Biological features, gene expression profile, and mechanisms of drug resistance of two- and three-dimensional hepatocellular carcinoma cell cultures.

Abstract

Hepatocellular carcinoma (HCC) is a common malignant tumor with insidious onset and rapid progression. Its treatment is often difficult owing to tumor resistance. In this study, we aimed to understand the different biological characteristics, gene expression profiles, and drug resistance mechanisms of HCC cells cultured under different conditions. A conventional adherence method and a liquid overlay technique were used to prepare two‐ and three‐dimensional cultures of Bel‐7402 and 5‐fluorouracil (5‐Fu)‐resistant Bel‐7402 (Bel‐7402/5‐Fu) cells. Morphological characteristics were assessed via microscopy, and cell cycle distribution and apoptotic rate were obtained using flow cytometry. Cell sensitivity to different concentrations of drugs was detected with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays. Gene expression profiles and signal transduction pathways of Bel‐7402 and Bel‐7402/5‐Fu cells under different culture conditions were determined using gene chips. Cells in three‐dimensional culture were suspended and they grew into dense multicellular spheroid (MCS) structures, aggregating with each other. In contrast to cells in the two‐dimensional culture, cell cycle arrest was observed in MCSs. The sensitivity of Bel‐7402 cells in the two‐dimensional culture to drugs at high concentrations was significantly higher than that of cells in the three‐dimensional culture (p < .05). The apoptotic rate of Bel‐7402 and Bel‐7402/5‐Fu cells was also higher in the two‐dimensional culture (p < .05). Signal transduction pathway analysis showed that after Bel‐7402 cells acquired resistance to 5‐Fu, CCND1, MCM2, and MCM3 gene expression was upregulated in the G1 to S cell cycle control signal transduction pathway, CDKN1C and CCNG2 gene expression was downregulated, and MCM2 and MCM3 gene expression was upregulated in the DNA replication signal transduction pathway. Therefore, the liquid overlay technique is a simple, low‐cost procedure to successfully construct three‐dimensional culture models of HCC. This study provides new information and methods for exploring the molecular mechanisms of liver cancer resistance, clinical treatment, development of molecular information, and interventional prevention.