Enhanced killing of chemo-resistant breast cancer cells via controlled aggravation of ER stress

Cancer Letters 282 (2009) 87–97

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Enhanced killing of chemo-resistant breast cancer cells via controlled aggravation of ER stress Hee-Yeon Cho a, Simmy Thomas a, Encouse B. Golden b, Kevin J. Gaffney c, Florence M. Hofman b, Thomas C. Chen d, Stan G. Louie e, Nicos A. Petasis c, Axel H. Schönthal a,* a

Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, 2011 Zonal Ave., HMR-405, CA 90089-9094, United States Department of Pathology, University of Southern California, Los Angeles, United States c Department of Chemistry, University of Southern California, Los Angeles, United States d Department of Neurosurgery, University of Southern California, Los Angeles, United States e Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, University of Southern California, Los Angeles, United States b

a r t i c l e

i n f o

Article history: Received 12 December 2008 Received in revised form 10 February 2009 Accepted 2 March 2009

Keywords: GRP78 CHOP GADD153 Doxorubicin resistance Paclitaxel resistance Trastuzumab resistance Endoplasmic reticulum stress Breast cancer Drug resistance Celecoxib

a b s t r a c t Moderate activity of the endoplasmic reticulum (ER) stress response system exerts antiapoptotic function and supports tumor cell survival and chemoresistance, whereas its more severe aggravation may exceed the protective capacity of this system and turn on its proapoptotic module. In this study, we investigated whether the combination of two pharmacologic agents with known ability to trigger ER stress via different mechanisms would synergize and lead to enhanced tumor cell death. We combined the HIV protease inhibitor nelfinavir (ViraceptÒ) and the cyclooxygenase 2 (COX-2) inhibitor celecoxib (CelebrexÒ) and investigated their combined effect on ER stress and on the viability of breast cancer cells. We found that this drug combination aggravated ER stress and caused pronounced toxicity in human breast cancer cell lines, inclusive of variants that were highly resistant to other therapeutic treatments, such as doxorubicin, paclitaxel, or trastuzumab. The anti-tumor effects of celecoxib were mimicked at increased potency by its non-coxib analog, 2,5-dimethyl-celecoxib (DMC), but were substantially weaker in the case of unmethylated-celecoxib (UMC), a derivative with superior COX-2 inhibitory efficacy. We conclude that the anti-tumor effects of nelfinavir can be enhanced by celecoxib analogs in a COX-2 independent fashion via the aggravation of ER stress, and such drug combinations should be considered as a beneficial adjunct to the treatment of drug-resistant breast cancers. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Nelfinavir (NFV; ViraceptÒ) belongs to the class of HIV protease inhibitors and has been used for over a decade in the treatment of HIV patients. More recently, it has come under investigation for potential repositioning as an anticancer agent. An early hint for such an additional application came from the observation that patients on highly active antiretroviral therapy [HAART] showed regression Abbreviations: CXB, celecoxib (CelebrexÒ); DMC, 2,5-dimethyl-celecoxib; NFV, nelfinavir (ViraceptÒ); UMC, unmethylated-celecoxib. * Corresponding author. Tel.: +1 323 442 1730; fax: +1 323 442 1721. E-mail address: [email protected] (A.H. Schönthal). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.03.007

of Kaposi’s sarcoma, a malignant tumor type frequently associated with HIV infection [1]. Subsequent studies demonstrated that NFV was able to cause cell cycle arrest, inhibited anchorage-independent growth, induced apoptosis in different tumor cell lines in vitro, and reduced xenograft tumor growth in various mouse models in vivo [2–9]. Clinical trials have recently been initiated (or completed [10]) to determine whether this tumor therapeutic potential can also be verified in patients with different types of advanced cancers. Several mechanisms have been proposed to explain the anti-tumor effects of NFV. For example, the accumulation of the cyclin-dependent kinase inhibitors p21 and p27 is thought to be involved in the drug’s ability to effect cell

