Article Figures & Data
Figures
- Fig. 1.
Effect of VPA on mRNA expression of MDTs in hCMEC/D3 cells. Cells were treated with VPA (300 μM, 600 μM) for 24 hours. Total RNA was extracted and subjected to RT-qPCR to determine changes in mRNA levels of ABCC1, ABCC2, ABCC4, ABCC5, and ABCG2. The mRNA levels of these genes were normalized to those of B2M and expressed as fold change over VC (0.1% DMSO). The data shown are the means ± S.D. of 3 independent experiments. *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey's post hoc test performed on log-converted values).
- Fig. 2.
Effect of VPA on mRNA expression of ABCG2 in hCMEC/D3 cells at different time points under study. Cells were treated with VPA (300 μM, 600 μM) for 6, 12, 24, and 48 hours. Total RNA was extracted and subjected to RT-qPCR to determine changes in mRNA levels of ABCG2. The mRNA level of ABCG2 was normalized to those with B2M and expressed as fold change over VC (0.1% DMSO). The data are the mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01 (two-way ANOVA with Tukey’s post hoc test performed on log-converted values).
- Fig. 3.
Effect of VPA on BCRP expression and functional activity in hCMEC/D3 cells. (A) Whole cell lysates from hCMEC/D3 cells treated with VC (0.1% DMSO) or VPA (300 μM, 600 μM) for 6, 12, 24, 48, and 72 hours were subjected to SDS-PAGE and western blot to measure BCRP expression. Bands of HSC were used to normalize the results. Mean densitometric values of the bands obtained from three independent experiments was used to calculate fold change over the VC. (B) Representative blot for 48-hour VPA treatment is shown. (C) hCMEC/D3 cells were treated with VC (0.1% DMSO) or VPA (300 μM, 600 μM) for 12, 24, 48, and 72 hours prior to the assay. Intracellular BPZ fluorescence was measured in the presence and absence of the BCRP inhibitor Ko143. Fluorescence intensities were normalized with total protein content. BCRP activity was determined as the ratio between the mean intracellular fluorescence of cells incubated with Ko143 and cells incubated without Ko143 in the VPA treated group, normalized to the corresponding ratio in the control (VC) group. Data are the means ± S.D. of four independent experiments. *P < 0.05, **P < 0.01 (two-way ANOVA with Tukey’s post hoc test performed on log-converted values).
- Fig. 4.
Effect of increasing concentrations of AEDs (A) PHT, (B) CBZ, (C) VPA, (D) LTG, (E) TPM, and (F) LEVI on vanadate-sensitive BCRP ATPase activity. The ATPase activity is expressed as nmol phosphate generated/mg protein/min. Lower dotted line represents baseline activity (in the presence of 2% v/v DMSO or water), and upper dotted line represents activity of fully activated membrane (in the presence of known BCRP activator sulfasalazine). Activation study measures stimulation of baseline ATPase activity indicating drug as BCRP substrate. Data are presented as means ± S.D from three independent experiments. Statistically significant differences between the baseline and drug-stimulated activity in the activation assay (*P < 0.05, **P < 0.01) were determined using one-way ANOVA with Dunnett’s post hoc test performed on log-converted values. Inhibition study measures decrease in maximally stimulated BCRP activity indicating interaction of drug with BCRP. Data are presented as means ± S.D from three independent experiments. Statistically significant differences between the maximal membrane activity and drug-treated activity in the inhibition assay (*P < 0.05) were determined using one-way ANOVA with Dunnett’s post hoc test performed on log-converted values.
- Fig. 5.
Competitive substrate efflux assay to study substrate relationship of PHT, VPA, and LTG with BCRP. hCMEC/D3 cells were co-incubated with either the assay substrate BPZ (250 nM) and VC (0.1% DMSO) or test AED [(A) PHT, (B) VPA, and (C) LTG] or an inhibitor of BCRP (1 µM Ko143), and intracellular accumulation of BPZ was measured. Results are reported as the fold increase in intracellular fluorescence in treated cells over control fluorescence. The data are the mean ± S.D. of five independent experiments. Statistical significance was calculated using one-way ANOVA with Dunnett’s post hoc test performed on log-converted values (**P < 0.01; BPZ+VC vs. BPZ+PHT/VPA/LTG or BPZ+VC vs. BPZ+Ko143).
