Therapeutic effect of α-fetoprotein promoter-mediated tBid and chemotherapeutic agents on orthotopic liver tumor in mice

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Abstract

Previous studies have shown that the application of Ad/AFPtBid significantly and specifically killed hepatocellular carcinoma (HCC) cells in culture and subcutaneously implanted in mice. This study was to test the therapeutic efficacy of Ad/AFPtBid in an orthotopic hepatic tumor model. Four weeks after implantation of tumor cells into the liver, nude mice were treated with Ad/AFPtBid alone or in combination with 5-fluorouracil (5-FU). Serum α-fetoprotein (AFP) was measured as a marker for tumor progression. The results showed that Ad/AFPtBid significantly inhibited Hep3B tumor growth. Ad/AFPtBid and 5-FU in combination was more effective than either agent alone. Tumor tissues of Ad/AFPtBid alone or combination treatment groups showed a decrease in cells positive for proliferation cell nuclear antigen, but an increase in apoptosis. Ad/AFPtBid did not suppress the hepatic tumor formed by non-AFP-producing hepatoma SK-HEP-1 cells or colorectal adenocarcinoma DLD-1 cells. The survival rate was higher in mice treated with Ad/AFPtBid plus 5-FU than those treated with either agent alone. No acute toxic effect was observed in mice receiving Ad/AFPtBid. Collectively, Ad/AFPtBid can specifically target and effectively suppress the AFP-producing orthotopic liver tumor in mice without obvious toxicity, indicating that it is a promising tool in combination with chemotherapeutic agents for treatment of AFP-producing HCC.

Introduction

Hepatocellular carcinoma (HCC) is the third commonest cancer worldwide.1 It is also one of the commonest malignancies in the developing countries of Africa and Asia including China. The incidence of HCC is also increasing in western countries.2 It is estimated that HCC would be the 13th leading cause of death by 2030.3 A major factor that contributes to such a gloomy prospect is the lack of effective therapeutic tools. HCC thus remains highly resistant to systemic chemotherapy.4 No effective systemic chemotherapy has been demonstrated an unequivocal and significant benefit in terms of survival.5

In HCC, there is an imbalance between the pro- and anti-apoptotic of Bcl-2 family members6 and defects in apoptosis signaling contribute to the chemotherapy resistance. For example, the expression of anti-apoptotic Bcl-xL and Mcl-1 is increased, whereas the expression of pro-apoptotic Bid and Bak proteins is decreased.7, 8, 9, 10 Owing to the limitation of the conventional chemotherapy, gene therapy that aims to upregulate pro-apoptotic Bcl-2 proteins or interfere with function of anti-apoptotic Bcl-2 proteins may sensitize HCC cells to chemotherapeutic agents as well as lower the tumoricidal dose required to kill the HCC cells.

Studies have shown that BH3 domain-only protein Bid and its activated form truncated Bid (tBid) are crucial in hepatocyte apoptosis induced by Fas/TNF-R1 signals.11, 12, 13, 14 Bid-dependent generation of oxygen radicals promotes death receptor activation-induced apoptosis in murine primary hepatocytes.14 Gene knockout mouse model indicated that Bid functioned upstream of either Bax or Bak to initiate mitochondrial dysfunction and cell death.15, 16 Others have shown that Bid can block the inhibitory effect of Bcl-2 on Fas-mediated apoptosis of HCC cells through oligomerizing Bak to release cytochrome c.17 In SK-HEP-1 cells, TNF-related apoptosis-inducting ligand is known to induce the translocation of Bax, which subsequently leads to the cleavage of Bid.18

Bid also has important functions in the development and chemotherapeutic sensitivity of HCC. Our previous experiments have shown that the level of Bid decreased in HCC tissues of patients except in poorly differentiated HCC in which cells may undergo a process of apoptosis or necrosis.19 The application of Ad/AFPtBid significantly and specifically killed α-fetoprotein (AFP)-producing Hep3B cells in vitro and in mice subcutaneously implanted with these cells. Furthermore, over-expression of Bid especially tBid significantly sensitized Hep3B cells to two commonly used HCC chemotherapeutic agents 5-fluorouracil (5-FU) and doxorubicin (Dox) in vitro.20, 21 It appears that the combination of gene therapy with existing chemotherapy or radiotherapy is likely to be more effective than separate approach alone.22, 23

The objective of this study is to test therapeutic efficacy of Ad/AFPtBid or its combination with 5-FU or Dox in an orthotopic hepatic tumor model, which represents naturally occurring HCC.24 Results showed that Ad/AFPtBid in combination with 5-FU significantly arrested the growth of liver tumor.

