His tagged Apaf 1 variants were immobilized on a spin column selleck products and then incubated Inhibitors,Modulators,Libraries with or without GST tagged TCTP. Silver staining of the eluates revealed that TCTP interacts with all of Apaf 1 variants, suggesting the interaction of TCTP at the site of Apaf 1 CARD. A parallel experiment using WD Repeat, Inhibitors,Modulators,Libraries protein lacking CARD and CED 4 domains of Apaf 1, confirmed that CARD domain serves the site for TCTP binding to Apaf 1. Therefore, it appears that TCTP itself interacts with CARD of Apaf 1 to assemble into the apoptosome without interrupting the procaspase 9 binding to Apaf 1 in apoptosome forming condition. Fragmented TCTP specifically interacts with Apaf 1 in etoposide induced cell death Since fragmented form of TCTP was a component of in vitro reconstituted apoptosome complex, cleaved TCTP may presumably operate in association with Apaf 1 in response to apoptotic trigger while full length TCTP interacts with Na,K ATPase.

To differ entiate the interaction between Na,K ATPase and TCTP upon etoposide treatment, we performed immunopre cipitation using anti Na,K ATPase antibodies following the Inhibitors,Modulators,Libraries adenoviral infection of N terminal Flag tagged TCTP. As shown in Figure 5A, Na,K ATPase interacted with full length TCTP and etoposide treatment had no effect on this binding. When the Apaf 1 interacting molecules were precipitated in parallel experimental settings, full length TCTP found to be associated with Apaf 1 in the TCTP overexpressing untreated cells. Treatment with etoposide resulted in additional interaction of Apaf 1 with short length Flag TCTP.

Taken together these findings Inhibitors,Modulators,Libraries suggest that full length TCTP binds to Apaf 1 CARD both in normal and apop totic conditions whereas C terminal cleaved TCTP spe cifically binds to Apaf 1 under apoptosome forming con ditions in TCTP overexpressed cells. It can be inferred that when apoptotic signaling is introduced, cleaved form of TCTP binds to Apaf 1 of apoptosome to interfere with the activation of mitochondria mediated Inhibitors,Modulators,Libraries cell death, while full length TCTP is responsible for Na,K ATPase binding. Discussion Genotoxic stress or DNA damage resulting from chemo therapy activates the intrinsic apoptotic pathway which includes a sequential cascade of events leading to cell death. Defects in the apoptotic pathways have been asso ciated with tumorigenesis as well as resistance against conventional chemotherapeutics.

Downregulation of target enzyme topoisomerase II, modulation of micro RNA, and acquisition of multiple drug resistance phenotype through induction of mdr scientific research 1 and ABC trans porter genes are the major known mechanism of resistance to etoposide treatment in tumor cells. In the present study, TCTP protected cancer cells from etoposide induced cytotoxicity via sequential regulation of major events of mitochondrial apoptosis.