PU-H71 effectively induces degradation of IjB kinase b
in the presence of TNF-a
Zhuling Qu • Shiduan Wang • Ruyang Teng •
Xuanlong Yi

Received: 21 May 2013 / Accepted: 27 September 2013 / Published online: 10 October 2013
© Springer Science+Business Media New York 2013

Abstract This study is to determine if PU-H71, a heat shock protein inhibitor, induces killing of malignant breast cells together with treatment of tumor necrosis factor-a (TNF-a). The related molecular mechanisms were also studied. A primary mammary epithelial cell line HMEC2595 cells and the highly metastatic breast cell line MDA-MB-231, the HER2-positive BT-474 cells, and the ER-positive MCF7 cells were treated with PU-H71 in the presence or absence of TNF-a. The effects of PU-H71 and TNF-a treatments on cells viabilities and on intracellular signaling pathway proteins were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- mide assay, apoptosis assays, immunoblot assays, and luciferase assays. It was found that TNF-a enhances the toxic effects of PU-H71 on tumor cells but not normal cells. PU-H71 treatments lead to degradation of IKKb. Moreover, PU-H71 down-regulates the NF-jB transcrip- tional activity induced by TNF-a treatment. The experi- mental results indicated PU-H71 effectively induces cell killing of malignant breast cells in the presence of TNF-a, possibly through a mechanism related to degradation of IKKb. It is suggested that combination of PU-H71 and TNF-a treatments might be an effective therapeutic strat- egy of breast malignancies.

Keywords PU-H71 · IKKb · TNF-a ·
NF-jB pathway

Z. Qu (&) S. Wang (&) R. Teng X. Yi
· · ·
The Affiliated Hospital of Medical College, Qingdao University,
Qingdao 266021, Shandong Province, China e-mail: [email protected]
S. Wang
e-mail: [email protected]
Introduction

The heat shock protein (Hsp90) is a chaperone protein responsible for the correct folding and functionalities of some proteins [1–4]. Such kinds of proteins are usually termed as client proteins of Hsp90. It has been reported that many of the Hsp90 client proteins are involved in key oncogenic pathways including proliferation, cell cycle pro- gression, inhibition of apoptosis, and metastasis [5]. In the presence of Hsp90 inhibitors, the chaperoning function of Hsp90 is blocked and the client proteins are misfolded, and then ubiquitinated and targeted for proteasomal degradation [6–8]. Such kinds of Hsp90 inhibitors have been found to be able to induce killing of many kinds of malignant cells [6]. PU-H71 is a novel purine-scaffold Hsp90 inhibitor [6], which was developed in the laboratory of Dr. Gabriela Chiosis at Memorial Sloan Kettering Cancer Center and was licensed to Samus Therapeutics, USA. PU-H71 is recently shown to have therapeutic effects on some malignancies [9– 12]. However, it needs to continue to study if PU-H71 can be applied in combination with other agents to generate a better method for treating breast malignancies.
It has been reported that tumor necrosis factor-a (TNF- a) plays important roles in cell proliferation, survival, differentiation, and apoptosis [13]. Because TNF-a may induce apoptosis of tumor cells, it may be used to induce killing of the tumor cells. However, TNF-a also induces activation of several other cellular pathways such as cell proliferation and survival processes, which may facilitate tumor development [14]. Therefore, other chemotherapy may be needed to increase the apoptosis induction of TNF- a, but decreases its activation function in the cell prolif- eration and survival processes. Therefore, novel reagents are necessary to be used in combination with TNF-a for treating malignancies.

