Fig 1: Assessment of PARP-1 and MGMT. (A) PARP-1 (B) MGMT (T98-G cells) (C) PARP-1 (U87-MG cells) concentration measurement by Elisa kit. (D) Expression of PARP-1 in T98-G cells by Western blotting. Lanes 1, 2, 3, and 4 are the control (no drug), naringin (243 μM), TMZ (212.5 μM), and naringin + TMZ (243 + 212.5 μM) groups, respectively, in the presentative Western blot picture of PARP-1 protein. The loading control used was GAPDH. For quantification of blots and images ImageJ software was used. (E) Expression of PARP-1 and MGMT by flow cytometry analysis. The percentage of protein expression is represented on the histogram. Results were presented as mean ± SD, n = 3. The D'Agostino-Pearson omnibus post hoc test was used to analyze the results’ normality of distribution. One-way ANOVA and Tukey’s multiple comparison tests were used for every possible comparison between the study groups. (*) p < 0.05, vs control; (#) p < 0.05 vs naringin; ($) p < 0.05 vs TMZ. Abbreviation: PARP-1 - Poly [ADP-ribose] polymerase 1, MGMT - O-6-Methylguanine-DNA Methyltransferase, TMZ - Temozolomide.
Fig 2: Mechanism depicting the pathway through which naringin in combination with TMZ causes chemosensitization of TMZ towards glioblastoma cells by inhibiting the DNA repair pathway (PARP-1 and MGMT) and causing apoptosis in tumor cells by increasing the expression of p53, caspase-3, PI3K and by decreasing the expression of Bcl-2. Abbreviation: TMZ - Temozolomide, PARP-1 - Poly [ADP-ribose] polymerase 1, MGMT - O-6-Methylguanine-DNA Methyltransferase, Bcl2 - B-cell lymphoma 2, Phospho PI3K - Phospho Phosphatidylinositol 3-kinase, Bax - Bcl-2 Associated X-protein, Bak - B-cell lymphoma 2 (BCL-2) antagonist/killer, APAF-1 - Apoptotic protease activating factor 1.
Fig 3: ATRA treatment reduces the expression of the TMZ resistance gene MGMT in GBM cell lines. Relative mRNA expression levels of MGMT in (A) U87-MG cells and (B) A172 cells following treatment with vehicle (DMSO) or 1 µM ATRA for 5 days. Expression was quantified by qPCR, normalized to the geometric mean of GAPDH and ACTB, and calculated relative to the vehicle control group (set to 1) using the ΔΔCt method. Data are presented as mean fold change ± SEM from three biological replicates. Statistical significance was determined by Student’s t-test. * p < 0.05, ** p < 0.01.
Supplier Page from Abcam for Human MGMT ELISA Kit