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cycle arrest, and the inhibition of intracellular signaling via the pro-survival phosphatidyl-inositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway might contribute to NFV’s recognized apoptosis-stimulatory effect [2,3,5,6]. Moreover, the pleiotropic biochemical and cellular effects of this drug also include induction of autophagy and endoplasmic reticulum (ER) stress, and it appears that these latter pathways may represent key mechanisms of nelfinavir-induced tumor cell death [3,5,9]. The ER stress response consists of a set of adaptive pathways that can be triggered by disparate perturbations in normal ER function, such as accumulation of misfolded proteins, lipid or glycolipid imbalances, or changes in the ionic conditions of the ER lumen (see Refs. [11,12] for reviews). The primary purpose of this system is to alleviate the stressful disturbance and restore proper ER homeostasis. In the case of severe or persistent ER stress, however, these pathways will trigger programmed cell death/apoptosis. One of the central pro-survival regulators of the ER stress response is glucose-regulated protein 78 (GRP78/ BiP), which has important roles in protein folding and assembly, in targeting misfolded proteins for degradation, in ER Ca2+-binding, and in controlling the activation of trans-membrane ER stress sensors [13]. On the other hand, CCAAT/enhancer binding protein homologous transcription factor (CHOP/GADD153) represents a critical executioner of the pro-apoptotic arm of the ER stress response [14,15]. Overall, this system can be viewed as a balance of interdependent ‘‘yin-yang” modules, where elevated levels of GRP78 attempt to restore ER homeostasis, and where unduly high levels of CHOP may gain dominance and tip the balance towards apoptosis [16]. The recently discovered feature of NFV to trigger ER stress appears to be based on the drug’s ability to inhibit proteases within the cellular proteasome [4,5,9]. As a consequence of blocked proteasome function, misfolded and other unneeded proteins accumulate, thus triggering ER stress (also called the unfolded protein response, UPR), which is revealed through elevated levels of the ER stress indicators GRP78 and CHOP. Similarly, the selective cyclooxygenase 2 (COX) inhibitor celecoxib (CXB) has been shown to stimulate GRP78 and CHOP expression in vitro and in vivo [17–20]. However, unlike NFV, CXB triggers ER stress via the inhibition of SERCA (sarcoplasmic/ER calcium ATPase), a transmembrane ER protein that pumps calcium from the cytosol into the ER [18,19,21]. Inhibition of SERCA results in the rapid depletion of calcium from the ER storage space and represents a well-known trigger for severe ER stress. It has recently become clear that CXB displays two very different pharmacologic functions, namely the selective inhibition of COX-2 and the COX-2 independent ability to cause apoptosis due to ER stress [22–26]. Using analogs of CBX, the two functions were dissected, demonstrating that the anti-tumor activity correlated with the ability to induce ER stress, whereas COX-2 inhibition potency did not correlate with anticancer properties. For example, 2,5-dimethyl-celecoxib (DMC) is unable to inhibit COX-2, yet it represents a more potent trigger for ER stress and tumor cell death than CXB [17,24,26]. Conversely, unmethylated-celecoxib (UMC) inhibits COX-2 more potently than CXB, but its ability to trigger ER stress and tumor cell death

is significantly reduced [17,24,26]. Thus, the experimental use of DMC and UMC has proven quite valuable to investigate the potential role of COX-2 in the anticancer mechanisms of CXB; in addition, due to its higher anticancer potency, DMC is being evaluated for its possible cancer therapeutic use in its own right [27,28]. Based on the ability of NFV and CXB (or its analogs) to trigger pro-apoptotic ER stress by distinctly different mechanisms, we reasoned that the combination of these compounds might result in further aggravated ER stress and ultimately more efficient tumor cell death. In this current study, we demonstrate that this is indeed the case and even applies to breast cancer cells that are highly resistant to conventional chemotherapy.

2. Materials and methods 2.1. Materials CXB was obtained as CelebrexÒ capsules from the pharmacy, or was synthesized in our laboratory according to previously published procedures [29]. DMC and UMC were synthesized in our laboratory according to previously published procedures (see Ref. [23] for DMC and Ref. [29] for UMC). These compounds were dissolved in DMSO at 100 mM (stock solution). Nelfinavir was derived either from ground pills of ViraceptÒ or used as pure powder supplied by Agouron Pharmaceuticals, Inc. (San Diego, CA), and dissolved in ethanol at 50 mM (stock solution). In the majority of experiments, the above drugs were added to the cell culture medium in a manner that kept the final concentration of solvent below 0.1%. 2.2. Cell lines and culture conditions The following human breast cancer cell lines were used: the trio of MCF7, MCF7/Dox (a doxorubicin/multidrug-resistant variant of MCF7), and MCF7/Tax (a Taxolresistant variant of MCF7) was kindly provided by Dr. Amadeo M. Parissenti [30]; the trio of BT-474, BT-1.0B and BT-1.0E (two trastuzumab-resistant variants of BT474) was kindly provided by Dr. Susan E. Kane [31]. All cell lines were propagated in DMEM (provided by the Cell Culture Core Lab of the USC/Norris Comprehensive Cancer Center and prepared with raw materials from Cellgro/MediaTech, Manassas, VA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a humidified incubator at 37 °C and a 5% CO2 atmosphere. During maintenance and propagation phase, 300 nM AdriamycinÒ or 6.7 nM TaxolÒ was added to MCF7/Dox and MCF7/Tax cells, respectively; similarly, 1 lM HerceptinÒ was added to BT-1.0B and BT-1.0E cells in order to maintain the drug-resistant phenotype. However, several days before and during experiments, all selective drugs were omitted. 2.3. MTT assays MTT assays were performed in 96-well plates as described in detail elsewhere [22]. All assays were repeated

