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

Abstract

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 a-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. ' 2006 Wiley-Liss, Inc.

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

“…[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.…”

“…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 …”

“…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.…”

“…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).…”

“…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.…”