Although the biological function of CD133 is not well understood, the CD133 currently serves as a useful marker for the isolation of brain cancer stem cells [1, 3]. The identification of brain cancer stem cells provides a powerful tool for the investigation of the tumorigenic process in the central nervous system, and will be crucial in developing therapies that use brain cancer stem cells as a target .
Our data indicated that a large number of CD133 positive cancer stem cells could be maintained without differentiation in 10% FBS culture medium after 3–6 passages. Recently, Kondo and his colleagues found that C6, an established malignant murine glioma cell line (which has been maintained for years in culture) contained a subpopulation of cancer stem cells . Furthermore, a distinct "side population" of cells with high drug efflux capacity were also found in the human glioblastoma cell line U-87, which is consistent with the findings of the study of C6 glioma cells . We propose that a minor subpopulation of cancer stem cells derived from glioblastoma tumor tissue may be maintained even in FBS containing medium which may be a significant character of cancer stem cells from normal stem cells.
Recent studies show that cancer and normal stem cells share the same self-renewal mechanisms, such as the Bmi-1 and Wnt canonical pathways [13, 14]. In the present study, we found thatCD133 positive cells expressed higher levels of CD90, CD44, Nestin, Msi1, MELK, GLI1 and PTCH. Particularly, Bmi-1, PSP, SHH, OCT4 and Snail were only expressed on CD133 positive cells; none of the five genes were detectable on CD133 negative tumor cells. The data suggests that these genes involved in self-renewal are expressed in CD133 positive cancer stem cells while not in CD133 negative cells. However, Hemmati et al. examined mRNA expression profiles of differentiated and undifferentiated spheres from six different brain tumours and found higher levels of musashi-1, Sox2, MELK, PSP, Bmi-1, and Nestin were not strictly associated with the undifferentiated spheres but differed strongly among the different specimens and these genes were sometimes even stronger expressed in the differentiated .
Bmi-1 functions as a transcriptional repressor that targets the INK4a locus and is necessary for self-renewal in the blood and brain. Parallel to these normal organ systems, Bmi-1 can also play an integral role in the malignant transformation of the HOX A9/MEIS – induced murine leukemia model  as well as in tumors of neural origin . These findings highlight the notion that the molecular regulation of self-renewal can be observed in both normal and cancer stem cells. Our observation of Bmi-1, SHH, Oct4, PSP and Snail expression only in the cancer stem cell component of a human brain malignancy may shed light on the dynamic nature of the cancer stem cell to exit the capacity to self renew as it differentiates into a daughter cell.
Cancer stem cells may be responsible for the initiation and maintenance of neoplastic tissue. Alternatively, neural progenitor or terminally differentiated cells may dedifferentiate to a stem-like state to form the tumor initiating cell. The tumor initiating and sustaining stem-like cells' resistance to chemo- and radiation therapy, may explain why such traditional therapies can shrink a tumor but often cannot completely eradicate it resulting in eventual recurrence. In this study, we tested four common chemotherapeutic agents on CD133 positive cancer stem cells. Consistent with our hypothesis, CD133 positive cancer stem cells are significantly resistant to four tested chemotherapeutic agents, including temozolomide, carboplatin, VP16 and Taxol than autologous CD133 negative cells.
Temozolomide is a new oral alkylating agent that is widely used for high-grade gliomas . The cytotoxicity of temozolomide is primarily due to the formation of O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication . Subsequent futile cell cycles of DNA lead to mismatched repairs and result in cancer cell death. . Current available data generated from investigations in cell lines and tumor samples, suggest that resistance to temozolomide is significantly linked to MGMT-mediated DNA repair in high-grade gliomas, primitive neuroectodermal tumors, and ependymomas . Our study showed CD133 positive cells had significant resistance to temozolomide compared to autologous CD133 negative cells, which was consistent with the results of higher MGMT expression in CD133 positive cells.