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several times at variable cell densities. The number of cells per well ranged from 3.0  103 to 8.0  103.

untransfected earlier [19].

cells

were

performed

as

described

2.4. Immunoblots 3. Results

Total cell lysates were prepared and analyzed by Western blot analysis as described earlier [17]. In brief, 50 lg of total cell lysate was separated by polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose membrane, blocked with milk, and probed with primary antibodies. We used fluorescence-conjugated secondary antibodies, which were imaged and documented with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE), according to protocols supplied by this manufacturer. Specific antibodies against GRP78, CHOP, PARP (fulllength and cleaved), caspase-7 (pro- and cleaved/activated forms), and actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) or from Cell Signaling Technologies (Beverly, MA). All immunoblots were repeated at least once with new lysates from a duplicate experiment to confirm the results. 2.5. Trypan blue cell count A quarter million cells were seeded into each well of a 6-well plate. After complete cell attachment, the medium was replaced with fresh medium containing various concentrations of drugs or solvent only. After various time points, the cells were trypsinized, suspended in phosphate-buffered saline (PBS), and mixed 1:1 with 0.4% trypan blue. The mixture was incubated at room temperature for a few minutes, and unstained (i.e., viable) cells were counted with the use of a hemacytometer under the microscope. 2.6. Cell death ELISA and DNA content measurements Cells were plated in 96-well plates, treated with the various drugs, and then analyzed for the presence of histone-complexed DNA fragments with the use of the Cell Death Detection ELISA Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. The kit was used in a manner as to specifically quantitate apoptosis rather than necrosis. DNA content of drug-treated cells and their distribution throughout the different phases of the cell cycle (G1, S, G2/ M, and sub-G0/G1) was determined by flow cytometry. Cells were fixed in 75% ethanol for 1 h, followed by incubation in 50 lg/mL propidium iodide and 0.1 mg/mL RNase A for 30 min at 37 °C. Subsequent FACS analysis was performed by a USC core lab. 2.7. Transfections and colony formation assays Cells were transfected with siRNA (obtained from Ambion Inc., Austin, TX) and either seeded for the evaluation of long-term survival after drug treatment, or harvested for Western blot analysis (to confirm knockdown of target gene expression), as described elsewhere [19]. Colony formation assays with siRNA-transfected or

