| Summary: | 碩士 === 國立陽明大學 === 解剖暨細胞生物學研究所 === 96 === Cancer stem cells (CSCs) have been identified in acute myeloid leukemia (AML), breast cancer and brain tumors, etc. These CSCs shared many properties including self-renewal and multi-potency with normal stem cells, and expressed a wide variety of transporters involved in drug efflux. Glioblastoma multiforme (GBM) is the most frequently found brain cancer. It is intrinsically resistant to conventional therapies, including radiotherapy and chemotherapy. Radiation therapy can be followed by recurrence due to existence of radiation-resistant cells. Although the existence of cancer stem cells in GBM has been previously demonstrated, there is little evidence to explain the difference between cancer stem cells and radio-resistant cells in GBM.
In this study, we used two GBM cell lines (GBM8901 and p't 3) derived from primary culture of GBM patients and a U87MG cell line (from ATCC). Cells were subjected to 10 Gy radiation and the survival cells maintained in MEM medium for 10 days. This process was repeated 3 times, and the final survival cells were used for experiments. The radio-resistant cells were characterized by investigating their growth rate, radiation survival curve, colony formation and migration ability, and their stemness-like properties by examining their neurospheres aggregation ability, CD133+, ALDH+, side population and stemness genes expression.
Our results show that radiation survival curve increased in GBM IR cells as compared to the parental cells. Although the growth rate and migration ability were lower, GBM IR cells colony formation numbers were higher than parental cells. In neurospheres aggregation ability test, we found that GBM IR cells were faster to form neurospheres. In the cell surface marker analysis, CD133+ cells percentage were almost 0% in GBM8901, GBM8901 IR, p't 3 and p't 3 IR cells. ALDH+ and side population (SP) were increased after GBM8901 was subjected to radiation. However, ALDH+ and SP were decreased in p't 3 IR. Experssion of several stemness genes such as Oct-4, Nestin, Sox-2 and KLF4 were increased in radio-resistant cells using RT-PCR analysis. We used chemotherapeutic agents including Etoposide (VP-16), Cisplatin and unknown drug－CSCK1 to treat all cell lines. When neurospheres were treated with 5 micromole CSCK1, there's no new neurospheres formation. Furthermore, in colony formation assay, all cell lines could only form tiny colonies after pretreating chemotherapeutic agents for 48 hours. We found CSCK1 could almost totally inhibit colonies formation. In contrast, we found that radiation treatment of our GBM cells increased the percentage of stem-like cells. Most importantly, GBM and GBM IR cells treated with CSCK1 were no longer able to form colonies in soft agar and neurospheres in serum free medium, suggesting that CSCK1 might be a potential chemotherapeutic agent for clinical treatment.
|
|---|
