Hepatocellular carcinoma ( HCC) is one of the most common malignancies worldwide with an annual occurrence of one million new cases. At present there is no effective treatment for HCC individuals that not amenable to curative therapies. Recent studies show the PI3K/ Akt/ mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Rapamycin ( a specific mTOR inhibitor) could lead to G1 arrest of many malignant cell lines, and currently analogs of rapamycin are being investigated as a cancer chemotherapeutic adjuvant. This study investigated rapamycin and chemotherapeutic agent 5-fluorouracil ( 5-Fu) in combination treatment induced apoptosis and cell senescence in hepatocarcinoma cell line SMMC-7721 cells. Treating SMMC-7721 cells with rapamycin plus 5-Fu led to not only apoptosis but also cell senescence, and the senescent cells exhibited significantly less clonogenic potential than 5-Fu individually treated cells. Further study showed rapamycin plus 5-Fu-induced senescence-like growth arrest was accompanied by down-regulation of AP-1 and NF kappa B transcription activity. These results suggest that inhibitors of mTOR may have anticancer potential when used together with some other chemotherapeutic agents, and that down-regulation of AP-1 and NF kappa B transcription activity might take part in a senescence-like growth arrest program induced by rapamycin plus 5-Fu.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, there is no effective treatment for HCC. It has been shown that sustained activation of telomerase is essential for the growth and progression of HCC, suggesting that telomerase is a rational target for HCC therapy. Here, we investigated the effects of siRNA-mediated knockdown of hTERT, the catalytic and rate-limiting subunit of telomerase, on the sensitivity of HCC cells to Cisplatin. While silencing of hTERT and the resultant inhibition of telomerase activity by infection with the recombinant adenoviruse expressing a hTERT siRNA (Ad-si/hTERT) alone did not affect the proliferation and viability of SMMC7721 and HepG2 HCC cells within 5 days, co-administration of Ad-si/TERT, but not the empty adenovirus vector, with cisplatin caused much greater extent of apoptosis in vitro under the same conditions and induced significantly more robust inhibition of SMMC7721 and HepG2 tumors growth in a mouse tumor xenograft model than cisplatin monotherapy. Our results demonstrated the synergistic effect between hTERT siRNA and Cisplatin in the suppression of HCC progression and indicated that the combination of hTERT-specific siRNA and cisplatin could be an effective therapy for HCC.

BACKGROUND:Hepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation.METHODS:This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu). We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively.RESULTS:Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase) mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells.CONCLUSION:These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.

Objective:To investigate the effect of homocysteine (HCY) in promoting senescence of cultured endothelial cells (EC) and the underlying mechanism. Methods: Bovine aortic endothelial cells were isolated by enzymatic digestion method from new born calf and were randomly divided into 4 groups. Cells in control group were not treated with HCY and those in the other 3 groups were treated with HCY with the final concentrations being 0.1, 0.5, and 1.0 mmol/L. The cultured cells were observed morphologically and stained with β-Gal; the cell cycle was examined with flow cytometric method (FCM) and the telomere length was analyzed by Southern blotting. Meanwhile, the contents of nitric oxide(NO), endothelin (ET), MDA and the activity of GSH-Px were determined in the supernatants. Results: Compared with control group, the 3 HCY groups had more β-Gal positive cells and shorter telomere length. Cells of G0/G1 phase in the 3 HCY groups were significantly increased than those of control group (P0.05) and the cells of S-phase were significantly decreased (P0.01); the contents of NO in supernatants of 3 HCY groups were significantly lower than that of control group(P0.05) and the contents of ET, Ox-LDL were markedly higher than that of control group(P0.05); the contents of MDA and the activity of GSH-Px were similar in all groups. Conclusion: It is indicated that homocysteine may accelerate senescence in endothelial cells, which is not related to oxygenation and reduction.