3.1. Cytotoxic effects of NFV, CXB, DMC, and UMC on drug-resistant breast cancer cell lines In our study, we used the following human breast carcinoma cell lines: MCF7 and two drug-resistant variants thereof, MCF7/Tax, which has been selected for increased resistance to paclitaxel (TaxolÒ), and MCF7/Dox, a highly multidrug-resistant variant that is unresponsive to doxorubicin (AdriamycinÒ), paclitaxel, and other chemotherapeutic agents. In addition, we used BT-474, a cell line overexpressing Her2/ neu, and two derivative cell lines resistant to trastuzumab (HerceptinÒ), named BT-1.0B and BT-1.0E. Although all these cell lines were characterized before [30,31], we confirmed their drug resistance pattern. Representative results of doxorubicin, paclitaxel, and trastuzumab treatments are shown in Fig. 1A, 1B, and 2A, respectively. Note that MCF7/Dox cells, due to their highly multidrug-resistant nature, are even more resistant to paclitaxel than the paclitaxel-resistant MCF7/Tax cell line (Fig. 1B), consistent with the dramatic drug resistance of these cells described earlier [30]. Next, MCF7, MCF7/Tax, and MCF7/Dox cells were treated with increasing concentrations of NFV, CXB, UMC, or DMC (see Chart 1 for previously established characteristics of the latter three agents) for 48 h and cell survival was analyzed by conventional MTT assays. As shown in Fig. 1C, NFV effectively reduced viability of all cell lines, although with somewhat varying potency. While the IC50 for MCF7 was slightly below 20 lM, MCF7/Tax and MCF7/Dox displayed somewhat increased resistance with IC50s of approximately 30 and 60 lM, respectively. When the three MCF7 lines were treated with CXB, small differences became apparent as well, where MCF7/Tax was slightly more resistant than MCF7, and MCF7/Dox was the most resistant, with IC50s of 55, 65, and 75 lM, respectively (Fig. 1D). When the same cells were treated with UMC, which is a CXB derivative with increased COX-2 inhibitory potency (Chart 1), substantially less cytotoxicity was observed; in fact, in the case of MCF7/Tax and MCF7/Dox, the respective IC50 was well above 100 lM (Fig. 1E). In contrast, DMC, which is a CXB analog lacking COX-2 inhibitory potency (Chart 1), exerted the greatest cytotoxic activity on all three MCF7 variants when compared to UMC or CXB (Fig. 1F), although once again, MCF7/Tax and MCF7/Dox were slightly more resistant than the parental MCF7 cells. BT-474, BT-1.0B, and BT-1.0E cells displayed very similar sensitivity towards NFV, CXB, UMC, and DMC, i.e., no one cell line was more or less sensitive, although the different drugs exerted different cytotoxic potencies. In several repetitions of these MTT assays, the IC50 for NFV was in the range of 15–25 lM (Fig. 2B), whereas the IC50s for CXB, UMC, and DMC were approximately 45–55, 75–85, and 30–35 lM (not shown, except for a representative example for DMC in Fig. 2C). Altogether, despite hugely varying sensitivities towards some currently used breast cancer therapeutic drugs, all of the above cell lines displayed cytotoxic sensitivity towards NFV, CXB, UMC, and DMC in a relatively narrow range of concentrations, and among the three CXB variants, the one without COX-2 inhibitory potential, DMC, turned out to be the most potent. 3.2. Correlation of drug cytotoxicity with induction of ER stress Since all of the above agents have been shown to trigger ER stress-induced apoptosis in other types of tumor cells, we next determined whether these events could also be observed in drug-resistant cell lines. Towards this end, the various cell lines were treated with increasing concentrations of individual drugs, and protein expression was analyzed by Western blot analysis. To reveal the extent of ER stress, we analyzed two major markers of this system, i.e., the defensive, pro-survival protein GRP78, and CHOP, a pro-apoptotic executor protein that is known to initiate ER stress-mediated cell death. As a readout for ongoing apoptosis, we investigated the proteolytic cleavage of PARP (poly ADP-ribose polymerase), a protein that is a known substrate of activated caspase-3 and a widely used, reliable marker for apoptosis.

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Fig. 1. Reduced cell growth and survival of MCF7 and drug-resistant variants. MCF7, MCF7/Tax, and MCF7/Dox cell lines were treated with the indicated concentrations of doxorubicin, paclitaxel, NFV, CXB, DMC, and UMC. After 48 h, conventional MTT assays were performed to reveal the overall cell viability in response drug treatment. Shown is percent survival (mean ± SD, n P 4), where the value from untreated control cells was set at 100%. Note log scale of xaxis in A and B.

As shown in Fig. 3, increasing concentrations of DMC or NFV resulted in the increased expression of GRP78 and CHOP and lead to pronounced cleavage of PARP. These effects were quite similar in MCF7 and MCF7/Dox cells and also in BT-474 and BT-1.0E cells, indicating that DMC and NFV were able to trigger ER stress and subsequent apoptosis even in the drug-resistant tumor cell variants. When these cells were treated with CXB or with UMC, very similar outcomes were observed, except that higher concentrations of CXB, and substantially higher concentrations of UMC, were required (not shown). This finding was in agreement with the cytotoxicity data shown Fig. 1, where the cytotoxic potential of these three agents was DMC > CXB > UMC. Taken together, these results show that highly drug-resistant breast cancer cells retained sensitivity towards the particular ER stress-inducing drugs used in our study.

3.3. Combination drug treatment enhances tumor cell death Because NFV and CXB/DMC/UMC are known to trigger ER stress via different molecular mechanisms, we postulated that their combined application might lead to enhanced ER stress and increased cell death. To test this assumption, all of the above cell lines were treated with NFV in combination with either CXB, DMC, or UMC at relatively low concentrations, so that potentially enhancing effects could emerge. We used drug concentrations that reduced cell viability only by approximately 10–30% when applied individually, and determined cell survival by MTT assays. As displayed in Fig. 4, in all cases the drug combinations were substantially more cytotoxic than individual drug treatments, and this could be observed even in the drug-resistant cell variants. As before, UMC and CXB displayed weaker potency than DMC, as higher con-

