Application of doxorubicin-induced rAAV2-p53 gene delivery in combined chemotherapy and gene therapy for hepatocellular carcinoma

Abstract

p53 gene transfer has been proposed as a potential therapeutic option for treatment of hepatocellular carcinoma (HCC). Compared to other commonly used gene transfer vectors such as adenovirus and retrovirus, recombinant adeno-associated virus serotype 2 (rAAV2) has shown promising results in human clinical trials. Significant enhancement in the gene transfer efficiency is needed, however, for HCC applications. In the present study, we applied chemotherapy drug Doxorubicin (DOX) to induce rAAV2 transduction of hepatomas. Using reporter assays, we showed that the DOX-treated hepatomas became more susceptible to rAAV2 infection in comparison to untreated controls: the permissiveness increased >350-fold and >120-fold for HepG2 (p53 wild-type) and Hep3B (p53 null) hepatomas, respectively. Using the induced permissiveness, we applied rAAV2-p53 transduction to restore p53 expression in the p53-null Hep3B hepatomas. Compared to rAAV2-p53 transduction alone, rAAV2-p53 transduction with DOX resulted in a >16-fold induction of p53 expression. The transduced Hep3B expressed as much as 380% more immunoreactive p53 in comparison to the wild-type p53 expression in the HepG2 hepatomas. Significantly, when Hep3B cells were treated with 0.5 muM of DOX and rAAV2-p53 (MOI = 10) for twelve hours, the cell viability dropped to 66% four days after the administration. This decrease in cell viability was similar to that of treatment with 1 microM of DOX alone in the absence of rAAV2. The 50% reduction in DOX administration--from 1 microM to 0.5 microM--revealed the antitumor property of the rAAV2-p53 transduction as well as the joint cytotoxicity of DOX and rAAV2-p53 against the p53-null hepatomas. We conclude that DOX mediates the enhancement effect on rAAV2 transduction of human hepatomas. Combined DOX and rAAV2-p53 administration may facilitate more efficient treatment for the HCC caused by p53 mutations.

Figures

Figure 1.

Schematics of the rAAV2 vector plasmids pAAV‑Luciferase‑EGFP and pAAV‑p53 used in this research. EGFP, enhanced green fluorescent protein. ITR, inverted terminal repeat. Pcmv, cytomegalovirus promoter. SV40 poly A, Simian Virus 40 late polyadenylation signal. Psv40, Simian Virus 40 promoter. hGH poly A, human growth hormone polyadenylation signal.

Figure 2.

Induction of reporter expression in rAAV2 transduced HepG2 and Hep3B hepatomas treated with Doxorubicin (DOX). (A) Reporter: luciferase. Luciferase activity in treated cells was normalized to the activity in untreated control cells. MOI = 10. Experiments were performed in triplicate wells. Bars indicate standard deviation. (B–E) Reporter: EGFP. Phase‑contrast (B and D) and fluorescence (C and E) micrographs of Hep3B hepatomas transduced with rAAV2 in the absence (B and C) or presence (D and E) of DOX. MOI = 50. Bar = 100 μm.

Figure 3.

MTS assay of cell viability/proliferation—indicated by mitochondrial activity—after treatment with DOX. The mitochondrial activity in untreated cells was assigned a value of 100. Experiments were performed in quadruplicate. Bars indicate standard deviation.

Figure 4.

Doxorubicin (DOX)‑induced rAAV2‑p53 transduction restores p53 expression in Hep3B (p53 null) hepatomas. (A) Induction of rAAV2‑p53 transduction in Hep3B hepatomas treated with various DOX doses. MOI = 10. (B) rAAV2‑p53 transduction of Hep3B hepatomas at different MOIs in the absence or presence of DOX. Control: wild‑type p53 expression in HepG2 hepatomas. Experiments were performed in quadruplicate. Bars indicate standard deviation. (C and D) Phase‑contrast and fluorescence micrographs of the rAAV2‑p53 transduced Hep3B hepatomas (DOX = 1 μM and MOI = 10) using FITC‑conjugated anti‑p53 antibody to detect p53 protein. These two images were taken under the same view. Bar = 100 μm.

Figure 5.

Hep3B viability after treatment with DOX alone or a combined DOX and rAAV2‑p53 vectors. MTS assay. The data of Hep3B treated with DOX alone (empty bars) are the same as those shown in Figure. The mitochondrial activity in untreated cells was assigned a value of 100. *, #, •, ⚈, ▴ and ◊ indicate p values <0.05. There were no statistical differences between the rAAV2‑p53 transduced and untransduced cells and between the rAAV2‑p53 transduced and reporter vector‑transduced cells at DOX = 0, 0.1, 0.2, 5 and 10 μM. p values were calculated using a one‑tailed t‑test, assuming unequal variances. Experiments were performed in quadruplicate. Bars indicate standard deviation.

Figure 6.

Kinetic cell‑growth assay to evaluate the change of Hep3B colonies in response to DOX and/or rAAV2‑p53 treatments. (A) Gross image of cloning rings (upper two panels) and the formation of Hep3B colonies (lower two panels, ~5 mm in diameter) in the wells of a 24‑well plate. Cells were seeded inside the ring space. Prior to performing the imaging of (A), culture media were removed to reveal the cell pellets. (B) A typical micrograph of a portion (approximately a quarter) of the Hep3B colony in (A), indicated by *. Magnification: ×40. (C, D, E, F, and G) The changes of Hep3B colonies in diameter over time in response to different DOX concentrations combined with reporter vector (rAAV2‑Luciferase‑EGFP, control) or rAAV2‑p53 transduction. Experiments were performed in quadruplicate. Bars indicate standard deviation.