Results

Liver tumors formed from intra-hepatic injection of tumor cells

Four weeks after tumor cell implantation, a small tumor (2 mm × 3 mm) could be observed on the surface of the lobe where tumor cells were injected. By the end of the study, as shown in Table 1, tumors formed in most of the mice (43/59, 72.88%) with Hep3B cell implantation were limited to the injected lobe as a single node, and a clear boundary between the tumor and normal tissues was obviously seen (Figure 1). For the rest of the mice (16/59, 27.12%) Hep3B tumors were not limited to the injected lobe but were found in other lobes as well. With SK-HEP-1 and DLD-1 cell implantation, 50% (8/16) and 40% (8/20) of mice had more than one tumor node in liver, respectively. No metastases of Hep3B and DLD-1 to other organs were observed in any of the animal tested. Metastases to abdominal cavity were observed in 31.25% (5/16) mice with SK-HEP-1 tumors (Figure 1c).

Table 1 Tumor profiles of mice implanted with different cancer cells
Figure 1
figure1

Orthotopic tumors in livers of the nude mice. (a) Tumor formed in the liver of nude mice; 2 × 106 Hep3B, 2 × 106 SK-HEP-1 or 3.5 × 106 DLD-1 cells were injected directly into the liver of the nude mice. In normal liver (control), PBS was injected in place of tumor cell implantation. Pictures were taken 8 weeks after cell implantation. (b) H&E staining of liver tumors. Original magnification × 200. Eight weeks after cell implantation, the implanted lobes of nude mice were collected, fixed with 4% paraformaldehyde and embedded in paraffin blocks for H&E staining. Scale bar=40 μm. (c) Metastases of SK-HEP-1 tumor in the abdominal cavity.

Serum AFP as a surrogate marker for Hep3B tumor progression

AFP was detected by ELISA in cultured Hep3B cells but not in cultured SK-HEP-1 or DLD-1 cells (Supplemental Table S1). In vivo, the determination of AFP was started at 5 weeks after Hep3B cell injection and it was weekly monitored until the end of the experiment. The AFP level was elevated with the increased period of tumor cell implantation, suggesting that the AFP level was correlated with Hep3B tumor growth in mice (Figure 2a). AFP was also detectable in Hep3B tumor tissues by immunohistochemical staining (Figure 2b). However, AFP was not detectable in both blood samples and liver tissue sections from normal mice and mice bearing SK-HEP-1 or DLD-1 tumors.

Figure 2
figure2

Serum AFP as a marker for Hep3B tumor progression (a) AFP value during Hep3B tumor growth. Numbers in bracket of all figures indicated mice of each group. *Indicates P<0.05. Error bars, s.e. (b) Immunohistochemical staining of AFP. Strong staining was observed in Hep3B tumors compared with absence in normal liver tissues. Sacle bar=40 μm.

Combination therapy of Ad/AFPtBid with 5-FU or Dox on Hep3B tumor

To evaluate the effect of Ad/AFPtBid and chemotherapeutic agents in combination on orthotopic tumors, Ad/AFPtBid was used alone or in combination with 5-FU or Dox. The extent of growth delay of the Hep3B xenograft tumors achieved with the various treatments is illustrated in Figure 3a. Tumor growth was significantly reduced after all treatments compared with the control group. The combination of Ad/AFPtBid and 5-FU was more effective than either agent alone. The tumor grew rapidly in the control animal treated with Ad/AFPlacZ and it reached the volume of 1000 mm3 at the end of the study. On the other hand, the average size of the tumor was about 380 mm3 for the Ad/AFPtBid-treated group and 11 mm3 for the combination of Ad/AFPtBid with 5-FU-treated group. All animals tested survived the experimental period of 8 weeks. In the control experiment, we have tested the effect of PBS and Ad/AFPLacZ alone on the growth of tumor in the same model and the result showed that neither of them had any significant effect on tumor growth (Supplemental Figure S1). In addition, the results of H&E, immunohistochemical and TUNEL staining of liver tissues also showed no difference between mice treated with PBS alone and Ad/AFPLacZ alone (data not provided). Thus, the effect of Ad/AFPLacZ on tumor growth can be excluded. As for SK-HEP-1 and DLD-1 tumors, there were no differences in tumor size between Ad/AFPtBid-treated groups and control group (Figure 3b). These results indicate that Ad/AFPtBid does not attack the non-AFP-producing SK-HEP-1 or DLD-1 xenografts, confirming that Ad/AFPtBid specifically attacks AFP-producing tumors only. Taken together, these studies show that Ad/AFPtBid has a specific and significant anti-tumor effect on AFP-producing tumors. Ad/AFPtBid in combination with 5-FU was much more powerful in the inhibition of the Hep3B xenografts than either agent alone.

