Expression of MGMT and chemosensitivity to TMZ in human malignant glioma cell lines.

<p><b>A, Expression of <i>MGMT</i> gene in human malignant glioma cell lines.</b> RNA was isolated from ten malignant glioma cell lines. RT-PCR was done to assess <i>MGMT</i> mRNA expression. Expression of <i>MGMT</i> mRNA was detected in T98G, YH-13 and LN-18. <b>B, Expression of MGMT protein in human malignant glioma cell lines.</b> Protein extracts were prepared from ten malignant glioma cell lines. MGMT expression was analyzed by immunoblotting. β-actin was used as a loading control. Expression of MGMT protein was detected in T98G, YH-13 and LN-18. <b>C, Expression and localization of MGMT protein in human malignant glioma cell lines.</b> Cultured cells were fixed and incubated with anti-MGMT antibody. Reactants were processed with the standard streptavidin-biotin immunoperoxidase method. Diaminobenzidine was used as the final chromogen. Expression and intranuclear localization of MGMT protein was observed in T98G, YH-13and LN-18. <b>D, Growth-inhibitory effect of TMZ in human malignant glioma cell lines.</b> One thousand cells per well were placed onto a 96-well tissue culture plate and incubated overnight. TMZ was added at concentrations of 0–800 µM and incubated for 0–7 days before an MTS assay was performed. T98G and LN18 did not exhibit growth inhibition by TMZ even at a longer exposure.</p

<i>In vitro</i> growth-inhibitory effect of co-treatment of ZOL with TMZ on MGMT-expressing malignant glioma cells.

<p><b>A, Growth-inhibitory effect of ZOL in human malignant glioma T98G cell line.</b> One thousand cells per well were placed onto a 96-well tissue culture plate and incubated overnight. ZOL was added at concentrations of 0–80 µM and incubated for 0–5 days before an MTS assay was performed. T98G did not exhibit substantial growth inhibition by ZOL (40 µM) at 120 hours. <b>B, Growth-inhibitory effect of TMZ and ZOL combination in human malignant glioma cell lines. </b><i>Upper</i> One thousand cells per well were placed onto a 96-well tissue culture plate and incubated overnight. TMZ (100 µM) and/or ZOL (40 µM) was added and incubated for 0–5 days before an MTS assay was performed. This figure is representative of three independent experiments. Bars, SD. *, P<0.05. <i>Lower</i> In the same experiment as <i>Upper</i>, an MTS assay after 120 hours incubation was highlighted. T98G and LN-18 exhibited significant growth inhibition by 100 µM TMZ plus 40 µM ZOL despite modest inhibition by each drug. <b>C, Isobologram analysis of growth-inhibitory effect of TMZ and ZOL combination in human malignant glioma T98G cell line.</b> Cells were treated with TMZ and/or ZOL as a single drug or in combination. Combination effect was analyzed at the 50% growth-inhibition ratio endpoints. The isobologram was based upon the dose-response curves, as determined by MTS assay. The X- and Y-axis indicate the ZOL and TMZ concentrations (µM), respectively. The isobologram plotted at the 50% endpoint was curved downward, suggesting synergistic interaction between TMZ and ZOL in T98G.</p

<i>In vivo</i> growth-inhibitory effect of co-treatment of ZOL with TMZ on MGMT-expressing malignant glioma xenografts.

<p><b>A, Anti-tumor effect of TMZ and ZOL combination in human malignant glioma LN-18 cell line.</b> BALB/cA nude mice (female, 5–6 weeks old) bearing LN-18 tumor were separated into four treatment groups; None, TMZ, ZOL and TMZ + ZOL. TMZ (10 mg/kg) was administered i.p. on day 1, 2, 3, 4, 5. ZOL (5 mg/kg) was injected i.p. on day 1, 3, 5 of each week for 2 weeks. At day 35, combination of TMZ and ZOL apparently inhibited tumor growth of LN-18. <b>B, Anti-tumor effect of TMZ and ZOL combination in human malignant glioma LN-18 cell line. </b><i>Left</i> BALB/cA nude mice bearing LN-18 tumor were treated with or without TMZ and/or ZOL. Tumor length and width were measured in situ with digital calipers once a week for 5 weeks (35 days). Tumor volume was calculated once a week for 5 weeks (35 days) according to the following equation: tumor volume (mm³)  = π/6×length× (width)². Points represent mean values ± SE. Combination of TMZ and ZOL significantly inhibited tumor growth of LN-18. <i>Right</i> In the same experiment as <i>Left</i>, body weight was measured once a week for 5 weeks (35 days). Points represent mean values ± SE. Approximate 10% body weight loss occurred in treated mice, whereas body weight was recovered during the observation period.</p

Apoptosis-mediated cell death of MGMT-expressing malignant glioma cells by co-treatment of ZOL with TMZ.

