Combined therapy with oncolytic adenoviruses encoding TRAIL and IL-12 genes markedly suppressed human hepatocellular carcinoma both in vitro and in an orthotopic transplanted mouse model

Abstract

Background: Gene-based virotherapy mediated by oncolytic viruses is currently experiencing a renaissance in cancer therapy. However, relatively little attention has been given to the potentiality of dual gene virotherapy strategy as a novel therapeutic approach to mediate triplex anticancer combination effects, particularly if the two suitable genes are well chosen. Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and interleukin-12 (IL-12) have been emerged as promising pharmacological candidates in cancer therapy; however, the combined efficacy of TRAIL and IL-12 genes for treatment of human hepatocellular carcinoma (HCC) remains to be determined.

Methods: Herein, we investigated the therapeutic efficacy of concurrent therapy with two armed oncolytic adenoviruses encoding human TRAIL gene (Ad-ΔB/TRAIL) and IL-12 gene (Ad-ΔB/IL-12), respectively, on preclinical models of human HCC, and also elucidated the possible underlying mechanisms. The effects of Ad-ΔB/TRAIL+Ad-ΔB/IL-12 combination therapy were assessed both in vitro on Hep3B and HuH7 human HCC cell lines and in vivo on HCC-orthotopic model established in the livers of athymic nude mice by intrahepatic implantation of human Hep3B cells.

Results: Compared to therapy with non-armed control Ad-ΔB, combined therapy with Ad-ΔB/TRAIL+Ad-ΔB/IL-12 elicited profound anti-HCC killing effects on Hep3B and HuH7 cells and on the transplanted Hep3B-orthotopic model. Efficient viral replication and TRAIL and IL-12 expression were also confirmed in HCC cells and the harvested tumor tissues treated with this combination therapy. Mechanistically, co-therapy with Ad-ΔB/TRAIL+Ad-ΔB/IL-12 exhibited an enhanced effect on apoptosis promotion, activation of caspase-3 and-8, generation of anti-tumor immune response evidenced by upregulation of interferon gamma (IFN-γ) production and infiltration of natural killer-and antigen presenting cells, and remarkable repression of intratumor vascular endothelial growth factor (VEGF) and cluster of differentiation 31 (CD31) expression and tumor microvessel density.

Conclusions: Overall, our data showed a favorable therapeutic effect of Ad-ΔB/TRAIL+Ad-ΔB/IL-12 combination therapy against human HCC, and may therefore constitute a promising and effective therapeutic strategy for treating human HCC. However, further studies are warranted for its reliable clinical translation.

Keywords: Dual gene virotherapy; Hepatocellular carcinoma; Interleukin-12; Oncolytic adenoviruses; Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Fig. 1

Graphical representation of the constructed and generated non-armed control oncolytic adenovirus (Ad-ΔB) and oncolytic adenovirus armed with human TRAIL gene (Ad-ΔB/TRAIL) or human IL-12 gene (Ad-ΔB/IL-12). TRAIL or IL-12 gene was incorporated in the E3 region of the viral backbone under the transcriptional control of the human cytomegalovirus (CMV) promoter

Fig. 2

Validation of in vitro oncolytic adenoviral replication efficiency and the expression patterns of the encoded TRAIL and IL-12 transgenes in human HCC cells. HuH7 cells were cultured and then treated with PBS, Ad-ΔB, or Ad-ΔB/TRAIL+ Ad-ΔB/IL-12 at a MOI of 5 for each virus. Two days post-treatment, the cell lysates and conditioned media were harvested and relative mRNA and protein levels of TRAIL (a and b), or IL-12 (c and d) were measured by using Q-RT-PCR and ELISA assays, respectively. Data are represented as mean ± SE. In addition, viral production was determined by measuring the expression of Ad E1A protein (e) by western blot assay using rabbit anti-Ad E1A primary antibody

Fig. 3

In vitro effects of Ad-ΔB and Ad-ΔB/TRAIL+Ad-ΔB/IL-12 on Hep3B and HuH7 human HCC cells and normal human liver (WRL68) cells. a Quantitative results of MTT assay showing the inhibitor effects on viability of Hep3B cells at 5 and 10 MOI (the multiplicity of infection) per virus; (b) Quantitative results of MTT assay showing the inhibitor effects on viability of HuH7 cells at 10 and 20 MOI per virus; and (c) Quantitative results of MTT assay showing the inhibitor effects on viability of WRL68 cells at 50 MOI per virus. Data are represented as mean ± SE. d Representatives of semi-quantitative assessment of cytotoxic potency by crystal violet cytopathic effect assay on Hep3B cells (left panel), HuH7 cells (middle panel), and WRL68 cells (right panel) at indicated MOIs. Ad-ΔB = non-armed control oncolytic adenovirus; Ad-ΔB/TRAIL+Ad-ΔB/IL-12 (or Ad-ΔB/TRAIL+IL-12) = combined therapy with two oncolytic adenoviruses encoding human TRAIL and IL-12 genes, respectively