- Fig. 6.
Effect of PPARα knockdown on VPA-induced ABCG2 expression and activity. Expression of PPARα was silenced by transient transfection of siPPARα (siRNA specific to human PPARα) in hCMEC/D3 cells. Then, cells were treated with VC (0.1% DMSO) or VPA (600 μM) for 24, 48, and 72 hours to check ABCG2 mRNA, protein and activity, respectively. SCR was used as nontargeting control. (A) RT-qPCR analysis of PPARα expression. mRNA level of PPARα was normalized to those with B2M. The data are the mean ± S.D. of three independent experiments. **P < 0.01, VC (SCR vs. siRNA); #P < 0.01, VPA (SCR vs. siRNA) (unpaired t test). (B) RT-qPCR analysis of ABCG2 expression. mRNA level of ABCG2 was normalized to those with B2M (C) Representative blot of PPARα and BCRP is shown; bands of HSC were used to normalize the protein expression. (D) Western blot analysis of BCRP expression and (E) analysis of BCRP functional activity. The data are the mean ± S.D. of the independent experiments. **P < 0.01, N.S., not significant (two-way ANOVA with Tukey’s post hoc test).
- Fig. 7.
Effect of PPARα antagonist MK886 on inductive effect of VPA on BCRP expression and activity. hCMEC/D3 cells were cultured with VC (0.1% DMSO) alone or VPA (600 μM) alone or VC/VPA in the presence of PPARα antagonist MK886 for 48 hours to check effect on ABCG2 protein and activity. (A) Representative blot of BCRP is shown; bands of HSC were used to normalize the protein expression. (B) Western blot analysis of BCRP expression and (C) analysis of BCRP functional activity. The data are the mean ± S.D. of three independent experiments. **P < 0.01, N.S., not significant (two-way ANOVA with Tukey’s post hoc test performed on log-converted values).
- Fig. 8.
Effect of VPA on expression of PPARα and its target genes. hCMEC/D3 cells were treated with VC (0.1% DMSO) or VPA (600 µM) for the indicated time points. (A) RT-qPCR was done to check the effect on PPARα mRNA expression. B2M was used as the reference gene for normalization. (B) Whole-cell lysates were subjected to western blot to measure change in PPARα protein expression. (C) Representative PPARα blot for 3 hours is shown. HSC protein bands served as a loading control. The data are the mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01; VC vs. VPA (two-way ANOVA with Tukey’s post hoc test performed on log-converted values). (D) RT-qPCR was done to check the effect of VPA on mRNA expression of PPARα target genes PDK4 and THBD at 24 hours. B2M was used as the reference gene for normalization. The data are the mean ± S.D. of three independent experiments. **P < 0.01; VC vs. VPA (unpaired t test performed on log-converted values).
- Fig. 9.
Effect of VPA treatment on PPARα translocation to nucleus. (A) hCMEC/D3 cells were incubated with VPA (600 µM) for 0, 3, and 6 hours. Isolated nuclear and cytoplasmic fractions of the cell lysate were subjected to western blot with PPARα antibody. Lamin A/C and GAPDH was used as nuclear and cytosolic marker respectively. (B) The bar graph illustrates the relative nuclear and cytosolic PPARα protein expression fold change at 3 and 6 hours w.r.t expression at 0 hours. The data are the mean ± S.D. of four independent experiments. *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s post hoc test performed on log-converted values). (C) hCMEC/D3 cells were treated with VPA (600 µM) for 0, 3, and 6 hours and subsequently fixed with 4% paraformaldehyde. Fixed cells were incubated with PPARα primary antibody and Alexa Fluor 488–conjugated secondary antibody. Nuclear staining was performed with DAPI. The intracellular distribution of the fluorescent tag was examined under a confocal microscope. Color in green (Alexa488-conjugated antibody) and blue (DAPI) represent PPARα and nucleus, respectively. Arrows indicate cells depicting the translocation. Average PPARα fluorescence ratios (nuclear to cytoplasm) at 3 and 6 hours compared with that at 0 hours were obtained from four independent experiments (80 cells per treatment group per experiment). (D) Bar graph shows relative quantification of nuclear: cytoplasmic ratios of PPARα fluorescence. **P < 0.01 (one-way ANOVA with Tukey’s post hoc test performed on log-converted values).
