Roles And Mechanisms Of TNFR1/TRADD/NF-kB Pathway And TRAIL/DR5Pathway In The Radiosensitization Of Glioblastoma Stem Cells

Part I Glioblastoma stem cells is resistant to radiotherapyObjective:To compare and analyze the difference of cell cycle, cell survival and apoptosis after radiation between glioblastoma stem cells (GSCs) and their parental differentiated glioma cells.Methods:(1) Glioblastoma (GBM) cell lines U87and U251were cultured in serum-free medium to induce to stem cell lines named as U87-sph and U251-sph. GBM stem cell line NCH-421k was cultured in differentiated medium for one week, and was termed NCH-421k-dif.(2) Stem cell markers of GSCs and their parental differentiated glioma cells were analyzed by flow cytometry, RT-PCR and immunofluorescence.(3) U87and U87-sph cells were treated with X-rays with different doses, and the apoptotic rates, cell cycles and survival rates were detected at different time points after irradiation. The apoptotic rates of U87and U87-sph after irradiation were also determined by Hoechst33342/PI staining assay.Results:(1) Two weeks after cultivation in serum-free medium supplemented with growth factors, U87and U251were both showed to grow in suspension and form spheres. Seven days after cultivation in differentiated medium containing ATRA, NCH-421k grew as adherent spindle-shaped fibroblast-like cell.(2) The stem cell specific marker CD133, nestin and Sox-2of U87-sph and U251-sph were upregulated compared with their parental differentiated glioma cells using flow cytometry and RT-PCR. Moreover, stem cell markers were decreased in NCH-421k-dif, while differentiated glioma cell marker GFAP was increased compared with NCH-421k. Additionally, U87-sph was also showed high expression of stem cell markers using immunofluorescence.(3) The survival rates of U87-sph were much higher than that of U87at different time points after irradiation with a series of doses. The G2/M phase arrest was significantly apparent in the U87cells especially at12h and24h after irradiation, but not in the U87-sph. The apoptotic rates of U87-sph and U251-sph after irradiation were significant lower than that of U87and U251, respectively. It was more common to see the apoptotic cells and necrotic cells in U87when staining with Hoechst33342/PI than in U87-sph.Conclusion:GSCs could be obtained from GBM cell lines when cultured in serum-free medium with growth factors. GBM stem cell lines could be induced differentiation when cultured in the medium containing ATRA. GSCs were more radioresistant than their parental differentiated glioma cells in vitro. Part II Establishment and identification of radioresistant glioblastoma stem cell lines and the expression of TRADD, DR5in the radioresistant glioblastoma stem cellsObjective:Establishment of radioresistant glioblastoma stem cell lines (GSC-R) and explore the underlying molecular mechanisms of radioresistance.Methods:Glioblastoma stem cells (GSC) NCH-421k, NCH-644and U87-sph were treated with long-term, escalating doses of X-ray irradiation, and the final survival radioresistant GSCs were termed NCH-421k-R, NCH-644-R and U87-sph-R, respectively. The success of establishment of radioresistant GSCs was identified through radiobiology methods. The proliferation curves and survival curves were graphed, and the radiobiological parameters, D0, Dq and SF2, were obtained and analyzed according to the survival curves. The cell cycle and apoptotic rates of GSC-Rs and their parental GSCs after irradiation were detected and analyzed. The expression of stem cell marker CD133in GSC-Rs was detected by flow cytometry. The mRNA expression levels of stem cell markers CD133, nestin, Sox-2and invasiveness markers MMP-2, MMP-9were determined by RT-PCR. The mRNA levels and protein levels of death receptor pathway molecules TRADD, DR5and other apoptosis-related molecules were detected by RT-PCR and Western blot, respectively.Results:The radiobiological parameters, D0, Dq and SF2, were all increased in NCH-421k-R, NCH-644-R and U87-sph-R. After irradiation, the G2/M phase arrests in three GSC-Rs were less apparent than their parental GSCs, and the apoptotic rates were also decreased. The doubling time of NCH-644-R and U87-sph-R was shorter than their parental GSCs, but there was no significant difference between NCH-421k-R and NCH-421k. The proportions of CD133positive cells were much higher in three GSC-Rs compared with parental GSCs. The mRNA expression