Figure 3
figure3

Effect of Ad/AFPtBid and 5-FU or Dox in combination on tumors in nude mice. Numbers in bracket of all figures indicated mice of each group. (a) 2 × 106 AFP-producing Hep3B cells were injected into the liver of nude mice. Four weeks after injection, mice were divided into six groups and treatments started. Four weeks after the treatment the mice were euthanized and the sizes of tumors were measured. (b) Two independent experiments were performed with non-AFP-producing human SK-HEP-1 hepatoma cells and DLD-1 colorectal adenocarcinoma cells. Each mouse was inoculated with 2 × 106 of SK-HEP-1 or 3.5 × 106 DLD-1 cells in liver. Ad/AFPtBid treatment was the same as in (a). Values are mean±s.e. with *indicates P<0.05. Error bars, s.e.

To evaluate the potential mechanisms for tumor repression, we performed H&E staining, immunohistochemical staining and in situ TUNEL staining on tumor sections. Hep3B tumor tissues of Ad/AFPtBid alone or combination treatment groups showed a decrease in cells positive for cell proliferative marker proliferation cell nuclear antigen (PCNA) (Figures 4A–G) but an increase in apoptotic cells in TUNEL staining (Figures 4A′–G′), compared with the tumor treated with Ad/AFPLacZ (Figures 4B and 4B′). Inflammatory cell infiltration, which may be an indicator of occurrence of apoptosis-related cell damages, was also increased in the tumor tissues of Ad/AFPtBid-treated mice (Figures 4c, e and g).

Figure 4
figure4

Immunohistochemical staining of PCNA, TUNEL staining and H&E staining of Hep3B tumors. The tumor was collected 4 weeks after treatments. (AG) Immunohistochemical staining for PCNA. (A′–G′) TUNEL staining. (ag) Histology of liver by H&E staining. Original magnification × 200. Sacle bar=40 μm.

The western blot analysis of apoptosis-related proteins in the tumor tissues (Figure 5) demonstrated that Hep3B tumor tissues of Ad/AFPtBid alone or combination treatment groups showed significantly increased levels of tBid, Bax, cleaved-caspase 9 and caspase 3, whereas there were no significant differences in the expression of Bcl-xL.

Figure 5
figure5

Western blot analysis of apoptosis-related proteins. Total protein of tumor tissues was isolated after treatment and was subjected to western blot analysis using anti-Bid, anti-Bax, anti-caspase 9, anti-caspase 3 and anti-Bcl-xL antibodies. Actin level was examined and served as the loading control.

Combination therapy of Ad/AFPtBid with 5-FU or Dox affects the survival rate of mice implanted with Hep3B cells

We observed the survival rate of mice receiving various treatments over a 6-month period after Hep3B implantation. There were no survivors in the control group and the Ad/AFPtBid in combination with Dox-treated group. However, Ad/AFPtBid in combination with 5-FU resulted in increased survival rate compared with the control group (Figure 6). On postmortem of the dead mice in the control group, the sizes of tumors were much bigger than those survived throughout the study period.

Figure 6
figure6

Survival rate of mice with orthotopic liver tumor. Mice with Hep3B implantation were treated with various protocols. Survival rate was calculated 6 month after Hep3B implantation. Survival analysis was performed by Kaplain–Meier survival analysis.

Assessment of the in vivo toxicity of Ad/AFPtBid

Compared with controls, there were no adenoviral dose-related toxic effects on total white blood cell count, white blood cell differential counts, function of liver (alanine aminotransferase and aspartate aminotransferase) and kidney (blood urea nitrogen and creatinine) (Supplemental Table S2). Differences in mouse daily activity, body weight and microscopic pathology of liver by H&E staining were not found among the groups tested (data not shown).