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direct analysis of cytotoxicity, whereas CFAs investigate the long-term impact of drug treatment and establish the extent to which individual cells are able to survive and spawn a colony of descendants within two weeks after a 48 h exposure to drug(s). MCF7 cells and their two drugresistant variants were treated with low concentrations of NFV and DMC, alone and in combination, and the number of viable cells in each culture was determined after 8, 24, and 48 h (Fig. 5A). At the 8 h time point, no drug effects on cell number could be detected. At the 24 h time point, individual drug treatments displayed only marginal effects, but the combination drug treatment resulted in a substantial reduction of the number of viable cells in MCF7 and MCF7/Tax cells; in comparison, MCF7/Dox cells were relatively unresponsive to drug treatment at this time point. However, at the 48 h time point, pronounced drug effects became apparent, particularly in response to combination treatment: in the MCF7 culture, no viable cells could be detected, and in the two drug-resistant variants the number of viable cells was reduced to about 15–20% as compared to untreated control cells (Fig. 5A). Thus, consistent with the MTT analysis shown in Fig. 4, combination drug treatment dramatically reduced the number of viable cells. A similar outcome was recorded when CFAs were performed. As shown in Fig. 5B, treatment of cells with NFV or DMC individually reduced long-term survival by 10–40%; however, when combined, the cytotoxic efficacy increased to 85–95% and took place in the drug-resistant MCF7 variants as well. Similar results were also obtained when NFV was combined with CXB, except that somewhat higher concentrations of CXB were required to match the potency of the NFV/DMC combination (not shown). The cleavage of PARP protein after cellular treatment with drugs, as shown above in Fig. 3, indicated that cell death was due to apoptosis. To confirm this, additional indicators of programmed cell death were investigated in MCF7 cells treated with NFV and DMC alone or in combination for various times. After 24 h of drug treatment, fluorescence-activated cell sorting (FACS) of propidium iodide-stained cells showed that individual drug treatments caused only a small fraction (<10%) of the cells to appear in the sub-G0/G1 phase of the cell cycle; however, combination drug treatment resulted in >40% of cells to show in this apoptotic phase (Fig. 6). Similarly, when apoptosis was measured by cell death ELISA after 48 h of drug treatment, it became apparent that combination drug treatment caused substantially more extensive cell death than individual drug treatment (Fig. 6B). Finally, apoptosis was also confirmed by investigating caspase-7, an ER stress-associated caspase [32]. Here as well, combination drug treatment triggered extensive activation of this enzyme (Fig. 6C), altogether establishing apoptosis as a major mechanism of cell death induced by these drug combinations. 3.4. Combination drug treatment aggravates ER stress

Fig. 2. Reduced cell growth and survival of BT-474 and trastuzumabresistant variants. (A) BT-474, BT-1.0B, and BT-1.0E cell lines were cultured in the presence of 1 lM trastuzumab (closed symbols). BT-474 cells were also cultured in the absence of any drug treatment (open circle). Cells were counted every 3 days. Shown is the change in cell number over the course of 15 days. Note that 1 lM trastuzumab is sufficient for growth inhibition of BT-474 cells, whereas the drugresistant variants are entirely unaffected. (B) Cells were treated with the indicated concentrations of NFV or DMC for 48 h. Thereafter, conventional MTT assays were performed to reveal the overall cell viability in response drug treatment. Shown is percent survival (mean ± SD, n P 4), where the value from untreated control cells was set at 100%.

centrations of these two agents were necessary to achieve similar outcomes; for this reason, we focused the subsequent studies primarily on DMC. The cytotoxic potency of the NFV and DMC combination was further confirmed with two additional procedures, cell counts and colony formation assays (CFAs). Counting the number of viable cells represents a very

We next investigated whether the pronounced cytotoxic combination effect of NFV and DMC would correlate with aggravated ER stress. Cells were treated with either drug alone, or with both in combination, and the ER stress markers GRP78 and CHOP were analyzed by Western blot analysis. As shown in Fig. 7, NFV or DMC by themselves displayed only marginal effects; however, when combined, there was severe induction of ER stress, as indicated by the pronounced increase in the amount of GRP78 and CHOP proteins in all cell lines tested. Furthermore, the highly elevated levels of pro-apoptotic CHOP corresponded with substantial cleavage of PARP, indicating the association of ER stress with extensive apoptosis under these combination treatment conditions (Fig. 7).

