ABSTRACT
Apoptin protein harbors tumor-selective cell death activity, which makes it a potential anticancer therapy candidate. This study reports an apoptin therapy approach based on protein transduction domain 4 (PTD4)-mediated transduction of recombinant apoptin protein. In vitro, the PTD4-apoptin fusion protein is located in the nucleus and induces cell death in, e.g., human hepatocarcinoma HepG2 cells. In normal human L-02 hepatocytes, PTD4-apoptin protein retained mainly cytoplasmic and did not induce detectable levels of cell death, illustrating that the PTD4 domain does not affect apoptin's tumor-selective characteristics. In vivo, liver, cervix and gastric carcinoma xenografts treated with PTD4-apoptin protein for 6 days via the tumor epidermis exhibited a significant tumor growth inhibition because of apoptin-mediated cell death. In addition, treatment of human hepatocarcinoma xenografts during 3 weeks showed that PTD4-apoptin protein has significant anticancer activity, whereas control treatment with PTD4-enhanced green fluorescence protein or saline did not. Cell death and disruption of the tumor integrity were apparent in the PTD4-apoptin transduced xenografted tumors. As important, although PTD4-apoptin protein could be detected in the epidermal tissue covering the subcutaneous tumor tissue and in several organs, such as liver and brain, of the treated mice, no tissue disruption or signs of cell death could be detected. Our in vivo data reveal that apoptin protein delivery constitutes a novel powerful and safe anticancer therapy.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Capsid Proteins/metabolism , Carcinoma, Hepatocellular/therapy , Carrier Proteins/metabolism , Liver Neoplasms/therapy , Recombinant Fusion Proteins/therapeutic use , Animals , Capsid Proteins/genetics , Carcinoma, Hepatocellular/pathology , Carrier Proteins/genetics , Cell Nucleus/metabolism , Cells, Cultured , Colony-Forming Units Assay , Female , Flow Cytometry , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , In Situ Nick-End Labeling , Liver Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Nuclear Localization Signals , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Stomach Neoplasms/therapy , Subcellular Fractions , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/therapyABSTRACT
The ligand-binding domain of the very low-density lipoprotein receptor (VLDL-R) contains eight cysteine-rich repeat sequences that have been postulated as ligand-binding sites. This is obviously different from that of low-density lipoprotein receptor (LDL-R) that includes seven similar repeats. To make clear the contribution of these repeats to ligand-binding and to explore the reason of both receptors' ligand-binding characteristic, the VLDL-R recombinants lacking different repeat(s) were constructed by oligonucleotide-directed mutagenesis and transfected into ldl-A7 cell. Ligand-binding results showed that repeat 1 and repeat 2 were the most important in binding with apoE-rich lipoprotein(VLDL and beta-VLDL). Repeat 3 and repeat 6 also important for binding VLDL. The results also showed that VLDL-R lacking LBR7 retained partly LDL-R ligand-binding properties. It suggests that LBR7 in VLDL-R may responsible for both receptors' ligand-binding properties differences.
