Abstract
The ezrin/radixin/moesin (ERM) family proteins act as linkers between the actin cytoskeleton and P-glycoprotein (P-gp) and regulate the plasma membrane localization and functionality of the latter in various cancer cells. Notably, P-gp overexpression in the plasma membrane of cancer cells is a principal factor responsible for multidrug resistance and drug-induced mutagenesis. However, it remains unknown whether the ERM proteins contribute to the plasma membrane localization and transport function of P-gp in human colorectal cancer cells in which the subcellular localization of ERM has yet to be determined. This study aimed to determine the gene expression patterns and subcellular localization of ERM and P-gp and investigate the role of ERM proteins in the plasma membrane localization and transport function of P-gp using the human colon adenocarcinoma cell line LS180. Using real-time reverse transcription polymerase chain reaction and immunofluorescence analyses, we showed higher levels of ezrin and moesin mRNAs than those of radixin mRNA in these cells and preferential distribution of all three ERM proteins on the plasma membrane. The ERM proteins were highly colocalized with P-gp. Additionally, we show that the knockdown of ezrin, but not of radixin and moesin, by RNA interference significantly decreased the cell surface expression of P-gp in LS180 cells without affecting the mRNA expression of P-gp. Furthermore, gene silencing of ezrin substantially increased the intracellular accumulation of rhodamine123, a typical P-gp substrate, with no alterations in the plasma membrane permeability of Evans blue, a passive transport marker. In conclusion, ezrin may primarily regulate the cell surface localization and transport function of P-gp as a scaffold protein without influencing the transcriptional activity of P-gp in LS180 cells. These findings should be relevant for treating colorectal cancer, which is the second leading cause of cancer-related deaths in males and females combined.
Highlights
According to the World Health Organization, cancer is the second most common cause of death worldwide and was responsible for an estimated 9.6 million deaths in 2018 [1]
We first measured the expression of ezrin, radixin, and moesin mRNAs in LS180 cells along with that of ABCB1 by real-time reverse transcription- polymerase chain reaction (RT-PCR) analysis
Higher levels of ezrin, radixin, and ABCB1 and lower levels of moesin mRNAs were observed in Caco-2 cells (Fig 1B)
Summary
According to the World Health Organization, cancer is the second most common cause of death worldwide and was responsible for an estimated 9.6 million deaths in 2018 [1]. One of the major problems with the treatment of CRC is the acquired multidrug resistance (MDR) of cancer cells, to a broad spectrum of structurally and functionally distinct conventional chemotherapeutic drugs and to molecular targeted drugs during the course of their treatment [5,6,7,8,9,10]. P-glycoprotein (P-gp/ABCB1/MDR1) belongs to the ATP-binding cassette (ABC) transporter superfamily, which actively exports structurally and functionally unrelated chemotherapeutic drugs from cancer cells to the extracellular space [13, 14]. Among the complex and multifactorial mechanisms underlying MDR, overexpression of P-gp in the cancer cell membrane has long been recognized as the principal factor responsible for MDR in a variety of cancers [15,16,17,18]
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