- Fig. 10.
Effect of VPA on PPARα recruitment to the ABCG2 promoter and promoter activation (A) hCMEC/D3 cells were treated with VC (0.1% DMSO) or VPA (600 µM) for 3 and 6 hours, and PPARα recruitment to the region of interest was assessed by ChIP-qPCR. A nonspecific rabbit IgG antibody was included as negative control. Recruitment is presented as percentage of total input. The data are the mean ± S.D. of three or four independent experiments. **P < 0.01 (VPA vs. VC) (unpaired t test performed on log-converted values). (B) 1% agarose gel image showing qPCR product from 3-hour treated samples. (C) Luciferase reporter gene construct containing the PPRE sequences of the ABCG2 promoter (pGL2-prom-ABCG2) or the pGL2 control vector was cotransfected with renilla-luciferase expression vector into HEK293 cells. Cells were treated with VC (0.1% DMSO) or VPA (600 µM) for 24 hours before measurement of luciferase activities. Firefly luciferase activities were normalized to renilla luciferase activities to consider changes in the transfection variability. Firefly/renilla activity of the VPA treated cells were then normalized to VC treatment (set as1) to obtain the relative luciferase activity fold change. Data are the mean of three independent experiments. **P < 0.01 (VPA-treated cells compared with VC treatment set to1; unpaired t test performed on log-converted values).
Additional Files
Data Supplement
- 568_supp_data.pdf -
Supplemental Table 1. Therapeutic plasma concentrations (reference range) of antiepileptic drugs in patients with epilepsy (Johannessen, 2004).
Supplemental Table 2. Sequence of primers used for qPCR.
Supplemental Table 3. Company source and assay IDs of the siRNAs used in this study.
Supplemental Table 4. Statistical analysis of data.
Supplemental Fig. 1. Effect of AEDs on hCMEC/D3 cell viability.
Supplemental Fig. 2. Effect of PHT, CBZ, LTG, TPM and LEVI on mRNA expression of MDTs in hCMEC/D3 cells.
Supplemental Fig. 3. siRNA validation data for 14 molecular factors at mRNA level.
Supplemental Fig. 4. Effect of silencing of molecular factors on VPA-induced ABCG2 mRNA.
Supplemental Fig. 5. Effect of MK886 on hCMEC/D3 cell viability.
Supplemental Fig. 6. Effect of known PPARα agonist on ABCG2 mRNA in hCMEC/D3 cells.
Supplemental Fig. 7. Binding of VPA to PPARα LBD in a TR-FRET competitive binding assay.
References.
- 568_supp_data.pdf -
Data Supplement
- 568_supp_data.pdf -
Supplemental Table 1. Therapeutic plasma concentrations (reference range) of antiepileptic drugs in patients with epilepsy (Johannessen, 2004).
Supplemental Table 2. Sequence of primers used for qPCR
Supplemental Table 3. Company source and assay IDs of the siRNAs used in this study.
Supplemental Table 4. Statistical analysis of data
Supplemental Fig. 1. Effect of AEDs on hCMEC/D3 cell viability.
Supplemental Fig. 2. Effect of PHT, CBZ, LTG, TPM and LEVI on mRNA expression of MDTs in hCMEC/D3 cells.
Supplemental Fig. 3. siRNA validation data for 14 molecular factors at mRNA level.
Supplemental Fig. 4. Effect of silencing of molecular factors on VPA-induced ABCG2 mRNA.
Supplemental Fig. 5. Effect of MK886 on hCMEC/D3 cell viability.
Supplemental Fig. 6. Effect of known PPARα agonist on ABCG2 mRNA in hCMEC/D3 cells.
Supplemental Fig. 7. Binding of VPA to PPARα LBD in a TR-FRET competitive binding assay.
References.
- 568_supp_data.pdf -
