Hsf-1 and POB1 Induce Drug Sensitivity and Apoptosis by Inhibiting Ralbp1

Sharad S Singhal,Sushma Yadav,Kenneth Drake,Jyotsana Singhal,Sanjay Awasthi

doi:10.1074/jbc.m708703200

Sharad S Singhal, Sushma Yadav + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m708703200

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Abstract

Hsf-1 (heat shock factor-1) is a transcription factor that is known to regulate cellular heat shock response through its binding with the multispecific transporter protein, Ralbp1. Results of present studies demonstrate that Hsf-1 causes specific and saturable inhibition of the transport activity of Ralbp1 and that the combination of Hsf-1 and POB1 causes nearly complete inhibition through specific bindings with Ralbp1. Augmentation of cellular levels of Hsf-1 and POB1 caused dramatic apoptosis in non-small cell lung cancer cell line H358 through Ralbp1 inhibition. These findings indicate a novel model for mutual regulation of Hsf-1 and Ralbp1 through Ralbp1-mediated sequestration of Hsf-1 in the cellular cytoskeleton and Hsf-1-mediated inhibition of the transport activity of membrane-bound Ralbp1.

Highlights

We have addressed this prediction in H358 nonsmall cell lung cancer cell line (NSCLC) by examining the effect of Hsf-1 and POB1 on the transport of and cellular accumulation and efflux of DOX and on apoptosis
The present studies demonstrate that Ralbp1, Hsf-1, and POB1 can form a ternary complex and for the first time show that Hsf-1 inhibits the transport activity of Ralbp1
The presence of Hsf-1 and POB1 is quite effective at abrogating the transport activity nearly completely

Summary

EXPERIMENTAL PROCEDURES

Materials—The human full-length Hsf-1 cDNA was a gift of Dr Nahid F. The liposomes reconstituted with purified recombinant Hsf-1, POB1, and Ralbp proteoliposomes were used for delivery of these proteins to cells in culture. Effects of Hsf-1 and POB1 Liposomes on 14C-DOX Accumulation in Lung Cancer Cell—H358 cells were harvested and washed with PBS, and aliquots containing 5 ϫ 106 cells (in triplicate for each time point) were inoculated into fresh medium. The cells were pelleted and resuspended in 80 ␮l of medium containing 4 ␮g of Hsf-1 or POB1 proteoliposomes or control liposomes and incubated at 37 °C for 24 h. Purified recombinant Ralbp, POB1, and Hsf-1 proteins (10 ␮g each) were cross-linked by incubation with 0.1 mM N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP; from Sigma) in a total volume of 0.5 ml in 10 mM sodium phosphate buffer, pH 7.4, for 30 min. Results were processed using CXP2.2 analysis software from Beckman Coulter

RESULTS

48 K 35 K

79 K 48 K 35 K 29 K

B IHC against anti-Ralbp1 IgG in NSCLC H358

DISCUSSION