Objective To evaluate the capability of the anti-tumor immunity induction by co- transfecting the breast cancer cells with MIP-1αand B7-1 gene.Methods SHZ-88 cells were inoculated in SD rats in order to establish rat breast cancer model.On day 10 after inoculation,the tumor size was documented,and rats were respectively vaccinated with SHZ-88,SHZ-88/B7-1,SHZ-88/MIP-1α,SHZ- 88/MIP-1α+B7-1 that were inactivated by mitomycin C on day 1 and 8.The therapeutic effect was tested in breast cancer SD rats by observing tumor volume and animal tumor-bearing life span in vivo.The im- mune functions in xenografts breast cancer rats were analyzed through checking the levels of whole blood CD4~+,CD8~+,CD4~+/CD8~+ ratio and the contents of serum IL-2 and IFN-γby flow cytometry and EIL- SA.Results The oncegenicity after subcutaneous inoculations of SHZ-88,B7-1,MIP-1αand MIP-1α+ B7-1 was 100%,30%,20% and 0 respectively,and the animal tumor-beating life span was prolonged in B7-1 group (63.80±1.64) d,MIP-1αgroup (64.20±1.92) d and MIP-1α+B7-1 group (89.00±2.55) d as compared with SHZ-88 group (53.40±1.14,P0.05).The peripheral blood CD4~+,CD8~+ and the ratio of CD4~+/CD8~+,and the contents of IL-2 and IFN-γin MIP-1α+B7-1 were the highest a- mong the groups.Fourteen,28 and 40 days after inoculation,the tumor size in MIP-1α+B7-1 group was less than other groups (P0.05).The antitumor effects had no significant difference after SHZ-88/MIP- 1α+B7-1 cells were injected into tumor center or contralateral axillary fossa (P0.05).Conclusion The co-transfection of MIP-1αgene and B7-1 gene could induce stronger anti-breast cancer effects.

Objective: To investigate telomerase activity and telomerase expression in the human gastric carcinoma cell line SGC7901 and its multi-drug resistant subcultured cell line SGC7901/VCR, and to search for a new target within the drug-resistance mechanism of the gastric carcinoma cell line. Methods: Silver staining TRAP(telomeric repeat amplification protocol) assay was used to detect telomerase activity. Semi-quantitative reverse transcriptase PCR was employed to detect hTERT, Mad1 and c-Myc mRNA expression. Plasmid (pGL3B-TRTP) was transfected into SGC7901/VCR and SGC7901. Expression of the reporter gene in the cell lysates was assayed by a dual luciferase reporter assay system. Results: Both telomerase activity and telomerase hTERT mRNA expression in SGC7901/VCR cells were higher than those in SGC7901 cells. hTERT promoter activity was significantly higher in SGC7901/VCR than in SGC7901. c-Myc mRNA expression was detected in SGC7901/VCR, but Mad1 mRNA was not. In SGC7901, both c-Myc and Mad1 mRNA expression could be detected and c-Myc mRNA expression was higher than that in SGC7901/VCR. Conclusion: Telomerase activity in SGC7901/VCR was higher than that in SGC7901, and the high level of telomerase hTERT gene transcription and expression in SGC7901/VCR was correlated with drug-resistance. Mad1 and c-Myc expression may contribute to hTERT transcription and telomerase activity.

OBJECTIVE:To investigate the relationship between promoter of ER methylation and ER mRNA expression and clinicopathologic features in hepatocellular carcinoma (HCC). And to observe the effects of 5-aza-2-deoxycytidine (5-aza-dc) in regulating ER mRNA expression. METHODS: Promoter methylation of the ER gene was determined by methylation-specific PCR (MSP) in HCC and non-tumor cases. And the expression of ER mRNA was investigated by RT-PCR. Two hepatic cancer cell lines SMMC-7721 and HepG2 were treated with DNA methyltransferase inhibitor, 5-aza-dc. RESULTS: Promoter of the ER gene was methylated in 60.0%(18/30)of HCC and 30.0%(9/30)of non-tumor tissues,respectively. Statistically significant association was found between the status of aberrant methylation in HCC and non-tumor case, χ2=5.455,P=0.037. The expression of ER mRNA was 40.0% (12/30) in HCC, while the expression of ER mRNA was 22.2% and 66.7% in methylated HCC cases and unmethylated HCC cases, respectively. An inverse correlation was found between promoter methylation and ER mRNA transcript levels in HCC, χ2=5.93,P=0.024. Meanwhile, there was a significant association between methylation of the ER gene and serum AFP level, χ2=12.13,P=0.001. The study shows that low micromolar concentrations of 5-aza-dc induce the ER mRNA expression of in HCC cancer lines. CONCLUSION: Promoter methylation of ER gene is a frequent event in HCC and may contribute to pathogenesis of HCC through silencing ER mRNA expression.

