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Mol Cancer Ther. 2006;5:170-178
© 2006 American Association for Cancer Research

Sensitization of TRAIL–resistant cells by inhibition of heat shock protein 90 with low-dose geldanamycin

Yulin Ma2, Vijayabaskar Lakshmikanthan2, Ronald W. Lewis2 and M. Vijay Kumar1,2

1 Department of Research, VA Medical Center and 2 Department of Surgery, Section of Urology, Medical College of Georgia, Augusta, Georgia

Requests for reprints: M. Vijay Kumar, Department of Research, VA Medical Center, One Freedom Way, Augusta, GA 30904. Phone: 706-721-6620; Fax: 706-721-2548. E-mail: vkumar{at}mail.mcg.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Due to its specificity and effectiveness, tumor necrosis factor-{alpha}–related apoptosis-inducing ligand (TRAIL) is being tested for cancer therapy. Inhibition of the function of heat shock protein 90 (HSP90) is under clinical trials for cancer therapy. However, some cancer cells are resistant to TRAIL, and at the dose required for inducing apoptosis, geldanamycin, a drug that inhibits HSP90 function, has shown adverse effects. Therefore, our working plan was to identify a sublethal dose of geldanamycin and combine it with TRAIL to induce apoptosis in TRAIL-resistant prostate cancer cells. Treatment of LNCaP with 250 nmol/L geldanamycin inhibited HSP90 function but did not induce significant apoptosis. However, combination of geldanamycin and TRAIL induced highly significant apoptosis in TRAIL-resistant LNCaP cells. In addition to inducing caspase activity and apoptosis, treatment with geldanamycin and TRAIL decreased inhibitor of {kappa}B (I{kappa}B) kinase (IKK) complex proteins, IKK{alpha}, IKKß, and IKK{gamma}. The loss of IKK affected I{kappa}B{alpha}/nuclear factor-{kappa}B (NF-{kappa}B) interaction and reduced nuclear transport of NF-{kappa}B, resulting in reduced NF-{kappa}B activity. Our data show increase in apoptosis using low, suboptimal dose of geldanamycin when used with TRAIL. These results provide a means to alleviate two problems: resistance to TRAIL and adverse effects of high-dose geldanamycin. [Mol Cancer Ther 5006;5(1):170–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The American Cancer Society indicated that cancer is now the primary cause of death in America, and 30,350 people are expected to die of prostate cancer in 2005. These statistics show the enormity of the problem and the necessity of having to design new treatment options for prostate cancer. Androgen deprivation therapy has been a common treatment for prostate cancer to induce apoptosis in androgen-responsive cells (1–4). However, androgen-insensitive cells are capable of undergoing apoptosis upon appropriate stimulus (5–8), which prompted investigation of several drugs to induce apoptosis in these cells. However, the tested drugs suffer from shortcomings, such as low potency, severe adverse effects, and/or lack of specificity. Among the new generation of drugs being tested, tumor necrosis factor-{alpha}–related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor-{alpha} family has shown promise. The advantages are that TRAIL induces apoptosis preferentially in cancer cells, and in animal experiments, TRAIL did not show appreciable adverse effects (9, 10). Therefore, we examined the response of prostate cancer cells to TRAIL and showed that most prostate cancer cells were responsive to TRAIL, although some cells, such as LNCaP, were resistant (11–15).

Heat shock proteins (HSP) are molecular chaperones that transport and stabilize proteins within the cell. HSPs have been shown to bind to several client proteins, a necessary event for the continued function of the client proteins. Most of HSP90 client proteins are prosurvival, antiapoptotic proteins, such as Akt and inhibitor of {kappa}B (I{kappa}B) kinase (IKK) complex, which are functional only when they are interacting with HSP90. Thus, continued functional integrity of HSP90 favors cell proliferation as seen by the correlation of its increased expression in cancer cells with poor prognosis and resistance to chemotherapy or radiation therapy (16). Therefore, it was hypothesized that the inhibition of the function of HSP90 would down-regulate the function of antiapoptotic proteins and promote apoptosis. The first molecule successfully used for inhibiting the function of HSP90 was benzoquinone ansamycin geldanamycin, which was later modified into 17-allylaminogeldanamycin. Both geldanamycin and 17-allylaminogeldanamycin bind to HSP90 and interfere with its interaction with client proteins. As these drugs induced cell death, they are in clinical trials for cancer therapy. However, animal experiments and clinical trials have shown that the doses of geldanamycin required for inducing apoptosis also resulted in severe adverse effects, such as hepatotoxicity (17–19). Therefore, we hypothesized that using low-dose geldanamycin would eliminate adverse effects. However, to induce effective apoptosis, it was necessary to use another apoptogenic drug, such as TRAIL.

