Enrichment Of Prostate Cancer Stem Cells By Chemoradiotherapy And The Mechanism Of Chemoresistance In Prostate Cancer Stem Cells

Part I Enrichment of prostate cancer stem cells by culture in serum-free medium, chemotherapy and radiotherapyObjective:The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. However, the CSCs are difficult to obtain for deeply research because of their very low proportion in tumor cells. Therefore, we used serum-free medium culture, chemotherapy and radiotherapy for prostate cancer stem cells enrichment from three lines of human prostate cancer cells, laying the foundation for the field of prostate cancer stem cells research.Methods: We cultured three commonly used human prostate cancer cell lines (DU145, PC-3and LNCaP) in the serum-supplemented medium (SSM) and the serum-free medium (SFM) supplemented with human epidermal growth factor (EGF), human basic fibroblast growth factor (bFGF) and recombinant human leukemia inhibitory factor (LIF), respectively. Flow cytometric analysis the proportion of CD133+/CD44+prostate cancer stem cells in these cell lines cultured under two different conditions. SFM culture, chemotherapy (docetaxel0.1μM) and radiotherapy (4Gy/times,2times/week, two weeks of continuous irradiation) were used for the enrichment of CD133+/CD44+prostate cancer stem cells in DU145cells. Flow cytometric analysis the proportion of CD133+/CD44+prostate cancer stem cells in DU145cells after enriched by the three methods.Results:Under normal culture conditions, CD133+/CD44+cells were only present in the DU145cell line, and comprised only a minor percentage (0.1%±0.01%) of the total population. After culture in SFM, a small amount of DU145cells and PC-3cells can survive and grow up by the formation of cell suspension spheres. Moreover, the proportion of CD133+/CD44+cells in DU145and PC-3had increased to10.3%and3.0%, respectively. The proportion had increased to9.8%enriched by chemotherapy and3.5%by radiotherapy in DU145.Conclusions:Under normal culture conditions, CD133+/CD44+cells were only detected in the DU145cell line with a minor percentage. After culture in SFM, CD133+/CD44+cells were detected in the DU145cell line and PC-3cell line with the number increased obviously. But the CD133+/CD44+cells were not detected in the LNCap cell line. CD133+/CD44+prostate cancer stem cells can be enriched effectively by SFM culture, chemotherapy and radiotherapy. Part Ⅱ Research for the characteristics of CD133+/CD44+prostate cancer stem cellsObjective:The CSCs hypothesis postulates that a small subpopulation of cancer cells drive tumor growth and metastasis, are the "seed of tumors". And that CSCs are more resistant to toxic injuries and chemoradiation therapy than differentiated daughter cells. There is increasing evidence that the initiation, growth, recurrence, and metastasis of cancers are related to the behavior of CSCs. The stem cell properties of CD133+/CD44+prostate cancer stem cells that were isolated via fluorescence-activated cell sorting (FACS) were evaluated by the in vitro and in vivo expriments.Methods: DU145cells were cultured in serum-free medium. Then CD133+/CD44+prostate cancer stem cells were isolated from the DU145cells with a Beckman Coulter MoFlo XDP flow cytometer. Colony-formation tests, transwell cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the characteristics of CD133+/CD44+DU145cells and parental DU145cells.20male BALB/c nude mice were divided randomly into2groups. Isolated CD133+/CD44+DU145cells (1X104) and parental DU145cells (1X106) were injected subcutaneously into the left flank of the mice (n=10/group). The tumor growth was measured and the tumor formation was assessed.Results:Colony-formation tests showed that the colony-formation efficiency (CFE) of CD133+/CD44+cells (68.5±4.7%) was3.5-fold greater than that of parental cells (19.7+3.4%)(P<0.001). Furthermore, as measured by the cell invasion assays, a four-fold greater proportion of CD133+/CD44+cells migrated through the Mattrigel-coated membrane compared to parental DU145cells (CD133+/CD44+cells versus the parental DU145cells,416±47versus109±24, P<0.001). In the xenograft model, our results showed that CD133+/CD44+cells initiated significantly larger tumors (10/10observations) than parental DU145cells (5/10observations). Tumors originating from CD133+/CD44+cells developed earlier and faster than those from parental cells.Conclusions:CD133+/CD44+cells displayed high clonogenicity and increased invasiveness. Moreover, CD133+/CD44+cells are more tumorigenic in vivo, they are prostate cancer stem cells. Part Ⅲ The mechanism of Notch-1in the chemoresistance of prostate cancer stem cellsObjective:Research on the drugresistance of CSCs has become a new field of stem cells. It is not clear that the mechanism of CSCs resistant to chemotherapy. Notch signaling pathway has a very important role in the proliferation and differentiation of stem cells. We explored the role of Notch-1in the CD133+/CD44+prostate cancer stem cells resistant to chemotherapy, in order to find a new target for the treatment of prostate cancer.Methods:MTT assays were used to detect the sensitivity of CD133+/CD44+DU145cells and parental DU145cells to docetaxel. The expression differences of Oct-4, Nanog, and Notch-1in CD133+/CD44+DU145cells and parental DU145cells were tested by real-time PCR. Sixteen male BALB/c nude mice bearing human DU145prostate tumors were divided randomly into two groups:①control group;②docetaxel group (10mg/kg/week, i.v., X3weeks). After three weeks, mice were euthanized and tumors were harvested. The expression of Notch-1and Jagged-1was evaluated by immunofluorescence staining. Xenograft tumors were cut into small pieces, and then mixed with collagenase IV. At the end of digestion, the cells were immunostained with PE-CD133/2and FITC-CD44antibodies and analyzed by fluorescence-activated cell sorting (FACS).Results:In MTT assays, CD133+/CD44+DU145cells survival was significantly higher than parental DU145cells (P<0.001). Real-time PCR analysis showed that CD133+/CD44+DU145cells preferentially expressed certain stem cell specific genes, such as Nanog and Oct-4. And Notch-1expression was significantly increased. In vivo experiment, our results showed that the expression of Notch-1and Jagged-1was signigicantly increased in docetaxel group compared with the control group. The increased protein expressions of Notch-1and Jagged-1in tumor specimens were confirmed by immunofluorescencecytochemical studies. And flow cytometry analysis revealed that the proportion of CD133+/CD44+prostate cancer stem cells in docetaxel group (2.6%) was significantly higher than that of the control group (0.1%)(P<0.001).Conclusions:CD133+/CD44+DU145cells resistanted to docetaxel and expressed Notch-1higher than that in parental DU145cells. Tumors after chemotherapy were highly expressed Notch-1and Jagged-1. The proportion of CD133+/CD44+cells in the tumors after chemotherapy increased significantly. CD133+/CD44+prostate cancer stem cells resistant to chemotherapy, Notch-1may play a role in the chemoresistance of prostate cancer stem cells.