Abstract
Tobacco smoke is an important risk factor for various human cancers, including esophageal cancer. How benzo [a]pyrene diol epoxide (BPDE), a carcinogen present in tobacco smoke as well as in environmental pollution, induces esophageal carcinogenesis has yet to be defined. In this study, we investigated the molecular mechanism responsible for BPDE-suppressed expression of retinoic acid receptor-beta2 (RAR-β2) in esophageal cancer cells. We treated esophageal cancer cells with BPDE before performing methylation-specific polymerase chain reaction (MSP) to find that BPDE induced methylation of the RAR-β2 gene promoter. We then performed chromatin immunoprecipitation (ChIP) assays to find that BPDE recruited genes of the methylation machinery into the RAR-β2 gene promoter. We found that BPDE recruited DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A), but not beta (DNMT3B), in a time-dependent manner to methylate the RAR-β2 gene promoter, which we confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the reduced RAR-β2 expression in these BPDE-treated esophageal cancer cell lines. However, BPDE did not significantly change DNMT3A expression, but it slightly reduced DNMT3B expression. DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and DNMT3A small hairpin RNA (shRNA) vector antagonized the effects of BPDE on RAR-β2 expressions. Transient transfection of the DNMT3A shRNA vector also antagonized BPDE's effects on expression of RAR-β2, c-Jun, phosphorylated extracellular signal-regulated protein kinases 1/2 (ERK1/2), and cyclooxygenase-2 (COX-2), suggesting a possible therapeutic effect. The results of this study form the link between the esophageal cancer risk factor BPDE and the reduced RAR-β2 expression.
Highlights
Tobacco smoke is an important risk factor for various human cancers, including esophageal cancer
The reason for reexpression of RAR-β2 after 24 h benzo [a]pyrene diol epoxide (BPDE) treatment may be because of BPDE-activated DNA repair mechanism and in our previous paper, we showed that BPDE treatment induced ATM expression, an early protein in the DNA repair pathway after esophageal cancer cells were insulted by BPDE and that ATM expression was associated with tobacco smoke exposure in esophageal cancer tissues [14,28]
The immunoprecipitated DNA confirmed that BPDE and DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A) bind to the RAR-β2 gene promoter
Summary
Tobacco smoke is an important risk factor for various human cancers, including esophageal cancer. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, has been shown to induce the methylation of the RAR-β2 gene promoters in murine lung cancer models [16].
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