| BACKGROUND&OBJECTIVE: One of the possible reasons of gliomas recurrence dues to Cancer stem-like cells (SLCCs). With their renewal ability and staying at the quiescent stage, cancer stem-like cells can leading to tumor recurrence and avoid the chemotherapy and radiotherapy. O~6methylguanine DNA methyltranferase (MGMT) can remove the DNA adduct and is the enzyme which can repair the damaged DNA. MGMT can affect the clinical chemotherapeutic effect of alkylation agent. The experiments aim to identify the glioma cell lines with MGMT low or negative expression. Glioma stem-like cells were obtained from glioma cell lines with medium culture of serum-free clone formation to characterize features of cancer stem cells.METHODS:We selected glioma cell lines U251, U373, SKMG-4, SF295, SKMG-1, U87, MGR1 and MGR2 with MGMT low or negative expression. Glioma stem-like cells U251G, U373G, SKMG-4G, SF295G, SKMG-1G, U87G, MGR1G and MGR2G were obtained from serum-free clone formation. Molecular markers for glioma stem-like cells (CD133, nestin, Sox-2, GFAP and TuJ1) were detected by immunofluorescent staining. Tumorigenicity of glioma stem-like cells was evaluated by subcutaneous xenograft model on nude mice. Tumors size were observed on every 2 days. Vernier gauges were employed to measure the tumors biggest diameter. RESULTS:Eight glioma stem-like cells (U251G, U373G, SKMG-4G, SF295G, SKMG-1G, U87G, MGR1G and MGR2G) were successfully established by means of serum-free clone formation. Glioma stem-like cells were propagating in the manner of suspending growth gliomaspheres. Gliomaspheres generated from all glioma stem-like cells showed high immunoreactivity for neural stem cell marker CD133, Nestin and Sox-2, but low for markers of differentiated lineages such as GFAP and TuJ1. Transplantation of 1×10~4 glioma stem-like cells and 1×10~4 glioma cell lines subcutaneously 6 weeks made immunocompromised nude mice generate tumors. The tumors biggest diameter of glioma stem-like cells was 1.4±0.7cm. The tumors biggest diameter of glioma cell lines was 0.6±0.4cm. The tumors biggest diameter of glioma stem-like cells are bigger than it of glioma cell lines(P<0.05).CONCLUSION:The glioma stem-like cells were obtained from serum-free clone formation and they had features of cancer stem cellsChapter II MGMT expression in glioma stem-like cellsBACKGROUND&OBJECTIVE: To explore the MGMT expression in glioma stem-like cells.METHODS:(1) RT-PCR was used to detect the MGMT gene in glioma stem-like cells and glioma cell lines; (2) Western blot was employed to detect the MGMT protein in glioma stem-like cells and glioma cell lines. (3) Methylation-specific PCR (MSP) was applied to detect the promoter methylation of MGMT in glioma stem-like cells and glioma cell lines. The results were analyzed by QauntityOne software and CORELDRAWX4 software.RESULTS:(1) RT-PCR showed that MGMT gene was negative in U251, SKMG-4, SF295, SKMG-1, U87, MGR1, MGR2 and was low erpression in U373 ; MGMT gene was positive expression in U251G, U373G, SKMG-4G, SF295G, SKMG-1G and negative erpression in U87G, MGR1G and MGR2G. (2)Western blot showed that MGMT protein was negative in all of the glioma cell lines ; MGMT protein was positive in U251G, U373G, SKMG-4G, SF295G, SKMG-1G and was negative in U87G, MGR1G, MGR2G. (3) MSP showed that MGMT promoter methylation in all of the glioma cell lines and demethylation in U373 ; MGMT promoter methylation was founded in all of the glioma stem-like cells and demethylation was in U251G, U373G, SKMG-4G, SF295G, SKMG-1G.CONCLUSION: MGMT positive and MGMT promoter demethylation can be founded in some of glioma stem-like cells. However, MGMT negative and MGMT promoter with non-demethylation can be also founded in some of glioma stem-like cells.Chapter III MGMT expression and Temozolomide resistance in glioma stem-like cellsBACKGROUND&OBJECTIVE: To investigate the role of MGMT expression in the TMZ resistance in glioma stem-like cells in vitro.METHODS:CCK-8 assay was applied to test the growth inhibition both of glioma stem-like cells and glioma cell lines by TMZ in different concentration. Half inhibiting concentration (IC50) was calculated. The results of CCK-8 assay statistical analyses were performed with the SPSS software package, version 16.0. The results of CCK-8 assay were showed in the form of(x|—)±s. P<0.05 was considered statistical significance.RESULTS:CCK-8 assay showed that the survival rate of glioma stem-like cells and glioma cell lines was decreased following the increasing concentration of TMZ. It showed that IC50 (glioma stem-like cells,μmol/l) : U251G(1527.23±74.71), U373G(1270.57±188.67), SKMG-4G(1570.84±99.69), SF295G(1502.84±74.57), SKMG-1G(1384.81±133.13), U87G(1520.89±277.64), MGR1G(1361.12±47.81), MGR2G(1455.99±243.65); U251(501.06±61.68), U373(592.36±25.69), SKMG-4(527.50±64.23), SF295(608.70±28.15), SKMG-1(597.21±60.75), U87(592.42±92.36), MGR1(431.87±85.18), MGR2(638.75±144.74). There