Entinostat reverses P-glycoprotein activation in snail-overexpressing adenocarcinoma HCC827 cells

Abstract

Epithelial-to-mesenchymal transition (EMT) in cancer cells facilitates tumor progression by promoting invasion and metastasis. Snail is a transcriptional factor that induces EMT, while P-glycoprotein (P-gp) is an efflux transporter involved in anticancer drug resistance, and P-gp efflux activity is stimulated in Snail-overexpressing lung cancer cells with EMT characteristics. Since the histone deacetylase (HDAC) inhibitor entinostat (Ent) reverses EMT features, our aim in this study was to determine whether Ent also suppresses P-gp activation in Snail-induced cells. First, we confirmed that Ent treatment reduced migration activity, downregulated E-cadherin and upregulated vimentin at the mRNA level in Snail-overexpressing cells, thus inhibiting EMT. Efflux and uptake assays using rhodamine123 (Rho123), a fluorescent P-gp substrate, showed that Ent also inhibited Snail-induced activation of P-gp. Moreover, P-gp activity was more strongly inhibited by Ent in Snail-overexpressing cells than in Mock cells. When we evaluated the uptakes of Rho123 by LLC-PK1 cells and P-gp-overexpressing LLC-GA5COL150 cells, Rho123 accumulation in LLC-GA5COL150 cells was significantly decreased compared with that in LLC-PK1 cells. Coincubation with Ent had no effect on Rho123 accumulation in either of the cell lines. Thus, Ent appears to be an inhibitor, but not a substrate, of P-gp at low concentration. Our results suggest that Ent treatment might suppress not only Snail-induced cancer malignant alteration, but also P-gp-mediated multidrug resistance.

Regulation of breast cancer resistance protein and P-glycoprotein by ezrin, radixin and moesin in lung, intestinal and renal cancer cell lines

“…[23,24] Moreover, the functional evaluation method of the efflux transporter suitable for each cell has already been established. [22][23][24] Based on those reports, this method to evaluate P-gp and BCRP activity has been selected in each cell. When Ezr or Msn expression was silenced in HCC827 cells, down-regulation of BCRP transport activity occurred; however, knockdown of Rdx did not affect the efflux rate of SN-38 ( Figure 2).…”

“…Overall, it seems that the regulation of P‐gp function by ERM proteins is tissue‐dependent . Additionally, our earlier report concluded that P‐gp function was enhanced without any increase in its mRNA or whole protein levels when epithelial–mesenchymal transition (EMT) was induced in non‐small‐cell lung cancer cells . Other researchers have reported that ERM protein expression was increased when EMT was induced in cancerous cells .…”

“…We conducted efflux assays and evaluated efflux rates as previously reported . Briefly, cells were washed twice using cold Dulbecco's phosphate‐buffered saline (PBS) and the uptake process was started in cold Opti‐MEM containing 5 μ m SN‐38 or 10 μ m Rho123 on ice.…”

“…We conducted efflux assays and evaluated efflux rates as previously reported. [23,24,28] Briefly, cells were washed twice using cold Dulbecco's phosphate-buffered saline (PBS) and the uptake process was started in cold Opti-MEM containing 5 lM SN-38 or 10 lM Rho123 on ice. After 30 min, cells were washed twice with cold PBS and incubated with 300 ll Opti-MEM at 37°C for 10 min.…”

“…[22] Additionally, our earlier report concluded that P-gp function was enhanced without any increase in its mRNA or whole protein levels when epithelial-mesenchymal transition (EMT) was induced in non-small-cell lung cancer cells. [23,24] Other researchers have reported that ERM protein expression was increased when EMT was induced in cancerous cells. [25][26][27] Based on these findings, it is considered that the membrane localization of P-gp is regulated by ERM proteins in lung cancer.…”