Fig 1.
Extracellular ATP upregulates the expression of MRP2 in enterocyte-like Caco-2 cells.
Human intestinal adenocarcinoma Caco-2 cells were stimulated with 100 μM ATP for 3 or 6 h, or with control vehicle only (Ctrl). (A) Stimulation with ATP significantly increased ABCC2 gene expression (encodes for MRP2) by 1.5 to 2-fold compared to non-stimulated Ctrl as determined by qRT-PCR analysis. Results are presented as the mean ± SEM of five individual experiments performed in duplicate. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison post-test. * p < 0.05, ** p < 0.01 as compared with Ctrl. (B) Typical Western blot result is showing enhanced MRP2 expression in ATP-stimulated cells. (C) Densitometric analysis revealed that 100 μM ATP induced MRP2 protein expression by more than 1.5-fold after 6 and 18 h of stimulation compared with control (Ctrl). Results are presented as the mean ± SEM of five separate experiments. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison post-test. * p < 0.05 as compared with Ctrl.
Fig 2.
ATP-dependent stimulation of MRP2 protein expression is strongly reduced in the presence of the P2 receptor antagonist Suramin.
Caco-2 cells were treated with 100 μM PPADS or Suramin 30 min prior to the addition of 100 μM ATP for 6 h. MRP2 expression was analyzed by Western blotting. ATP stimulated the expression of MRP2 as compared to non-stimulated (N-S) cells, whereas the addition of Suramin prior to the ATP stimulation strongly decreased MRP2 expression compared to ATP-stimulated cells in the presence of vehicle (DMSO (-)) only. The presented blot is typical of three separate sets of experiments.
Fig 3.
MRP2 expression is regulated by MEK/ERK signaling cascade.
Caco-2 cells were pretreated with NFκB (2 μM, Bay, BAY11-7082), MEK1/2 (10 μM, U0, U0126), PI3K (20 μM, LY, LY294003) and p38 (20 μM, SB, SB203580) inhibitors for 30 minutes and stimulated with 100 μM ATP for 6 h. (A) A typical Western blot against MRP2 is displayed from which (B) densitometry analysis showed a significant reduction in MRP2 expression in the presence of U0126, a selective MEK 1/2 inhibitor. Cells pretreated with U0126 led to a 75% reduction in the expression of MRP2 compared to ATP-stimulated cells only (-). Results are presented as the mean ± SEM of three separate experiments performed in duplicate. Statistical significance was determined by an unpaired t-test, where * p < 0.05 vs. non-stimulated (N-S) or ATP-stimulated cells as indicated on figure.
Fig 4.
Stimulation of Caco-2 cells with ATP increases resistance to the anti-cancer drug etoposide.
Caco-2 cells were incubated with increasing concentrations of etoposide for 84 h in the presence or absence of 100 μM ATP added every 24 h. MTT cell viability assay was used to determine sensitivity to the drug. (A) A dose-response curve was fitted to the data to determine the toxicity (IC50 value) of the drug. Results are presented as a non-linear survival curve of a typical response. (B) IC50 and RR values are presented on the histogram and results represent the means ± SEM of three to four independent experiments performed in triplicate. Statistical significance was determined by unpaired t-test, where * p < 0.05 as compared with Ctrl.
Fig 5.
The down-regulation of MRP2 expression by shRNA renders Caco-2 cells more sensitive to etoposide.
(A) Western blot analysis was used to assess the down-regulation of MRP2 protein expression in the presence of two shRNAs directed against the protein. Down-regulation was achieved by lentiviral infection of Caco-2 cells as previously described [31]. shRNA directed against MRP2 (sh305 and sh307) abolished protein expression by 90–100% comparatively to cells expressing a non-targeting shRNA (shNT). (B) Caco-2 cells stably expressing shNT or shMRP2 (#305) were incubated with the cytotoxic drug etoposide for 84 h. Sensitivity to the anti-cancer drug was determined by the MTT cell viability assay. A dose-response curve was fitted to the data to determine the toxicity (IC50 value) of the drugs. The non-linear survival curves are presented as the mean ± SEM of four experiments performed in triplicate. Statistical significance was calculated using multiple t-test comparisons, where * p < 0.05 as compared with shNT. Inhibition of human shMRP2 expression reduced the resistance of Caco-2 cells to etoposide compared to control cells. IC50 and RR values are presented in Table 1.
Table 1.
IC50 and relative resistance (RR) values measured in response to etoposide, cisplatin and doxorubicin treatment of Caco-2 cells stably expressing non-target shRNA (shNT) or shMRP2.