3.5. Aggravated ER stress affects combination drug-induced tumor cell death The above results clearly showed that aggravated ER stress was closely aligned with drug-induced cell death in the various breast cancer cell lines. In order to determine whether ER stress would play a causal role in these events, we used siRNA to block the expression of GRP78 or of CHOP, the two major executors of the protective and pro-apoptotic function, respectively, of the ER stress response system. MCF7 cells and their two drug-resistant derivatives were transfected with siGRP78 (or an siRNA against green fluorescent protein, GFP, as a control) and treated with the NFV/DMC combination for 48 h; thereafter, long-term cellular survival was determined by CFAs. As shown in Fig. 8A, cells transfected with siGRP78 displayed significantly (p < 0.05) reduced survival after combination drug treatment as compared to cells transfected with control siGFP,

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Chart 1. Chemical structures and activities of CXB, UMC, and DMC. CXB has one methyl group at the C-4 (p) position of its terminal phenyl ring; this substitution is lacking in UMC; DMC has two methyl groups (at the 2- and 5-positions). The listed COX-2 inhibitory potency (IC50) of these compounds is derived from two earlier studies that used human recombinant COX-2 in vitro [27,29]; comparative ER stress-inducing potency is from Ref. [17]; apoptosisinducing potency is from Refs. [17,24,26].

Fig. 3. Increased expression of markers for ER stress and apoptosis in response to treatment with NFV or DMC. MCF7, MCF7/Dox, BT-474, and BT-1.0E cell lines were treated with increasing concentrations of DMC (top panels) or NFV (bottom panels) and cell lysates were analyzed by Western blot with specific antibodies to GRP78 (a pro-survival ER stress indicator protein), CHOP (a pro-apoptotic ER stress indicator protein), and PARP (proteolytic cleavage of PARP indicates ongoing apoptosis). To verify equal loading in each case, the blots were also probed with an antibody to actin. f.l.: full length PARP; cl.: cleaved PARP.

revealing increased chemosensitization of these cells when GRP78 function was blocked. Conversely, siRNA against CHOP resulted in increased survival of drug-treated cells, i.e., without the ability to induce pro-apoptotic CHOP protein during ER stress, the cells became more resistant to drug treatment (Fig. 8B). As a control for the efficiency of siGRP78 and siCHOP, the expression levels of the targeted proteins were documented by Western blot analysis. As shown in Fig. 8C, siGRP78 effectively blocked GRP78 expression in

all three transfected cell lines. Furthermore, siGRP78 also prevented the increase of GRP78 protein levels in response to treatment with DMC, indicating that siGRP78 not only suppressed the basal level of this protein, but also its induction by drug treatment. Similarly, after transfection of siCHOP, there was no detectable expression of CHOP protein after treatment of cells with DMC, i.e., CHOP protein levels, which generally are below detection limits in untreated cells, remained undetectable even after drug treatment (Fig. 8C). Taken together, these results indicate that

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Fig. 4. Enhancement of NFV cytotoxicity by CXB, DMC, and UMC. The various cell lines were treated with NFV alone or in combination with CXB, DMC and UMC as indicated for 36 h and MTT assays were performed to measure changes in cell viability. The following drug concentrations were used: MCF7 (20 lM NFV, 30 lM DMC, 40 lM CXB, 60 lM UMC), MCF7/Tax and MCF7/Dox (30 lM NFV, 40 lM DMC, 50 lM CXB, 70 lM UMC), BT-474, BT-1.0B and BT-1.0E (15 lM NFV, 30 lM DMC, 40 lM CXB, 60 lM UMC). Shown is percent survival (mean ± SD, n P 4), where the value from untreated control cells was set at 100%.

blockage of the major ER stress effector proteins GRP78 or CHOP resulted in significantly altered chemosensitivity of tumor cells, which took place even in otherwise drug-resistant breast cancer cells and thereby established ER stress as a major determinant in this process.

4. Discussion One of the most pressing and unresolved problems in cancer therapy is the development of drug resistance, oftentimes against multiple treatment regimens simultaneously, which results in grim prognosis for the affected patients. As exemplified by the multidrug-resistant MCF7/Dox breast cancer cell line used in our study, tumor cells may become resistant to several conventional anticancer drugs, such as doxorubicin and paclitaxel, even when drug concentrations are increased by two orders of magnitude. In order to treat such resilient cancers, novel treatment modalities are urgently needed. Most tumor cells appear to harbor chronic ER stress, as indicated by continuously elevated expression levels of GRP78, a major pro-survival component of the ER stress response system. Besides supporting cell survival within a hostile tumor microenvironment (hypoxia, low glucose

