Effects Of Lovastatin On The Characteristics Of Breast Cancer Stem Cells

Objective: To study the effects of Lovastatin(LV) on the biological characteristics of human triple-negative breast cancer stem-like cells(BCSLCs) in vitro, and on xenograft growth of human triple-negative breast cancer cells MDA-MB-231 in nude mice. Methods: 1、 Breast cancer stem-like cells(BCSLCs) were enriched by ultra-low adherence suspension culture from human triple-negative breast cancer cells and non-triple-negative breast cancer cells. The expression of breast cancer stem cell surface markers(CD44/CD24) was detected by flow cytometry. The tumorigenicity of BCSCLs was confirmed by subcutaneous tumor formation in nude mice. 2、 Parental breast cancer cells or BCSLCs were treated with LV or vehicle under normoxia or hypoxia. Cell proliferation, cell migration, and cell apoptosis were detected by MTT assay, scratch assay, and flow cytometry, respectively. 3、 MDA-MB-231 cells were treated with different concentrations of LV. The protein expression level of breast cancer stem cell surface marker CD44, EMT-related nuclear transcription factor β-catenin, and epithelial cell marker E-cadherin were analyzed by Western blotting. 4、 One week after subcutaneous injection of MDA-MB-231 cells, the nude mice were treated with different concentrations of LV(2 mg/kg or 10mg/kg) or normal saline, twice weekly for three weeks. The tumor volume was measured weekly; the xenograft tumors were removed and weighed when the animals were sacrificed. The tumor tissues were embedded in paraffin, and histopathological and immunohistochemical analyses were performed. Results: 1、 BCSLCs were enriched from triple-negative and non-triple-negative breast cancer cells using ultra-low adherence suspension culture technique. Cell surface markers were identified by flow cytometry to obtain a purity of about 80% of CD44+/CD24 low cells in MDA-MB-231-derived BCSLCs; in nude mice tumorigenicity experiment, compared with MDA-MB-231 parental cells, the tumor-forming ability of MDA-MB-231 stem-like cells was stronger. These data indicate the BCSLCs we separated and amplified possess the properties of breast cancer stem cells. 2、 Compared with parental cells and BCSLCs derived from non-triple-negative breast cancer cells, LV preferentially suppressed proliferation and migration and promoted apoptosis of parental cells and BCSLCs derived from triple-negative breast cancer cells; Compared with parental cells, LV preferentially suppressed proliferation of BCSLCs derived from MDA-MB-231 cells. 3、 LV inhibited the level of the proteins related with the breast cancer stem cell characteristics in cultured MDA-MB-231 cells, which indicate that LV could inhibit the stemness of triple-negative breast cancer cells in vitro. 4、 In a nude mice subcutaneous xenograft model, LV inhibited the weight and volume of the tumors formed from triple-negative breast cancer MDA-MB-231 cells. Immunohistochemical staining showed that the protein levels of CD44 and CD138 in tumor tissue specimens were significantly decreased in LV-treated mice compared with the control group. These data indicate that LV could inhibit the tumorigenic ability and the stemness of triple-negative breast cancer cells in vivo. Conclusions: 1、 BCSLCs with breast cancer stem cell properties were enriched and amplified from triple-negative and non-triple-negative breast cancer cells using ultra-low adherence suspension culture technique. 2、 Compared with non-triple-negative breast cancer, LV can preferentially inhibit proliferation and migration and promote apoptosis of cancer stem cells derived from triple-negative breast cancer cells in vitro. 3、 LV can inhibit the tumorigenic ability and reverse the stemness properties of MDA-MB-231 cells in vivo.