Endocrine Abstracts (2001) 2 P22

The influence of IGFBP-3 on colonic epithelial cell proliferation and apoptosis

TM David1, S Khalaf, D Davies2, S Bustin3 & PJ Jenkins2


Bart's and The London, Queen Mary College; and ICRF Laboratories2, Lincoln's Inn Fields


Background. Recent studies have linked high serum levels of IGF-1 and low levels of IGFBP-3 with an increased risk of colorectal cancer. BP3 has been shown to have IGF-1 independent effects on cell proliferation in breast and prostate cells, but its effects on colon cancer cells is largely unknown. Aims. To determine: 1) the effects of BP-3 on colonic epithelial cell proliferation and apoptosis, and 2) whether any effects are independent of IGF-1. Methods. HT-29 cells were cultured with different concentrations of BP-3 (10, 20, 50, 100 and 200ng/ml), ± IGF-1 (20ng/ml), ± sodium butyrate (5mM), an inducer of apoptosis, in 96 well plates for 72 hours in serum free media. Proliferation was assessed by the MTS assay and apoptosis assessed by cell morphology and FACs analysis using propidium iodide (PI) and annexin staining. Results. There was no increase in cell proliferation in response to any concentration of IGFBP-3. BP-3 alone induced apoptosis at 100 and 200ng/ml, an effect which was markedly increased on co-incubation with butyrate. Co-incubation with IGF-1 significantly attenuated these apoptotic effects. The table below shows the mean±SEM of 3 experiments at 48h incubation with 200ng/ml BP-3. The MTS results are expressed as a percentage of media-only.

P-3IGF-1BP-3 +
IGF-1
ButBP-3 +
But
BP3+ IGF-
1+But
MTS
Assay
87.3±2.6*114±3.8**105±12.4*43.3±5.2**31.0±7.0**36.5±7.5**
PI %
apoptotic cells
8.8±2.14.7±0.63.7±0.466.0±12.782.7±6.739.8±3.4
Annexin %
apoptotic cells
12.2±2.14.9±0.74.9±0.228.3±4.230.3±2.627.5±5.2

Conclusions. BP-3 exerts pro-apoptotic, IGF-1 independent effects on colonic epithelial cells, but which can be prevented by IGF-1. These findings suggest that BP-3 and IGF-1 may exert their differential effects via separate pathways and also help explain recent epidemiological studies linking low levels of BP-3 in the colon to a higher risk of colorectal cancer.

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