PURPOSE: ApcMin/+ (Min, multiple intestinal neoplasia) is a point mutation at codon 850 in the murine homolog of human APC tumor suppressor gene. Mutations and hypermethylation of APC gene has recently been reported in up to 45% of sporadic breast cancers. Our recently established ApcMin/+ mammary epithelial cell line [MinMG] from ApcMin/+ Min mouse, exhibit shorter population doubling time and 25% higher proliferation within a week, increased expression of β-catenin and cyclin D1 proteins, compared to normal Apc+/+ C57MG cell line [Apc+/+ C57BL/6J mouse]. In accordance with the two-hit theory of carcinogenesis, MinMG cells are susceptible to a “second hit” and thus prone to malignant transformation. This study was designed to evaluate preventive efficacy of the dietary agent Curcumin [CUR], on N-Ethyl-N-nitrosourea [ENU] induced carcinogenesis of MinMG mammary epithelial cells.

EXPERIMENTAL DESIGN: To evaluate the in vitro preventive efficacy of CUR on ENU induced hyperproliferation and transformation, MinMG cells were treated with 20uM CUR for 24hr prior to addition of 20uM ENU for 2hr. The treated cells with parallel controls were further grown for 48hr in regular medium and cell proliferation was measured. Cells were further maintained in regular medium for number of passages to monitor the difference in proliferation rate, at regular weekly interval. Cell proliferation (cell count & MTT assay), Cell cycle analyses, and Anchorage Independent Growth (AIG), served as surrogate endpoint biomarkers to evaluate preventive efficacy of CUR for ENU induced carcinogenic transformation of MinMG cells.

RESULTS: MinMG cells exhibit a 3 fold increase in cell proliferation rate induced by 2hr exposure of 20uM ENU compared to untreated cells that persisted for 19 weeks of regular passaging post-treatment. MinMG cells pretreated with 20uM CUR prior to ENU exposure show significant preventive modulation in cell proliferation (12 fold decrease), ratio of quiescent vs proliferative phases of cell cycle (G1:S+G2/M=115% in favor of G1), and number of soft agar colonies (50% reduction), compared to corresponding control. These results show correlation with altered expression of Ki67, β-catenin and cyclin D1 proteins in MinMG cells.

CONCLUSIONS: Our results suggest ApcMin/+ mutation in MinMG cells render them at high risk for carcinogenic transformation. This in vitro pre-clinical model validates its use for Apc+/− initiated risk for mammary carcinogenesis. Pretreatment of cells with CUR reduced ENU induced hyperproliferation and transformation in MinMG cells indicating its preventive efficacy for carcinogenic transformation. Thus our data indicates that CUR is efficacious in preventing ENU induced high risk of hyperproliferation and carcinogenic transformation of ApcMin/+ MinMG mammary cells. [Support: NIH-RO1 CA122394]

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2870.