Metformin, a Type II diabetic treatment drug, which inhibits transcription of gluconeogenesis genes, has recently been shown to lower the risk of some diabetes-related tumors, including breast cancer. mammospheres but not in the bisphenol A mammospheres, suggesting different mechanisms of action of the bisphenol A on human breast carcinoma cells. In addition, these results support the use of 3-dimensional human breast cancer stem cells as a means to screen for potential human breast tumor promoters and breast chemopreventive and chemotherapeutic brokers. Introduction Metformin, a Type 2 diabetic treatment drug, which inhibits transcription of gluconeogenesis genes , has recently been shown to lower the risk of some diabetes-related tumors, including breast cancer C. However, not all studies demonstrate this response  possibly due to confounding factors. Although patients with diabetes are at high risk for cancers of the liver, pancreas, endometrium, breast, colon, and bladder, it is usually not clear as to whether the positive effects of metformin against certain cancers affects the cancer, directly or indirectly, by inhibiting the diabetic state. In addition, it is usually not clear whether metformin might affect other cancers in non-diabetic individuals. ENMD-2076 Moreover, metformin inhibited the growth of breast cancer cell lines in vitro. However, in some cases, it inhibited non-transformed cells at comparable concentrations C. Recently, it has been exhibited that cancer stem cells sustain the growth of tumors and are resistant to therapy. MCF-7 mammospheres have been shown to enrich breast cancer stem cells expressing CD44+CD24?/low , . Assuming the concept of cancer stem cells as the tumor-initiating or tumor-sustaining cells of any tumor or permanent cell line C, the objective of this study was to determine the effects NFIL3 of several known epigenetic-acting chemicals, such as endocrine disrupting- or tumor promoting chemicals (phenol red , TCDD ,  and bisphenol A ), compared to estrogen’s effect ENMD-2076 on the growth of ENMD-2076 MCF-7 mammospheres. These chemical ENMD-2076 Ctreated mammospheres were uncovered to metformin at various non-cytotoxic concentrations. In effect, this series of experiments was designed to test the hypothesis that metformin might be reducing the risk to certain cancers by affecting the breast cancer stem cells in these mammospheres. ENMD-2076 The results, in general, exhibited that metformin reduced the expression of Oct4 in E2- and TCDD- treated human breast cancer stem cells in MCF-7 mammospheres, but not in the bisphenol A-treated mammospheres, suggesting a different mechanism of action of the bisphenol A on the breast cancer stem cells self-renewal ability. In addition, the study supports the use of 3-dimensional mammospheres to screen for potential human breast tumor promoters or cancer chemopreventive or chemotherapeutic brokers. Results The mammosphere formations of human breast cell lines The mammospheres were generated from the ER positive human breast cancer cell line, MCF-7, M13SV1, M13SV1 R2 and M13SV1 R2N1, in phenol red-containing MEBM and phenol red-free MEBM. In both media, the cells efficiently formed compact mammospheres (Physique 1). MCF-7 cells were constantly capable of forming mammospheres through repeated subcultures in medium with phenol red (data not shown). ER- negative human breast cancer cell lines, MDA-MB-231 cells (Physique 1E) and SK-BR-3 cells (data not shown), failed to form mammospheres in both phenol red-contained MEBM and phenol red-free MEBM. Rather, they formed aggregated clusters of cells. It suggests that the estrogen receptor status of breast cells affected the formation and maintenance of mammospheres. Physique 1 ER positive (ACD and FCH) and unfavorable (E) human breast cells in phenol red-contained (Expert) or phenol red-free MEBM (FCH), expression level of mRNA in passaged MCF-7 mammospheres (I), and several ER+ breast … Flow cytometric analysis of MCF-7 mammospheres As stated above, MCF-7 cells efficiently formed mammospheres and this ability was maintained through repeated subcultures.