In some cancer cells, treatment with TRAIL results in some paradoxical responses. For example, TRAIL induces apoptosis by activating the death-inducing signaling complex, leading to the activation of caspase-8 and truncation of Bid. At the same time, in some cancer cells (typically TRAIL-resistant cells, such as LNCaP), TRAIL activates prosurvival proteins, such as nuclear factor-{kappa}B (NF-{kappa}B). NF-{kappa}B is a member of the Rel family, which consists of transcriptional activators (p65/RelA, p50/ NF-{kappa}B1, p52/ NF-{kappa}B2, RelB, and c-Rel) that form homodimers or heterodimers. These proteins are regulated by their interaction with one or more of the inhibitor subunits I{kappa}B{alpha}, I{kappa}Bß, or I{kappa}B{varepsilon}. In unstimulated cells NF-{kappa}B is sequestered in the cytoplasm in an inactive state due to its interaction with I{kappa}B. Upon stimulation by cytokines, I{kappa}B is phosphorylated and subsequently ubiquitinated and degraded by 26 S proteasome. NF-{kappa}B thus released from I{kappa}B is translocated into the nucleus, where it is responsible for the transcriptional activity of the target gene. NF-{kappa}B has been implicated in transformation and tumorigenesis, and recently, several laboratories have shown that NF-{kappa}B actively inhibits apoptosis by recruiting and activating antiapoptotic proteins that suppress apoptotic pathways. Thus, inhibition of NF-{kappa}B sensitizes cancer cells to apoptosis-inducing drugs (20). Furthermore, NF-{kappa}B is known to block tumor necrosis factor-{alpha}– or TRAIL-induced apoptosis by interfering directly with the function of receptor-associated proteins, such as decoy receptors, RIP, FADD, and caspase-8 (21–23).

As discussed above, phosphorylation of I{kappa}B is a key step in the regulation of NF-{kappa}B function. Phosphorylation of I{kappa}B is controlled by IKK group of proteins. The IKK complex comprises mainly two catalytic subunits IKK{alpha} (IKK1) and IKKß (IKK2) and a regulatory subunit, IKK{gamma} (also known as NEMO, NF-{kappa}B essential modulator, or IKK-associated protein 1, IKKAP1). Gene targeting studies have shown that IKKß and IKK{gamma} subunits of the IKK are required for the activation of NF-{kappa}B by proinflammatory stimuli (24). However, for the activation of NF-{kappa}B by receptor-mediated pathway, such as the tumor necrosis factor family, IKK{alpha} is required. Under certain stimuli, IKK{alpha} has been shown to affect the function of NF-{kappa}B by direct phosphorylation of I{kappa}B{alpha} (25, 26). The importance of IKK proteins in survival and tumorigenesis was shown when tissue-specific deletion of IKKß prevented tumor initiation and progression in a mouse model (27). Furthermore, in these experiments, depending upon the cell type and the context, IKKß contributed to tumor initiation and promotion by suppressing apoptosis. Thus, IKK proteins are considered effective targets for the development of drugs for cancer therapy (28). However, none of the known inhibitors of IKK are potent enough or devoid of cross-reaction with other proteins of the family. Therefore, methods to interfere with the function of IKK proteins have great potential in cancer therapy. In this article, we show that the inhibition of the function of HSP90 by geldanamycin and treatment with TRAIL induced significant apoptosis. This was especially impressive as LNCaP are resistant to TRAIL, and treatment with low, sublethal dose of geldanamycin was able to facilitate TRAIL-mediated apoptosis. Furthermore, we observed that the induction of apoptosis correlated with decreased IKK proteins and down-regulation of NF-{kappa}B activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Prostate carcinoma cell line, LNCaP (passage 24), was obtained from the American Type Culture Collection (Rockville, MD) and were grown in RPMI 1640 supplemented with 2 mmol/L L-glutamine, 4.5 g/L glucose, 10 mmol/L HEPES, 1.5 g/L sodium bicarbonate, 1 mmol/L sodium pyruvate, and 10% fetal bovine serum (Hyclone, Logan, UT) and grown in the presence of 5% CO2 at 37°C. Cells were treated with geldanamycin (Alexis Biochemicals, San Diego, CA) for 48 hours or with 200 ng/mL TRAIL (Biomol Research Laboratories, Inc., Plymouth Meeting, PA) for 4 hours. In combination experiments, cells were treated with geldanamycin for 44 hours, and TRAIL was added 4 hours before terminating the experiment. Upon completion of the experiments, cells were harvested, and total proteins, cytosol, or nuclear fractions were isolated as described below.

