Study On Impact Of Chemically Modified Cyclopamine On Breast Cancer Cell Growth, Invasion And Radiosensitivity

A larger number of studies have found that cyclopamine, a specific inhibitor of hedgehog signal pathway, reduces growth and proliferation of cancer cells. Futhermore, cyclopamine can increase chemosensitivity and radiosensitivity of tumor cells. This study was designed to investigate the in vitro and in vivo effects and relevant mechanisms of SCJ-26 and SCJ-43, two soluble structural and chemically modified derivatives of cyclopamine that is extracted from Eranthis hyemalis, on cell growth, migration, invasion and radiosensitivity in human breast cancer MDA-MB-231 and MCF-7 cell lines.In this study, a series of methods, including MTT assay, in vitro cellular adhesion assay, clonogenic survival assay, in vitro scratch assay and Boyden chamber assay, were employed to determine cell growth and proliferation, invasion and migration. Flow cytometry and Western blot assays were used to determine alteration of cell cycle progression and expression of proteins related to cell cycle or apoptosis. In animal studies, female nude-mice breast cancer transplanted models were employed, moreover, lung metastasis models were established by injection of cancer cells from mouse tails.In both MCF-7 and MD A-MB-231 cell lines, like cyclopamine, SCJ-26 and SCJ-43 significantly inhibited cell growth, reduced cell adhesion, and decreased migratory and invasive ability in a dose-dependent fashion. The anti-breast cancer might be associated with SCJ-26 and SCJ-43-caused G2 arrest, down-regulation of Cyclin D1、Cyclin B1 and Bcl-2. SCJ-26 and SCJ-43 were more active than cyclopamine at the same concentration. However, SCJ-26 and SCJ-43 did not affect growth of breast epitheral MCF-10A cells with the doses which significantly inhibited growth and proliferation of breast cancer cells.As observed by clone survival assay, both SCJ-26 and SCJ-43 increased the radiosensitivity of MDA-MB-231 and MCF-7 cells by enhancing inhibition of cell survival caused byχ-ray irradiation. SCJ-mediated radiosensitivity might be associated with G2 arrest and apoptosis induction. Furthermore, a futher decrease of Cyclin B1, a futher increase of BAX and down-regulation of DNA repair proteins, such as XRCC1, Ku-70 and FEN-1, in the cells pre-treated with SCJ-26 and SCJ-43 and subsequent radiation. SCJ-26 and SCJ-43 mediated radiosensitization was observed to be more effective than cyclopamine under the same concentration.Female nude-mice breast cancer transplanted models were successfully established by injecyion of MDA-MB-231 and MCF-7 cells. Compared to untreated, fewer SCJ-26 and SCJ-43 fed nude mice developed focal tumors, the mean volume of focal tumor was significantly smaller. Liver metastasis of focal tumor was only found in blank control mice (2/6), rather than in SCJ-26 or SCJ-43 fed nude mice. A dose-dependent inhibition of tumor growth was observed with SCJ-26 or SCJ-43 treatment, which was more effective than cyclopamine. As observed in nude-mice lung metastasis models with cells injection from mouse tails, fewer SCJ fed nude mice developed lung metastasis compared with untreated control mice. A dose-dependent inhibition of lung metastasis was observed with SCJ-26 and SCJ-43, which were more active compared to cyclopamine as well.In conclusion, both SCJ-26 and SCJ-43 as new anti-cancer compounds not only inhibit breast cancer cell growth, adhesion, invasion and migration in vitro, but also reduce tumor growth and metastasis in nude-mice in vivo. Furthermore, SCJ-26 and SCJ-43 may enhance radiosensitivity by affecting DNA damage repair proteins and apoptosis-related proteins. These studies, for the first time, indicate that SCJ-26 and SCJ-43, two new derivatives of cyclopamine, are promsing anti-breast cancer compounds under higher toxicity doses and work as novel radiosensitizers under non-toxic doses. Although the exact mechanisms need to be further studied, multiple working pathways, involved in cell cycle progression, apoptosis and DNA damage repair, have been identified in the current study. More important, SCJ-26 and SCJ-43 works more effectively and successfully than their parental cyclopamine.