Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

Abstract

AIM:To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity. METHODS: In this study, we designed a secretory protein by adding a secretory signal peptide (SP) to the N terminus of Transactivating Transcription (TAT)-apoptin (SP-TAT-apoptin), to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model. RESULTS: In human liver carcinoma HepG2 cells, SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48 h, however, it translocated into the nuclear compartment and caused massive apoptotic cell death, as determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not, however, cause any cell death in non-malignant human umbilical vein endothelial cells (HUVECs). Most importantly, the conditioned medium from Chinese hamster ovary (CHO) cells transfected with SP-TAT-apoptin also induced significant cell death in HepG2 cells, but not in HUVECs. CONCLUSION: The data demonstrated that SP-TATapoptin induces apoptosis only in malignant cells, and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.

Apoptin sensitizes radiation-induced cell death via classic mitochondrial, caspase and p53-dependent signaling in HepG2 cells

“…This is consistent with the results of our previous study ( Fig. 1a) (9), indicating that a lentivirus-apoptin expression system is capable of dramatically up-regulating apoptin expression in HepG2 cells. Apoptin expression in HepG2 cells was further confirmed by Western blot analysis (Fig.…”

“…in our previous study, we demonstrated that apoptin promoted HepG2 cell apoptosis by a secretory transactivating transcription-apoptin fusion protein system (9). in the present study, concurrent treatment of HepG2 radio-resistant cells with radiation and apoptin lentivirus was shown to further increase apoptotic cell death compared to either treatment alone, suggesting that apoptin sensitizes radio-resistant HepG2 cells to radiation.…”

“…HepG2 cells prepared as above were collected at the desired time periods and stained with annexin V and Pi (Molecular Probes) following the manufacturer's protocol. Early apoptotic cells were analyzed immediately by flow cytometry as described previously (9).…”

“…our previous study showed that secretory transactivating transcription apoptin fusion protein induces cell apoptosis in hepatocellular carcinoma HepG2 cells (9,10). in the present study, we sought to explore the anti-tumor effect of a combination of rT and apoptin gene therapy in radio-resistant hepatocellular carcinoma HepG2 cells, and to further investigate the mechanisms of apoptosis.…”

“…This protein was first isolated from the chicken anemia virus, and its potential anti-cancer effects appear to be tumor specific; transformed cells are specifically targeted, while primary and non-transformed cells are spared (6). The apoptotic activity of apoptin is suggested to occur via a p53-independent, Bcl-2-enhanced mechanism (7,8).our previous study showed that secretory transactivating transcription apoptin fusion protein induces cell apoptosis in hepatocellular carcinoma HepG2 cells (9,10). in the present study, we sought to explore the anti-tumor effect of a combination of rT and apoptin gene therapy in radio-resistant hepatocellular carcinoma HepG2 cells, and to further investigate the mechanisms of apoptosis.…”