Preparation of Cell Lysates and Western Blotting
Cells were harvested by trypsinization and washed, and cell pellets were resuspended in lysis buffer (1x PBS, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA, 0.5 µg/µL leupeptin, 1 µg/µL pepstatin, 1 µg/µL phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin). Cells were incubated over ice for 30 minutes and centrifuged at 10,000 x g at 4°C for 10 minutes. The supernatant was collected, and the protein concentration was estimated using Bio-Rad protein reagent (Bio-Rad Laboratories, Hercules, CA).

For extracting nuclear proteins, cells were washed with cold PBS or HBSS, and the cells were released using trypsin. Cells were lysed in ice-cold lysis buffer [10 nmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.1% Triton X-100, and protease inhibitor cocktail] by passing through a No. 27 gauge needle, and the mixture was centrifuged. The supernatant was collected as cytosolic fraction, and the pellet was resuspended in nuclear extraction buffer [20 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 0.42 mol/L NaCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 1.0% Igepal CA-630, 25% (v/v) glycerol, and protease inhibitor cocktail], and the membrane were disrupted using a No. 27 gauge needle. The nuclear extract was separated by centrifugation, and the supernatant was saved at –80°C until further experimentation.

Proteins (50 µg, unless stated otherwise) were separated on NuPAGE 10% Bis-Tris gels (Novex precast mini gels, Invitrogen, Carlsbad, CA) at 100 V for 1 hour in the presence of 1x MES-SDS running buffer (Invitrogen). Separated proteins were transferred to (polyvinylidene difluoride) membranes (Bio-Rad Laboratories) at 42 V for 2.5 hours using a Novex XCell II blotting apparatus in MES transfer buffer in the presence of NuPAGE antioxidant. Transfer of the proteins to the polyvinylidene difluoride membrane was confirmed by staining with Ponceau S (Sigma, St. Louis, MO). The blots were blocked in 5% nonfat dry milk in TBST, washed twice for 10 minutes each with TBS containing 0.01% Tween 20, and incubated for overnight at 4°C with primary antibody diluted in TBST containing 0.5% milk. The list of antibodies and their source is indicated in Table 1 . Immunoreactive bands were visualized using enhanced chemiluminescence detection system (Amersham, Pharmacia Biotech, Arlington Heights, IL) on Alpha Innotech 8900 Image Analyzer. Signals were digitized, and the data were corrected for loading using the values for actin.


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  		Table 1. Antibodies used in these experiments

 
Measurement of Apoptosis
The M30 Apoptosense kit (Peviva AB, Bromma, Sweden) was used for measuring apoptosis. This is an ELISA that measures the generation of a neoepitope of cytokeratin 18 that is cleaved by the caspases activated in response to treatment. This assay measures early apoptotic changes and is more sensitive compared with other assays, such as Annexin V. This assay is specific to apoptosis and does not measure necrotic changes. Upon completion of the experiments, cells were harvested, and total protein was extracted as described above. Protein extract was added to 96-well plate coated with mouse monoclonal M30 antibody, and horseradish peroxide tracer solution was added to the wells and incubated for 4 hours, as described by the supplier. Color was developed by adding tetramethyl benzidine solution, and the absorbance was determined at 450 nm on a Spectra MAX 340 microplate reader (Molecular Devices, Menlo Park, CA). Standard solution supplied by the supplier was used for generating standard curves.

