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Different involvement of promoter methylation in the expression of organic cation/carnitine transporter 2 (OCTN2) in cancer cell lines.

ABSTRACT: 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 OCTN2 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 OCTN2 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.

SUBMITTER: Qu Q

PROVIDER: S-EPMC3797819 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Different involvement of promoter methylation in the expression of organic cation/carnitine transporter 2 (OCTN2) in cancer cell lines.

Qu Qiang Q Qu Jian J Zhan Min M Wu Lan-Xiang LX Zhang Yi-Wen YW Lou Xiao-Ya XY Fu Li-Juan LJ Zhou Hong-Hao HH

PloS one 20131016 10

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 furthe ...[more]

PMID: 24146874

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Project description:Primary carnitine deficiency is caused by impaired activity of the Na(+)-dependent OCTN2 carnitine/organic cation transporter. Carnitine is essential for entry of long-chain fatty acids into mitochondria and its deficiency impairs fatty acid oxidation. Most missense mutations identified in patients with primary carnitine deficiency affect putative transmembrane or intracellular domains of the transporter. Exceptions are the substitutions P46S and R83L located in an extracellular loop close to putative glycosylation sites (N57, N64, and N91) of OCTN2. P46S and R83L impaired glycosylation and maturation of OCTN2 transporters to the plasma membrane. We tested whether glycosylation was essential for the maturation of OCTN2 transporters to the plasma membrane. Substitution of each of the three asparagine (N) glycosylation sites with glutamine (Q) decreased carnitine transport. Substitution of two sites at a time caused a further decline in carnitine transport that was fully abolished when all three glycosylation sites were substituted by glutamine (N57Q/N64Q/N91Q). Kinetic analysis of carnitine and sodium-stimulated carnitine transport indicated that all substitutions decreased the Vmax for carnitine transport, but N64Q/N91Q also significantly increased the Km toward carnitine, indicating that these two substitutions affected regions of the transporter important for substrate recognition. Western blot analysis confirmed increased mobility of OCTN2 transporters with progressive substitutions of asparagines 57, 64 and/or 91 with glutamine. Confocal microscopy indicated that glutamine substitutions caused progressive retention of OCTN2 transporters in the cytoplasm, up to full retention (such as that observed with R83L) when all three glycosylation sites were substituted. Tunicamycin prevented OCTN2 glycosylation, but it did not impair maturation to the plasma membrane. These results indicate that OCTN2 is physiologically glycosylated and that the P46S and R83L substitutions impair this process. Glycosylation does not affect maturation of OCTN2 transporters to the plasma membrane, but the 3 asparagines that are normally glycosylated are located in a region important for substrate recognition and turnover rate.

Dataset Information

Different involvement of promoter methylation in the expression of organic cation/carnitine transporter 2 (OCTN2) in cancer cell lines.

Publications

Different involvement of promoter methylation in the expression of organic cation/carnitine transporter 2 (OCTN2) in cancer cell lines.

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