The transcription factor nuclear factor-kappaB (NF-jB) plays important roles in various cellular processes, such as cell proliferation, survival, apoptosis pathways, which are important for development of many human malignancies [15–17]. The human NF-jB transcription factors are bound by the inhibitory jB (IjB) proteins under unstimulated conditions [18]. Cellular stimuli, including TNF-a, lead to NF-jB activation. Activation of IjB kinases (IKKa and IKKb) leads to IjB phosphorylation and subsequent ubiquitin-dependent degradation by the cellular proteaso- mal pathway [19, 20]. The released NF-jB transcription factor then enters into the nucleus to regulate expression of genes encoding cytokines, cytokine receptors, and apop- totic regulators [21, 22].
IKKb is an Hsp90 client protein [23, 24], therefore we hypothesize that combined treatments with PU-H71 and TNF-a may lead to a promising antitumor effects if PU- H71 can decrease the survival signaling properties of TNF-
a. In this study, we have determined the effects of PU-H71 and TNF-a treatments on multiple cell lines, including the highly metastatic cell line MDA-MB-231, the HER2- positive BT-474 cells, and the ER-positive MCF7 cells. It is found that the combined treatments result in killing of all of these three types of malignant cells, but much less toxicity to the normal cells. It is also indicated that such a killing may be related to the decreases in IKKb levels in the presence of PU-H71. Our results suggest that combi- nation of PU-H71 and TNF-a might be an effective ther- apeutic strategy to treat malignant cells.

Materials and methods

Cell lines and reagents

Three breast cancer cell lines, including the highly meta- static cell line MDA-MB-231, the HER2-positive BT-474 cells, and the ER-positive MCF7 cells, and a primary mammary epithelial cell line (HMEC2595), were provided by Shanghai Cell Biology Institute (Shanghai, China). MDA-MB-231 cells were grown in DMEM (Sigma- Aldrich Co. Ltd, Irvine, CA) containing 10 % FBS (Hy- clone), 100 U/mL penicillin, and 100 lg/mL streptomycin. BT-474 cells were grown in RPMI 1640 (Sigma-Aldrich Co. Ltd, Irvine, CA) supplemented with L-glutamine, 10 % FBS, 100 U/mL penicillin, and 100 lg/mL streptomycin. The MCF-7 cells were cultured in alpha minimal essential medium (a-MEM) (Sigma-Aldrich Co. Ltd, Irvine, CA) containing 10 % FBS, 100 U/mL penicillin, and 100 lg/ mL streptomycin. The culture conditions for HMEC2595 followed supplier recommendations. Cells were cultured at 37 °C with 5 % CO2 and 100 % humidity. PU-H71 (In- vivoGen, San Diego, CA) and TNF-a were dissolved in
DMSO. Recombinant Human TNF-a was purchased from Promega (Madison, WI, USA). Bortezomib was purchased from LC Lab (Woburn, MA) and dissolved in DMSO.

Cell treatments and the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrozolium bromide (MTT) assay

Cells at a density of 1 9 105 cells/well were seeded into six- well plates in medium and were cultured for 24 h. The cells were then treated with vehicle control (DMSO, 0.016 %, v/v), TNF-a (10 ng/mL), PU-H71 (0.05 lM), or TNF-a
(10 ng/mL) and PU-H71 (0.05 lM). At the end of each experiment, cells were incubated with 0.5 mg/mL MTT at 37 °C for 4 h. The MTT kit was purchased from Life Technologies, USA. The supernatants were discarded, and 100 lL of DMSO was added into each well. The plates were shaken for 10 min. The growth status and morphological changes of the cells were detected under an inverted micro- scope. The absorbance was determined at 540 nm using a Synergy HT microplate reader (Molecular Devices, Sunny- vale, USA). Viability of treated cells was expressed relative to control cells treated with DMSO (relative viability).

Apoptosis assay

Cells at a density of 1 9 105 cells/well were cultured in six-well plates in medium supplemented with 10 % calf serum for 24 h, followed by addition of DMSO (0.016 %, v/v), TNF-a (10 ng/mL), PU-H71 (0.05 lM), or TNF-a
(10 ng/mL) and PU-H71 (0.05 lM). After 48 h, cells were pelleted by centrifugation, washed once with PBS, fixed by incubation in 4 % paraformaldehyde for 30 min at room temperature, and then washed again with PBS. The fixed cells were resuspended in PBS that contained Hoechst 33258 (5 lg/mL; Sigma-Aldrich Co. Ltd, Irvine, CA), followed by an incubation at room temperature for 15 min in the dark. Aliquots of cells were placed on glass slides and examined for cells with apoptotic morphology (nuclear condensation and chromatin fragmentation) via fluores- cence microscopy. To quantify the apoptosis, 300 nuclei from random microscopic fields were analyzed. Data are presented as the mean percentages of apoptotic cells.