Discussion

Orthotopic xenotransplant model is a useful tool to study liver cancer. It had high fidelity to the actual environment in liver, where a portal system and drug-detoxifying enzymes exist. The organ environment has profound effects on the response of tumor cells to chemotherapy. Studies have shown that response to chemotherapeutic agents varies depending on whether the tumor was ectopic or orthotopic.25 The similarity between our in vivo mouse model and human HCC is reflected by the following observations. First, intra-hepatic metastases were found in our model. The intra-hepatic metastasis is an important and frequent event in HCC, which accounts for tumor recurrence and low survival rate.26, 27 Second, our SK-HEP-1 orthotopic tumor was able to metastasize to extra-hepatic organs. Extra-hepatic metastasis also occurs in HCC.28 Third, in our model the tumor growth positively correlates with the level of AFP in serum. AFP is known to be expressed in majority of human HCC but not detected in normal hepatocytes.29 It is a valuable marker for not only the diagnosis of HCC but also the monitor of therapeutic response.30, 31 Therefore, our model highly resembles human HCC and it is a suitable in vivo model to study the efficacy of anti-HCC agents.

Ad/AFPtBid is a recombinant replication defective adenovirus, which carries pro-apoptotic tBid gene driven by a hepatoma-specific AFP promoter. As described in our previous work, the recombinants of adenovirus were constructed by using the Adeno-X expression system, according to the manufacture's protocol (Clontech, Palo Alto, CA, USA). AFP promoter was inserted to generate liver cancer cell-specific gene expression.20 Recombinant adenoviruses have gained increasing usage in gene therapy because the vector system combines a high rate of gene transduction that is not cell cycle dependent and the safety of transient expression.32 Bid is a pro-apoptotic Bcl-2 family protein, tBid is its activated form. They link the death signals between death receptor pathway and mitochondrial pathway.33 Hepatocytes require a Bid (tBid)-dependent mitochondrial feed-forward amplification loop that releases cytochrome c, oligomerizing Apaf-1 and caspase-9 to activate sufficient effector caspases to execute apoptosis.11, 12, 13, 14 Our previous in vitro study showed that the application of Ad/AFPtBid specifically killed Hep3B cells that are known to produce AFP, and significantly sensitized Hep3B cells to 5-FU and Dox.20, 21 In this study, we confirmed that Ad/AFPtBid could specifically suppress the AFP-producing HCC but not attack non-AFP-producing tumor in an orthotopic mouse model. Application of Ad/AFPtBid can significantly increase the effect of other chemotherapeutic agents such as 5-FU, resulting in a synergistic interaction between Ad/AFPtBid and 5-FU in our model. The effectiveness of the combination therapy of 5-FU and interferon-α on advanced HCC has been reported.34, 35 It is noted that the dose of 5-FU used in this study is 40 mg kg−1 daily for 3 days, which is much less than the amount of 5-FU used in other cases. Generally, 5-FU was given at 60–66 mg kg−1 daily in treatment of either human HCC or gastric tumors in animals.36, 37 Moreover, the application of 5-FU was often associated with some side effects including myelosuppression, gastrointestinal and skin toxicity.38 However, because of the low dose of 5-FU used, these toxic effects were not found in our mouse model. Therefore, the combination of Ad/AFPtBid and 5-FU can achieve potent anti-tumor effect while reducing the possible side effects associated with 5-FU. We also tested the combination effect of Ad/AFPtBid and Dox on HCC. The dose of Dox we used is a conventional dose in treatment of HCC.39 The combination of Ad/AFPtBid with Dox or Dox treatment alone showed significant effect on tumor regression. However, the mice in the combination of Ad/AFPtBid with Dox-treated group failed to show better therapeutic response than Dox treatment alone group because the mice that received Dox exhibited serious weight loss in this study. It is known that Dox can result in heavy body weight loss.40, 41 Further experiments using lower dose of Dox are needed to explore the possibility of its combination with Ad/AFPtBid for treatment of HCC.

Hep3B tumors showed a significant number of TUNEL-positive cells after treatment with Ad/AFPtBid, indicating that Ad/AFPtBid induced tumor regression through its pro-apoptotic effects. In accordance with this, the western blot analysis showed that Ad/AFPtBid treatment resulted in increased expressions of tBid, Bax, cleaved-caspase 9 and caspase 3. Our previous study showed that the activities of cytochrome c and subsequent caspase 8, caspase 9, caspase 3 were significantly increased in Ad/AFPtBid-infected Hep3B cells.20 Studies have shown that 5-FU executed its therapeutic effect through activation of caspases in colorectal, pancreatic and liver cancer cells including Hep3B HCC cells. 5-FU treatment can also cause a significant increase in the cleavaged Bid in Hep3B cells.21, 42, 43 Moreover, 5-FU is a cell cycle phase-specific agent.44, 45 Our recent results showed that tBid primarily induced Hep3B cells from G0/G1 phase to apoptosis, but 5-FU mainly reduced Hep3B cells in the G2/M phase. The double pro-apoptotic effects of Ad/AFPtBid and 5-FU and the complementary effects of tBid and 5-FU on different phases of Hep3B cells may contribute to the better therapeutic result of Ad/AFPtBid in combination with 5-FU observed in this study. In addition, this sensitization of HCC cells was accompanied by inflammatory cell infiltration. These findings are in accordance with the study by Geoerger et al.,46 which showed that anti-tumor effects in vivo were associated with apoptosis induction and tumor inflammatory cell infiltration. Usually, infection by an adenoviral vector encoding apoptosis-inducing molecules can induce potent inflammatory reactions that may contribute to regression of malignancies.47, 48

