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Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selection - PubMed

  • ️Thu Jan 01 1998

Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selection

J Rüschoff et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known cancer preventives, which have been largely attributed to their antiproliferative and apoptosis-inducing activities. In this study, we show that microsatellite instability (MSI) in colorectal cancer cells deficient for a subset of the human mismatch repair (MMR) genes (hMLH1, hMSH2, and hMSH6), is markedly reduced during exposure to aspirin or sulindac [or Clinoril, which is chemically related to indomethacin (Indocin)]. This effect was reversible, time and concentration dependent, and appeared independent of proliferation rate and cyclooxygenase function. In contrast, the MSI phenotype of a hPMS2-deficient endometrial cancer cell line was unaffected by aspirin/sulindac. We show that the MSI reduction in the susceptible MMR-deficient cells was confined to nonapoptotic cells, whereas apoptotic cells remained unstable and were eliminated from the growing population. These results suggest that aspirin/sulindac induces a genetic selection for microsatellite stability in a subset of MMR-deficient cells and may provide an effective prophylactic therapy for hereditary nonpolyposis colorectal cancer kindreds where alteration of the hMSH2 and hMLH1 genes are associated with the majority of cancer susceptibility cases.

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Figures

Figure 1
Figure 1

Analysis of microclone assay for MSI. MSI in subclones of HCT116 (A and B) and SW480 (C and D). MSI analysis at Mycl1 locus in HCT116 cells performed by large-scale subcloning (A) and microclone subcloning (B). MSI analysis at the Mycl1 locus in wild-type SW480 control cell line performed by large-scale subcloning (C) and microclone subcloning (D).

Figure 2
Figure 2

Effect of long-term sulindac treatment on MSI frequency. MSI frequency in HCT116 cells after 16 weeks of 400 μM sulindac treatment comparing Mycl1 locus (A, untreated; B, treated) and APC locus (C, untreated; D, treated). Thirteen microsatellite loci (1, BAT25; 2, BAT26; 3, BAT40; 4, D13S175; 5, APC/D5S346; 6, Mfd28/D10S89; 7, D3S1287; 8, Mfd15/D17S250; 9, D2S123; 10, D18S58; 11, TATA box-binding protein; 12, Mycl1; 13, Rb) and three cell lines were examined after treatment with sulindac (400 μM). (E) HCT116 (hMLH1−). (F) Lovo (hMSH2−). (G) HCT15/DLD1 (hMSH6−). (H) HEC-1-A (hPMS2−). The primary panel of five diagnostic microsatellite loci recommended by the NCI/ICG-HNPCC (20) corresponds to numbers 1, 2, 5, 8, and 9. In E-H the empty bars indicate MSI in untreated controls and filled bars indicate MSI after treatment with sulindac.

Figure 3
Figure 3

Time and concentration dependence of NSAID treatment on MSI frequency. (A) Concentration dependence of MSI reduction by aspirin. (B) Concentration dependence of MSI reduction by sulindac, in HCT116 (hMLH1−) cells after 12 weeks of treatment. (C) Time dependency of MSI frequency (filled diamond) and apoptotic index (squares) in HCT116 cells during treatment with sulindac (400 μM). Comparison of apoptotic index assay (derived from spectrophotometric ELISA readings) to TUNEL assay (where apoptotic cells are counted under a microscope) we estimate that an apoptotic index of 0.25 corresponds to approximately 30% apoptotic cells. For untreated cells the percentage of apoptotic cells is 1% or lower.

Figure 4
Figure 4

Genetic selection of MSI via apoptosis. Representative gel analysis of MSI alterations at the Mycl1 locus in untreated HCT116 (hMLH1−) nonapoptotic (A) and apoptotic cells (B) and HCT116 (hMLH1−) nonapoptotic (C) and apoptotic (D) cells after a 12-week treatment with sulindac (400 μM). (E) Graphic analysis of the MSI frequency alteration in HCT116 (hMLH1−) cells at the BAT26, APC, and Mycl1 loci during a 12-week sulindac treatment (400 μM).

Figure 5
Figure 5

Kinetics analysis of the MSI phenotype recovery after withdrawal of sulindac. HCT116 cells were treated with sulindac (400 μM) for 16 weeks and then at time 0 sulindac was withdrawn. MSI at three loci (BAT26, APC, and Mycl1) was followed for an additional 3.5 months.

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