Anti-FADD polyclonal antibody [Alexa Fluor® 647]

PurposeThe current study was designed to evaluate the toxicity of the secondary metabolites of Lactobacillus plantarum against the human breast cancer cell line (MCF-7) and the Drosophila melanogaster.MethodsIn this study, toxicity analyses of secondary metabolites of Lactobacillus plantarum were analyzed on breast cancer cells, and the Drosophila melanogaster. After application, in the MCF-7 cell line, expression levels of RRAS-2, TP53, BCL-2, APAF-1, CASP-3, FADD, CASP-7, BOK genes; in D. melanogaster; expression levels of RAS64B P53, BUFFY, DARK, DECAY, FADD, DRICE, and DEBCL genes were determined by RT-PCR. In addition, analysis of L. plantarum secondary metabolite was performed by GC-MS method and molecular binding poses of secondary metabolites and human enzymes were investigated in silico.ResultsDrosophila melanogaster being used as a model organism where some of the human genes were preserved. The IC50 value of the secondary metabolite in the MCF-7 cell line was determined to be 0.0011 mg/ml. Lethal concentration 50 (LC50) and 99 (LC99) values of secondary metabolites against fruit fly adults were 0.24 mg/ml and 0.54 mg/ml, respectively. The expression levels of BCL-2 and BUFFY genes which are anti-apoptotic in human and fruit flies have been reduced, and at the same time, increased expression of DECAY, FADD, RAS64B apoptotic genes in D. melanogaster.ConclusionThe substance detected in the secondary metabolite content and encoded as L13 (3-phenyl-1, 2, 4-benzotriazine) has been observed to have high binding affinity in the studied genes.

BackgroundMechanisms by which Smac mimetics (SMs) interact with proteasome inhibitors (e.g., bortezomib) are largely unknown, particularly in multiple myeloma (MM), a disease in which bortezomib represents a mainstay of therapy.MethodsInteractions between the clinically relevant IAP (inhibitor of apoptosis protein) antagonist birinapant (TL32711) and the proteasome inhibitor bortezomib were investigated in multiple myeloma (MM) cell lines and primary cells, as well as in vivo models. Induction of apoptosis and changes in gene and protein expression were monitored using MM cell lines and confirmed in primary MM cell populations. Genetically modified cells (e.g., exhibiting shRNA knockdown or ectopic expression) were employed to evaluate the functional significance of birinapant/bortezomib-induced changes in protein levels. A MM xenograft model was used to evaluate the in vivo activity of the birinapant/bortezomib regimen.ResultsBirinapant and bortezomib synergistically induced apoptosis in diverse cell lines, including bortezomib-resistant cells (PS-R). The regimen robustly downregulated cIAP1/2 but not the canonical NF-B pathway, reflected by p65 phosphorylation and nuclear accumulation. In contrast, the bortezomib/birinapant regimen upregulated TRAF3, downregulated TRAF2, and diminished p52 processing and BCL-X-L expression, consistent with disruption of the non-canonical NF-B pathway. TRAF3 knockdown, ectopic TRAF2, or BCL-X-L expression significantly diminished birinapant/bortezomib toxicity. The regimen sharply increased extrinsic apoptotic pathway activation, and cells expressing dominant-negative FADD or caspase-8 displayed markedly reduced birinapant/bortezomib sensitivity. Primary CD138(+) (n=43) and primitive MM populations (CD138(-)/19(+)/20(+)/27(+); n=31) but not normal CD34(+) cells exhibited significantly enhanced toxicity with combined treatment (P<0.0001). The regimen was also fully active in the presence of HS-5 stromal cells or growth factors (e.g., IL-6 and VEGF). Finally, the regimen was well tolerated and significantly increased survival (P<0.05 and P<0.001) compared to single agents in a MM xenograft model. Combined treatment also downregulated cIAP1/2 and p52 while increasing PARP cleavage in MM cells in vivo.ConclusionsOur data suggest that birinapant and bortezomib interact synergistically in MM cells, including those resistant to bortezomib, through inactivation of the non-canonical NF-B and activation of the extrinsic apoptotic pathway both in vitro and in vivo. They also argue that a strategy combining cIAP antagonists and proteasome inhibitors warrants attention in MM.