levels, acidity), GRP78 may also contribute to the chemoresistance of cancer cells [33]. As long as there is only moderate ER stress, the pro-survival module of this system dominates and at the same time suppresses its pro-apoptotic function (such as CHOP expression). However, if ER stress becomes too severe, the protective efforts of the ER stress response (including increased levels of GRP78) are being overwhelmed and its pro-apoptotic module (including increased levels of CHOP) gains dominance [34,35]. As presented in greater detail elsewhere [16], it might be possible to pharmacologically manipulate this ‘‘yin-yang” balance of ER stress-regulated cell survival vs. cell death in a tumor-specific fashion with little or no toxic side effects to the organism as a whole. In support of such a view, it has been demonstrated that severe ER stress can be triggered in tumor tissues of mice treated with celecoxib (CXB) or nelfinavir (NFV) in monotherapy; such treatments resulted in significant anti-tumor outcomes, but had no noticeable side effects in the animals [4,19,20]. In the present study, we reasoned that the combination of two agents that are able to trigger ER stress via different mechanisms might result in further aggravated ER stress, leading to significantly enhanced tumor cell death. Indeed,

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Fig. 5. Reduced cell growth and survival when NFV is combined with DMC. MCF7 and MCF7/Tax cells were treated with 15 lM NFV and 30 lM DMC alone or in combination. MCF7/Dox similarly received 20 lM NFV and 30 lM DMC. (A) Viable cells were determined by trypan blue exclusion and were counted after 8, 24, and 48 h. Shown are cell numbers at the three different time points (mean ± SD; n = 3). (B) Cells were treated with drugs for 48 h and cell survival was determined by colony formation assay after an additional 12-day culture in the absence of drugs. Shown is percent colony formation (mean ± SD, n = 3), where the number of colonies in untreated control cultures was set to 100%.

we found that the combination of NFV with CXB or its analogs displayed greatly increased anticancer activity, and the underlying mechanism of this drug synergy appeared to involve the aggravation of ER stress. The direct involve-

ment of ER stress mechanisms could be established with the use of siRNA directed at GRP78; knock-down of this pro-survival ER stress component resulted in significantly (p < 0.05) increased chemosensitization towards combination drug treatment (Fig. 8), consistent with several other prior studies that established a role for GRP78 in supporting the chemoresistance of tumor cells (see detailed Ref. in [36]). Conversely, knock-down of the pro-apoptotic ER stress component CHOP resulted in increased chemoresistance (Fig. 8), further confirming the critical role of ER stress in mediating cell death in response to combination treatment with drugs, such as NFV and DMC, that are able to aggravate this cellular system. A potentially critical role for COX-2 inhibition in the observed processes is minimized by the fact that MCF7 and BT-474 cells only express very low levels of COX-2 protein and do not display elevated levels of prostaglandin E2 (PGE2). For example, several studies have demonstrated that detection of COX-2 in MCF7 cells is below detection limits when total cell lysates are analyzed by Western blot analysis [37,38], although the preparation of concentrated nuclear extracts allows the detection of these very low COX-2 levels [39]. In our studies, we were unable to detect COX-2 expression in MCF7 and BT-474 cells by Western blot (not shown), in agreement with these earlier studies. Perhaps more convincingly, a role for COX-2 was also excluded through our comparative use of CXB, UMC, and DMC (see below). We have shown before [17] that among the three CXB variants used, DMC (a COX-2 inactive analog) is the most potent with regards to the ability to trigger ER stress; CXB is noticeably weaker, and UMC (a CXB analog with greater COX-2 inhibitory potency than CXB itself) has the weakest ER stress-inducing potency. This differential also holds true when these agents are applied to the various breast cancer cell lines tested in this study (not shown for all combinations; because DMC consistently displayed the strongest effects, we primarily showed results with DMC). Similarly, when CXB, DMC, or UMC were combined with NFV, all three compounds were able to further aggravate ER stress; however, as observed in monotherapy fashion, substantially higher concentrations of UMC were required to achieve this effect; CXB was more potent than UMC, but DMC consistently was the most effective (Fig. 7; results not shown for CXB and UMC). Thus, the COX-2 inhibitory potency of CXB and its two analogs was inversely correlated with their ability to aggravate ER stress in combination with NFV. Similarly, subsequent tumor cell death was closely aligned with the aggravation of ER stress, but not at all with the COX-2 inhibitory potential of CXB, DMC, or UMC, neither when these agents were used alone nor when they were combined with NFV. Altogether, these data indicate that the observed combination drug effects were COX-2 independent, and were entirely consistent with several earlier studies that used CXB, UMC, or DMC in monotherapy fashion and found that induction of apoptosis by these compounds bore no correlation to their COX2 inhibitory potency (see Refs. in the legend to Chart 1). It is noteworthy that in our combination experiments we used various drug concentrations that were sub-optimal when administered in monotherapy fashion; this al-