<p><b>A, Apoptotic Cell Death with TMZ and ZOL combination in human malignant glioma cell lines.</b> Cells were treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours. The cytoplasmic histone-associated DNA fragments, which are indicative of ongoing apoptosis, were quantitatively measured by using the photometric enzyme-immunoassay method. Combination of TMZ and ZOL synergistically induced apoptotic cell death in T98G and LN-18. Apoptotic response of LN-229 to TMZ (100 µM) was served as a positive control. Bars, SD. *, P<0.05. <b>B, Apoptotic cell death by TMZ and ZOL combination in human malignant glioma T98G cell line.. </b><i>Left</i> Cells were treated with TMZ (100 µM) and ZOL (40 µM) for 72 hours and stained by Hoechst 33342. Apoptosis was evidenced by chromatin margination in the cell nucleus. Scale bar represents 100 µm. <i>Right</i> Cells were treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours and stained by Propidium Iodide. Cell death was more frequent in the combination treatment with Temozolomide and ZOL. Scale bar represents 100 µm. <b>C, Apoptotic cell death by TMZ and ZOL combination in human malignant glioma T98G cell line.</b> Cells were treated with TMZ (100 µM) and/or ZOL (40 µM) for 72 hours and stained by Annexin-V-FLUOS and Propidium Iodide solution. Phosphatidylserine on the outer leaflet of apoptotic cell-membranes was apparent in the combination treatment with Temozolomide and ZOL. Scale bar represents 100 µm. <b>D, Effect of TMZ and ZOL combination on cleavage of caspase-3 or PARP in human malignant glioma cell lines.. </b><i>Upper</i> Cells were treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours. Expression of cleaved caspase-3 and PARP were analyzed by immunoblotting. <i>Lower</i> Relative densitometric units of the cleaved caspase-3 and PARP bands in each cell treated with or without TMZ and/or ZOL. The density of the None band is set arbitrarily at 1.0. Bars, SD. Activities of caspase-3 and PARP were prominent in the combination treatment with Temozolomide and ZOL.</p

Effect of co-treatment of ZOL with TMZ on MGMT expression in MGMT-expressing malignant glioma cells.

<p><b>A, Expression of <i>MGMT</i> gene in human malignant glioma cell lines.</b> RNA was isolated from cells treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours. <i>MGMT</i> mRNA expression was assessed by RT-PCR. <i>GAPDH</i> was used as a loading control. ZOL down-regulated <i>MGMT</i> mRNA expression in T98G and LN-18. <b>B, Expression of MGMT protein in human malignant glioma cell lines. </b><i>Upper</i> Protein extracts were prepared from cells treated with or without TMZ (100 µM) and/or ZOL (40 µM) for 72 hours. MGMT expression was analyzed by immunoblotting. β-actin was used as a loading control. <i>Lower</i> Relative densitometric units of the Ras-GTP bands in each cell treated with or without TMZ and/or ZOL. The density of the None band is set arbitrarily at 1.0. Bars, SD. ZOL down-regulated MGMT protein expression in T98G and LN-18.</p

A diagram of the proposed mechanism showing anti-tumor effect of TMZ in combination with ZOL against malignant glioma cells expressing MGMT.

<p>According to the results of the present study, ZOL inhibits the activity of Ras and the expression of MGMT in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. The molecular mechanism leading to the pathway indicated by the dotted arrow remains to be determined.</p

Additional file 2: Table S1. of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Molecular and clinical characteristics of Cohort 1 (n = 758). Table S2. Molecular and clinical characteristics of GBM cohort (n = 453). Table S3. Univariate and multivariate Cox regression analyses for Group A (IDH mutated-TERT mutated) tumors in Cohort 1 (n = 155). Table S4. Univariate and multivariate Cox regression analyses for Group B (IDH mutated-TERT wild-type) tumors in Cohort 1 (n = 131). Table S5. Univariate and multivariate Cox regression analyses for Group C (IDH wild-type-TERT wild-type) tumors in Cohort 1 (n = 237). Table S6. Univariate and multivariate Cox regression analyses for Group D (IDH wild-type-TERT mutated) tumors in Cohort 1 (n = 235). Table S7. Univariate and multivariate Cox regression analyses for GBM in Cohort 1 (n = 260). Table S8. Univariate and multivariate Cox regression analyses for GBM in Cohort 2 (n = 193). Table S9. Background of combined GBM cohort stratified by TERT and MGMT status (n = 453). Table S10. Survival time and WHO grade in each molecular subgroup of Cohort 1 (n = 758). (XLSX 254 kb

Additional file 1: of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Supplementary Information. (DOCX 141 kb

Additional file 3: Figure S1. of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Distributions of molecular alterations according to histology in Cohort 1. Figure S2. Kaplan-Meier analysis for Group A cases stratified by 1p/19q status. Figure S3. Kaplan-Meier analyses for GBM cases in Cohorts 1 and 2. (PPTX 172 kb