Immunoblotting assays

Cells at a density of 1 9 105 cells/well were cultured in six- well plates in medium supplemented with 10 % calf serum for 24 h, followed by addition of DMSO (0.016 %, v/v), TNF-a (10 ng/mL), PU-H71 (0.05 lM), or TNF-a (10 ng/mL) plus PU-H71 (0.05 lM). After 48 h, cells were pelleted by cen- trifugation, washed twice with PBS. Total proteins were har- vested from cells, separated on 10 % SDS/PAGE gels, and then subjected to immunoblot analyses. The primary

antibodies against IKKa (about 85 kDa), IKKb (about 90 kDa), and b-actin were purchased from Santa Cruz, USA (anti-IKKa, cat # sc-7606, 1:200; anti-IKKb, cat # sc-8014, 1:200; anti-b-actin, cat # sc-130301, 1:10,000). The primary antibody against the phosphorylated IjBa (p-IjBa) was pur- chased from cell signaling, USA. Secondary antibodies used in this study were goat anti-mouse IgG-HRP (cat # sc-2005, 1:10,000, Santa Cruz, USA). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The immuno- blot experiments were repeated at least three times. Image quantifications were performed using ImageQuant software.

Luciferase assays

Cells were plated onto 24-well plates and incubated for 20 h at 37 °C in 5 % CO2. The control pGL3 vector DNA (0.2 lg) (purchased from Clontech Co.) or similar amounts of Luciferase-NF-jB reporter vectors (Clontech Co., USA) was cotransfected into cells with the pRL-SV40 vector using the effectene transfection reagent (Qiagen, USA) for 20 h. Cells were then treated with DMSO (0.016 %, v/v), TNF-a (10 ng/mL), PU-H71 (0.05 lM), or TNF-a (10 ng/mL) plus PU-H71 (0.05 lM) for 24 h. Luciferase activity was mea- sured using the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer’s protocol.

Quantitative reverse transcription-PCR (RT-PCR)

Total RNAs were harvested from cells using the RNeasy Kit (Qiagen, USA) according to the manufacturer’s instructions. The RT-PCR experiments were repeated at least three times. RNA (1 lL) was reverse transcribed into cDNA using ran- dom primers in a Reverse Transcription II system (Promega, USA) according to the manufacturer’s instructions. Expres- sion of mRNAs was quantified by quantitative PCR using an ABI Prism Sequence Detection System (Applied Biosys- tems). An assay reagent containing premixed primers and a VIC labeled probe (Applied Biosystems; cat. no. 4310884E) was used to quantify expression of endogenous GAPDH mRNA. Amplification of the BCL2 cDNAs and the endoge- nous GAPDH cDNA was monitored by changes in FAM and VIC fluorescent intensities, respectively, with the ABI 7900 software. The relative amounts of BCL2 transcript were normalized to the amount of GAPDH mRNA in the same sample. The primers for BCL2 are 50-AAATCCATGCAC CTAAACCTTTTG and 50-CAAATTCTACCTTGGAGG-
GAAAAAAC. The probe sequence is CCGTGGGCCCT CCA GATAGCTCAT.

Statistical analyses

The experimental data are expressed as mean ± SD. Sta- tistical software (SPSS10.0) was used for independent
sample t tests, followed by one-way variance analysis. In all analyses, P \ 0.05 was considered statistically significant.

