Abstract
Current treatment approaches for hepatocellular carcinoma (HCC) have a narrow therapeutic index and alternate modes of treatment are thus required. We have utilized a gene delivery vector containing inducible caspase 9 (iCasp9) gene, which is a synthetic analogue based on the mammalian caspase 9 and fused to a human FK506 binding protein that allows its conditional dimerization to a synthetic, small molecule [chemical inducer of dimerization, AP20187] and results in target cell apoptosis. In our studies, we have tested these synthetic vectors based on an adeno-associated virus platform for their potential anti-tumorigenic effect in human HCC cells in vitro and in a HCC tumor model developed in nude mice. Our data demonstrates that the iCasp9-AP20187 bioconjugate is able to trigger terminal effectors of cellular apoptosis and presents a viable approach for the potential treatment of HCC.
Highlights
Hepatocellular carcinoma (HCC) is a malignancy of the liver
An inducible caspase 9 gene, which is a synthetic analogue based on the mammalian caspase 9 fused to a human FK506 binding protein (FKBP) that allows conditional dimerization to a synthetic, bioinert small molecule [chemical inducer of dimerization (CID), AP20187], has been tested to switch-off genes during T cell therapy.[22,23] iCasp[9] is nonimmunogenic, since apart from a single amino acid substitution
In order to further characterize the tumor regression at the molecular level, in response to inducible caspase 9 (iCasp9)/AP20187 treatment, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL, Roche, Basel, Switzerland) assay was performed. This test detects DNA strand break in cells undergoing apoptosis and has been used widely as a biomarker for diagnostic evaluation of apoptosis along with caspase-3.39,40 Our screening revealed that tumor tissue from the iCasp9/ AP20187 treated group were markedly positive for TUNEL staining signifying massive cell death as compared to the tumor tissue harvested from the control group
Summary
Hepatocellular carcinoma (HCC) is a malignancy of the liver. About 600,000 new cases of HCC are reported annually, making it the fifth most common cause of all cancers affecting humans.[1−3] The mortality of this condition is very high; approximately 250,000 deaths occur annually due to HCC.[3]. These data indicate that the rate of tumor attenuation was slightly reduced in the low dose group (1 mg/kg body weight) as compared to animals that received a higher dose (2 mg/kg bodyweight) of AP20187 (Figure 3). In order to further characterize the tumor regression at the molecular level, in response to iCasp9/AP20187 treatment, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL, Roche, Basel, Switzerland) assay was performed This test detects DNA strand break in cells undergoing apoptosis and has been used widely as a biomarker for diagnostic evaluation of apoptosis along with caspase-3.39,40 Our screening revealed that tumor tissue from the iCasp9/ AP20187 treated group were markedly positive for TUNEL staining (red color) signifying massive cell death as compared to the tumor tissue harvested from the control group
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