Abstract
BackgroundUrinary bladder cancer is one of the most fatal and expensive diseases of industrialized world. Despite the strenuous efforts, no seminal advances have been achieved for its clinical management. Given the importance of metabolic reprogramming in cancer cell survival and growth, we have herein employed 3-BrPA, a halogenated derivative of pyruvate and historically considered inhibitor of glycolysis, to eliminate bladder cancer cells with highly oncogenic molecular signatures.MethodsBladder cancer cells were exposed to 3-BrPA in the absence or presence of several specific inhibitors. Cell viability was determined by MTT and flow-cytometry assays; cell death, signaling activity and metabolic integrity by Western blotting and immunofluorescence; mutant-gene profiling by DNA sequencing; and gene expression by RT-sqPCR.Results3-BrPA could activate dose-dependent apoptosis (type 1 PCD) and regulated necrosis (type 3 PCD) of T24 (grade III; H-RasG12V; p53ΔY126), but not RT4 (grade I), cells, with PARP, MLKL, Drp1 and Nec-7-targeted components critically orchestrating necrotic death. However, similarly to RIPK1 and CypD, p53 presented with non-essential contribution to 3-BrPA-induced cellular collapse, while reactivation of mutant p53 with PRIMA-1 resulted in strong synergism of the two agents. Given the reduced expression of MPC components (likely imposing mitochondrial dysfunction) in T24 cells, the suppression of constitutive autophagy (required by cells carrying oncogenic Ras; also, type 2 PCD) and derangement of glucose-homeostasis determinants by 3-BrPA critically contribute to drug-directed depletion of ATP cellular stores. This bioenergetic crisis is translated to severe dysregulation of Akt/FoxO/GSK-3, mTOR/S6, AMPK and MAPK (p44/42, p38 and SAPK/JNK) signaling pathways in 3-BrPA-treated T24 cells. Sensitivity to 3-BrPA (and tolerance to glucose deprivation) does not rely on B-RafV600E or K-RasG13D mutant oncogenic proteins, but partly depends on aberrant signaling activities of Akt, MAPK and AMPK kinases. Interestingly, MCT1- and macropinocytosis-mediated influx of 3-BrPA in T24 represents the principal mechanism that regulates cellular responsiveness to the drug. Besides its capacity to affect transcription in gene-dependent manner, 3-BrPA can also induce GLUT4-specific splicing silencing in both sensitive and resistant cells, thus dictating alternative routes of drug trafficking.ConclusionsAltogether, it seems that 3-BrPA represents a promising agent for bladder cancer targeted therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0399-9) contains supplementary material, which is available to authorized users.
Highlights
Urinary bladder cancer is one of the most fatal and expensive diseases of industrialized world
None of the Akt, Glycogen synthase kinase (GSK)-3β, AMPKα and p44/42 Mitogen-activated protein kinase (MAPK) respective phosphorylation profiles figured with high Molecular weight (MW) forms, strongly reflecting settings of cellular bioenergetic crisis (Fig. 2j) and likely indicating the harmful effects of post-translational modifications in kinase signaling functions in 3-BrPA-treated T24 and T24-X cells during regulated necrosis
Brackets and asterisks denote the high MW forms of Akt (a) and p44/42 MAPK (b) kinases. (c-e) MTT cytotoxicity assays of T24 cells, seeded at low confluency and exposed to the indicated doses of 3-BrPA for 24 h, in the absence or presence of 100 μM LY294002 (c), 100 μM U0126 (d) and 1 mM 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) (e)
Summary
Reagents and chemicals 3-BrPA, PJ-34, Nec-1, Nec-5, Nec-7, Mdivi-1, CsA and UK-5099 were obtained from Sigma-Aldrich (Missouri, USA). DNA sequencing chromatograms (three independent experiments) of Exon 7 - Intron 7/8 - Exon 8 (upper panel) and Exon 9 - Intron 9/10 - Exon 10 (lower panel) RT-sqPCR (unspliced) products of 411 and 336 bp, respectively, derived from T24 cells (~60 % confluency) after their exposure to 75 μM of 3-BrPA for 24 h (see Fig. 7e and Additional file 9: Table S1). The p53-target genes PUMA and NOXA remained rather unharmed, while BAX, TIGAR and SESTRIN-1 ones followed (necrotic) dose-driven reduction of their expression levels, in response to the drug, further corroborating the engagement of p53-independent cyto/genotoxic routes in 3-BrPA-treated bladder cancer cells (Fig. 3). Note the cytoplasmic retention and non-nuclear compartmentalization of AIF protein (employment of a rabbit polyclonal antibody used at a dilution of 1:200), in control (−) cells and in ones exposed to 3-BrPA (related to Fig. 2a-b).
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