LS180

Gene and Protein Expression Analysis of PD-L1 and the EMR Proteins in LS180 Cells

Colorectal cancer (CRC) is the third most prevalent and the second most fatal cancer in the world. The currently available treatment options of surgery, chemotherapy, and radiotherapy have poor clinical benefits, thus necessitating the development of new therapeutic approaches. Programmed cell death protein ligand-1 (PD-L1), an immune checkpoint protein that is upregulated in several cancer cells, plays an important role in immune evasion by cancer cells.

Kobori et al. investigated whether ezrin, radixin, and moesin (ERM) proteins, which act as scaffolds linking plasma membrane proteins to the actin cytoskeleton, influence PD-L1 expression on the cell surface of human colorectal adenocarcinoma cells (LS180). They initially examined the gene expression levels of PD-L1, ezrin, radixin, and moesin in LS180 and Caco-2 cells. RT-qPCR analysis revealed higher PD-L1 gene expression in LS180 cells compared to that in Caco-2 cells (Fig. 1a). Ezrin gene expression was comparable between the two cell lines (Fig. 1b). Radixin gene expression was lower in LS180 cells compared to that in Caco-2 cells (Fig. 1c). Moesin gene expression was only detected in LS180 cells but not in Caco-2 cells (Fig. 1d), which was consistent with the previous report that Caco-2 cells do not express moesin. Therefore, the authors used LS180 cells for the subsequent experiments to examine the role of ERM proteins in PD-L1 expression. Western blot analysis also showed detectable protein expression levels of PD-L1 in addition to ezrin, radixin, and moesin in the whole-cell lysates of LS180 cells (Fig. 1e).

Fig. 1. Gene and protein expression profiles of programmed cell death ligand-1 (PD-L1), ezrin, radixin, and moesin (ERM) in LS180 cells and Caco-2 cells (Kobori T, Tanaka C, et al., 2021).

Effects of Hydroxytyrosol on Expression of Apoptotic Genes and Activity of Antioxidant Enzymes in LS180 Cells

Colorectal cancer is one of the most frequent tumors in developed countries. Hydroxytyrosol, a powerful antioxidant, is able to exert control over oxidative stress, proliferation inhibition, and apoptosis induction. In the present work, the modulation induced by hydroxytyrosol on apoptotic gene expression (BAX, BCL2, CASP3, P53, PPARG, and NFE2L2) and antioxidant enzyme activity in human colorectal LS180 cancer cells was studied. Methods include treating LS180 cells with hydroxytyrosol, analyzing gene expression via real-time PCR, and measuring enzyme activity calorimetrically. The study found that Hydroxytyrosol causes a significant up-regulation of BAX expression at all the tested concentrations (Fig. 2A). BCL2 expression significantly increased (Fig. 2B). BAX:BCL2 ratio (Fig. 2C) and CASP3 expression (Fig. 2D) are significantly higher in all treated groups. P53 expression is not significantly altered after hydroxytyrosol treatment (Fig. 3A), while NFE2L2 expression is significantly down-regulated at 50 μM (Fig. 3B). Hydroxytyrosol treatment results in a significant down-regulation of PPARG expression at all the concentrations used (Fig. 3C).

Fig. 2. Expression of BAX, BCL2, and CASP3 and BAX:BCL2 ratio in groups treated with 50, 100, and 150 μM hydroxytyrosol and controls in the LS180 cell line (Hormozi M, Marzijerani A S, et al., 2020).

Fig. 3. Expression of P53, NFE2L2, and PPARG genes in groups treated with 50, 100, and 150 μM hydroxytyrosol and controls in the LS180 cell line (Hormozi M, Marzijerani A S, et al., 2020).