Abstract. Period circadian regulator (Per)1 and Per2 genes are involved in the molecular mechanism of the circadian clock, and exhibit tumor suppressor properties. Several studies have reported a decreased expression of Per1, Per2 and Per3 genes in different types of cancer and cancer cell lines. Promoter methylation downregulates Per1, Per2 or Per3 expression in myeloid leukemia, breast, lung, and other cancer cells; whereas histone deacetylase inhibitors (HDACi) upregulate Per1 or Per3 expression in certain cancer cell lines. However, the transcriptional regulation of Per1 and Per2 in cancer cells by chromatin modifications is not fully understood. The present study aimed to determine whether HDACi regulate Per1 and Per2 expression in gastric cancer cell lines, and to investigate changes in chromatin modifications in response to HDACi. Treatment of KATO III and NCI-N87 human gastric cancer cells with sodium butyrate (NaB) or Trichostatin A (TSA) induced Per1 and Per2 mRNA expression in a dose-dependent manner. Chromatin immunoprecipitaion assays revealed that NaB and TSA decreased lysine 9 trimethylation on histone H3 (H3K9me3) at the Per1 promoter. TSA, but not NaB increased H3K9 acetylation at the Per2 promoter. It was also observed that binding of Sp1 and Sp3 to the Per1 promoter decreased following NaB treatment, whereas Sp1 binding increased at the Per2 promoter of NaB-and TSA-treated cells. In addition, Per1 promoter is not methylated in KATO III cells, while Per2 promoter was methylated, although NaB, TSA, and 5-Azacytidine do not change the methylated CpGs analyzed. In conclusion, HDACi induce Per1 and Per2 expression, in part, through mechanisms involving chromatin remodeling at the proximal promoter of these genes; however, other indirect mechanisms triggered by these HDACi cannot be ruled out. These findings reveal a previously unappreciated regulatory pathway between silencing of Per1 gene by H3K9me3 and upregulation of Per2 by HDACi in cancer cells.
The knowledge about the role of calcium‐regulated pathways in cancer cell growth and differentiation could be useful for the development of new therapeutic approaches to diminish its mortality. The ATP2A genes encode for SERCA pumps, which modulate cytosolic Ca2+ concentration, regulating various cellular processes including cell growth. ATP2A3 gene transcriptional down‐regulation has been reported in gastric and colon cancer, but there is still a lack of understanding about the epigenetic processes regulating its transcription. In this work, we report that butyrate, trichostatin A, and 5‐azacytidine treatments increase SERCA3 expression, increased apoptosis, and decreased cell viability of the KATO‐III gastric carcinoma cell line. We analyzed the methylation profile of the ATP2A3 gene promoter CpG island, finding clones with methylated status through −280 to −135 promoter region, harboring Sp1 and AP‐2 binding sites, which could have a role in transcriptional repression. Post‐translational modifications of histones show a major role in the ATP2A3 transcriptional regulation, and our results show histones marks linked to transcriptional repression associated with the −262 to −135 region, this repressive context changed to transcriptional permissive through SERCA3 re‐expressing conditions. These results suggest that the nucleotide sequence from −280 to −135 position is an ATP2A3 epigenetic regulatory CpG region in KATO‐III cells. Analyses of online‐databases show a decreased SERCA3 expression in gastric and colon tumors, as well as overall survival results, showed that high SERCA3 expression could serve as a favorable prognostic marker for colon and gastric cancer patients.
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