16蛋白,MTT结果显示细胞生长受到明显抑制,到第六天Ad-AFP-p16对肝癌细胞Bel-7402和HepG2的生长抑制率分别为(94.42±11.70)%和(94.99±6.74)%,DNA片段化分析和流式细胞术分析证实p16能诱导肝癌细胞发生显著凋亡,感染后48hBel-7402和HepG2的凋亡率分别为39.57%和39.75%。感染Ad-AFP-p16的肝癌细胞体内成瘤受到明显抑制,对照组、Ad-GFP组、Ad-AFP-p16组以及Ad-CMV-p16组瘤体体积分别为(1.54±0.65)cm3、(1.71±1.01)cm3、(0.25±0.39)cm3和(0.25±0.45)cm3。结论:AFP启动子驱动p16基因表达可以诱导肝癌细胞发生凋亡。     

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。     

TIP30 mutant derived from hepatocellular carcinoma specimens promotes growth of HepG2 cells through up-regulation of N-cadherin

TIP30 is a tumor suppressor whose expression is altered in human liver, prostate, lung, colon, and breast cancers. Mice lacking TIP30 spontaneously developed hepatocellular carcinomas (HCC) and other tumors at a higher incidence than wild-type mice. Somatic missense mutations in the TIP30 gene were identified in human HCC tissue specimens, which resulted in instability or abnormal cellular distribution of TIP30 protein in cells. Here, we show that TIP30 mutants are able to promote cell growth and invasion and inhibit cisplatin-induced apoptosis in the HCC cell line HepG2 negative for endogenous TIP30. Moreover, one of the TIP30 mutants can dramatically accelerate tumor formation in immunodeficient mice. Analysis of gene expression in HepG2 cells, ectopically expressing either wild-type TIP30 or mutant TIP30, by Affymetrix GeneChip array, real-time quantitative PCR, and Western blotting assays reveals that TIP30 mutants can alter expression of genes involved in the regulation of tumorigenesis. This includes up-regulation of expression of N-cadherin and c-MYC and down-regulation of expression of p53 and E-cadherin. N-cadherin knockdown with small interfering RNA in HepG2 cells expressing mutant TIP30 resulted in a profound reduction in cell viability. Taken together, our data indicate that somatic mutations in the TIP30 gene may abolish its native tumor-suppressor activity and gain oncogenic activities partially through up-regulation of N-cadherin, thereby potentiating the pathogenesis of HCC in patients.     

MicroRNA-15a-5p suppresses cancer proliferation and division in human hepatocellular carcinoma by targeting BDNF

We examined the expression pattern and functional roles of microRNA 15a-5p (miR-15a-5p) in human hepatocellular carcinoma (HCC). Possible miR-15a-5p aberrant expression in HCC cell lines or clinical HCC specimens was examined by quantitative real-time PCR (qRT-PCR). In HCC HepG2 and SNU-182 cells, miR-15a-5p was ectopically overexpressed by lentiviral transduction. Its effect on HCC proliferation, cancer division, and in vivo tumor growth were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle assay, and tumorigenicity assay, respectively. The targeting of miR-15a-5p on its downstream gene, brain-derived neurotrophic factor (BDNF), was examined by dual-luciferase assay, qRT-PCR, and Western blot, respectively. BDNF was then overexpressed in HepG2 and SNU-182 cells to evaluate its selective effect on miR-15a-5p in HCC modulation. MiR-15a-5p is aberrantly downregulated in in vitro HCC cell lines and in vivo HCC clinical specimens. Ectopic overexpression of miR-15a-5p suppressed cancer proliferation, induced cell cycle arrest in HepG2 or SNU-182 cells in vitro, and inhibited HCC tumor growth in vivo. MiR-15a-5p selectively and negatively regulated BDNF at both gene and protein levels in HCC cells. Forced overexpression of BDNF effectively reversed the tumor suppressive functions of miR-15a-5p on HCC proliferation and cell division in vitro. Our study demonstrated that miR-15a-5p is a tumor suppressor in HCC and its regulation is through BDNF in HCC.     

