ABSTRACT
Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer’s disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
ABSTRACT
Objective: To evaluate the inhibitory role of a novel oncolytic adenovirus (OA), GP73-SphK1sR-Ad5, on the growth of hepatocellular carcinoma (HCC). Methods: GP73-SphK1sR-Ad5 was constructed by integrating Golgi protein 73 (GP73) promoter and sphingosine kinase 1 (SphK1)-short hairpin RNA (shRNA) into adenovirus serotype 5 (Ad5), and transfecting into HCC Huh7 cells and normal human liver HL-7702 cells. The expression of SphK1 and adenovirus early region 1 (E1A) was detected by quantitative real-time PCR (qRT-PCR) and western blot, respectively. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and apoptotic rate was determined by flow cytometry. An Huh7 xenograft model was established in mice injected intratumorally with GP73-SphK1sR-Ad5. Twenty days after injection, the tumor volume and weight, and the survival time of the mice were recorded. The histopathological changes in tumor tissues were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). Results: Transfection of GP73-SphK1sR-Ad5 significantly upregulated E1A and downregulated SphK1 in Huh7 cells, but not in HL7702 cells. GP73-SphK1sR-Ad5 transfection significantly decreased the viability and increased the apoptotic rate of Huh7 cells, but had no effect on HL7702 cells. Intratumoral injection of GP73-SphK1sR-Ad5 into the Huh7 xenograft mouse model significantly decreased tumor volume and weight, and prolonged survival time. It also significantly decreased the tumor infiltration area and blood vessel density, and increased the percentages of cells with nucleus deformation and cells with condensed chromatin in tumor tissues. Conclusions: GP73-SphK1sR-Ad5 serves as a novel OA and can inhibit HCC progression with high specificity and efficacy.