siRNA-mediated inhibition of hTERT enhances chemosensitivity of hepatocellular carcinoma.

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 adenovirus expressing a hTERT siRNA (Ad-si/hTERT) alone did not affect the proliferation and viability of SMMC7721 and HepG2 HCC cells within five days, co-administration of Ad-si/hTERT, 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.

Synergistic effect of mTOR inhibitor rapamycin and fluorouracil in inducing apoptosis and cell senescence in hepatocarcinoma cells.

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 G(1) 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.

Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma cells.

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.

CpG island methylator phenotype association with elevated serum alpha-fetoprotein level in hepatocellular carcinoma.

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.

Disseminated tumor cells homing into rats' liver: a new possible mechanism of HCC recurrence.

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 specific to murine sry gene. 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.

[The in vivo antitumor activity of murine liver tumor vaccine expressing MIP-1alpha].

OBJECTIVE: To observe the in vivo antitumor activity of murine liver tumor vaccine expressing MIP-1alpha mediated by recombinant adenoviral vector. METHODS: The infection efficacy was measured by GFP expression 48 hours after infection of Hepa1-6, and the number of cells was counted daily for 14 days. 5 x 10(6) modified Hepa1-6 cells were inoculated subcutaneously to C57BL/6 mice and the tumor-free animals were rechallenged by 2 x 10(6) wild-type Hepa1-6 cells or syngenic EL4 cells four weeks later. The tumor volume was measured twice a week. RESULTS: Adenoviral vectors could efficiently infect Hepa1-6 cells in vitro, and the in vitro growth rate of AdmMIP-1alpha modified Hepa1-6 cells was not affected; however the in vivo tumorigenicity was significantly decreased, compared with that of control vector modified Hepa1-6. Rechallenge of the tumor-free mice four weeks after administration of AdmMIP-1alpha with the parental Hepa1-6 cells resulted in significant inhibition of tumor growth, but there was no significant difference when rechallenged with EL4. CONCLUSIONS: The liver cancer cells expressing mMIP-1alpha mediated by recombinant adenoviral vector decrease tumorigenicity and elicit specific immunological protection, and could be used as an effective liver tumor vaccine.

[Gene transfer of murine Flt3 ligand mediated by adenoviral vector efficiently induces growth inhibition of murine liver cancer].

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.