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Nelfinavir induces TRAIL receptor upregulation in ovarian cancer cells
Biochemical and Biophysical Research Communications 377 (2008) 1309–1314
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Nelfinavir induces TRAIL receptor upregulation in ovarian cancer cells Ansgar Brüning *, Marianne Vogel, Petra Burger, Martina Rahmeh, Andrea Gingelmaier, Klaus Friese, Miriam Lenhard 1, Alexander Burges 1 University Hospital Munich, Department of Obstetrics/Gynecology, Maistrasse 11, 80337 Munich, Germany
a r t i c l e
i n f o
Article history: Received 29 October 2008 Available online 8 November 2008
Keywords: Ovarian cancer Nelfinavir TRAIL
a b s t r a c t HIV protease inhibitors are currently being discussed to be useful as new and alternative anti-cancer agents, especially as second line treatments for chemo-resistant human cancer types. Among three clinically applied HIV protease inhibitors tested, we found a high efficacy of nelfinavir on ovarian cancer cells, accompanied by apoptosis (annexin binding) and necrosis (propidium iodide permeability). In vitro, at concentrations used to induce cell death in ovarian cancer cells, nelfinavir had no effect on the cellular viability of fibroblasts or peripheral blood mononuclear leukocytes. Nelfinavir sensitized ovarian cancer cells to treatment with an apoptosis-inducing TRAIL receptor antibody due to upregulation of the TRAIL receptor DR5 as shown by RT-PCR and FACScan analysis. We conclude that nelfinavir, an already approved drug, is a highly efficient agent against ovarian cancer cells and could sensitize ovarian cancer cells to TRAIL treatment, either therapeutically applied or endogenously produced by cells of the immune system. Ó 2008 Elsevier Inc. All rights reserved.
HIV protease inhibitors, originally designed to inhibit cleavage of HIV envelope precursor proteins [1] and in current use for HIV patients, have recently been discussed to be of potential use in human cancer treatment [2]. The exact action mechanism of these drugs on human cancer cells remains unclear, but it seems to be a combination of proteasome inhibition, cell stress induction, influence on cell signalling cascades, and autophagy [2–4]. This new action mechanism of cancer cell death could be of special interest for the treatment of ovarian cancer, because treatment of this cancer entity is often limited by a rapidly acquired chemoresistance, resulting in cancer recurrence and high mortality of the disease [5,6]. Ovarian cancer is primarily treated by a combination of carboplatin and taxol, drugs which cause intracellular cell damage and thus activation of the so called intrinsic pathway of apoptosis [7]. This pathway is often accompanied by a destabilization of the mitochondrial membrane potential and subsequent activation of cell-degrading caspases [7]. The involvement of caspases is further common to the extrinsic pathway of apoptosis, in which caspases are activated after binding of extracellular ligands such as FasL, TNF (tumor necrosis factor), or TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) to their corresponding receptors [7]. The identification of HIV protease inhibitors as potential agents against human cancer cells could be of special interest for the * Corresponding author. Fax: +49 89 5160 4916. E-mail address: [email protected] (A. Brüning). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.10.167
treatment of highly refractory ovarian cancer. Therefore, we tested the effect of ViraceptÒ (nelfinavir), KaletraÒ (lopinavir and ritonavir), and InviraseÒ (saquinavir) on ovarian cancer cells with different chemo-resistance and p53 mutation status.