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Fig. 6. Increased apoptosis when NFV is combined with DMC in MCF7 cells. MCF7 cells were treated with 15 lM NFV and 30 lM DMC alone or in combination for various times. (A) At 24 h, apoptosis was analyzed by the propidium iodide method using flow cytometry. The sub-G0/G1 fraction to the left of each graph is indicative of cells undergoing apoptosis. Percentage shown is the average of two independent measurements. (B) After 48 h, apoptosis was determined by cell death ELISA. Shown is the relative increase in apoptotic cell death after drug treatment. (C) After 20 h treatment, activation of caspase-7 was analyzed by Western blot analysis of the pro-caspase (inactive) form and the proteolytically cleaved (activated) form of this enzyme. Actin was used as a loading control.

Fig. 7. Aggravated ER stress and enhanced apoptosis when NFV is combined with DMC in drug-resistant cells. The various cell lines were treated for 20 h with the following concentrations of NFV and/or DMC: MCF7 and BT-474 (20 lM NFV, 40 lM DMC), MCF7/Dox (30 lM NFV, 50 lM DMC), and BT-1.0E (20 lM NFV, 30 lM DMC). Cell lysates were analyzed by Western blot as described in the legend to Fig. 3.

lowed the more obvious emergence of increased toxicity under combination treatment and also supported calculation of the combination index (CI), which consistently was <1.0, indicating drug synergy (not shown). The use of variable concentrations in the different experiments also helped to establish the reproducibility of the observed drug combination effects, as the enhancing effect of drug combination treatment was consistently observed under different experimental conditions and variable concentrations of each drug.

A further important outcome of our study was the finding that NFV, when combined with DMC (or CXB or UMC; results not shown) further aggravated ER stress and increased cell death in drug-resistant breast cancer cells as well. In the case of trastuzumab-resistant cells, the effective cytotoxic drug concentrations essentially were the same as the ones used on the respective non-resistant parental tumor cell line. In the case of highly multidrugresistant (mdr) MCF7/Dox cells, slightly higher concentrations of NFV and CXB (or DMC or UMC) were necessary to

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Fig. 8. Altered chemosensitivity after knock-down of GRP78 or CHOP. (A) MCF7, MCF7/Tax, and MCF7/Dox cells were transfected with siRNA directed at GRP78 or GFP (green fluorescent protein; used as a control). Transfected cells were treated with a combination of 15 lM NVF and 30 lM DMC for 48 h or with vehicle only as a control. (B) MCF7/Dox cells were transfected with siRNA directed at CHOP or GFP and treated with 15 lM NVF and 30 lM DMC alone or in combination for 48 h. In all cases, cell survival was determined by colony formation assay. Shown is percent colony formation (mean ± SD, n P 3), where the number of colonies in control cultures was set to 100%. Asterisk (*) indicates statistically significant (p < 0.05) difference between the number of colonies obtained from drug-treated cells transfected with siGRP78 or CHOP as compared to siGFP (control). (C) Knock-down of basal level GRP78 expression was confirmed by Western blot analysis of all transfected cell lines. In addition, transfected MCF7/Dox cells were treated with 50 lM DMC to stimulate GRP78 and CHOP expression; note that the presence of siRNA suppressed DMC-stimulated levels of both proteins. (The GRP78 blot is somewhat over-exposed to emphasize the large difference between siGFP-transfected and siGRP78-transfected cell cultures.) These experiments were repeated with very similar outcomes.

achieve the same extent of tumor cell death as in the nonresistant parental line, MCF7. When put in perspective, however, this small increase in required drug concentrations for mdr cells was tiny (up to 1.5-fold) when compared to the extent of their resistance to doxorubicin or paclitaxel (>100-fold). Therefore, collectively, our results suggest that the drug combinations used in our study should be explored further with regards to their potential therapeutic benefit for difficult-to-treat cancers, such as advanced and drug-resistant breast cancer. Conflict of interest The authors declare no conflict of interest. Acknowledgements We are grateful to Dr. Amadeo M. Parissenti (Northeastern Ontario Regional Cancer Centre, Sudbury, Ontario, Canada) and Dr. Susan E. Kane (Beckman Research Institute of the City of Hope, Duarte, California) for providing drugresistant cell lines. Funding for this project was received from the L.K. Whittier Foundation via the USC/Norris Comprehensive Cancer Center; the funding source had no involvement in study design or execution, in writing of

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