Results

TNF-a enhances the toxic effects on tumor cells of PU-H71

Three breast cancer cell lines, including the highly meta- static cell line MDA-MB-231, the HER2-positive BT-474 cells, and the ER-positive MCF7 cells, and a primary mammary epithelial cell line (HMEC2595), were treated with TNF-a, PU-H71 (Fig. 1a), or both of them for 24 or 48 h. The treatment with DMSO served as a drug vehicle control. The cells were analyzed for differences in cell killing upon various treatments via number counting of living cells in the presence or absence of the above compounds.
Results showed that the treatments with the drug vehicle control (DMSO) did not significantly affect cell viability of all of these four types of cells, including the primary mammary epithelial cell line (HMEC2595, Fig. 1b) and three breast cancer cell lines MDA-MB-231 (Fig. 1c), BT- 474 (Fig. 1d), and MCF7 (Fig. 1e). Treatments with TNF- a had slight effects on cell viability of all of these four types of cells, leading to about 10–22 % reduction in cell numbers at day 3 (Fig. 1b–e). Treatments with PU-H71 decreased viabilities of the three breast cancer cell lines MDA-MB-231 (Fig. 1c), BT-474 (Fig. 1d), and MCF7
(Fig. 1e) by *35–80 % at day 2 and up to 95 % at day 3, but slightly decreases for the primary mammary epithelial cell line at day 3 (HMEC2595, Fig. 1b). When treated with TNF-a and PU-H71 together, the viabilities of the three breast cancer cell lines MDA-MB-231 (Fig. 1c), BT-474 (Fig. 1d), and MCF7 (Fig. 1e) were reduced by more than 88 % at day 2 and by 96 % at day 3. Combined treatments with TNF-a and PU-H71 only decreased the viability of the normal cells by 22 % (HMEC2595, Fig. 1b), suggesting that such dosages of TNF-a and PU-H71 are not toxic to normal cells. The above results suggest that TNF-a enhances the toxic effects on tumor cells of PU-H71.

TNF-a enhances the apoptosis induced by PU-H71

Since TNF-a enhances the toxic effects on tumor cells of PU-H71, it was determined that the effects of the drugs on apoptosis in all of these four types of cells. The cells were treated with either vehicle control (DMSO), TNF-a (10 ng/ mL), PU-H71 (0.05 lM), or both of TNF-a (10 ng/mL) and PU-H71 (0.05 lM). To quantify the apoptotic inci- dence, we used a fluorescence microscopic assay following staining of the drug-treated cells with Hoechst 33258.

Fig. 1 Cell treatments with DMSO, TNF-a, PU-H71, or
TNF-a and PU-H71 together. A primary mammary epithelial cell line (HMEC2595) and three breast cancer cell lines (MDA- MB-231, BT-474, and MCF7)
were treated with either vehicle control (DMSO), TNF-a (10 ng/ mL), PU-H71 (0.05 lM), or
both of TNF-a and PU-H71. Cell counts in each condition were determined by trypan blue exclusion at the time points indicated. a Structure of PU- H71. b HMEC2595 cells;
c MDA-MB-231 cells; d BT- 474 cells; e MCF7 cells. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrozolium bromide (MTT) assay
immediately before (day 0) and after 1, 2, or 3 days of incubation with the drugs.
Values are mean ± SD for three experiments. It is considered not significant, when P [ 0.05 versus control (DMSO) cell viability of each treatment.
Asterisk is considered as a significant difference, when
P \ 0.05 versus corresponding control

As shown in Fig. 2, treatment with DMSO or TNF-a resulted in only slightly increased effects on apoptosis of all of these four types of cells. PU-H71 caused apoptosis of MDA-MB-231, BT-474, and MCF7 cells with the inci- dences between 48 and 65 %, although it did not alter the apoptotic incidence of the normal HMEC2595 cells sig- nificantly when compared with the DMSO treatment. It is worthy to note that the presence of TNF-a increased the PU-H71-induced apoptosis, with the incidences up to 95 % in comparison with the treatments with PU-H71 alone. An anticancer drug bortezomib (an inhibitor of the 26S pro- teasome that is effective to induce apoptosis in the breast cancer cells) [25] served as a positive control of apoptosis induction in the experiment. These results indicated that TNF-a significantly elevated the apoptosis induced by PU- H71, although it alone did not result in a significant induction of apoptosis.
PU-H71 treatments lead to degradation of IKKb