Experimental procedures Cells and cell culture. The established ovarian carcinoma cell lines SKOV-3 (ATCC HTB 77; p53 null), OVCAR-3 (ATCC HTB 161; p53 mutant), A2780 (ECACC 93112519; p53 wild-type), and the BJ6-hTERT human foreskin fibroblast cell line were kindly provided by G. Saretzki, Newcastle, UK. The ovarian cancer cell line OV-GH-5 was established at the Women’s Hospital Großhadern (GH), LMU Munich as a cancer cell line from the ascites of an untreated ovarian cancer patient (OV-GH-5) with informed consent and approval by the local ethics committee. Cells were used at passage numbers between 22 and 33. Cells were cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum and antibiotics at 37 °C in a humidified atmosphere with 5% CO2. All cell culture reagents were from Invitrogen, Karlsruhe, Germany. Cell counting was performed with a hemocytometer. Isolation of peripheral blood mononuclear leukocytes (PBML). Ten milliliter heparinised blood of a healthy donor was centrifuged for 10 min at 996g. The buffy coat was layered on a Biocoll separating solution (1.077 g/ml; Biochrom, Berlin, Germany) and centrifuged for 20 min at 823g. Recovered cells were washed 3-times with PBS at 463 g and resuspended in cell culture medium.
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Drugs and drug treatment. ViraceptÒ and InviraseÒ were from Roche, Basel Switzerland, and KaletraÒ from Abbott, Wiesbaden, Germany. Additional amounts of nelfinavir (ViraceptÒ) were generously provided by Pfizer, Groton, CT. HIV protease inhibitors were dissolved in DMSO and kept at 20 °C as stock solutions. In control experiments, cells received the same DMSO concentration as used to apply the DMSO-dissolved HIV protease inhibitors. Apoptosisinducing anti-TRAIL receptor antibody (anti-h-Trail R2, R&D Systems, Wiesbaden, Germany) was used at concentrations between 12.5–100 ng/ml. Carboplatin, provided by the University Hospital Pharmacy, Clinic Großhadern, Munich, was applied at a concentration below or equal to the maximal serum concentration of 100% PPC (peak plasma concentration, 15.8 lg/ml) as reached in clinical chemotherapy for ovarian cancer. Cell survival assays (ATP-TCA and MTT assay). To test viability of cancer cells, 5000 cells in a total volume of 200 ll were plated in flat bottom 96-well plates (Nunc, Wiesbaden, Germany) and incubated with the indicated cytostatic drugs for 72 h at 37 °C. For cell extraction, 50 ll tumor cell extraction buffer (DCS Innovative Diagnostik-Systems, Hamburg, Germany) was added to each well, mixed thoroughly and incubated for 20 min at room temperature. Using a MicroLumat LB 96P bioluminometer (EG&G Berthold, Bad Wildbad, Germany), Luciferin–Luciferase agent (DCS Innovative Diagnostik-Systems, Hamburg, Germany) was added automatically to each sample and analyzed for bioluminescence. Annexin binding assay. FITC-labelled annexin V (Biocat, Heidelberg, Germany) was applied on viable cells as recommended by the supplier in combination with propidium iodide. FACScan analysis was performed using a BectonDickinson FACS analyzer (BectonDickinson, Heidelberg, Germany). Annexin V recognizes the exposition of phosphatidylserine at the outer cell membrane and is regarded as a specific and early event of apoptosis. Propidium iodide (PI) is impermeable for intact cell membranes and stains necrotic cells or apoptotic cells with secondary necrosis. FACScan analysis was performed using a BectonDickinson FACS analyzer (BectonDickinson, Heidelberg, Germany). Western blot analysis. Western blot analysis was performed as recently described [8]. Anti-bid, caspase 8, and PARP antibodies were from Cellsignal (NEB, Frankfurt, Germany), anti-actin antibody from Santa Cruz Biotechnology (Heidelberg, Germany). RT-PCR analysis. RT-PCR analysis was performed as recently described [8]. Primer pairs for DR5 were DR5F (50 -GATCCCA CTGAGACTCTGAGAC-30 ) and DR5R (50 -GGACATGGCAGAGTCTG CAT-30 ). Results The ovarian cancer cell lines SKOV3 (p53 negative, carboplatinsensitive), OVCAR3 (p53 mutant, carboplatin-resistant), and A2780 (wild-type p53, carboplatin-sensitive) were treated for 72 h with the HIV protease inhibitors nelfinavir, lopinavir, and saquinavir. Assessment of cell viability by a bioluminescent ATP assay revealed nelfinavir to be the most effective cell death-inducing drug among the tested protease inhibitors (Fig. 1). No obvious differences in the reaction of the ovarian cancer cells with respect to p53 mutation status or chemo resistance status could be observed. FACScan analysis of OVCAR3 and A2780 cells treated with annexin-FITC to detect apoptotic cells (FL2 setting; Fig. 2) and propidium iodide to detect the occurrence of necrotic cells (FL1 setting; Fig. 2) revealed that cell death induced by nelfinavir is associated with apoptotic as well as necrotic cell death (Fig. 2). However, the extent of cell death as viewed by phase contrast microscopy (Fig. 2) appeared to be significantly higher than detectable by FACScan analysis, indicating further cell death mechanisms induced by nelfinavir. Autophagy, as indicated by extensive vacuolization of
Fig. 1. HIV protease inhibitors induce cell death in ovarian cancer cells. The ovarian cancer cell lines SKOV-3 (A), OVCAR-3, (B) and A2780 (C) were treated for 72 h with the HIV protease inhibitors nelfinavir (ViraceptÒ), lopinavir (KaletraÒ), and saquinavir (InviraseÒ) at the indicated concentrations and cell viability was analyzed by a chemo-sensitivity assay (ATP-assay).
OVCAR3 cells (Fig. 2) might represent one of these mechanisms. We further extended our study on non-malignant control cells, including peripheral blood mononuclear leukocytes (PBML) and BJ6 fibroblasts, an immortalized human fibroblast cell line. Fig. 2 shows that nelfinavir, used at the same concentrations to induce cell death in ovarian cancer cells, exerts no detectable apoptotic or necrotic effects on PBML or fibroblasts. To get a better insight into the cell death mechanism induced by nelfinavir, we applied a commercially available antibody array (RnD Systems) to screen for the expression of apoptosis-related proteins in cell extracts of A2780 cells treated with (+) or without () nelfinavir (Fig. 3). The antibody array is based on membranebound capture antibodies, spotted in duplicate on carrier membranes (Fig. 3). Unexpectedly, most apoptosis-related proteins, including those of the bcl family, appeared to be uninfluenced by nelfinavir, although a significant increase in the occurrence of the cleaved form of caspase 3 clearly indicated the occurrence of apoptosis (Fig. 3). This reveals a non-classical, but yet unidentified mechanism of apoptosis induced by nelfinavir. Besides activation of caspase 3, the most prominently regulated protein appeared to be the TRAIL receptor 2 (DR5, death receptor 5), indicating a potential TRAIL-sensitizing effect of nelfinavir. Fig. 4a shows that incubation of A2780 cells with nelfinvar enhanced sensitivity of this cell line to treatment with an apoptosis-inducing TRAIL receptor antibody (RnD Systems, DR5-AB, Clone 71903). The combination effect
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Fig. 2. Nelfinavir induces cell death in ovarian cancer cells but not in non-malignant fibroblasts or PBML. OVCAR3 cells (A), A2780 cells (B), BJ6 cells (C), and PBML (D) were treated for 48 h either with or without 10 lg/ml nelfinavir and analyzed by FACScan analysis with FITC-conjugated annexin to reveal apoptosis, and propidium iodide to reveal necrotic cell death. FL-1 setting (propidium iodide): 575 nm; FL-2 setting (FITC): 530 nm. Corresponding figures of OVCAR3 cells (20 lens), A2780 cells (20 lens), BJ6 cells (20 lens), and PBML (40 lens) treated with or without nelfinavir are shown by phase contrast microphotographs.
