Transformation of non-tumorigenic prostate epithelial cells by a common food-derived carcinogen

Abstract

3901

BACKGROUND: Heterocyclic amines in cooked meats, the most common being 2-amino-1-methyl-6-phenylimidazopyridine (PhIP), are suspected etiological factors in prostate carcinogenesis. PhIP is converted to the proximate carcinogen, N-hydroxy-PhIP (N-OH-PhIP). Previous published studies showed that treatment of C3H/M2 mouse fibroblasts with N-OH-PhIP can result in loss of contact inhibition; however, other evidence of possible tumorigenicity was not provided. Similar studies have not been performed in any epithelial cell type to our knowledge. We, therefore, treated immortalized, non-tumorigenic prostate epithelial cells, RWPE-1, with N-OH-PhIP to transform the cells. METHODS: RWPE-1 cells were synchronously treated with 0.1-0.4 μM N-OH-PhIP four times the first week, and asynchronously treated twice in the second week. Matrix metalloproteinase-9 (MMP-9) activity, a marker of the transformed state used in previous studies, was determined by gelatin zymography using concentrated cell culture medium. Clonogenicity was determined by soft agar assay. RESULTS: MMP-9 activity was reduced in a dose-dependent manner after the first week of treatment. Compared to untreated control cells, treatment with N-OH-PhIP decreased MMP-9 activity by 28% at 0.1 μM, 78% at 0.3 μM and 85% at 0.4 μM. The mechanism for this effect of N-OH-PhIP on MMP-9 activity is unknown, but was not due to reduced MMP-9 protein levels (determined by immunoblotting), elevated tissue inhibitor of metalloproteinase-1, or a direct effect on enzyme activity. By the end of the fourth week, large foci of piled up cells (loss of contact inhibition) were observed in the 0.4 μM-treated cells, though MMP-9 activity was still below control levels. We established 5 clones. After several more passages, all clones had elevated MMP-9 activity compared to control cells except for clone 4 that had little detectable levels of MMP-9 activity. Paradoxically, only clone 4 demonstrated clonal growth in soft agar similar to A549 lung adenocarcinoma cells (positive control). CONCLUSION: These results suggest that a common diet-derived compound found in cooked meats can transform prostate epithelial cells in vitro, though studies in nude mice will be needed to confirm this. This is the first report suggesting that N-OH-PhIP treatment reduces MMP-9 activity. Previous studies have transformed RWPE-1 cells with cadmium and arsenic. MMP-9 was used as a biomarker of transformation instead of soft agar cloning. When treated cells demonstrated elevated MMP-9 activity, the cells were then injected into nude mice and tumorigenicity was established. Only clone 4, which has reduced MMP-9 activity, grew in soft agar. This paradox cannot be explained at this time.