PMC

3-Cl-AHPC-mediated activation of NF-κB canonical and noncanonical pathways. 3-Cl-AHPC enhanced binding of HSP90 to Cdc37 and the binding of this complex to IKKα and activation of IKKα and IKKβ in NF-κB canonical pathway, and is summarized in the text. In the NF-κB noncanonical pathway, 3-Cl-AHPC exposure resulted in the rapid decrease in c-IAP1 and activation of NIK. Enhanced NIK expression occurred within 6 h of 3-Cl-AHPC exposure with the increased binding of NIK to TRAF3, activation of IKKα, phosphorylation of NF-κB2p100, its subsequent processing through the proteasome pathway and the increase in p52 levels

TNFα mediates IKKβ phosphorylation/activation. (a) TNFα (10 ng/ml) induces phosphorylation of IKKβ but not of IKKα. (b) TNFα has no effect on cIAP1, XIAP and p-Bad protein levels. (c and d) TNFα and 3-Cl-AHPC-mediated apoptosis in MDA-MB-468 and KG-1 cell lines at 24 h. The cells were treated with TNFα (10 ng/ml) 2 h before adding 3-Cl-AHPC (1 μM). Apoptosis was analyzed by flow cytometry as described in Materials and Methods section. Error bars indicate standard deviations

3-Cl-AHPC apoptosis induction requires degradation of c-IAP1. (a) 3-Cl-AHPC decreases TRAF3 binding with c-IAP1. (b) 3-Cl-AHPC induces loss of c-IAP1 and XIAP expression in MDA-MB-468 and KG-1 cells. (c) Overexpression of c-IAP1 inhibited 3-Cl-AHPC-mediated apoptosis in pcDNA3-Flag-c-IAP1 expression vector stably transfected MDA-MB-468 cells. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1 μM) for 24 h as described in Materials and Methods section. Apoptosis was assessed by flow cytometry. ^** significantly different from 3-Cl-AHPC-treated vector cells (P-value is <0.01 as determined by t-test). (d) Overexpression of c-IAP1 blocked 3-Cl-AHPC-mediated NIK stabilization and (e) densitometric analysis of NIK protein expression

Knockout (KO) and knockdown (KD) of IKKs attenuates 3-Cl-AHPC-mediated apoptosis in MEF and MDA-MB-468 cells. (a and b) Knockout of IKKα, IKKβ and IKKγ inhibited 3-Cl-AHPC-mediated apoptosis in MEF cells. (c and d) Knockdown of IKKα and IKKβ inhibited 3-Cl-AHPC-mediated apoptosis in IKKα-KD and IKKβ-KD MDA-MB-468 stably transfected cells for 24 h. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1 μM) for the indicated time. Apoptosis of cells was assessed by flow cytometry. Columns represent mean of three independent experiments, and error bars indicate standard deviations. ^** significantly different from sh-vector and MEF wild-type 3-Cl-AHPC treated cells (P-value is <0.01 respectively, as determined by t-test)

Knockdown of IKKα and IKKβ decreases IKK and NF-κB phosphorylation levels, 3-Cl-AHPC-mediated NIK stabilization and binding of TRAF3 with NIK in the NF-κB noncanonical pathway. (a) phosphorylation of IKKα, IKKβ in knockdown (IKKα-KD and IKKβ-KD) MDA-MB-468 cells and (b) loss of IKKα but not of IKKβ expression inhibited the phosphorylation of NF-κB2p100 and generation of p52 in IKKα-KD cells. (c) 3-Cl-AHPC increases the expression of NIK at 24 h and the densitometric analysis of NIK levels (right panel). (d) Proteasome inhibitor MG132 (20 μM) increased NIK expression more than the increase noted with 3-Cl-AHPC. Cells were grown and pre-incubated with MG132 for 2 h then exposed to vehicle or 3-Cl-AHPC (1 μM) for 24 h. (e) 3-Cl-AHPC-mediated TRAF2 and TRAF3 expression in cells. (f) TRAF3 increased binding with NIK in 3-Cl-AHPC-treated cells. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1 μM) for various indicated times

Interactions of Cdc37 with HSP90 and IKKα are essential for 3-Cl-AHPC-mediated NF-κB activation and apoptosis. (a) 3-Cl-AHPC-mediated Cdc37 increased binding with HSP90 and IKKα and 3-Cl-AHPC induction of Cdc37 protein expression after 3-Cl-AHPC exposure. (b) Phosphorylation of IKKα and IKKβ in Cdc37-KD cells. (c) Loss of Cdc37 expression inhibited 3-Cl-AHPC- and TNF-mediated NF-κB activation. (d) Knockdown of Cdc37 expression inhibited 3-Cl-AHPC-mediated apoptosis, and apoptosis was analyzed by flow cytometry. (e) Inhibition of Cdc37 expression in cells (Cdc37-KD) blocked HSP90 binding with IKKα. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1 μM) for various time; immunoprecipitations and immunoblots were performed as described in Material and Methods section. Columns represent mean of two independent experiments, and error bars indicate standard deviations. ^* and ^** significantly different from 3-Cl-AHPC-treated sh-vector cells (P-value is <0.05 and <0.01, respectively, as determined by t-test)

3-Cl-AHPC-mediated phosphorylation of IKKα and IKKβ, and activation of the NF-κB canonical and noncanonical pathways. (a) 3-Cl-AHPC activates NF-κB in KG-1 cells; cells were transduced with lentiviral NF-κB reporter (GFP) particles transiently for 48 h and then treated with 1 μM 3-Cl-AHPC for 24 h. (b) NF-κB activation inhibitor JSH-23 blocks 3-Cl-AHPC-mediated apoptosis. Induction of apoptosis and cell death was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining; the percentage of apoptotic cells corresponds to the sum of percent noted in upper right (late apoptotic cells, annexin V and PI-positive cells) and lower right (early apoptotic cells, annexin V positive, PI-negative) quadrants. KG-1 cells were exposed to 1 μM 3-Cl-AHPC for 24 h. (c) 3-Cl-AHPC enhances phosphorylation of IKKα and IKKβ. (d) 3-Cl-AHPC induces phosphorylation of NF-κBp65/RelA at Ser276. (e) 3-Cl-AHPC enhances phosphorylation of NF-κB2p100 followed by processing to p52 in both cell lines. (f) 3-Cl-AHPC induces increased RelB expression and the RelB binding with NF-κB2p100/p52. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1.0 μM) for various times as described in Materials and Methods section. Columns represent mean of two independent experiments. Error bars indicate standard deviations. ^* and ^** significantly different from control cells; and JSH-23+3-Cl-AHPC from 3-Cl-AHPC treated cells, respectively (P-value is <0.05 and <0.01 as determined by t-test)