PURPOSE:CpG island methylator phenotype (CIMP) involves hypermethylation targeted toward the promoters of multiple genes. To gain insight into the role of epigenetic aberration of tumor-related genes in hepatocarcinogenesis, we determined a hypermethylation profile in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN:We examined the promoter methylation status of nine genes in 50 HCCs, 50 paired nontumor tissues, and 6 normal liver tissues by methylation-specific PCR. CIMP+ was defined as having five genes that are concordantly methylated. RESULTS:The frequency of promoter methylation of nine genes in 50 HCCs varied from 10% in P53 to 94% in c-Myc. The methylation status of P14, P15, P16, ER, RASSF1A, WT1, and c-Myc was significantly correlated with HCC and nontumor tissues (P<0.05). Hypermethylation of one or more genes was found in 96% of HCC. CIMP was more frequent in HCC than in nontumor tissues (70% and 12%, P<0.001). There is a significant association between CIMP and methylation of P14, P15, P16, ER, RSAAF1A, and WT1 (P<0.05) and serum alpha-fetoprotein (AFP) level (P=0.017). CIMP+ was more frequent in HCC with AFP>or=30 microg/L than those with AFP<30 microg/L (P=0.005). In addition, the promoter hypermethylation of P15 and P16 was associated with elevated serum AFP levels in 35 HCC samples with CIMP+ (P<0.05). CONCLUSIONS:Positive correlation of CIMP and AFP levels in HCC suggests that CIMP can serve as a molecular marker of late-stage HCC development.

Objective:To study the correlation of postoperative early recurrence with both telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression in hepatocellular carcinoma (HCC). Methods:Telomerase activity was detected by ELISA-based telomeric repeat amplification protocol (ELISA-TRAP) in 60 HCC tissues and adjacent non-tumor liver tissues at a distance of 2 cm from HCC nodules. Expression of hTERT mRNA was measured by reverse transcriptional polymerase chain reaction (RT-PCR). Five normal liver tissues were used as control. The correlation of clinicopathological features with both telomerase activity and hTERT mRNA expression was analyzed statistically. Results: The positive rates of telomerase activity and hTERT mRNA expression were 86.7% (52/60) and 90% (54/60), respectively, in HCC tissues, and 40% (24/60) and 43.3% (26/60), respectively, in adjacent non-tumor liver tissues. Neither telomerase activity nor hTERT mRNA expression was detected in 5 normal liver tissues. Telomerase activity and hTERT mRNA expression in adjacent non-tumor liver tissues were closely associated with the postoperative early recurrence and the biologically malignant behaviors of the tumor itself such as infiltration of inflammatory cells around fibrous capsule, portal venous invasion, and intrahepatic metastasis. Conclusions:Telomerase activity and hTERT mRNA expression in adjacent non-tumor liver tissues of HCC may serve as useful predictors of postoperative early recurrence.

Objective: To study the possible protective effects of tea polyphenols (TP) on the cultured bovine endothelial cells impaired by homocysteine . Methods: Bovine aortic endothelial cells were cultured and treated with a concentrated homocysteine and TP for 72 h . The cultured cells and the contents in the medium were studied by FCM , meanwhile nitric oxide (NO), endothelin(ET),GSH-Px and MDA contents were determined. Results:(1) TP inhibit the aortic endothelial cells proliferation induced by homocysteine ; (2) Vascular relaxing factor NO content in culture media in TP group was significantly higher than that of homocysteine group; (3) Vasoconstrictor ET was markedly lower than that of homocysteine group ; (4) There were no differences in the activity of GSH-Px and contents of MDA between TP group and homocysteine group. Conclusion: TP diminish the harmful effects of homocysteine on the aortic endothelial cells.

目的:探讨原发性肝癌(HCC)经皮肝穿刺细针活检端粒酶活性检测的临床意义.方法:25例直径1.2～3cm的HCC进行了无水酒精注射治疗(PEIT).PEIT治疗前及PEIT治疗后1周在B超引导下经皮肝穿刺细针活检,所得微量肝癌组织采用非放射性同位素银染端粒重复序列扩增法(TRAP)行端粒酶活性检测及病理学检查.3例正常肝脏组织及5例肝硬变组织作为对照研究.结果:PEIT治疗前,病理学检查诊断率为80%(20/25),端粒酶活性阳性率为84%(21/25);PEIT治疗后1周,病理学检查未发现肿瘤细胞,3例端粒酶活性阳性(12%),其中1例经手术治疗后临床治愈,2例未手术治疗者1年内复发.其余22例端粒酶转阴的患者1年内未复发.3例正常肝脏组织及5例肝硬变组织端粒酶活性检测均阴性.结论:经皮肝穿刺活检微量肝脏组织端粒酶活性检测可应用于小肝癌的辅助诊断,在原发性肝癌PEIT治疗的疗效评价及预后判断方面具有一定的应用价值.