IKKb expression is reported to be reduced by Hsp90 inhibitors in some cell types [23, 24]. To determine if PU- H71 inhibits the expression of IKKb in HMEC2595, MDA- MB-231, BT-474, and MCF7 cells, the cells were treated with either vehicle control (DMSO), TNF-a (10 ng/mL), PU-H71 (0.05 lM), or both of TNF-a (10 ng/mL) and PU- H71 (0.05 lM). The total proteins were extracted and the expression levels of IKKb were determined using immu- noblot analysis, with the cellular b-actin protein serving as a loading control. The mean normalized level of IKKb protein bands relative to that of b-actin band from the same con- dition was all calculated and subjected to statistical analyses. Representative blots were shown in Fig. 3. As shown in Fig. 3, treatment with DMSO or TNF-a did not detectably affect IKKb expression in all of these four types of cells.

Fig. 2 Detection of phenotype-dependent apoptosis induced by treatments with DMSO, TNF-a, PU-H71, or TNF-a plus PU-H71. A primary mammary epithelial cell line (HMEC2595) and three breast cancer cell lines (MDA-MB-231, BT-474, and MCF7) were treated with either vehicle control (DMSO), TNF-a (10 ng/mL), PU- H71 (0.05 lM), both of TNF-a (10 ng/mL) and PU-H71, or Bortezomib (0.01 lM). Cells were harvested 48 h later. Hoechst 33258-stained cells were examined for apoptotic characteristics (nuclear margination and chromatin condensation) using a fluores- cence microscope. Apoptotic incidence was calculated. Data were expressed as mean ± SD for three independent experiments
PU-H71, in the absence or presence of TNF-a, did not result in decreased expression of IKKb in the normal HMEC2595 cells. However, the treatments with PU-H71 decreased expression of IKKb by up to 70, 95, and 82 % in MDA-MB-231, BT-474, and MCF7 cells, respectively, according to the calculated values of the IKKb bands rel- ative to the b-actin bands. Surprisingly, the presence of TNF-a did not affect the effects of PU-H71 detectably. These results indicated that PU-H71 significantly decreased IKKb expression, although TNF-a did not affect such effect of PU-H71.
In these experiments, the effects of PU-H71 on other major NF-jB regulators, such as IKKa, were also detected. As shown in Fig. 3, the levels of IKKa were not signifi- cantly altered, except that the IKKa levels were slightly decreased in the BT-474 cells. To determine if the decreased IKKb expression resulted in reduced phosphor- ylation of the IKK target IjBa, levels of the phosphory- lated IjBa (p-IjBa) were detected. As shown in Fig. 3, PU-H71 decreased the levels of p-IjBa in MDA-MB-231, BT-474, and MCF7 cells, respectively, but not in the nor- mal HMEC2595 cells. These results are consistent to the

Fig. 3 PU-H71 decreases expression of IKKb. Cells were treated with DMSO, TNF-a (10 ng/mL), PU-H71
(0.05 lM), or both of TNF-a (10 ng/mL) and PU-H71 for 40 h. Whole-cell extracts were prepared and immunoblot analysis was performed to analyze the expression of
⦁ IKKa, IKKb, p-IjBa, and b-actin. The cellular b-actin served as a loading control. a HMEC2595 cells; b MDA-
MB-231 cells; c BT-474 cells;
d MCF7 cells

