Abstract 581: Complementary reactivation of tumor suppressor genes in breast cancer cells by curcumin and curcumin O-glucuronide

Abstract

Abstract Purpose: Curcumin appears to be a promising chemopreventive agent for various human cancers including breast cancer (BC). However, the molecular mechanism(s) by which it functions in vivo have not been fully elucidated. Recently, we have shown curcumin to be an effective hypomethylating agent in acute myeloid leukemia cells. Here, we investigated the ability of curcumin and its major metabolite, curcumin O-glucuronide (COG) to epigenetically modulate tumor suppressor gene expression in BC cells. Method: MCF-7 and MDA-MB-231 BC cells were treated with curcumin and COG (0.1 to 20 μM) for up to 96 hrs. MCF-7 and MDA-MB-231 cell engrafted tumor-bearing nude mice were treated with i.p. doses of 100 mg/kg curcumin or oral doses of 300 mg/kg nanoemulsion curcumin (NEC). The mRNA levels of RASSF1A, TIMP-3, DNMT1, microRNA29b, and microRNA221/222 were assessed by RT-PCR and protein levels were measured by Western Blot. DNA methylation levels were assessed using LC-MS. The in vitro anti-proliferative activities of curcumin, COG and NEC and the in vivo anti-tumor growth activities of curcumin and NEC were evaluated. Results: Curcumin induced significant global DNA hypomethylation in MCF-7 cells and reactivated RASSF1A expression in both MCF-7 and MDA-MB-231 cells. In vivo reactivation of RASSF1A by curcumin was confirmed in MCF-7 engrafted tumor tissues in mice. Curcumin reactivated TIMP-3 expression in MCF-7 engrafted tumor tissues, but down-regulated TIMP-3 expression in MCF-7 cells in vitro. We have evidence that this discrepancy was due to differential metabolism of curcumin to COG in vitro (no COG formation) and in vivo (COG level > curcumin), since COG itself reactivated TIMP-3 in MCF-7 cells. Notably, COG down-regulated microRNA221/222, two oncogenic microRNAs that suppress TIMP-3 expression, which may account for TIMP-3 reactivation in MCF-7 cells by COG. Importantly, COG chemically inhibited DNMT1 methylation activity and up-regulated microRNA29b, a central regulator of DNMTs in MCF-7 and MDA-MB-231 cells, which may be responsible for its hypomethylating activity. Curcumin elicited anti-tumor growth effects in both MCF-7 (70%) and MDA-MB-231 (27%) cell engrafted tumor bearing nude mice after four weeks of daily i.p. injection. Notably, NEC inhibited MDA-MB-231 cell engrafted tumor growth up to 50%, nearly twice that of curcumin. Conclusion: Curcumin reactivated RASSF1A and TIMP-3 tumor suppressor genes in vivo presumably through its conversion to COG and COG's modulation of DNA methylation and microRNAs. NEC is a more potent formulation for MDA-MB-231 cell engrafted tumor growth in nude mice when compared to curcumin. Further evaluation of NEC for BC prevention or treatment either alone or in combination with clinically-used agents is warranted. Supported by R21 CA135478 [ZL] and biomedical mass spectrometry laboratory (KKC and ZL). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 581. doi:1538-7445.AM2012-581