NT4p53(N15)Ant融合基因重组腺病毒的构建及其体外抑瘤作用

背景与目的:有研究发现P53氨基端15肽与穿膜肽(antennapedia,Ant)的融合肽能迅速穿透细胞膜,直接、快速引起乳腺癌、胰腺癌细胞坏死而非凋亡,但对正常细胞几乎没有毒性。本研究构建缺陷型腺病毒载体表达NT4p53(N15)Ant融合基因,研究其体外抑瘤作用。方法:应用Ad-EasyTM系统,在大肠杆菌同源重组,构建NT4p53(N15)Ant腺病毒表达载体,在HEK-293细胞内成功包装并鉴定后,感染肝癌细胞株HepG2,用倒置相差显微镜、透射电镜观察细胞形态学变化、四甲基偶氮唑蓝(MTT)比色实验及培养基中的乳酸脱氢酶(lactatedehydrogenase,LDH)测定研究其体外抑瘤作用。结果:Ad-NT4p53(N15)Ant对肝癌细胞株HepG2有明显抑制作用,48、72、96h细胞存活率分别为36.67%、20.47%、17.82%,与空病毒处理组比较差异有统计学意义(P<0.05);电镜观察到感染的HepG2细胞胞膜、核膜破坏,胞浆内出现囊泡状物质,染色质聚集。Ad-NT4p53(N15)Ant处理HepG2细胞24、48、72、96h时培养液中LDH释放分别为94、236、267、313U/L,较空病毒处理组明显增加,其差异有统计学意义(P<0.05)。结论:构建的腺病毒载体Ad-NT4p53(N15)Ant感染HepG2细胞后能抑制细胞增殖。     

Reduction of TIP30 correlates with poor prognosis of gastric cancer patients and its restoration drastically inhibits tumor growth and metastasis

Gastric cancer is an aggressive cancer with poor prognosis. Identification of precise prognostic marker and effective therapeutic target is important in the treatment of gastric cancer. TIP30, a newly identified tumor suppressor, appears to be involved in multiple functions including tumorigenic suppression, apoptosis induction and diminishing angiogenic properties. Here, the level of TIP30 expression was determined in gastric cancer, and the impact of its alteration on cancer biology and clinical outcome was investigated. We found that TIP30 protein was absent or reduced in gastric cancer cell lines. There was also a loss or substantial decrease of TIP30 expression in 106 cases of gastric tumors as compared with that in normal gastric mucosa (p<0.05), which was significantly associated with inferior survival duration. In a Cox proportional hazards model, TIP30 expression independently predicted better survival (p<0.05). We also restored TIP30 protein expression in human gastric cancer-derived cells AGS and MKN28 lacking endogenous TIP30 protein to study the effects of TIP30 expression on cell proliferation, cell kinetics, tumorigenicity and metastasis in BALB/c nude mice and found that adenoviral-mediated restoration of TIP30 expression led to downregulation of cyclin D1, Bcl-2, Bcl-xl, but to upregulation of p27, Bax, p53, caspase 3 and 9 expression, cell cycle G0/G1 arrest and apoptosis in vitro, and dramatic attenuation of tumor growth and abrogation of metastasis in animal models. Taken together, the present work revealed a novel function of TIP30, which can possibly be used as an independent prognostic factor and a potential therapeutic target for gastric cancer.     

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.     

PJ34, an inhibitor of PARP-1, suppresses cell growth and enhances the suppressive effects of cisplatin in liver cancer cells

It has been suggested that poly(ADP-ribose) polymerase-l (PARP-l) plays an important role in DNA repair, cell death and proliferation, as well as in the stabilization of the genome. Pharmacological inhibition or genetic ablation of PARP-1 had a beneficial outcome in cancer chemotherapy since the cancer cells lacked PARP-1 and were sensitive to chemotherapeutic DNA damage. As a novel potent specific inhibitor of PARP-l, PJ34 has been reported to enhance chemotherapeutic effects in certain types of tumors. In a previous study, we found that PARP-1 expression was significantly increased in human hepatocellular carcinoma (HCC) compared to its surrounding liver tissue. This study investigated whether or not the inhibition of PARP-1 activity by PJ34 produces suppressive effects on human liver cancer cells and sensitizes the tumor cells to chemotherapeutic agents. We conclude that PJ34 significantly suppresses HepG2 cell growth in a dose-dependent manner, and inhibits HepG2 cell-derived tumor growth in nude mice. The suppressive effects of PJ34 are associated with increased cell apoptosis. Furthermore, PJ34 enhances suppressive effects of cisplatin in HepG2 cells. These results suggest that PJ34 may be developed into an effective agent for the treatment of human HCC.     

Chemokine receptor CCR7 enhances intrahepatic and lymphatic dissemination of human hepatocellular cancer

Despite many pathophysiological analyses, the process of tumor dissemination of hepatocellular carcinoma (HCC) remains vague. In diverse tumor entities, expression of the chemokine receptor, CCR7, has been linked to tumor dissemination and poor prognosis. Therefore, we evaluated, whether CCR7 exerts similar effects in human HCC. CCR7 expression analysis was performed in vitro on human hepatoma cell lines (Huh7, Hep3B, wt HepG2, p53 dominant negative transfected HepG2). In addition, CCR7 expression was evaluated in 39 patients with hepatocellular cancer and correlated with both, tumor and patients characteristics. Human hepatocellular carcinoma samples and hepatoma cell lines displayed variable intensities of CCR7 expression. In patients, CCR7 expression was significantly associated with progressed local tumors (P = 0.02) and lymphatic metastasis (P = 0.02). Strong expression of CCR7 promotes intrahepatic and lymphatic HCC dissemination.