Fig. 4 PU-H71 down-regulates the NF-jB transcriptional activity induced by TNF-a treatment. a Luciferase reporter gene assay. HMEC2595 cells, MDA-MB-231 cells, BT-474 cells, and MCF7 cells were plated onto 24-well plates and incubated for 20 h at 37 °C in 5 % CO2. The control pGL3 vector DNA (0.2 lg) or similar amounts of Luciferase-NF-jB reporter vectors was cotransfected into cells with the pRL-SV40 vector using the effectene transfection reagent. Cells were then treated with DMSO (0.016 %, v/v), TNF-a (10 ng/ mL), PU-H71 (0.05 lM), or TNF-a (10 ng/mL) plus PU-H71
(0.05 lM) for 24 h. Luciferase activity was measured in cell lysates. Each experiment was repeated thrice with similar results. Asterisk is considered as a significant difference, when P \ 0.05 versus corre- sponding control. b Quantitative RT-PCR analysis of the mRNA expression levels of BCL2 mRNA. Total RNAs were harvested from cells and the RT-PCR experiments were repeated at least three times. Amplification of the BCL2 cDNAs and the endogenous GAPDH cDNA was monitored by changes in FAM and VIC fluorescent intensities, respectively, with the ABI 7900 software. The relative amounts of BCL2 transcript were normalized to the amount of GAPDH mRNA in the same sample. Asterisk is considered as a significant difference, when P \ 0.05 versus corresponding control. Data were expressed as mean ± SD for three independent experiments

reduced levels of IKKb in the MDA-MB-231, BT-474, and MCF7 cells.

PU-H71 down-regulates the NF-jB transcriptional activity induced by TNF-a treatment

Next, we performed a luciferase reporter gene assay to determine whether TNF-a induces transcriptional activity of NF-jB and, if so, whether PU-H71 affects the induction of NF-jB transcriptional activity by TNF-a. HMEC2595, MDA-MB-231, BT-474, and MCF7 cells were transfected with a control pGL3 vector DNA or similar amounts of
Luciferase-NF-jB reporter vectors together with the pRL- SV40 vector for 20 h. Cells were then treated with DMSO (0.016 %, v/v), TNF-a (10 ng/mL), PU-H71 (0.05 lM), or
TNF-a (10 ng/mL) plus PU-H71 (0.05 lM) for 24 h. Luciferase activity was measured using the Dual-Lucifer- ase Reporter Assay. The transcriptional activity of NF-jB in the cells transfected with the Luciferase-NF-jB reporter vectors was calculated as folds of those in the relative cells transfected with the empty vector.
As shown in Fig. 4a, treatments with TNF-a resulted in fourfold higher luciferase activities in HMEC2595 cells and about 7.5-fold higher luciferase activities in MDA- MB-231 cells, BT-474 cells, and MCF7 cells, when com- pared with the DMSO conditions. Treatments with PU-H71 did not lead to detectable alteration of the luciferase activities in HMEC2595 cells, but reduced the luciferase activities in MDA-MB-231 cells, BT-474 cells, and MCF7 cells by about 75 % in comparison with the DMSO con- ditions. Combination of PU-H71 with TNF-a inhibited the TNF-a-induced NFkB activity (or BCL2 transcription) in HMEC2595 cells down to the level of DMSO control. However, the activities in MDA-MB-231 cells, BT-474 cells, and MCF7 cells were reduced by about 75–85 %, in comparison with the DMSO conditions. These results indicated that PU-H71 down-regulates the NF-jB tran- scriptional activity induced by TNF-a treatment.
To determine whether PU-H71 affects the induction of NF-jB transcriptional activity by TNF-a, real-time PCR was performed to detect the mRNA levels of an NF-jB target, BCL2 gene that is involved in caspase-dependent apoptosis. As shown in Fig. 4b, TNF-a increased tran- scription of BCL2 gene in MDA-MB-231 cells, BT-474 cells, and MCF7 cells. Upon treatments with TNF-a and PU-H71 together, the BCL2 transcript levels in HMEC2595 cells were not significantly changed, but the levels in MDA- MB-231 cells, BT-474 cells, and MCF7 cells were reduced significantly, in comparison with the DMSO conditions. These results indicated that PU-H71 down-regulates the transcriptional activity of the NF-jB down-stream targets.