Immunocytochemical Examination of NF-{kappa}B/p65 and NF-{kappa}B/p50
LNCaP cells were treated with geldanamycin and/or TRAIL as described above. At the end of the treatment, the cells were fixed with 100% methanol at –20°C for 10 minutes. After being washed with PBS, the samples were blocked with blocking buffer at room temperature for 1 hour. The cells were incubated with anti-NF-{kappa}B/p65 or anti-NF-{kappa}B/p50 antibody overnight at 4°C. The signal was visualized using secondary antibodies of appropriate specificity conjugated to Alexa Fluor 488 or Alexa Fluor 594 (4 µg/mL, room temperature for 1 hour; Molecular Probes, Eugene, OR). Nuclei were stained using 4',6-diamidino-2-phenylindole (Molecular Probes). The fluorescence was examined in Carl Zeiss Axiophot Fluorescent microscope, and the images were captured digitally.

Transfection of NF-{kappa}B Luciferase Reporter and Luciferase Assay
LNCaP cells were grown to log phase, and 3.5 x 106 cells in 350 µL were mixed with luciferase reporter, pNF-{kappa}B-Luc construct containing four copies of NF-{kappa}B response element. Cells were subject to two cycles of 200 V of 10-millisecond pulse length, with a pulse interval of 500 milliseconds using Electroporator 830 (BTX, Inc., San Diego, CA). Electroporated cells were allowed to incubate in the cuvette for 15 minutes at room temperature before plating them into dishes. Cells were treated and harvested as described above and processed for the presence of luciferase activity. Luciferase activity was assayed using a kit from Promega Co. (Madison, WI). The firefly luciferase reporter assay was initiated by adding an aliquot of lysate to Luciferase Assay Reagent II, as described by the manufacturer. The assay was conducted in 96-well plates and the luminescence signals were measured (Fluorstar Optima, BMG Lab Technologies, Durham, NC). Luciferase activity was expressed per unit total protein. PEGFP-N1 vector (Biosciences, Clontech, San Jose, CA) or Renilla luciferase (Promega) was used as transfection controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Geldanamycin Induced Apoptosis in Prostate Cancer Cells
To determine the effects of geldanamycin on prostate cancer cells, LNCaP cells were treated with increasing concentrations (range, 10–500 nmol/L) of geldanamycin. The apoptotic response of the cells was measured using M30 Apoptosense ELISA, which measures the generation of a neoepitope of cytokeratin 18 generated due to the effects of activated caspases. Results showed that at lower doses (10, 50, or 100 nmol/L) geldanamycin did not induce apoptosis in LNCaP cells (Fig. 1A ). Increasing the dose of geldanamycin to 250 nmol/L showed marginal increase in apoptosis, whereas 500 nmol/L geldanamycin induced ~7-fold apoptosis compared with controls. As our research plan was to identify a dose that is sufficient to affect HSP and HSP-related functions but not high enough to cause significant apoptosis by itself, all further experiments were conducted with 250 nmol/L geldanamycin.


Figure 1
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  		Figure 1. Effect of geldanamycin and TRAIL on LNCaP cells. A, LNCaP cells were treated with increasing concentrations of geldanamycin (GA) for 48 h. B, LNCaP cells were treated with geldanamycin (250 nmol/L) for 44 h at which time increasing concentrations of TRAIL was added. Cells were harvested 4 h after TRAIL treatment. C, cells were treated with geldanamycin (250 nmol/L) and/or 200 ng/mL TRAIL (T). D, DU145 cells were treated as described for LNCaP. Apoptosis was measured in the cell lysates by M30 apoptosense ELISA assay.

 
Earlier reports from our and other laboratories have indicated that LNCaP are resistant to TRAIL. As one of the goals of these experiments was to determine whether disruption of the function of HSP sensitizes LNCaP to TRAIL, cells were treated with 250 nmol/L geldanamycin and increasing concentrations of TRAIL. Apoptosis assay showed no significant apoptosis until the concentration of TRAIL was raised to 200 ng/mL TRAIL, in combination with geldanamycin (Fig. 1B). Increasing the concentration of TRAIL to 400 ng/mL induced maximum apoptotic response, although no difference in apoptotic response was noted between cells treated with 200 or 400 ng/mL TRAIL. Therefore, all subsequent experiments were conducted using 200 ng/mL TRAIL and 250 nmol/L geldanamycin. Finally, combination experiments confirmed that treatment of LNCaP cells with geldanamycin and TRAIL induced synergistic apoptosis compared with geldanamycin or TRAIL alone (Fig. 1C). These experiments indicated that low-dose, subapoptotic dose of geldanamycin, which by itself did not induce significant apoptosis, sensitized TRAIL-resistant LNCaP cells.

