Selective activation of TNFR1 and NF-κB inhibition by a novel biyouyanagin analogue promotes apoptosis in acute leukemia cells

Background: Acquired resistance towards apoptosis is a hallmark of cancer. Elimination of cells bearing activated oncogenes or stimulation of tumor suppressor mediators may provide a selection pressure to overcome resistance. KC-53 is a novel biyouyanagin analogue known to elicit strong anti-inflammatory and anti-viral activity. The current study was designed to evaluate the anticancer efficacy and molecular mechanisms of KC-53 against human cancer cells. Methods: Using the MTT assay we examined initially how KC-53 affects the proliferation rates of thirteen representative human cancer cell lines in comparison to normal peripheral blood mononuclear cells (PBMCs) and immortalized cell lines. To decipher the key molecular events underlying its mode of action we selected the human promyelocytic leukemia HL-60 and the acute lymphocytic leukemia CCRF/CEM cell lines that were found to be the most sensitive to the antiproliferative effects of KC-53. Results: KC-53 promoted rapidly and irreversibly apoptosis in both leukemia cell lines at relatively low concentrations. Apoptosis was characterized by an increase in membrane-associated TNFR1, activation of Caspase-8 and proteolytic inactivation of the death domain kinase RIP1 indicating that KC-53 induced mainly the extrinsic/death receptor apoptotic pathway. Regardless, induction of the intrinsic/mitochondrial pathway was also achieved by Caspase-8 processing of Bid, activation of Caspase-9 and increased translocation of AIF to the nucleus. FADD protein knockdown restored HL-60 and CCRF/CEM cell viability and completely blocked KC-53-induced apoptosis. Furthermore, KC-53 administration dramatically inhibited TNFα-induced serine phosphorylation on TRAF2 and on IκBα hindering therefore p65/NF-κΒ translocation to nucleus. Reduced transcriptional expression of pro-inflammatory and pro-survival p65 target genes, confirmed that the agent functionally inhibited the transcriptional activity of p65. Conclusions: Our findings demonstrate, for the first time, the selective anticancer properties of KC-53 towards leukemic cell lines and provide a detailed understanding of the molecular events underlying its dual anti-proliferative and pro-apoptotic properties. These results provide new insights into the development of innovative and targeted therapies for the treatment of some forms of leukemia

Alport syndrome: Proteomic analysis identifies early molecular pathway alterations in Col4a3 knock out mice

Aim: Alport syndrome (AS) is the second most common hereditary kidney disease caused by mutations in collagen IV genes. Patients present with microhaematuria that progressively leads to proteinuria and end stage renal disease. Currently, no specific treatment exists for AS. Using mass spectrometry based proteomics, we aimed to detect early alterations in molecular pathways implicated in AS before the stage of overt proteinuria, which could be amenable to therapeutic intervention. Methods: Kidneys were harvested from male Col4a3−/− knock out and sex and age-matched Col4a3+/+ wild-type mice at 4 weeks of age. Purified peptides were separated by liquid chromatography and analysed by high resolution mass spectrometry. The Cytoscape bioinformatics tool was used for function enrichment and pathway analysis. PPARα expression levels were evaluated by immunofluorescence and immunoblotting. Results: Proteomic analysis identified 415 significantly differentially expressed proteins, which were mainly involved in metabolic and cellular processes, the extracellular matrix, binding and catalytic activity. Pathway enrichment analysis revealed among others, downregulation of the proteasome and PPAR pathways. PPARα protein expression levels were observed to be downregulated in Alport mice, supporting further the results of the discovery proteomics. Conclusion: This study provides additional evidence that alterations in proteins which participate in cellular metabolism and mitochondrial homeostasis in kidney cells are early events in the development of chronic kidney disease in AS. Of note is the dysregulation of the PPAR pathway, which is amenable to therapeutic intervention and provides a new potential target for therapy in AS