Fig. 4

In vivo antitumor effects of Ad-ΔB and Ad-ΔB/TRAIL+Ad-ΔB/IL-12 on an orthotopic transplanted mouse model of human HCC. The model was induced by intrahepatic implantation of 2 × 10⁶ human Hep3B/fluc cells into the left liver lobes of mice in athymic nude mice. Positive HCC-bearing mice were thereafter randomized into three groups (n = 10/group) and through their tail veins they were treated with: PBS alone (Group 1), Ad-ΔB (Group 2), and Ad-ΔB/TRAIL+Ad-ΔB/IL-12 (Group 3) at a dosage regimen of 1 × 10¹⁰ VP in 200 μL PBS of each virus; repeated three times every other day. Next, the mice were monitored and photographed (a) weekly by in vivo bioluminescence imaging to determine the pattern of tumor response to the subjected treatments (b) until the end of study (day 14 post-the last treatment dose)

Fig. 5

Histopathological, immunohistochemical and apoptosis assessments of the harvested tumor tissues from different animal groups. Three days post-treatment of HCC-bearing mice with PBS, Ad-ΔB, or Ad-ΔB/TRAIL+Ad-ΔB/IL-12 (at a dosage regimen of 1 × 10¹⁰ VP in 200 μL PBS of each virus; repeated three times every other day), the mice were euthanized under general anesthesia and their liver tumor tissues were harvested and prepared for histopathological, immunohistochemical (IHC) and apoptosis assessments, and measurement of intra-tumor levels of IFN-γ and VEGF. a Photographs of H&E: hematoxylin and eosin staining of tumor tissues for histopathology; TUNEL: Terminal uridine deoxynucleotidyl transferase dUTP nick end labeling staining assay of apoptotic cells in tumor tissue; NK1.1: IHC staining of infiltrated natural killer cells; CD11b: IHC staining of recruited antigen-presenting cells (dendritic cells and macrophage); and CD31: IHC staining of tumor CD31-positive microvessels endothelial cells. Original magnification: × 400. b and c are quantitative ELISA assays of the intratumor expression levels of IFN-γ and VEGF, respectively, at their protein levels. d The mean microvessel density for each treatment group was determined by counting CD31-positive vessels in 10 high-power fields. Each experiment was performed at least three times, and data shown are from representative experiments. Values of (b), (c) and (d) represent the mean ± SE

Fig. 6

Validation of intra-tumor oncolytic adenoviral replication and the expression patterns of the encoded TRAIL and IL-12 transgenes. Three days post-treatment of HCC-bearing mice with PBS, Ad-ΔB, or Ad-ΔB/TRAIL+Ad-ΔB/IL-12 (at a dosage regimen of 1 × 10¹⁰ VP in 200 μL PBS of each virus; repeated three times every other day), the mice were euthanized under general anesthesia and their liver tumor tissues were harvested and investigated to validate intratumor viral replication and expression of TRAIL and IL-12 transgenes. a and b represents intra-tumor TRAIL expression at its mRNA and protein level, respectively, and (c) and (d) represents intra-tumor IL-12 expression at its mRNA and protein level, respectively. Data are represented as Mean ± SE. e represents the expression of Ad E1A protein (an indicator of viral production) by western blot assay

Fig. 7

In vitro and in vivo apoptotic findings. a Flow cytometric analysis of apoptosis induction in HCC cells-treated Ad-ΔB/TRAIL plus Ad-ΔB/IL-12 (Ad-ΔB/TRAIL+IL-12), in comparison with cells treated with control Ad-ΔB or PBS, by using Annexin-V and propidium iodide (PI) fluorescence staining assay. Each scatter plot demonstrates the percentage of early apoptotic cells (Annexin-V⁺cells, bottom right quadrant) and late apoptotic cells (PI⁺cells, upper right quadrant). b Western blot analysis showing the intra-tumor expression levels of cleaved caspase-3 (left panel) and caspase-8 (right panel) at day 3 after treatment of HCC-bearing mice with PBS, Ad-ΔB, or Ad-ΔB/TRAIL + Ad-ΔB/IL-12 (Ad-ΔB/TRAIL+IL-12) at a dosage regimen of 1 × 10¹⁰ VP in 200 μL PBS of each virus; repeated three times every other day