To confirm the induction of apoptosis in these treatments, cell extracts were subject to Western blot analyses. As expected from Fig. 1, treatment with 250 nmol/L geldanamycin alone did not induce caspase-3 activity (Fig. 2A ). Combination of geldanamycin and TRAIL induced significant caspase-3 activity as seen by the presence of caspase-3 cleaved products of 19, 17, and 12 kDa. When LNCaP were treated with TRAIL alone, only a 19 kDa, cleaved fragment was observed, which correlated with low level induction of apoptosis in this group (Fig. 1). Similarly, combined treatment with geldanamycin and TRAIL induced caspase-7 activity, whereas geldanamycin alone did not activate this caspase (Fig. 2A, middle). To show the effect of activated caspases on their substrate, activation of poly(ADP-ribose) polymerase was examined. As expected from apoptosense assay and caspase activation results, poly(ADP-ribose) polymerase was cleaved into 85-kDa fragment only when cells were treated with both geldanamycin and TRAIL (Fig. 2A, bottom). The above results confirmed our earlier findings that LNCaP are resistant to TRAIL and showed that low-dose geldanamycin sensitized these cells to TRAIL by activating caspases and inducing apoptosis.


Figure 2
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  		Figure 2. Treatment of LNCaP cells with geldanamycin and TRAIL induced caspase activation. A, LNCaP were treated with low-dose geldanamycin (GA) and TRAIL as described in Fig. 1, and the cell extracts were analyzed by Western blots for the activation of caspase-3, caspase-7, or poly(ADP-ribose) polymerase (PARP). Blots were stripped and reprobed with antibody for actin, to be used as loading control. B, caspase-3 was analyzed by Western blots using extracts from DU145 cells. Representative data from at least three replicates.

 
The above results were obtained using LNCaP prostate cancer cells that are responsive to androgens and are wild type for the expression of antiapoptotic protein Bcl2. Therefore, it was of interest to determine the effect of geldanamycin/TRAIL on DU145 prostate cancer cells that are insensitive to androgens and do not express Bcl2. Treatment with geldanamycin or TRAIL alone induced 3-fold or 6-fold apoptosis in DU145, suggesting that these cells are sensitive to these drugs (Fig. 1D). Combination treatment increased apoptotic response further compared with individual treatments. Furthermore, just as in LNCaP cells, combined treatment with geldanamycin and TRAIL induced significant caspase-3 activity (Fig. 2B). As the response of both the cell lines was similar, all further experiments were conducted with LNCaP cells.

Geldanamycin Sensitized LNCaP to TRAIL by Affecting the IKK Complex
Geldanamycin treatment specifically disrupts the function of HSP90, resulting in the loss of binding of HSP90 to client proteins. Published data showed that treatment of cells with geldanamycin affected the interaction between HSP90 and the IKK protein complex, leading to the loss of function of the IKK proteins and their degradation due to proteasomal activity. Therefore, experiments were conducted to determine whether geldanamycin-mediated induction of apoptosis by TRAIL is aided by the disruption of IKK proteins. As a first step, LNCaP cells were treated with increasing concentrations of geldanamycin, and the cell extracts were analyzed for the expression of the IKK{alpha}, IKKß, and IKK{gamma}. Results indicated dose-dependent decrease in the levels of all three IKK proteins (Fig. 3A ). Reduction in IKK{alpha} levels was noticed with 50 nmol/L geldanamycin, leading to significant reduction of IKK{alpha} with 250 nmol/L geldanamycin. Further increase in the concentration of geldanamycin resulted in further loss of IKK{alpha}. It is known that the function of IKKß and IKK{gamma} depends upon the availability and normal function of IKK{alpha}. Similar to IKK{alpha}, IKKß levels reduced significantly with increasing doses of geldanamycin starting with 50 nmol/L geldanamycin. However, the response of IKKß protein to geldanamycin was significantly higher compared with IKK{alpha}, as it completely disappeared with 250 nmol/L geldanamycin. The response of IKK{gamma} was similar to that described for IKK{alpha} and IKKß. Thus, the levels of all three major proteins of the IKK complex were significantly reduced with increasing concentrations of geldanamycin. However, under these conditions, there was no change in the levels of HSP90 (Fig. 3A), suggesting that the reduction in the levels of IKK proteins is due to their degradation and not due to changes in the levels of HSP90 (19). Next, the effect of combined treatment with geldanamycin and TRAIL on the IKK proteins was examined. As observed above (Fig. 3A), 250 nmol/L geldanamycin reduced IKK{alpha} about 2.5-fold, whereas TRAIL did not affect IKK{alpha} (Fig. 3B). However, combination of geldanamycin and TRAIL resulted in 28-fold reduction in IKK{alpha} compared with control and 11-fold reduction compared with geldanamycin alone (Fig. 3B). Similarly, IKKß and IKK{gamma} showed dramatic reduction in geldanamycin-treated cells in the absence or presence of TRAIL. However, once again, under these conditions, HSP90 did not alter.


