Selective Inhibition of Hepatoma Cells Using Diphtheria Toxin A under the Control of the Promoter/Enhancer Region of the Human α‐Fetoprotein Gene

Abstract

We constructed a plasmid containing human α α α α-fetoprotein (AFP) promoter/enhancer to direct the cell type-specific expression of diphtheria toxin fragment A (DTA), designated as pAF-DTA, to AFP-producing hepatocellular carcinoma cells. The transfection was carried out with cationic liposomes (DMRIE-C) and the expression of the DTA gene was confirmed by a northern blot analysis. When pAF-DTA was transfected, the growth of AFP-positive HuH-7 cells was inhibited, whereas growth inhibition was not observed in AFP-negative MKN45 cells. In this experiment, the secretion of AFP was similarly suppressed, but the secretion of carcinoembryonic antigen from MKN45 was not altered. pAF-DTA could also exert its growth inhibitory effect on PLC, a cell line with a low level of AFP. However, no inhibitory effect of pAF-DTA was observed on the proliferation of primary hepatocyte cells. Furthermore, transfection experiments in which HuH-7 and splenic stromal cells were co-cultured revealed the growth inhibition by pAF-DTA to be selective in HuH-7 cells. Finally, the growth of HuH-7 transplanted on BALB/c nu/nu mice was inhibited by the direct injection of pAF-DTA/liposome complex into a tumor mass. These results suggest that use of pAF-DTA may be potentially useful as a novel approach for the selective treatment of tumor cells producing AFP even at low levels, without affecting other types of cells.

Hypoxia-Regulated Expression of Attenuated Diphtheria Toxin A Fused with Hypoxia-Inducible Factor-1α Oxygen-Dependent Degradation Domain Preferentially Induces Apoptosis of Hypoxic Cells in Solid Tumor

“…It should be noted, however, that we could not observe a clear tumor growth inhibition after multiple injection of pVEGFpro-DT-A W153F /ODD-EBBS/liposome complex into a tumor mass. This contrasts to the case of direct injection of a-fetoprotein promoter/ enhancer-driven DT-A expression construct/DMRIE-C complex into hepatoma in which significant growth retardation was observed (33). However, our results rather seem to reflect the hypoxia specificity of our expression construct.…”

“…Nevertheless, several strategies that limit its toxicity have been developed and those include targeted delivery of DT-A to specific cells or tissue-specific expression, such as DT-A immunotoxin (30), DT-A fused to peptide ligands for cell-specific receptor (32), and DT-A expression construct under the control of a regulatory element or tissue-specific promoter (31,33,34). With regard to cancer gene therapy, a-fetoprotein promoter and prostate-specific antigen promoter-regulated expression of DT-A gene led to selective killing of hepatocarcinoma cell lines and prostate cancer cell lines, respectively, by using a liposomal gene transfer system (33,34). In other cases, however, although preferential killing of target tumor cells could be shown, nonspecific cytotoxicity could not be abolished due to the background expression of DT-A (35).…”

“…It directly binds NAD + and catalyzes the transfer of ADP ribose from NAD + to elongation factor 2, which irreversibly inhibits elongation factor 2 (28). DT-A gene has been considered to be applicable for cancer gene therapy (29)(30)(31)(32)(33)(34). Because DT-A leads to rapid cell cycleindependent death, it might be useful to kill hypoxic tumor cells if its expression is properly regulated.…”

Progress toward liver‐based gene therapy

“…Because the liver is a main site of metastasis from gastrointestinal malignant tumors, not only carcinomas originating in the liver, but also other types of cancers such as colon or pancreas cancers, can be treated by gene therapy through the liver. Earlier attempts were made to deliver genes into cancer cells aiming at direct destruction of the target cells by inhibiting oncogene activity, 91,92 restoring tumor suppressor function, 93,94 introducing prodrug‐activating enzymes, 95,96 and expressing cytotoxic 97 or apoptotic genes 98 . Blocking the production of specific gene products can be achieved by delivering antisense oligonucleotides, 99 small interfering RNA, 100 short hairpin RNA 101 or ribozymes 102 .…”

Development of Gene Therapy Using Prostate‐specific Membrane Antigen Promoter/Enhancer with Cre Recombinase/LoxP System for Prostate Cancer Cells under Androgen Ablation Condition

“…18) Three micrograms of DNA plasmid and 3 µg of DMRIE-C (Life Technologies) in 50 µl of OPTI-MEM (Life Technologies) were gently mixed and incubated at room temperature for 30 min. 19) Next, the plasmid-liposome complex was directly injected into the inoculated tumor. After 48 h, tumors were excised and X-Gal staining and luciferase expression assay were performed.…”