The other important finding of this study was that Ad/AFPtBid did not produce any obvious side effects in term of the activity of mice, the blood cell number, liver and renal functions. However, data obtained by Choi et al.49 showed a dose-dependent toxicity response to systemically administered adenoviral vectors. Our Ad/AFPtBid was specifically designed to kill AFP-producing tumor cells and it did not attack non-AFP-producing cells. This feature of our therapeutic device can leave normal cells intact and thus may contribute to the lack of obvious toxicity in our study. Therefore, Ad/AFPtBid seems to be a relatively safe anti-HCC agent.

Taken together, our findings show that Ad/AFPtBid can effectively and specifically kill AFP-producing liver caner cells in vivo and that Ad/AFPtBid can significantly sensitize liver cancer cells to 5-FU. The combination of Ad/AFPtBid gene therapy with 5-FU results in tumor regression, apoptosis, inflammatory cell infiltration and better survival in our model. Moreover, the therapeutic effect was observed at lower vector doses with no detectable toxicity. This combination approach may improve the therapeutic benefits of some chemotherapeutic agents such as 5-FU, rendering Ad/AFPtBid a promising alternative tool in combination with chemotherapeutic agents for treatment of AFP-producing HCC.

Materials and methods

Cell culture

The human HCC cell line Hep3B, SK-HEP-1 and human colorectal adenocarcinoma cell line DLD-1 were obtained from American Type Culture Collection (Rockville, MD, USA). Hep3B and SK-HEP-1 cells were maintained in Dulbeco's modified Eagle medium (Invitrogen, Carlsbad, CA, USA), DLD-1 was maintained in RPMI medium 1640 (Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum at 37 °C with 5% CO2. Exponentially growing cells were collected by trypsin-EDTA (Invitrogen) and washed once with PBS. The cell pellet was suspended in PBS and kept in ice before the intra-hepatic injection.

Establishment of orthotopic liver tumor model

Male BALB/c athymic (nu/nu) nude mice (4–5 weeks) were provided by the animal house of the Chinese University of Hong Kong and acclimated to laboratory conditions 1 week before tumor implantation. Nude mice were kept under pathogen-free conditions, fed standard food and given free access to sterilized water. Housing and all other procedures were performed according to protocols approved by the Animal Experimentation Ethics Committee of our institute. The institutional guideline for the welfare of animals was strictly followed during the study.

The procedure for establishment of orthotopic liver tumor model followed the previous description.24 Briefly, the mice were anesthetized by intra-peritoneal injection of 80 mg kg−1 ketamine and 10 mg kg−1 xylazine (Alfasan, Woerden, the Netherlands). After anesthesia, nude mice were placed in a supine position. A small transverse incision below the sternum was made to expose the liver. Hep3B cells (2 × 106), suspended in 50 μl of PBS, were slowly injected into the upper left lobe of the liver, so that a transparent bleb of cells could be seen through the liver capsule. After injection, a small piece of sterile gauze was placed on the injection site, and light pressure was applied for 1 min to prevent bleeding. The abdomen was then closed with a 6–0 silk suture. Gross tumors were formed 4 weeks after tumor cell implantation. Two independent experiments were performed with non-AFP-producing SK-HEP-1 and DLD-1 cells. Each mouse was inoculated with 2 × 106 SK-HEP-1 or 3.5 × 106 DLD-1 cells in liver as control experiments.