Figure 3
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  		Figure 3. Treatment with increasing concentrations of geldanamycin and/or TRAIL decreased the levels of IKK proteins. A, LNCaP cells were treated with increasing concentrations of geldanamycin (GA) for 48 h, and the levels of IKK{alpha}, IKKß, IKK{gamma}, or HSP90 were determined by using specific antibodies. The blots were stripped and reprobed with actin for loading controls. B, cells with treated with 250 nmol/L geldanamycin and/or 200 ng/mL TRAIL as described in Fig. 1. Cell extracts were analyzed for the presence of IKK{alpha}, IKKß, and IKK{gamma} or HSP90 by Western blots. C, to confirm the effects of treatment on the formation of IKK complex, IKK{gamma} was immunoprecipitated, and the presence of IKK{alpha} and IKKß in the immunoprecipitate (IP) was examined by Western blots. Representative data from at least three replicates. Con, control.

 
Among the IKK proteins, both IKK{alpha} and IKKß possess catalytic subunits and therefore are involved in phosphorylation functions of the IKK complex. To maintain their function, IKK proteins have to continue to exist as a complex. We hypothesized that loss of interaction with HSP90 would affect interactions with each other and affect their function. Therefore, specific IKK{gamma} antibody was used for immunoprecipitation, and the proteins were analyzed for the presence of other IKK proteins. Results showed significant decrease in the binding of IKK{alpha} and IKKß to IKK{gamma} in geldanamycin-treated cells (Fig. 3C). This effect was further increased in cells treated with both geldanamycin and TRAIL, indicating that treatment with geldanamycin affected the complex formation of the IKK proteins.

Disruption of IKK Proteins Affected the Function of I{kappa}B{alpha}
In unstimulated cells, I{kappa}B{alpha} binds to NF-{kappa}B, thus sequestering NF-{kappa}B in the cytoplasm. In cells stimulated with cytokines, IKK proteins are activated, which phosphorylate I{kappa}B{alpha}, releasing the NF-{kappa}B from the I{kappa}B{alpha}/NF-{kappa}B complex, which then is translocated into the nucleus. To examine whether treatment with geldanamycin and/or TRAIL affected NF-{kappa}B, nuclear transport of NF-{kappa}B was examined along with its interaction with I{kappa}B{alpha}. In cells treated with geldanamycin, NF-{kappa}B was not seen in the nuclear extract (Fig. 4, top , lanes 2 and 4), indicating its continued sequestration in the cytoplasm. These results were supported by immunoprecipitation with NF-{kappa}B, which showed continued binding of NF-{kappa}B to I{kappa}B{alpha} in geldanamycin-treated cells (Fig. 4, middle, lanes 2 and 4). Treatment with TRAIL increased the level of nuclear NF-{kappa}B (Fig. 4, top, lane 3), which correlated with reduced interaction between I{kappa}B{alpha} and NF-{kappa}B (Fig. 4, middle, lane 3). However, treatment with both geldanamycin and TRAIL decreased TRAIL-induced nuclear transport of NF-{kappa}B compared with TRAIL alone. Examination of the levels of phosphorylated I{kappa}B{alpha} in these cells showed increase in the phosphorylation of I{kappa}B{alpha} in TRAIL-treated cells (Fig. 4, bottom, lanes 3 and 4), which agreed with published data that TRAIL induced NF-{kappa}B activity in LNCaP cells. It was interesting to note that although the phosphorylation of I{kappa}B{alpha} did not decrease dramatically in geldanamycin plus TRAIL-treated cells, the nuclear level of NF-{kappa}B was reduced.


