Cell-specific expression of the analgesic-antitumor peptide coding sequence under the control of the human α-fetoprotein gene promoter and enhancer

Abstract

The aim of the present study was to construct a gene-modified hepatocellular carcinoma (HCC)-specific analgesic-antitumor peptide (AGAP) expression vector regulated by the α-fetoprotein (AFP) promoter and enhancer, in order to evaluate its effect. The AFP promoter is generally used in HCC-specific gene therapy strategies. However, this approach is limited by the weak activity of the AFP promoter. Linking the AFP enhancer and promoter has been shown to generate a stronger and more HCC-selective promoter. The AGAP DNA fragment was amplified from the total RNA of the Chinese scorpion, Buthus martensii Karsch. The fragment was subsequently cloned into the pAFP plasmid with the minimal essential DNA fragment, which included the AFP gene promoter and enhancer, to construct the recombinant plasmid, pAFP-AGAP. The plasmid was transfected into HepG2 cells and the mRNA expression levels of AGAP were detected by reverse transcription polymerase chain reaction (RT-PCR). In addition, Cell Counting Kit 8 (CCK-8) was used to analyze the cytotoxicity of plasmid transfection. The length, position and orientation of the inserted AGAP gene were all confirmed to be correct; thus, the recombinant vector was successfully constructed. Using RT-PCR and CCK-8 analysis, the mRNA expression levels of AGAP and the cytotoxicity in AFP-producing human HCC cells were determined. The AFP promoter and enhancer were found to specifically accelerate the expression of the target genes within the cells that were positive for AFP. Therefore, the method used in the present study was demonstrated to be a novel integration of traditional Chinese medicine with western medicine.

Modification of the hTERT promoter by heat shock elements enhances the efficiency and specificity of cancer targeted gene therapy

“…Previous studies have investigated a number of tumour-specific promoters in a variety of models with promising results. These include the CEA promoter in CEA-positive lung carcinoma [23], the AFP promoter in AFP positive hepatocellular carcinoma [24] and the stressinducible grp78/BiP promoter in fibrosarcoma [25]. One promising tumour-specific promoter that has been demonstrated to target a broad range of cancer types is the hTERT promoter [26].…”

In-Situ-Generated Vasoactive Intestinal Peptide Loaded Microspheres in Mussel-Inspired Polycaprolactone Nanosheets Creating Spatiotemporal Releasing Microenvironment to Promote Wound Healing and Angiogenesis

“…After incubation for 2 h, 10 μL CCK-8 (Dojindo, Kyushu, Japan) solution was added into each well. After incubation at 37 °C for 3 h, the mean optical density value of the culture medium was quantitated at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader (Thermo Varioskan Flash, USA) …”

“…After incubation at 37 °C for 3 h, the mean optical density value of the culture medium was quantitated at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader (Thermo Varioskan Flash, USA). 32 2.6. Animal Experiment in Vivo.…”