One of the important mechanisms of drug resistance is the expression of ATP-binding cassette transporter protein, such as BCRP1. BCRP1 accounts for chemoresistance of some clinical cancers including acute myeloid leukemia, non-small cell cancer, and breast cancer. In both breast carcinoma and acute myeloid leukemia, putative cancer stem cells have been isolated and characterized. The evidence suggests that the "side population (SP)" phenotype is associated with high expression levels of BCRP1 . The exclusion of Hoechst 33342 dye defines the pluripotential side population (SP) originally reported in hematopoietic stem cells. Their high drug efflux capacity correlates with the strong expression of the drug-transporter protein BCRP1. Therefore, SP has become a common approachto identify putative adult stem cells  and cancer stem cells . BCRP1 has been shown to be specifically expressed in the fetal neural stem cell by microarray and immunocytochemical analysis . We found that CD133 positive cells express higher levels of BCRP1, which indicate that BCRP1 may also play an important part in the drug resistance of CD133 positive cells. BCRP1 over-expressing tumor cells, however, are only resistant to mitoxantrone, adriamycin, daunorubicin, etoposide, topotecan, and irinotecan. They are not resistant to Taxol and vincristine . In the present study, CD133 positive cells derived from three cell lines are all resistance to carboplatin and Taxol. Therefore, we proposed that both anti-apoptosis factors and BCRP1 contribute the drug resistant property on CD133 positive cancer stem cells.
Apoptosis mechanisms are hypothesized to play an important role in the resistance of tumor cells to chemotherapy . Most studies indicate that increased levels of the anti-apoptotic protein Bcl-2 correlates with chemotherapy resistant disease and decreased overall survival . The inhibitor of apoptosis protein (IAPs) family are anti-apoptotic proteins that bind and inhibit caspases 3, 7 and 9, and thereby prevent apoptosis . Growing evidence also indicates that IAPs also modulate cell division, cell cycle progression and signal transduction pathways . In this study, we found that Bcl-2, FLIP, BCL-XL and all of the IAPs we tested, including Class I IAPs (XIAP, cIAP1 and cIAP2); Class II IAP (NAIP) and Class III IAP (survivin) expressed higher mRNA levels in CD133 positive cells than in CD133 negative cells. The data supports the hypothesis that apoptosis pathways contribute to drug resistance of CD133 positive cancer stem cells.
Bcl-2 promotes prolonged but not indefinite cell survival under apoptotic stress from radiation, chemotherapy drugs, and toxins . The Bcl-2 family is made up of both suppressors (e.g. Bcl-2, Bcl-XL and MCL-1) and inducers (e.g. Bax, Bad, Bak and Bid) of apoptosis. AML leukemic cells recovered from patients with minimal residual disease have demonstrated high levels of Bcl-2 and Bcl-XL , suggesting a significant role of these genes in resistance to therapy.
ATRA has been shown to increase sensitivity to ARA-C by downregulating Bcl-2 and Bcl-XL in vitro . As a clinical corollary, retinoids given with chemotherapy can improve the survival of AML patients. Bcl-2 expression in patient samples of AML cells correlated with decreased rates of remission and event-free survival . Furthermore, a low Bcl-2/Bax ratio was associated with prolonged survival . These findings support the notion the inhibition of apoptosis is directly associated with response to chemotherapy and hence clinical outcome.
Neural stem cells (NSCs) express the markers nestin and CD133, and differentiate into neurons, astrocytes and oligodendrocytes at a clonal level . An important characteristic of NSCs, not fully understood, is their migratory ability and their tropism to brain pathology. High expression levels of functional chemokine receptor CXCR4 have been found on human neural stem cells . These findings suggest that CXCR4 may play a significant role in directing NSC migration during CNS development. Similar to normal NSCs, CD133 positive cancer stem cells showed 337.8 times increase on the expression levels of CXCR4 than did CD133 negative cells, which suggests CD133 positive cancer stem cells have higher capability of migration and may play an important role in glioma invasion.
Finally, CD133 expression levels are higher in recurrent tumor tissue than in autologous primary tumor tissue, which suggests that CD133 positive cancer stem cells may play an important role in the tumor's ability to resist chemotherapies and radiation therapy.
Taken together, because of higher BCRP1, MGMT and IAPs levels, CD133 positive cancer stem cells may be resistant to conventional chemotherapy and contribute to disease relapse. Since normal tissue stem cells and cancer stem cells have many similar properties, further studies should focus on the difference between CD133positive normal tissue stem cells versus CD133 positive cancer stem cells in order to find unique pathways to differentiate these two cell populations. These differences may be exploited with the aim of targeting the CD133 positive cancer stem cell population either biochemically or immunologically without harming normal tissue stem cells. Delineating the molecular genetics and epigenetics involved in the propagation and differentiation of these stem cell populations will be useful in defining the therapeutic window to target only the cancer stem cell.