Discussion

There are two groups of Hsp90 inhibitors recently in advanced developmental stages, including the geldanamy- cin-based inhibitors, such as 17-AAG and 17-DMAG, and the novel purine-scaffold Hsp90 inhibitor PU-H71. 17-AAG and 17-DMAG are in phase I/phase II clinical trials for patients with advanced malignancies [26–32]. The purine-scaffold Hsp90 inhibitors have also been designed and are in advanced preclinical and phase I clinical eval- uation, since they may have even less cell toxicity due to their purine-based scaffold [27, 33].

In this study, we have determined the effects of PU-H71 and TNF-a treatments on multiple cell lines. It is found that the combined treatments result in enhanced killing of malignant cells. It is also indicated that such a killing may be related to the decreases in IKKb levels in the presence of PU-H71 and TNF-a. Our results suggest that combina- tion of PU-H71 and TNF-a might be an effective thera- peutic strategy to treat breast malignancies.
Combined treatments with TNF-a and PU-H71 had not significantly decreased the viability of the normal HMEC2595 cells, but leads to obvious toxicity on malig- nant MDA-MB-231 cells, BT-474 cells, and MCF7 cells. This result may be related to the properties of Hsp90. As one of the most abundant proteins in the cytoplasm, Hsp90 constitutes *1–2 % of the total proteins [34], although some Hsp90 may translocate to the nucleus in response to stress and other environmental stimuli [35–38]. Under normal conditions of the normal cells, there is an abundant Hsp90 chaperone reservoir, which can buffer proteostasis against environmental stress. However, under extreme environmental conditions, such as conditions in malignant cells, the chaperone reservoir is rapidly exhausted. There- fore, effects of PU-H71 may be increased correspondingly. In the other words, function of Hsp90 inhibitors can then affect the relationship between genotype and phenotype, and thus influencing human health, disease and evolu- tionary processes. This reason also explains why PU-H71 did not affect IKKb expression in normal cell.
Hsp90 is composed of a highly conserved amino-ter-
minal domain, a middle domain, followed by a carboxy- terminal domain. Hsp90 binds ATP in its amino-terminal domain, hydrolyzing it upon interaction with clients and co-chaperoning molecules. Structurally unrelated inhibitors of Hsp90, such as PU-H71, replace ATP and specifically destroy Hsp90 function. The strong conservation of the ATP-binding pocket allows PU-H71 to serve as extremely useful and specific tools for affecting HSP90 function. This may explain why TNF-a did not enhance the effect of PU- H71 on IKKb expression, while they exert a combined effect on viability of malignant MDA-MB-231 cells, BT- 474 cells, and MCF7 cells, possibly by playing roles in different cellular signaling pathways or processes.
When cells are treated with TNF-a, NF-jB is activated through RIP-mediated activation of IjB kinase and then released, allowing for its translocation into the nucleus and activation of its target genes [18]. Many cellular targets of NF-jB, such as BCL2, Bcl-xL, cIAP-1, A20, cIAP-2, and XIAP, have antiapoptotic functions. Therefore, many kinds of tumor cells are resistant or tolerant to TNF-a-induced apoptosis, although TNF-a may lead to apoptosis via the FADD-Caspase pathway and the TRAF2-JNKK2 pathway. The NF-jB activity is considered as a major factor of TNF- a resistance in cancer cells [39]. Our results clearly show
that inhibition of the NF-jB pathway by PU-H71 signifi- cantly sensitizes the TNF-a-treated cells to induced cell death, supporting the combined effects of PU-H71 and TNF-a on tumor cells through a mechanism related to the NF-jB pathway. Although our results suggest that PU-H71 may function by leading to degradation of IKKb, some other pathways or proteins may be also affected by PU- H71.

Acknowledgments This work was supported by the Shandong province tackle key problems in science and technology program (Grant No. Y2006C14) and Qingdao municipal science and technol- ogy commission (Grant No. 05-1-NS-74).

Disclosures All authors declare no financial and non-financial competing interests.

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