were statistical significance of IC50 between glioma stem-like cells and glioma cell lines (P=0.00<0.05), but no statistical significance of IC50 between MGMT positive expression glioma stem-like cells and MGMT negative expression glioma stem-like cells(P=0.94>0.05).CONCLUSION:Glioma stem-like cells showed more TMZ resistant. However, there were no obvious difference between MGMT positive expression glioma stem-like cells and MGMT negative expression glioma stem-like cells on TMZ resistance.ChapterⅣRegulatory mechanism of NF-κB regulates MGMT expression in glioma stem-like cellsBACKGROUND&OBJECTIVE: To investigate the expression relationship between NF-κB and MGMT , to explore the regulatory mechanism of inhibiting NF-κB can regulate MGMT to reverse TMZ resistance in MGMT positive expression glioma stem-like cellsMETHODS:(1) CCK-8 assay was applied to investigate the sensitivity of glioma stem-like cells (U251G, U373G, SKMG-4G, SF295G and SKMG-1G) and glioma cell lines(U251, U373, SKMG-4, SF295 and SKMG-1) to MG-132 (Proteasome inhibitor that can inhibit activation of NF-κB) combine with Temozolomide. (2) RT-PCR was employed to explore the MGMT and NF-κB genes in glioma stem-like cells(U251G, U373G, SKMG-4G, SF295G and SKMG-1G) and glioma cell lines(U251, U373, SKMG-4, SF295 and SKMG-1) treated with different medicines(control, TMZ, MG-132 and TMZ+MG-132 ). (3) Western blot was employed to explore the MGMT and NF-κB protein in glioma stem-like cells (U251G, U373G, SKMG-4G, SF295G and SKMG-1G)and glioma cell lines (U251, U373, SKMG-4, SF295 and SKMG-1) treated by different medicine(control, TMZ, MG-132 and TMZ+MG-132 ). The results of CCK-8 assay statistical analyses were performed with the SPSS software package, version 16.0. The results of CCK-8 assay were showed in the form of (x|—)±s. P<0.05 was considered statistical significance. The results of RT-PCR and western blot were analyzed by QauntityOne software and CORELDRAWX4 software.RESULTS:(1) TMZ had the synergy effect on MGMT positive expression glioma stem-like cells (U251G, U373G, SKMG-4G, SF295G and SKMG-1G) when combined with MG-132. However, there was no the same synergy effect on glioma cell lines(U251, U373, SKMG-4, SF295 and SKMG-1). The probibility of kill of different medicines(%): (2) RT-PCR showed that MGMT positive expression glioma stem-like cells (U251G, U373G, SKMG-4G, SF295G and SKMG-1G): control group : NF-κB gene and MGMT gene were positive expression ; TMZ : NF-κB gene was not down-regulated and MGMT gene was down-regulated ; MG-132: NF-κB gene and MGMT gene were down-regulated ; TMZ+MG-132 : NF-κB gene and MGMT gene were down-regulated.(3) Western blot showed that MGMT positive expression glioma stem-like cells (U251G, U373G, SKMG-4G, SF295G and SKMG-1G): control group : NF-κB protein and MGMT protein were positive expression ; TMZ : NF-κB protein was not down-regulated and MGMT protein was down-regulated ; MG-132: NF-κB protein and MGMT protein were down-regulated ; TMZ+MG-132 : NF-κB protein and MGMT protein were down-regulated.(4) RT-PCR showed that glioma cell lines(U251, U373, SKMG-4, SF295 and SKMG-1): control group : NF-κB gene was positive expression and MGMT gene was negative expression in U251, SKMG-4, SF295 and SKMG-1 except U373 with low expression ; TMZ : NF-κB gene was not down-regulated and MGMT gene was negative expression; MG-132 : NF-κB gene was down-regulated and MGMT gene was negative expression ; TMZ+MG-132 : NF-κB gene was down-regulated and MGMT gene was negative expression .(5) Western blot showed that glioma cell lines(U251, U373, SKMG-4, SF295 and SKMG-1): control group : NF-κB gene was positive expression and MGMT gene was negative expression ; TMZ : NF-κB gene was not down-regulated and MGMT gene was negative expression; MG-132 : NF-κB gene was down-regulated and MGMT gene was negative expression ; TMZ+MG-132 : NF-κB gene was down-regulated and MGMT gene was negative expression .CONCLUSION:(1) TMZ has the synergy effect on MGMT positive expression glioma stem-like cells when combined with MG-132. However, there was no the same synergy effect on glioma cell lines. (2) NF-κB and MGMT are positive expression in MGMT positive expression glioma stem-like cells, but NF-κB is positive expression and MGMT is negative expression in glioma cell lines. Although NF-κB can not be down-regulated by TMZ, MGMT can be down-regulated by TMZ. MGMT expression is regulated by NF-κB. MGMT expression down-regulate can be achieved by reducing NF-κB expression so that the resistance of TMZ in MGMT positive expression glioma stem-like cells can be reversed. It suggested that NF-κB and MGMT have positive correlation.
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