levels of stem cell markers and invasiveness markers were also increased in three GSC-Rs. The mRNA and protein expression levels of death receptor pathway molecules TRADD, DR5and apoptosis inhibitory molecule cFLIP were both increased, but the expression of DR4and Bcl-2were heterogeneous in three GSC-Rs.Conclusion:Three radioresistant glioblastoma stem cells were established. The expression of stem cell markers was upregulated in these GSC-Rs. The mRNA and protein levels of TRADD and DR5were both increased in GSC-Rs. Part III Roles and mechanisms of TNFR1/TRADD/NF-κB pathway and TRAIL/DR5pathway in the radiosensitization of glioblastoma stem cellsObjective:To explore the roles and mechanisms of TNFR1/TRADD/NF-κB pathway and TRAIL/DR5pathway in the radioresistance and radiosensitization of glioblastoma stem cells (GSCs).Methods:Human apoptosis PCR Array was applied to test and analyze the difference of mRNA expression levels between U87and U87-sph after irradiation with single dose. Correlation between TRADD expression in human glioma samples and prognosis was evaluated in REMBRANDT database. The expression of TRADD in GBM tissues and normal brain tissues was detected by Western blot. The mRNA and protein expression levels of TRADD and DR5in GSCs and their parental differentiated glioma cells were determined by RT-PCR and Western blot, respectively. At different time point after irradiation with a single dose of8Gy, the expression of the death receptor pathway related proteins, apoptosis-associated proteins and NF-κB pathway proteins in U87and U87-sph was detected by Western blot. After the treatment with dose escalation of PDTC (inhibitor of NF-κB) or TRAIL, the apoptotic rates of U87and U87-sph were determined by flow cytometry. After the treatment with dose escalation of TRAIL, the expression levels of NF-κB pathway proteins and apoptosis-related proteins in U87and U87-sph were detected by Western blot. U87, U87-sph and U251-sph were divided into4groups:PDTC alone, radiation alone, radiation combined with PDTC and control group. The apoptotic rates of U87and U87-sph in four groups were determined by flow cytometry, and the expression levels of NF-κB pathway proteins and apoptosis-related proteins in U87-sph and U251-sph were detected by Western blot. U87, U87-sph and U251-sph were divided into4groups:TRAIL alone, radiation alone, radiation combined with TRAIL and control group. The apoptotic rates of U87and U87-sph in four groups were determined by flow cytometry, and the expression levels of NF-κB pathway proteins and apoptosis-related proteins in U87-sph and U251-sph were detected by Western blot. Results:The results of Human apoptosis PCR Array showed that the mRNA expression levels of TRADD and DR5in U87-sph was significantly upregulated after irradiation, but not in U87. The expression level of TRADD was positively correlated with tumor grade in gliomas and negatively with patient prognosis. TRADD protein was highly expressed in GBM tissues. The mRNA and protein expression levels of TRADD were both upregulated in GSCs compared with their parental differentiated glioma cells. However, the expression of DR5was of the opposite trends. After irradiation with a single dose of8Gy, the expression of TRADD, TNFR1and DR5protein was significantly increased time-dependent in U87-sph, and NF-κB pathway proteins were significantly activated in U87-sph compared with U87cells. U87-sph was less sensitive to TRAIL-induced apoptosis than U87cells. However, there was no significant difference in apoptotic rates between U87and U87-sph when treated with dose escalation of PDTC. NF-κB and IκB were significantly activated in U87-sph compared with U87when treated with dose escalation of TRAIL. When U87-sph was treated with radiation whether combined with PDTC or TRAIL, the apoptotic rates were significantly increased compared with radiation alone or PDTC (TRAIL) alone. When U87-sph and U251-sph were treated with radiation combined with PDTC, the activities of NF-κB and IκB were inhibited compared to radiation alone. When U87-sph and U251-sph were treated with radiation combined with TRAIL, the NF-κB pathway was still activated, whereas, the expression of DR5was upregulated and cFLIP was downregulated compared to other groups.Conclusion:TNFR1/TRADD/NF-κB pathway played a crucial role in the radioresistance of GSCs. Interfering the pathway with PDTC and TRAIL might be an adjuvant for glioma radiotherapy.