Abstract 5242: Differential, dose-dependent, effects of curcumin/analogs, on long (L) versus short (S) isoforms of a cancer stem cell marker, DCLK1, in colon and pancreatic cancer cells: biological impact
Authors: Sarkar Shubhashish , Connell Malaney O. , Singh Pomila 
Published: July 2016

Curcumin, a dietary agent, has potent anti-inflammatory/anti-cancer activities, but is much less bioavailable, unlike some of its more potent synthetic analogs, such as EF24. In recent years, inhibitory effects of curcumin/analogs on DNMT1 have been described, resulting in de-methylation and re-expression of several genes, including tumor suppressor genes. Double-Cortin Like Kinase 1 (DCLK1) marks colon cancer stem cells, and is required for colon-carcinogenesis in mice. We recently reported a critical role of DCLK1 for maintaining tumorogenic/metastatic potential of human colon cancer (CRC) cells (Kantara et al, Can Res 2014). More recently we reported that the 5’α promoter of hDCLK1-gene is epigenetically silenced in CRCs due to hypermethylation, while a novel short (DCLK1-S) isoform is transcriptionally up-regulated in response to oncogenic pathways, including NFκB, from an alternate β promoter, located in intron V (O’Connell and Sarkar et al, Sci. Rep 2015). Since curcumin potently inhibits NFκB pathway, and can potentially de-methylate epigenetically silenced promoters, we examined possible re-expression/loss of expression of L/S isoforms, respectively, in colon/pancreatic cancer cells, in vitro and in vivo, in response to curcumin/EF24, using several molecular approaches and bioassays. Surprisingly, both agents had dose-dependent effects on demethylation. Low concentrations of curcumin (10μM) and EF24 (0.5μM) inhibited DNMT1, resulting in re-expression of DCLK1-L in both colon/pancreatic cancer cells in vitro. However, higher concentrations of curcumin (&gt;20μM) and EF24 (&gt;1.0μM) caused autophagic death of the cells, and re-expression of DCLK1-L was not measured. NFκB activation and DCLK1-S expression, on the other hand, were increasingly decreased in all cell lines in a dose-dependent manner, in response to curcumin/EF24. Low doses were equally effective against DNMT1/NFκB in vivo, resulting in re-expression of DCLK1-L and loss of DCLk1-S, in xenografts of colon/pancreatic cancer cells. However, the high doses used in this study continued to be effective against the two enzymes, suggesting that the high dose used was not enough for causing autophagic death in vivo, and may need to be increased to replicate cell-death effects observed in vitro. Even though DCLK1-L was re-expressed in the xenografts at both does, in vivo, the size and weight of the tumors was reduced in a dose-dependent manner, mimicking the loss of expression of DCLKL1-S. Our results thus show, for the first time, a dose-dependent epigenetic effect of curcumin/EF24 on cancer cells, which could significantly impact treatment decisions. Low to moderate doses of curcumin/EF24 may allow re-expression of normal/silenced genes, such as DCLK1-L, while reducing growth promoting pathways (such as NFκB/DCLK1-S).

Citation Format: Shubhashish Sarkar, Malaney O. Connell, Pomila Singh. Differential, dose-dependent, effects of curcumin/analogs, on long (L) versus short (S) isoforms of a cancer stem cell marker, DCLK1, in colon and pancreatic cancer cells: biological impact. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5242.