Figure 6

Topotecan alters the proteomic/metabolic profile of GBM cell lines and decreases the levels of HIF-1α. (A) Global SUMO-1 pull-down in line U251, probed with an anti-SUMO-1 antibody. D = DMSO (0.1%), T = Topotecan 1 μM. Input = precleared lysate, IP = immunoprecipitated lysate. Mass spectrometry detected 82 proteins co-immunoprecipitated with SUMO-1 in only the DMSO control condition, 20 proteins in only the topotecan condition, and 97 proteins in both conditions. The top 10 attenuated pathways and functions in topotecan-treated cells based on mass spectrometry results are reported; Fisher’s Exact Test was used to assess significance; x-axes represent the −log(enrichment P-value). (B) Levels of lactate, a by-product of glycolysis, decreases in a dose-dependent manner with topotecan treatment in lines U251, LN229, and Mz18. (C) Activity of glucose-6-phosphate dehydrogenase, an enzyme in the pentose phosphate pathway, decreases in a dose-dependent manner with topotecan treatment in lines U251, LN229, and Mz18. (D) Topotecan effects a dose-dependent decrease on levels of HIF-1α protein in GBM lines U251, LN229, and Mz18 at concentrations of 1 μM and 10 μM. Representative immunoblots are shown. The bands corresponding to HIF-1α (~116 kDa) were cropped in each lane and the total intensities were measured. Densities were normalized to corresponding actin levels and expressed as a fold difference relative to the control (DMSO). Data in B-D are means (±SD) from n = 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 vs. DMSO.