Different Involvement of Promoter Methylation in the Expression of Organic Cation/Carnitine Transporter 2 (OCTN2) in Cancer Cell Lines

<div><p>Organic cation/carnitine transporter 2 (OCTN2) is responsible for the cellular uptake of the antineoplastic agent, oxaliplatin. Epigenetic modification is a possible mechanism of altered drug-transporter expression in cancers, leading to altered efficacy of chemotherapeutic drugs. However, the mechanisms governing OCTN2 regulation are not completely understood. In this study, the low levels of OCTN2 in HepG2 and LS174T cells were elevated by the demethylating reagent, decitabine (DCA). To further reveal the epigenetic mechanism of down-regulation of OCTN2, we found that Region-1 within the <i>OCTN2</i> promoter (spanning −354 to +85) was a determinant of OCTN2 expression in a luciferase reporter assay. Moreover, methylation-specific PCR (MSP) and bisulfite genomic sequencing showed that the degree of individual methylated CpG sites within this region was inversely correlated with the levels of OCTN2 in different cancer cells. Application of DCA to HepG2 and LS174T cells reversed the hypermethylation status of the <i>OCTN2</i> promoter and increased OCTN2 expression, enhancing cellular uptake of oxaliplatin. Thus, we identified that promoter methylation is responsible for epigenetic down-regulation of OCTN2 in HepG2 and LS174T cells. Given the essential role of OCTN2 in cancer cell uptake of chemotherapeutics, and thus treatment efficacy, pretreatment with a demethylating reagent is a possible strategy for optimizing pharmacotherapies against cancers.</p></div

Methylation profiles around the transcriptional start site of <i>OCTN2</i>.

<p>(A), the genomic sequence spanning −325 to −92 bp in promoter Region-1 of <i>OCTN2</i>. Eighteen CpG sites were located and marked. (B), bisulfite genomic sequencing analysis of Region-1 of <i>OCTN2</i> in HepG2 cells. Bisulfite modified DNA was amplified by BSP as described under <i>Materials and methods</i> and then sequenced. Pointed star represents methylated CpG sites. (C), DNA methylation profiles of individual methylated CpG dinucleotides in four cancer cell lines. After BSP, five clones were randomly selected and sequenced. The methylated sequences were checked for alignment using MethBLAST. The open and closed circles represent unmethylated or methylated cytosines, respectively.</p

Effects of DCA on mRNA and protein levels of OCTN2 in cancer cell lines.

<p>(A), analysis of mRNA levels of OCTN2 in DCA-treated cancer cell lines. After treatment with 0.5 and 1 μM DCA, total RNA in cancer cells was purified by TRIzol reagent for cDNA synthesis and the mRNA levels of OCTN2 were analyzed by Quantitative RT-PCR. (B), analysis of protein levels of OCTN2 in DCA-treated cancer cell lines. Total proteins were extracted and protein levels of OCTN2 were analyzed by western blotting. The relative expression of mRNA and protein was normalized to β-actin. Data presented represent the mean ± S.D. of three independent experiments. Significant difference from control group of 0.1% DMSO is denoted with asterisks (*, <i>p</i><0.05; **, <i>p</i><0.01).</p

Methylation profiles of <i>OCTN2</i> in DCA-treated HepG2 cells.

<p>(A), bisulfite genomic sequencing analysis of DCA-treated HepG2 cells. After the treatment of DCA, DNA was extracted and subjected to bisulfite modification. Bisulfite modified DNA was amplified by BSP as described under <i>Materials and methods</i> and then sequenced. Pointed star represents methylated CpG sites. (B), DNA methylation profiles of individual methylated CpG dinucleotides in DCA-treated HepG2 cells. After BSP, five clones were randomly selected and sequenced. The methylated sequences were checked for alignment using MethBLAST. The open and closed circles represent unmethylated or methylated cytosines, respectively.</p

Methylation effects on transcriptional activity of the OCTN2 promoter in a luciferase reporter assay.

