Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.

Introduction The lifetime risk for developing colorectal cancer (CRC) is approximately 7%. In the USA over 140,000 people a year develop CRC, with approximately 51,000 deaths/year. Epidemiological studies indicate that long-term aspirin usage reduces the incidence of CRC, and may be protective against other non-CRC related adenocarcinoma, including esophageal cancer. We have investigated the mechanism by which aspirin exhibits toxicity to CRC by synthesising novel analogues of aspirin in an effort to identify functionally significant moieties. We previously suggested that “di-aspirin” (bis-carboxyphenol succinate, BCS) and analogues thereof retain relatively specific toxicity to colorectal cancer cells cultured in vitro. The cellular functions of the Inhibitors of Apoptosis (IAPs) correlate with suppression of apoptotic cell death, tumor progression and metastases. Aspirin triggered apoptosis correlates with its capacity to decrease expression of Survivin, a member of the IAP family. Survivin exhibits dual functions; as an inhibitor of apoptosis and regulator of mitosis. We hypothesised that studying the alterations in the expression of IAPs would give an insight into the cytotoxic nature of the compounds. Experimental procedure A cell viability assay (MTT) was employed to investigate the cytotoxicity of the analogues in various CRC (SW480, HCT116 and LoVo) and non-CRC cell lines (A549, MCF7 and EA.hy926). Expression levels of IAPs (c-IAP1, cIAP2, XIAP, Livin and Survivin) were examined in the SW480 cell line by immunoblotting. Survivin expression was further examined by immunofluorescence analysis. Changes in Survivin expression in the CRC and non-CRC cell lines was also determined by immunoblotting. Summary of data Herein, we show that the “di-aspirin” analogues are relatively more toxic to colorectal cancer cells compared to non-colorectal cancer cell lines. In addition we suggest that these analogues can potently reduce the expression of Survivin in comparison to other IAPs. Conclusion We conclude that the analogues of aspirin used in the study promote cell death in CRC and non-CRC cell lines. We propose that the compounds induce apoptosis by decreasing the expression of Survivin. We also propose that the anti-proliferative activity of “di-aspirins” and their analogues have potential benefit in tackling human cancer. Citation Format: Chandra S. Kankipati, Christopher J. Perry, Iain D. Nicholl. Aspirin, di-aspirin and their toxicity to colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 335. doi:10.1158/1538-7445.AM2014-335