Targeted expression of BikDD combined with metronomic doxorubicin induces synergistic antitumor effect through Bax activation in hepatocellular carcinoma

Abstract

Conventional chemotherapy is commonly used to treat advanced non-resectable hepatocellular carcinoma (HCC) but this treatment modality has not demonstrated convincing survival benefit in HCC patients. Our previous studies indicated that targeted expression of therapeutic BikDD driven by a liver cancer-specific α-fetoprotein promoter/enhancer (eAFP) in the VISA backbone (eAFP-VISA-BikDD) significantly and specifically kills HCC cells in multiple orthotopic animal models. To enhance its therapeutic efficacy, we combined eAFP-VISA-BikDD with chemotherapeutic agents and found that eAFP-VISA-BikDD plus doxorubicin (Dox) or 5-fluorouracil (5-FU) demonstrated synergistic cytotoxicity in HCC cells. Specifically, the combination of eAFP-VISA-BikDD plus Dox markedly induced apoptosis via increased Bax mitochondrial translocation and cytoplasmic cytochrome c release. Compared with either agent alone, a low dose of Dox combined with eAFP-VISA-BikDD induced better antitumor effect and prolonged longer survival of mice in two orthotopic liver cancer xenograft models. Our findings provide strong preclinical support for evaluating the combined therapy of eAFP-VISA-BikDD and Dox in a clinical setting as a treatment option for HCC.

Keywords: combination therapy; hepatocellular carcinoma; metronomic chemotherapy; orthotopic animal model; synergistic antitumor effect.

Figure 1. Cytotoxic effects of eAFP-VISA-BikDD plus Dox against human HCC cell lines in vitro

Huh7 A. PLC/PRF/5 B. and Tong/HCC C. cells were transfected with indicated concentrations of eAFP-VISA-BikDD by using extruding DOTAP:cholesterol liposomes. Transfection mixtures were removed after 4 hours, and fresh DMEM complete medium containing the indicated concentration of Dox was added for an additional 72 hours followed by cytotoxicity analysis by WST-1 assay. Relative cell viability was normalized to untreated cells (set as 100%) and expressed as mean ±SD.

Figure 2. Cytotoxic effects of eAFP-VISA-BikDD plus 5-FU against human HCC cell lines in vitro

Huh7 A. PLC/PRF/5 B. and Tong/HCC C. cells were transfected for 4 hours with indicated concentrations of eAFP-VISA-BikDD by using extruding DOTAP:cholesterol liposomes. Transfection mixtures were removed after 4 hours, and fresh DMEM complete medium containing the indicated concentration of 5-FU was added for an additional 72 hours followed by cytotoxicity analysis by WST-1 assay. Relative cell viability was normalized to untreated cells (set as 100%) and expressed as mean ±SD.

Figure 3. Cytotoxic effects of combining eAFP-VISA-BikDD and Dox in normal liver cell lines

THLE-2 A. and MIHA B. cells were transfected with control (Ctrl) or eAFP-VISA-BikDD for 4 hours. Transfection mixtures were then replaced by fresh complete medium containing the indicated concentration of Dox for an additional 72 hours. At the end of incubation, cytotoxic effects were evaluated by WST-1 assay. Relative cell viability was normalized to untreated cells (set as 100%). Data represent mean ±SD. N = 3.

Figure 4. Effects of eAFP-VISA-BikDD combined with DOX on apoptosis-related protein expression in HCC cell lines

Huh7 A. and Tong/HCC B. cells were transfected with control (Ctrl) vector or eAFP-VISA-BikDD. After 4 hours of incubation, transfection mixtures were replaced by fresh DMEM complete medium or DMEM complete medium containing Dox (0.1 μM) for 72 hours. Cell lysates were collected for Western blot analysis. C. Huh7 cells were transfected for 4 hours with vector control (Ctrl) or eAFP-VISA-BikDD. After incubation, the transfection mixture was removed then replaced with DMEM complete medium or DMEM complete medium containing Dox (0.1 μM) for additional 12–48 hours. Cells were harvested at the indicated time points and subjected to immunoprecipitation with anti-Bcl-xL and anti-Mcl-1 antibodies. D, E. Cells were treated as described above and lysates were harvested at 24 hours after Dox administration and subjected to immunoprecipitation by anti-Bik antibody in 1% CHAPS lysis buffer. Equal amounts of proteins from different treatment were separated by SDS-PAGE and then incubated with anti-BiK antibody. The anti-Bik polyclonal antibody recognizes both wild-type and mutant form Bik. F. Supernatant was harvested from precipitated immune complexes from (D) and (E) and subjected to secondary immunoprecipitation with anti-Bax (6A7) antibody. G. Bax mitochondrial translocation and cytochrome c release in combination therapy. Huh7 cells were transfected with control vector or eAFP-VISA-BikDD for 4 hours and then incubated with DMEM complete medium or DMEM complete medium containing Dox for additional 36 hours. Cell lysates were harvested and subjected to subcellular fractionation experiments. Western blot analysis was performed to determine the levels of Bax and cytochrome c. Antibody to cytochrome c oxidase IV antibody (COX IV) was used as mitochondrial loading control.

Figure 5. Antitumor effect of eAFP-VISA-BikDD combined with Dox in an orthotopic xenograft mouse model

A. Huh7 cells were orthotopically inoculated into the left liver lobe of SCID mice. One week after tumor inoculation, mice were randomly divided into four groups for treatment. Mice received systemic intravenous injection of control vector or eAFP-VISA-BikDD encapsulated in liposomes twice a week for four weeks. Dox (0.5 mg/kg) or normal saline was administered once a week by tail vein injection for four weeks. Tumor growth was monitored weekly by in vivo imaging system. B. Thirty-seven days after initial treatment, mice were sacrificed and liver tumors were dissected and weighed. Data represent mean ±SEM. N = 5. Representative images of tumor tissues from mice after the indicated treatment are shown on the right. C. Survival rate of mice after the indicated treatment. n.s., not significant. D. Detection of apoptosis in liver tissues by TUNEL assays. Tissues collected from (A) were fixed with 10% neutral buffered formalin, and TUNEL-positive cells were stained and counted from four randomly selected fields per section. Arrows indicate TUNEL-positive apoptotic cells. Quantitation is shown below. Data represent mean ±SD.

Figure 6. Antitumor effect of eAFP-VISA-BikDD combined with Dox in an orthotopic syngeneic mouse model

A. Left, ML-1 cells were orthotopically inoculated into the left liver lobe of BALB/c mice. One week after tumor inoculation, mice were randomly divided into four groups for treatment. Mice received systemic intravenous injection of control vector or eAFP-VISA-BikDD encapsulated in liposomes twice a week for four weeks. Dox (0.5 mg/kg) or normal saline was administered once a week by tail vein injection for four weeks. Thirty-seven days after initial treatment, mice were sacrificed and liver tumors were dissected and weighed. Data represent mean ±SEM. N = 5. Representative images of tumor tissues from mice after the indicated treatment are shown on the right. B. Survival rate of mice after the indicated treatment. n.s., not significant. C. Detection of apoptosis in liver tissues by TUNEL assays. Tissues collected from (A) were fixed with 10% neutral buffered formalin, and TUNEL-positive cells were stained and counted from four randomly selected fields per section. Arrows indicate TUNEL-positive apoptotic cells. Quantitation is shown below. Data represent mean ±SD.