Dual regulation of P-glycoprotein expression by trichostatin A in cancer cell lines

Abstract

Background: It has been reported that the histone deacetylase inhibitor (iHDAc) trichostatin A (TSA) induces an increase in MDR1 gene transcription (ABCB1). This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp). It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a translational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation.

Methods: A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry has been used in this study.

Results: The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a translational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5' end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used.

Conclusions: The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we demonstrate that TSA in fact, regulates differentially both ABCB1 promoters, downregulating the upstream promoter that is responsible for active P-glycoprotein expression. These results suggest that iHDACs such as TSA may in fact potentiate the effects of antitumour drugs that are substrates of Pgp. Finally, we also demonstrate that TSA upregulates RUNDC3B mRNA independently of the ABCB1 promoter in use.

Figure 1

TSA effect on Pgp levels and activity in pancreatic cancer cells.A. MDR1 mRNA levels determined by real time RT-PCR. IMIM-PC-1, IMIM-PC-2 and RWP1 cell lines were treated or not with 1 μM TSA or 7.5 μM SAHA for 24 h. GAPDH mRNA was also determined as internal control. Results are shown as the mean ± SD of at least three independent experiments with three replicates in each experiment. B. Western blot analysis of Pgp expression in IMIM-PC-1, IMIM-PC-2 and RWP1 pancreatic carcinoma cell lines treated or not with 1 μM TSA for 24 h. As a positive control the Pgp expressing K-562/Adr cell line was included. β-Actin is included as internal control. C. Pgp activity in the pancreatic carcinoma cell lines IMIM-PC-1, IMIM-PC-2 and RWP1 treated or nontreated with 1 μM TSA for 24 h and in the presence or absence of 2.5 μM verapamil (Pgp inhibitor). Pgp activity was estimated as daunomycin accumulation determined by flow cytometry. Daunomycin accumulation was also determined in the L1210R cell line for positive control.

Figure 2

Amplification by RT-PCR of the long 5^′UTR MDR1 cDNA.A. Diagram showing two ABCB1 promoters and the MDR1 mRNA isoform produced by each promoter. B. RT-PCR amplification with F1 and R1 primers of the long 5^′ UTR of the MDR1 cDNA. Lane 1. Molecular markers.Lanes 2–7. IMIM-PC-1, IMIM-PC-2, RWP-1, Hs 766 T, BxPC-3 and PANC-1 cell lines. Lane 8.y 9, K-562/Adr and HTC-15 (positive controls).

Figure 3

TSA effects on MDR1 mRNA in different pancreatic carcinoma cell lines. Hs-766 T, BxPC-3, IMIM-PC-1, IMIM-PC-2, PANC-1 and RWP1cell lines were treated for 0–24 hours with 1 μM TSA. As internal control GAPDH mRNA was also determined. Results are shown as the mean ± SD of at least three independent experiments with three replicates in each experiment.

Figure 4

Differential effect of TSA on ABCB1 alternative promoters. Group A cell lines (Hs-766 T, IMIM-PC-1, IMIM-PC-2, RWP1, PANC-1, MCF-7, K-562, HT-29, HT-29/M6, SW620 and BxPC-3) expressing the short 5^′UTR MDR1 mRNA and group B cell lines (HTC-15, K5-62/Adr and MCF-7/Adr) expressing the long 5^′UTR MDR1 mRNAs were treated for 24 hours with 1 μM TSA. As internal control GAPDH mRNA was also determined. Results are shown as the mean ± SD of at least three independent experiments with three replicates in each experiment.

Figure 5

TSA dual regulation of ABCB1 promoters.A. Western blot analysis of Pgp expression in K-562 (lane 1), K-562/Adr (lane 2), negative and positive control respectively and the colon carcinoma cell lines Colo 320 (lane 3), DLD-1 (lane 4) and LS 174 T (lane 5). β-Actin is included as internal control. B. RT-PCR amplification with F1 and R1 primers of the long 5^′ UTR of the MDR1 cDNA. Lane 1. Molecular markers. Lanes 2 and 3.K562/Adr and HTC-15 (positive controls). Lane 4, 6 and 8, Ls174T, Colo320 and DLD1 untreated cell lines. Lane 5, 7 and 9, Ls174T, Colo320 and DLD1 cell lines treated with 1 μM TSA for 24 h. C. Pgp activity in the erythroleukaemia cell lines K-562 (negative control), K-562 (d20) and K-562/Adr (positive control) treated or no treated with 1 μM TSA for 24 h and in the presence or absence of 2.5 μM verapamil (Pgp inhibitor). Pgp activity was estimated as daunomycin accumulation determined by flow cytometry. D. MDR1 mRNA levels determined by real time RT-PCR. K-562, K-562 (d450) and K-562/Adr cell lines were treated or not with 1 μM TSA for 24 h. GAPDH mRNA was also determined as internal control. Results are shown as the mean ± SD of at least three independent experiments with three replicates in each experiment.

Figure 6

Effect of TSA on RUNDC3B mRNA expression.A. Diagram showing the overlapping of ABCB1 intron and RUNDC3B exons. B. Hs-766 T, BxPC-3, IMIM-PC-1, IMIM-PC-2, PANC-1, RWP1, HTC-15,MCF-7/Adr, LS 174 T, DLD-1 and Colo 320 cell lines were non treated or treated for 24 hours with 1 μM TSA and RUNDC3B mRNA expression were determined. As internal control GAPDH mRNA levels were also determined. Results are shown as the mean ± SD of at least three independent experiments with three replicates in each experiment.