Figure 4
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  		Figure 4. Geldanamycin (GA) and/or TRAIL treatment affected the interaction between I{kappa}B{alpha} and NF-{kappa}B. LNCaP cells were treated as described, and the nuclear protein was isolated and analyzed for the presence of NF-{kappa}B in the nucleus (top). Cytosolic fractions were used to immunoprecipitate NF-{kappa}B, and the binding of phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}) to the NF-{kappa}B was examined by Western blots (middle). Furthermore, phosphorylated I{kappa}B{alpha} was examined in cytosolic fractions (bottom). Actin was used as loading control (Con).

 
To further examine the effects of treatment on nuclear translocation of NF-{kappa}B, immunocytochemical methods were used (Fig. 5 ). LNCaP cells were treated as above, and the locations of NF-{kappa}B/p65 and NF-{kappa}B/p50 were examined by differential fluorescence staining and the nuclei were stained with 4',6-diamidino-2-phenylindole. Results showed that in control and low-dose geldanamycin-treated cells, NF-{kappa}B/p65 and NF-{kappa}B/p50 were limited to cytoplasm (Fig. 5). Cells treated with TRAIL alone showed nuclear staining for both NF-{kappa}B/p65 and NF-{kappa}B/p50, suggesting that TRAIL promoted nuclear translocation of NF-{kappa}B/p65 (supports the data in Fig. 4). It was of interest to note that treatment with geldanamycin inhibited TRAIL-induced transport of NF-{kappa}B/p65 and NF-{kappa}B/p50 to the nucleus. These results support the Western blot data presented above and suggest that combination treatment affected NF-{kappa}B activity.


Figure 5
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  		Figure 5. Immunocytochemical examination of NF-{kappa}B/p65 and NF-{kappa}B/p50 LNCaP cells were treated with vehicle (C), low-dose geldanamycin (GA), TRAIL, or combination (GA + T). The cells were fixed with methanol and stained with anti-NF-{kappa}B/p65 antibody (green), anti-NF-{kappa}B/p50 antibody (red), or 4',6-diamidino-2-phenylindole (blue). NF-{kappa}B/p65 and NF-{kappa}B/p50 showed cytoplasmic staining in the cells treated with vehicle, geldanamycin, and combination, and nuclear staining in the cells treated with TRAIL. Magnification, x400.

 
Treatment of LNCaP Cells with Geldanamycin Affected the Function of NF-{kappa}B
As the above results showed that the treatment affected nuclear transport of NF-{kappa}B, experiments were conducted to examine the effect of treatment on NF-{kappa}B activity. NF-{kappa}B luciferase reporter construct containing four copies of NF-{kappa}B response elements was transfected into LNCaP and treated as above. Assay for luciferase activity showed significant reduction in the luciferase activity in geldanamycin-treated cells compared with control cells (Fig. 6 ). Maximum luciferase activity was noted in cells treated with TRAIL, which agreed with the results of phosphorylation of I{kappa}B{alpha} and translocation of NF-{kappa}B into the nucleus. Treatment with both geldanamycin and TRAIL reduced luciferase activity, which is significant especially compared with significantly higher activity in cells treated with TRAIL alone. The results confirm that in addition to inducing apoptosis, combination of geldanamycin and TRAIL reduced NF-{kappa}B activity significantly.