<p>Constructed pGL4.17 luciferase vector containing unmethylated and methylated promoter regions were transfected into COS-7 cells with the pRL-TK control vector. Forty-eight h later, the cells were washed and assayed for luciferase activity. Relative luciferase activity was measured in triplicate wells. Data presented represent the mean ± S.D. of three independent experiments. Significant difference from the treatment group of pGL4.17 is denoted with asterisks (*, <i>p</i><0.05; **, <i>p</i><0.01). Significant difference from the group of methylated region in the same region is denoted with a pound sign (#, <i>p</i><0.05).</p

Demethylation of OCTN2 by DCA influenced cell viability to oxaliplatin.

<p>Cancer cells were pre-treated with 1 μM DCA for 1 week, seeded in 96-well plates at 1×10⁴ cells/well in 100 μl DMEM and 10% FBS. After 8 h, the cells were exposed to oxaliplatin at various concentrations (0.1, 0.3, 1.0, 3.3, 10, 33, 100 or 330 μM) for 48 h in fresh culture medium. Cell viability was determined by a MTS cell proliferating assay kit. Each concentration was determined in three wells. Data presented represent the mean ± S.D. of three independent experiments. (A), the cell viability at each concentration was displayed. Significant differences from DMSO treatment are denoted with an asterisk (*, <i>p</i><0.05). (B), IC₅₀ values were calculated by SPSS 13.0 software. Data presented represent the mean ± S.D. of three independent experiments. Significant differences from non-treated cells are denoted with an asterisk (*, <i>p</i><0.05).</p

Methylation status of CpG islands was analyzed by MSP.

<p>(A), computational analysis revealed three putative CpG islands within the OCTN2 genomic sequence by Methyl-primer software as described in <i>Material and Methods</i>. (B), MSP analysis for CpG islands of OCTN2 in 1 μM DCA treated and non-treated cancer cell lines. MSP was performed with the specific primers to amplify the target CpG islands fractions and run on a 2% agarose gel. All of MSP and BSP analysis were repeated at three times. U: unmethylated sequence; M: methylated sequence.</p

Telmisartan PKs of 48 health males with different genotypes.

<p>Data were shown as mean±SD;</p>*<p><i>P</i><0.05; **<i>P</i><0.01.</p><p>PKs, pharmacokinetics; <i>ORM1</i>, orosomucoid 1; <i>ABCC2</i>, ATP-binding cassette, sub-family C, member 2; <i>ABCG2</i>, ATP-binding cassette, sub-family G, member 2; <i>ABCB1,</i> ATP-binding cassette, sub-family B, member 1; <i>SLCO1B3,</i> solute carrier organic anion transporter family, member 1B3; SNP, single nucleotide polymorphisms; AUC_(0–48) the area under the plasma concentration-time curve (AUC) from 0 to 48 h; AUC_(0–∞), AUC from 0 to ∞; C_max, the peak concentration in plasma; CL/F, clearance; T_1/2, elimination half-life; T_max, the time to C_max.</p

The impacts of <i>ORM1</i> genotypes on the PKs and BP change(%) after 40 mg telmisartan.

<p>A, Concentration; B, AUC_(0–∞); C, SBP change(%) from the baseline; D, DBP change(%) from the baseline.</p

Summary of Genetic Variations of SNPs in this Study.

a<p>data published on hapmap; b, data calculated in this study, N = 48; N/A, no data found in hapmap.</p><p>MAF: Minor Allele frequencies; CHB: Han Chinese in Beijing, China; ASW: African ancestry in Southwest USA; CEU: Utah residents with Northern and Western European ancestry from the CEPH collection <i>ORM1</i>, orosomucoid 1; <i>ABCC2</i>, ATP-binding cassette, sub-family C, member 2; <i>ABCG2</i>, ATP-binding cassette, sub-family G, member 2; <i>ABCB1,</i> ATP-binding cassette, sub-family B, member 1; <i>SLCO1B3,</i> solute carrier organic anion transporter family, member 1B3; SNP, single nucleotide polymorphisms; AUC_(0–48) the area under the plasma concentration-time curve (AUC) from 0 to 48 h; AUC_(0–∞), AUC from 0 to ∞; C_max, the peak concentration in plasma; CL/F, clearance; T_1/2, elimination half-life; T_max, the time to C_max.</p