AIM:To detect the origin of hepatocellular carcinoma (HCC)recurring and attempt to propose a new recurrent mechanism.METHODS:Orthotopic liver allotransplantation was performed on male rats with HCC- induced by diethylnitrosamine using female donors.Metastatic tumors in transplanted livers were obtained.A DNA probe that exhibits specificity for the rat Y chromosome was generated by using a set of primers specificto murine srygene.In situ hybridization (ISH) for Y chromosome was used to detected the origin of HCC recurring. Male HCC tissue was designed to be positive control. ISH on female tissue and using non-labeled with DIG probe was thought to be negative control.RESULTS: Positive marks were seen through ISH for Y chromosome in recurrent tumor tissue and positive control.No signal was detected in both negative controls.CONCLUSION: Recurrent HCC after liver transplantation originated from disseminated tumor cells in recipients.Extrahepatic cells homing into liver may be a new HCC recurrence mechanism. Likewise, it implicates that this mechanism is responsible for HCC recurring after hepatectomy.

To study the clinical significance of telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression for differential diagnosis of malignant and benign liver lesions.

Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity; the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion: Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.

目的:将重组腺病毒介导的FLT3配体(FLT3 ligand,FL)与阿霉素(adriamycin,ADM)联合治疗小鼠肝癌,探索基因治疗和化疗相结合治疗肿瘤的新方法.方法:构建携带鼠FL的重组腺病毒载体(AdmFL),单独用其或ADM以及两者联用对实验性肝癌小鼠进行治疗,观察抗肿瘤效果.结果:(1) FL+ADM组小鼠肝癌生长抑制率最大值为56.5%,明显高于ADM组和FL组的27.5%和26.6%(P<0.01);(2) FL+ADM组小鼠早期死亡率为8.3%,明显低于ADM组的37.5%(P<0.01);FL+ADM组小鼠长期生存率为86.4%,明显高于ADM组和FL组的50.0%和41.5%(P<0.01).结论:(1)FL和ADM治疗小鼠肝癌具有协同作用, FL可显著增强化疗效果;(2)FL能明显降低化疗不良反应.

OBJECTIVE:To observe the in vivo therapeutic effects of murine Flt3 ligand (mFL) mediated by recombinant adenoviral vector on murine liver cancer.METHODS:Murine liver cancer cell line Hepal-6 was infected with adenovirus in vitro. The infection efficacy was measured by green fluorescence protein (GFP) expression and the amount of mFL in supernatant was measured by ELISA 48 hrs following infection of Hepal-6. AdmFL, Ad-null, and PBS were added into the culture of Hepal-6 cells, the number of cells was counted every other day for 14 days. A murine liver cancer model was established by subcutaneous inoculation of Hepal-6 cells. A single dosage of 1 x 10(9) expression forming unit (efu) of Ad-mFL, Ad-null or PBS was injected intratumorally. The tumor volume and survival rate were measured twice a week. Twenty days after treatment of adenoviral vectors, three treated mice with their tumor disappearing were killed and their spleen was taken. The splenocytes from these tumor free mice were adoptively transferred to naive mice to whom Hepal-6 cells were inoculated 3 days thereaftter and then the tumor volume was measured once a week.for 4 weeks. 38 days after administration of adenoviral vectors, tumor free animals were rechallenged by parental Hepal-6 cells or syngenic EL4 lymphoma cells at the opposite sites of the original inoculation sites, and the tumor volume was also measured once a week for 4 weeks.RESULTS:Adenoviral vector efficiently infected Hepal-6 cells in intro, and lead to the secretion of high levels of mFL protein (80.5 +/- 7.3 ng/10(6)/24 h) in the supernatant. The growth of Hepal-6 tumor was significantly inhibited by one single intratumoral administration of Ad-mFL in 90% of the treated mice, and the tumor gradually grew in the two other groups. Two of the 7 mice (30%) in PBS group died in 17 days, 14% still lived in 37 days, and all died within 45 days. The mice in the Ad-null group began to die since the 21(st) day, only 11% of them were still alive in 60 days. All mice in the Ad-mFL group were alive at the 60(th) day after tumor implantation. Adoptive transfer of splenocytes to the animals receiving Ad-mFL treatment protected effectively them against a subsequent challenge with the identical tumor cells. Rechallenge of the Ad-mFL cured mice with the parental Hepal-6 cells resulted in complete inhibition of tumor growth. The growth of inoculated EL4 lymphoma cells gradually grew equally in the controls and the experimental mice.CONCLUSION:FL gene transfer mediated by recombinant adenoviral vector has potent therapeutic effects on Hepal-6 liver cancer, and develops long-lasting specific antitumor immunity, which may become a potent cancer gene therapy candidate for further clinic application.