Administration of Ad/AFPtBid and chemotherapeutic agents to tumor-bearing mice

Four weeks after Hep3B cell implantation, the mice were randomized into six groups: (i) Ad/AFPLacZ, (ii) Ad/AFPtBid, (iii) 5-FU and Ad/AFPLacZ, (iv) 5-FU and Ad/AFPtBid, (v) Dox and Ad/AFPLacZ and (vi) Dox and Ad/AFPtBid. The first group was treated with Ad/AFPLacZ as control. The administration of 1 × 1010 p.f.u. of Ad/AFPtBid through tail vein was applied every 2 days for a total of three times under sterile conditions. 5-FU (Sigma-Aldrich, St Louis, MO, USA) was administrated intra-peritoneal at 40 mg kg−1 for 3 consecutive days after the first injection of Ad/AFPtBid. Dox (Toronto Research Chemicals, North York, ON, Canada) was administrated intra-peritoneal at a single dose of 10 mg kg−1 immediately after the first injection of Ad/AFPtBid. Mice were continuously monitored for their activity until they were euthanized 4 weeks after treatment. The liver was removed intact and examined for tumors. Tumors were measured in two orthogonal dimensions by external caliper and volume was estimated by the formula (length (mm) × width (mm)2)/2.50 Liver tissues were subjected to H&E staining and immunohistochemical detection of PCNA and AFP. In situ determination of apoptosis was performed using TUNEL staining.20 Another batch of mice as grouped above was observed for survival over a 6-month period after tumor cell injection. Mortality was recorded for all animals.

Serum AFP measurement

Blood samples (10 μl) were collected from the tail vein weekly. Serum samples were kept at −80 °C and the levels of AFP in the serum were determined by ELISA kit (GenWay Biotech, San Diego, CA, USA) for the monitoring of tumor progression.

Immunohistochemical staining

The preparation of the tissues and the immunohistochemical staining of PCNA and AFP were performed according to the manufacture's instruction (The Vector MOM Immunodetection kit, Vector Laboratories, Burlingame, CA, USA). Briefly, antibodies against PCNA and AFP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies of PCNA and AFP were used at a working dilution of 1:600 and 1:40, respectively. After tissue sections were washed with PBS, a biotinylated anti-mouse IgG was applied for 30 min. After washing with PBS, ABC reagent conjugated with horseradish peroxidase (VECTASTAIN Elite ABC kit, Vector Laboratories) was applied for 30 min. Finally, staining was visualized by DAB substrate (DAB Substrate kit, Zymed Laboratories, South San Francisco, CA, USA). Photos were captured using Nikon microscope equipped with 3CCD camera (DC-330, DAGE-MTI, Mississauga, Ontario, Canada). Images were analyzed by the MetaMorph Imaging System (Universal Imaging Corp., PA, USA).

Western blot analysis

Western bolts were performed according to previous publication.20 The antibody against Bid was obtained from Becton Dickinson Pharmingen (Rockville, MD, USA). The antibodies against Bax, caspase-9 and caspase-3 were obtained from Cell signaling (Cell Signaling Technology, Danvers, MA, USA). The antibodies against Bcl-xL and actin were obtained from Santa Cruz Biotechnology.

The acute toxicity of Ad/AFPtBid in mice

Six-week-old male BALB/c mice were randomized into five groups and each group contained five mice. Ad/AFPtBid was administered by (i) single dose of 1 × 1010 p.f.u., (ii) single dose of 1 × 1011 p.f.u., (iii) three doses of 1 × 1010 p.f.u. every 2 days and (iv) three doses of 1 × 1011 p.f.u. every 2 days. Ad/AFPLacZ was given as control. Three doses of 1 × 1010 p.f.u. adenovirus every 2 days were previously determined to confer clinical benefit to the animals.20 Mice were monitored daily for their activity (digestive, respiratory and circulation systems) and weighed every day. On Days 4 and 7 after Ad/AFPtBid administration, blood was collected from tail vein for hematology analysis. At the same time, retro-orbital blood collection was performed for the assessment of hepatic enzymes alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine. Microscopic examinations of H&E staining of livers were performed to observe the pathological change.

Statistical analysis

The difference in mean of the tumor volume between groups was compared for statistical significance, using the ANOVA test by SPSS 15.0. P<0.05 was considered statistically significant. The survival rate of animals after various treatment protocols was calculated using Kaplain–Meier survival analysis.

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Acknowledgements

This work was supported by Research Grants Council of the Hong Kong Special Administrative Region (No. CUHK 4534/06M). We are grateful to Ji Miao and Suk Ying Chun (Department of Surgery, The Chinese University of Hong Kong) for their help in providing adenovirus for the experiments.

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Correspondence to G G Chen.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on Gene Therapy website

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Keywords

  • adenovirus
  • apoptosis
  • hepatocellular carcinoma
  • tBid
  • 5-fluorouracil

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