S110, a 5-Aza-2'-deoxycytidine-containing dinucleotide, is an effective DNA methylation inhibitor in vivo and can reduce tumor growth

Abstract

Methylation of CpG islands in promoter regions is often associated with gene silencing and aberrant DNA methylation occurs in most cancers, leading to the silencing of some tumor suppressor genes. Reversal of this abnormal hypermethylation by DNA methylation inhibitors is effective in reactivating methylation-silenced tumor suppressor genes both in vitro and in vivo. Several DNA methylation inhibitors have been well studied; the most potent among them is 5-aza-2'-deoxycytidine (5-Aza-CdR), which can induce myelosuppression in patients. S110 is a dinucleotide consisting of 5-Aza-CdR followed by a deoxyguanosine, which we previously showed to be effective in vitro as a DNA methylation inhibitor while being less prone to deamination by cytidine deaminase, making it a promising alternative to 5-Aza-CdR. Here, we show that S110 is better tolerated than 5-Aza-CdR in mice and is as effective in vivo in inducing p16 expression, reducing DNA methylation at the p16 promoter region, and retarding tumor growth in human xenograft. We also show that S110 is effective by both i.p. and s.c. deliveries. S110 therefore is a promising new agent that acts similarly to 5-Aza-CdR and has better stability and less toxicity.

Figure 1

Chemical structures of (A) 5-Aza-CdR and (B) S110

Figure 2

S110 is better tolerated than 5-Aza-CdR as demonstrated by relative body weight (A), percent survival (B), and percent weight loss (C) in normal tumor-free mice. Animals were treated according to the dosages and schedules listed in Table 1. A: Six animals per group treated with S110 maintained body weights better and survived longer (B) compared to those treated with 5-Aza-CdR. C: Histogram illustrating the effects of S-110 and 5-Aza-CdR treatment on body weight for groups 5 and 6 only, which received 5 mg/kg 5-Aza-CdR or a molar equivalent of S110 three times a week. Graphed is the average percent weight loss, therefore negative values are indicative of weight gain. Animals receiving 5-Aza-CdR began losing weight more rapidly than those receiving S-110.

Figure 3

S110 is effective at slowing EJ6 tumor growth in vivo (A), inducing p16 expression (B), and decreasing methylation (C) by IP administration. Mice were treated by IP injections of PBS, 5-Aza-CdR at 5mg/kg, or S110 at 10mg/kg daily for 6 days. A: Tumors were measured with calipers, and tumor volumes (TVs) were calculated by the following formula: TV = LD²/2 (where L is the longest diameter and D is the shortest diameter). The fold differences in tumor growth among the various mice groups were calculated using RTVs, which are calculated as follows: RTV = TV₆/TV₀, where TV₆ is the tumor volume in mm³ at day 6 and TV₀ is the tumor volume in mm³ at day 0 (initial treatment). B: Tumors were harvested on day 6, and total RNA extracted for RT-PCR analysis. Quantitative real time RT-PCR analysis for the p16 gene was done with normalization against the GAPDH reference gene. C: Tumors were harvested on day 6, and genomic DNA extracted for Ms-SNuPE analysis. Both 5-Aza-CdR and S110 are effective at reducing DNA methylation at the p16 gene promoter in the xenografts. The numbers of tumors and p values in each group are labeled above the histograms. The changes in percent methylation level in 5-Aza-CdR- and S110-treated groups are statistically significant to the PBS control treatments by the Student t-test. Results are shown as means + one standard deviation.

Figure 4

S110 is equitoxic to 5-Aza-CdR in vivo by IP administration Mice were weighed at the beginning and the end of treatment to determine toxicity. The percent weight change for each mouse was calculated with the following formula: [(W₆-W₀)/W₀] × 100% (where W_n is the mouse weight on day n). The average percent weight change ± one standard deviation is plotted, and the number of mice in each group is labeled accordingly.

Figure 5

S110 is effective at slowing EJ6 tumor growth in vivo by subcutaneous (SQ) administration every seven days (Q7D). Eight mice each were treated by SQ injections of PBS, 5-Aza-CdR at 5mg/kg, or S110 at 12.2mg/kg every 7 days for 25 days. Results are given as the average of tumor volumes with one standard deviation.