Objective:To prepare murine liver tumor vaccine by infection of recombinant adenoviral vector carrying murine Flt3 ligand and to observe its in vivo antitumor activity. Methods:The infection efficacy was measured by GFP expression 48 h following infection of Hepa1-6, and the amount of mFL in supernatant at day 0, 2, 4, 6, 8 was measured by ELISA. The cells number was counted daily for 14 d. Modified Hepa1-6 cells (5×10 6) were subcutaneously inoculated into C57BL/6 mice, and they were rechallenged by 2×10 6 wild-type Hepa1-6 cells or syngenic EL4 cells 4 weeks later. The tumor volume was measured twice a week. Results:Adenoviral vectors efficiently infected Hepa1-6 cells in vitro, and led to the secretion of high levels of mFL protein (71.1±6.3 ng/ml) 2 d after infection. The in vitro growth rate of AdmFL modified Hepa1-6 cells was not affected, while the in vivo tumorigenesis was significantly decreased compared with that of control vector modified Hepa1-6. Rechallenge of the mice 4 weeks after administration of Ad mFL with the parental Hepa1-6 cells resulted in significant inhibition of tumor growth, while there was no difference when rechallenged with EL4. Conclusion:The liver cancer cells modified by recombinant adenoviral vector carrying murine flt3 ligand can decease tumorigenesis and elicit specific immunological protection, it can be used as an effective liver tumor vaccine.

目的:构建小鼠趋化因子MIP1-α(mMIP1-α)的重组腺病毒载体并在小鼠肝癌细胞中表达,为肝癌基因治疗提供实验基础.方法:PCR扩增含有mMIP1-α的质粒,将腺病毒骨架质粒pAdEasy-1以及线性化的重组穿梭质粒pTrack-CMV- mMIP1-α共转化BJ5183受体菌并在其中发生同源重组,利用重组前后抗性的改变筛选出重组子,重组腺病毒质粒经过293细胞的包装、扩增和纯化后,感染小鼠肝癌细胞株Hepa1-6,一步法提取细胞总RNA,RT-PCR检测mMIP1-α的mRNA.琼脂糖凝胶打孔法体外观察感染上清中mMIP1-α对小鼠脾细胞的趋化活性.结果:得到了携带mMIP1-α的重组腺病毒,纯化后滴度为4×1011 efu/ml,在感染后的Hepa1-6细胞中检测到mMIP1-α的表达,上清中mMIP1-α的趋化指数为6.3.结论:成功地构建了携带mMIP1-α的重组腺病毒载体,为下一步应用于肝癌的基因治疗提供了基础.

目的:探讨细针穿刺肝活检组织行端粒酶活性检测用于肝细胞癌(HCC)诊断的可行性.方法:HCC共28例,每例HCC肿瘤切除后立即行肝肿瘤模拟细针穿刺.应用非放射性同位素银染的端粒重复序列扩增法(TRAP),分别对28例模拟肝穿组织和28例肿瘤切取组织行端粒酶活性检测.结果:模拟肝穿组织与肿瘤切取组织的端粒酶检测结果完全一致,阳性率均为82%(23/28).结论:模拟肝穿组织与肿瘤切取组织在HCC端粒酶活性检测方面具有同等的效果.该结果提示,B超引导下经皮肝穿刺活检行端粒酶活性检测在HCC的诊断、治疗效果评价和预后的判断方面具有一定的应用前景.

Objective: To search for new alternative splicing isoforms of human Flt3 lignd (hFL). Methods: Freshly isolated peripheral blood mononuclearcytes (PBMC) from healthy donors were separated by ficoll-hypaque gradient sedimentation. Total RNA was extracted by TRIzol reagents, and the extracellular portion of hFL was amplified by RT-PCR. PCR products were cloned into a plasmid and sequenced. Analysis was performed by Blast software. Results: Sequence analysis showed that there were 2 newly identified alternative splicing isoforms of hFL, one was a cytosine insertion at 434 bp and the other was 53 bp deletion between 426 and 478 bp. Conclusion: There exist 2 new alternative splicing isoforms of hFL.