Figure 6
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  		Figure 6. Effect of treatment on NF-{kappa}B activity. LNCaP was transfected with luciferase reporter construct containing four copies of NF-{kappa}B binding sites by electroporation (two cycles of 200 V of 10-millisecond pulse length, with a pulse interval of 500 milliseconds using Electroporator 830 from BTX). Luciferase activity was assayed using a kit from Promega. Luciferase activity was expressed per unit total protein. Columns, mean for at least three experiments with a minimum of three replicates; bars, SE. NTC, nontransfected controls; C, untreated controls transfected with luciferase reporter construct; GA, geldanamycin-treated cells; GA + T, cells treated with geldanamycin and TRAIL.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Prostate cancer is a multifocal disease with clones of androgen-sensitive and androgen-refractory cells (1–4). Commonly used androgen deprivation therapies induce apoptotic cell death in androgen-sensitive cells, whereas many patients with androgen-refractory cancer die due to lack of effective chemotherapeutic agents (5–8). However, the androgen-refractory prostate cancer cells retain the capacity to undergo apoptosis (29–31). In prostate cancer cells, apoptosis has been induced by variety of agents, such as staurosporine (31, 32), levostatin (30), thapsigargin (5), okadaic acid (33), mifepristone (13, 34), and camptothecin (35, 36). In the last few years, reports from other and our laboratory have shown induction of apoptosis in prostate cancer cells using TRAIL, although some prostate cancer cells, such as LNCaP, are resistant (10, 11, 15). We have previously shown that combined treatment of LNCaP with TRAIL and mifepristone can partially overcome the resistance to TRAIL (12). Therefore, to further improve apoptotic response of TRAIL-resistant cells, we have been exploring newer agents to act synergistically with TRAIL. In this article, we have shown increased apoptotic response of LNCaP cells to TRAIL when the function of HSP90 was inhibited.

HSPs function by binding to client proteins, thus protecting the client proteins from ubiquitin proteasome–mediated degradation. As cancer cells continue to proliferate due to the increased activity of prosurvival proteins, it was hypothesized that disruption of the interaction between HSP and client proteins would result in the degradation of the client proteins (37). For this purpose, geldanamycin and 17-allylaminogeldanamycin, specific inhibitors of HSP90, are being tested as therapeutic agents (38). Although both these drugs induced apoptosis, they have shown significant adverse effects, such as hepatotoxicity, raising concerns about their feasibility as therapeutic drugs (17, 18). Therefore, we hypothesized that an effective therapeutic advantage would be to identify a combination treatment that would require lower dose of geldanamycin but still induce higher level of apoptosis. Our results show that combined treatment with TRAIL and geldanamycin induced high level apoptosis, although at this low dose, geldanamycin by itself did not induce significant apoptosis. The significance of this data is not only increased apoptosis in geldanamycin-treated and TRAIL-treated cells but also the fact that this was achieved using low, suboptimal dose of geldanamycin in LNCaP cells that are resistant to TRAIL. These results imply clinical benefits as both geldanamycin and TRAIL are being individually explored as therapeutic agents.

HSP90 affects cell survival through its effects on two different pathways: (a) interfering with apoptosis at the mitochondrial level and (b) by altering the function of survival proteins, such as IKK complex. Our results showed that inhibition of HSP90 function and treatment with TRAIL affected mitochondrial functions as seen by increased activation of caspase-3, caspase-7, and their substrate poly(ADP-ribose) polymerase. In addition to direct effects on mitochondria-mediated apoptosis, we expected that inhibition of HSP90 would affect prosurvival client proteins. Dose-dependent decrease of IKK{alpha}, IKKß, and IKK{gamma} in geldanamycin-treated cells confirms the importance of continued binding of IKK proteins to HSP90 (19). It was interesting to note that under these conditions, the levels of HSP90 did not alter, indicating that (a) the low-dose geldanamycin used in these experiments did not affect the levels of HSP90 and that (ii) the reduction in the levels of IKK proteins was not due to reduced availability of HSP90. This suggestion was supported by immunoprecipitation data, which showed loss of interaction among IKK proteins in geldanamycin-treated cells. As IKK proteins regulate the function of NF-{kappa}B activity mediated through I{kappa}B{alpha}, our data confirm the effects of treatment on this axis. Immunocytochemistry and Western blots showed inhibition of nuclear translocation of NF-{kappa}B in geldanamycin-treated cells, which was responsible for the reduced activity of NF-{kappa}B, as measured by luciferase reporter assays.

Thus, in these experiments, we have shown that inhibition of HSP90 with low-dose geldanamycin along with treatment with TRAIL has several advantages for prostate cancer treatment. It is of particular significance that we were able to increase apoptosis while reducing the dose of geldanamycin and sensitizing TRAIL-resistant cancer cells.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/27/05; revised 10/ 8/05; accepted 10/25/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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