Abstract 4365: Epigenetic and biological effects of 5-azacytidine and RRX-001 on DLD-1 colorectal cancer cell lines
Authors: Kokelaar Rory , Jones Huw , Beynon John , Harris Dean , Jenkins Gareth 
Published: July 2017

Introduction. Extramural vascular invasion (EMVI) in rectal tumours is an important risk factor for disease recurrence and death. Recent studies have demonstrated CpG island hypermethylation phenotype (CIMP) to be an independent risk factor for developing EMVI. This study investigates the cellular processes linking hypermethylation with EMVI by an in vitro model of rectal cancer, and assess the biological and epigenetic effects of known and putative demethylating agents.

Methods. Hypermethylated DLD-1 human colorectal cancer cells were cultured for three days and treated daily with demethylating agents 5-azacytidine (AZA) or RRx-001 across a dose range (0.5µM to 5µM). Standard tests of population doubling (RPD) and cytotoxicity (trypan blue) were performed across the dose range for each compound. Treated cells were subjected to a 500µm gel-insert scratch migration assay and video microscopy was used to record time-to-convergence (TC). Methylation specific PCR was performed on harvested cells across the dose range and compared to controls for eight genes associated with CIMP (SOCS1, MINT-1, hMLH, NEUROG1, THBD, HAND1, ADAMTS1, IGFBP3).

Results. 5-azacytidine and RRx-001 both demonstrated &gt;50% reduction in RPD at a dose of 2µM, with a linear dose-dependent retardation of population growth from 0.1µM to 1.0µM. Trypan blue test also demonstrated linear increase in cytotoxicity for both agents up to 1µM. Mean TC was 38.6hrs +/- 2.4hrs (1SD) for control, solvent control (DMSO), and 0.5µM AZA & RRx-001. Mean TC for 1.0µM and 2.0µM AZA was 52.0hrs +/- 3.0hrs and 91.0hrs +/- 3.75hrs, respectively (p=&lt;0.001). Mean TC for 1.0µM and 2.0µM RRx-001 was 57.0hrs +/- 2.0hrs and 96.0hrs +/- 6.0hrs, respectively (p=&lt;0.005).

Both AZA and RRx-001 demethylate 5 out of 8 CpG islands across the dose spectrum. hMLH, SOCS, and IGFBP3 were resistant to demethylation for both compounds at all doses. MINT, HAND, and THBD were partially demethylated by both agents, but ADAMTS1 was demethylated by AZA only. In each of the genes that demonstrated CpG island demethylation the demethylation was incomplete, with strong methylated bands present despite an additional demethylated band also being demonstrated. RRx-001 demonstrated a dose-dependent demethylation of NEUROG1 and THBD as integrated density of demethylated bands decreased against increasing dose of drug.

Conclusions. This study demonstrates the demethylating ability of both agents against the methylome of 5 out of 8 genes associated with CIMP, which is a known to be associated with EMVI; an independent risk factor for poor prognosis in rectal cancers. The biological and demethylating effects occur at doses below those that may be considered cytotoxic, suggesting that demethylation its self is reducing the rate of cell division and migration of cancer cells. Further work is required to quantify the effects of both agents on the methylome of DLD-1 cells, and to explore the mechanisms of action of both compounds.

Citation Format: Rory Kokelaar, Huw Jones, John Beynon, Dean Harris, Gareth Jenkins. Epigenetic and biological effects of 5-azacytidine and RRX-001 on DLD-1 colorectal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2017-4365