Synergistic killing in pancreatic cancer cell lines of the combination of a MEK inhibitor and DNA methylation inhibitor.

Abstract

e15025 Background: A growing body of evidence implicates aberrant DNA methylation in the etiology of a number of cancers, including pancreatic. Previous reports link defective ERK signaling with reduced DNA methylation via DNMT1. Here, we examined the ability of the MEK inhibitor U0126 to alter expression of DNMTs in k-RAS mutant and wild type pancreatic cancer cell lines and assess additive cytotoxicity of U0126, the hypomethylating agent 5-Azacytidine and two HDAC inhibitors. Methods: DNMT levels were quantified in AsPC1 and BxPC3 lines 2 days after adding 25 umolar U0126 using real-time RT-PCR. Significance was calculated using a Student t-test. For survival, agents were added singly and in combination: U0126, 25 umol; 5-Aza, 10 umol; Valproic acid 1 mmol and Trichostatin-A 5 umol. Survival was assessed using a colorimetric MTS method at 1, 2, 3, and 4 days. Expected cell survival and synergism were calculated via relative risk ratios, and significance via a Student t-test. Results: U0126 treatment reduced expression of DNMT1 significantly in AsPC1: 2.06 ± 0.3 vs. 1.09 ± 0.08, p = 0.004 (mean ± S.D., arbitrary units). No change was seen in BxPC3. Increased expression of de novo DNMT3a and DNMT3b was seen: AsPC1 1.28 ± 0.3 vs. 3.13 ± 0.8, p = 0.02; BxPC3 1.78 ± 0.3 vs. 4.14 ± 1, p = 0.02; AsPC1 1.26 ± 0.3 vs. 2.66 ± 0.4, p = 0.006, respectively. DNMT3b in BxPC3 was not altered significantly. U0126 and 5-Aza were individually cytotoxic in both cell lines, and synergism was observed in combination. In AsPC1, the observed vs. expected combination survival was 13% ± 3% vs. 29% ± 2%, p = 0.001; for BxPC3, 7% ± 2% vs. 62% ± 3%, p = 0.002, both at 4d. No synergism was found when adding Valproic acid or Trichostatin-A. Conclusions:Treatment of k-RAS mutant pancreatic cancer cell lines with the MEK inhibitor U0126 results in significantly reduced expression of DNMT1 and increased DNMT3a and DNMT3b. U0126 combined with 5-Aza produced synergistic cytotoxicity, which may be due to enhanced hypomethylation activity. Although no significant change in expression of DNMT1 was found in the BxPC3 cells, we noted synergistic cytotoxicity when treated with both U0126 and 5-Aza.