Identification The Cancer Stem Cells In Breast Medullary Carcinoma Bcap-37Cell Line And The Effects Of Huaier Granules On Breast Cancer Stem Cells

Breast cancer is the most common malignant tumor of women in China with thesecond highest mortality rates among all tumors. Moreover, its incidence rate is increasingyear by year. The medullary carcinoma is a special type of breast cancer, which usuallyexhibits a better prognosis. However, there is still confusion about its unique pathologicalfeatures and better prognosis. Here, a further study on medullary carcinoma was carried out.Moreover, the targeted treatment of breast cancer is always difficult in clinical trials. Thescreening of anti-tumor drugs for cancer stem cells has become a research hotspot.CSCs are a subgroup of cancer cells with self-renewal, multi-directional differentiationability, which are related to tumorigenesis, metastasis, and recurrence. The selection ofCSCs is the first step for the studying of CSCs. Previous research has showed that the CSCsisolation can be performed in flow-cytometry with CSC markers, however,the CSCsmarkers of breast medullary carcinoma has not been reported. Therefore, an appropriateCSCs marker for breast medullary carcinoma is the basis for the further research. Recentstudies also showed that CSCs had obvious positive correlation with tumor recurrence.Therefore, it is premising to screen drugs targeting CSCs for clinical treatment.Here in this study, we chose breast medullary carcinoma cell line Bcap37as cell model,PROCR/ESA (PROCR, endothelial protein C receptor. ESA,(epithelial specific antigen) assorting marker, and we investigated the percentage of PROCR~+/ESA~+in Bcap37andexamined the malignant behavior of breast CSCs in breast medullary carcinoma. To provethe PROCR~+/ESA~+cells were of CSCs characteristics, the tumorigenicity assays inNOD/SCID mice, colony formation assays in vitro, cell cycle assays and stemness-relatedgene expression profile were studied. In order to verify the inhibitory effect on CSCs of theHuaier granules, the PROCR~+/ESA~+cells were treated with the Huaier granules. Anobserving reduction of the clone formation ability was found after administration. Besides, we chooses another breast cancer cell line--SUM159for further study, especially ininhibiting effect on tumor stem cells of Huaier granules. The Huaier granules sharplyreduced colony-forming and mammospheres-forming ability of breast cancer SUM159cells, and with a obvious reduction in the proportion ALDH1highin SUM159cell line.Results of the preliminary study indicated inhibiting effect on breast CSCs of Huaiergranules, which provided a premising drug for CSCs-targeting therapy and providing a newtreatment method for clinical treatment.Methods&Results1. Identification the cancer stem cells in breast medullary carcinoma Bcap-37celllineMethods: CD24/CD44and PROCR/ESA cells of breast medullary carcinomaBcap-37cell line were sorted by flow cytometry. The breast CSCs characteristics of thesesubpopulations were identified through Clone formation assay, cell cycle experiment,stemness-related gene expression profile as well as xenografts in NOD/SCID mice. Weintended to identified whether PROCR~+/ESA~+was an appropriate breast CSCs marker forbreast medullary carcinoma Bcap-37cell line, rather than CD24/CD44.Results: The results of flow cytometry showed that the percentage of CD44~+/CD24-/lowsubpopulation was about52.7%,whereas PROCR~+/ESA~+was3.2%. Cloning formationassays showed that there were no difference between the number of CD44~+/CD24-/lowandNon CD44~+/CD24-/lowcells, whereas PROCR~+/ESA~+cells formed more clones thanPROCR-/ESA-cells (P<0.05), suggesting that PROCR~+/ESA~+cells exhibited strongself-renewal ability. Cell cycle experiments showed that most PROCR~+/ESA~+cells enteredinto the cell division cycle of G2and S phase from G1in48hours, while PROCR-/ESA-cells almost stayed in G1phase. The q-RT-PCR assays of stemness related gene expressionshowed that PROCR~+/ESA~+cells expressed higher level of Nanog, OCT4and Bim1thanPROCR-/ESA-cells (P<0.05). Xenografts in NOD/SCID mice also showed that, comparedwith PROCR-/ESA-cells, PROCR~+/ESA~+cells generated more xenografts, with largertumor volume(P<0.05)2. The effects of Huaier granules on the breast cancer stem cellsMethods: Clone formation assays of PROCR~+/ESA~+of Bcap-37cells were observedwith or without Huaier granules treatment.CCK-8assay was used in determination of IC50 values, and the morphological images, and cells clones and clustering were observed beforeand after drug administration. The ratio of ALDH1highcells was detected by flow cytometrywith or without Huaier granules administration.Results: Huaier granules treatment obviously decreased the clone of PROCR~+/ESA~+ofBcap-37cells(P<0.05)； Sum-159cells were treated with Huaier granules at theconcentrations of0mg/ml,1mg/ml, and10mg/ml, respectively. The IC50value ofHuaier granules in Sum-159cells was approximately10mg/ml. As the concentrationincreased, the number of colonies gradually reduced (p <0.05), and the number anddiameter of mammospheres were also decreased (p <0.05). With the application of Huaiergranules in10mg/ml concentration, the ratio of ALDH1highcells was decreased to1.5%with administration, compared of2.8%without administration. However, with theapplication of1μg/ml paclitaxel, the ratio of ALDH1highcells was increased from2.8%before administration to9.1%after administration.Conclusion:1) The PROCR~+/ESA~+cells of Bcap-37which were sorted by flow cytometryexhibited obviously breast CSCs biobehavior, such as tumorigenity andmulti-differentiation ability. We found PROCR~+/ESA~+can worked as a CSCs marker inbreast medullary carcinoma cell line.2) Huaier granules significantly inhabited the cloning and clustering abilities of humanbreast cancer cell lines Bcap37and Sum-159, and reduced the ratio of ALDH1highcells,which indicated that Huaier granules may inhibit or kill breast cancer stem cells.