of nelfinavir with TRAIL was further analyzed on the OV-GH5 cell line, a carboplatin- and TRAIL-sensitive ovarian cancer cell line of low passage number, derived from the ascites of an untreated ovarian cancer patient established at our laboratory. Nelfinavir did not seem to cooperate or sensitize to carboplatin treatment (Fig. 4b), but a cooperative effect for nelfinavir and the DR5 antibody could be observed (Fig. 4b). FACScan analysis for TRAIL receptor expression with a non-apoptosis-inducing DR5 antibody showed time-dependent upregulation of DR5 by nelfinavir at the cell membrane of OVGH5 cells (Fig. 4c), explaining its sensitizing effect to the apoptosis-inducing DR5 antibody. DR5 was found to be upregulated at the mRNA level, as shown by RT-PCR analysis
(Fig. 4d). However, FACScan as well as RT-PCR analysis revealed that DR5 upregulation was a late effect, obtained after prolonged incubation with nelfinavir. Fig. 4e further shows that combination of nelfinavir with the DR5 antibody significantly enhanced cleavage of PARP, associated with cleavage of caspase 8 and bid. Discussion Among three HIV protease inhibitors tested in our study, nelfinavir proved to be the most effective drug against ovarian cancer cells. Most noticeable, nelfinavir acted on carboplatin-sensitive (A2780, OVGH5, SKOV3) as well as on carboplatin-resistant ovarian cancer
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Fig. 3. Antibody array analysis for apoptosis-relevant proteins influenced by nelfinavir. A Human Apoptosis Proteome ProfilerArray (R&D Systems) comprising 35 apoptosis-relevant protein antibodies was incubated with cell extracts from A2780 cells treated either with (+) or without () nelfinavir.
cells (OVCAR3) and could thus be of interest to be used as a second line treatment in case of recurrent, chemo-resistant ovarian cancer. Cell death was independent of the p53 status and was induced in A2780 p53 wild-type cancer cells as well as in p53 mutant SKOV3 and OVCAR3 cell lines. Further, and of special interest for clinical purposes, nelfinavir had no effect on the cell survival of fibroblasts or peripheral blood mononuclear leukocytes when applied at concentrations used to induce apoptosis in ovarian cancer cells. As already discussed by Gills et al. [4], the cell death mechanism of nelfinavir is not well understood and involves mechanisms of apoptosis, necrosis, and autophagy. In fact, our FACScan data (Fig. 2) confirm induction of apoptosis (annexin binding) and necrosis (PI staining). Western blot analysis further revealed PARP cleavage and caspase 3 activation (antibody array). Extensive vacuolization of OVCAR3 cells by nelfinavir (Fig. 2) further indicated induction of autophagy, although less in A2780 cells. Thus, induction of ovarian cancer cell death by nelfinavir is mediated by a yet not well understood pleiotropic effect and needs further independent studies. Besides its effect as a single agent, a sensitizing effect could be observed for co-treatment of nelfinavir with an apoptosis-inducing
antibody against the TRAIL receptor DR5. Efficacy and potential use of TRAIL treatment against gynecological cancers has recently been shown by us [9] and others [10–12]. However, not all ovarian cancer cells lines respond to TRAIL treatment as shown in our present study, and drugs that could sensitize to TRAIL treatment by e.g. TRAIL receptor upregulation could be rather useful to enhance and broaden efficacy of TRAIL treatment against ovarian cancer cells. TRAIL, either in its membrane-bound form or as a trimeric soluble peptide, is able to induce apoptosis in target cells by causing receptor trimerization [7] and thus activation of either the TRAIL receptor 1 (DR4, death receptor 4) or TRAIL receptor 2 (DR5). In contrast to e.g. the tumor necrosis factor TNF, which is far less effective on cancer cells than TRAIL and causes severe systemic side effects, TRAIL appears to be rather well tolerable, except an often observed negative effect on liver cells [13,14]. Therefore, several human monoclonal antibodies have been developed that can induce trimerization and activation of either DR4 or DR5 to induce TRAIL receptor-mediated apoptosis in human cancer cells [15–17], similar to that induced by us with a commercially available mouse monoclonal antibody. Interestingly, whereas normal human body cells are rather insensitive to TRAIL [13], the expression of TRAIL receptors and sensitivity to TRAIL is found predominantly associated with cancer cells, thus representing a potential Achilles heel of cancer cells. Bortezomib, a specific proteasome inhibitor, has previously been shown to induce upregulation of TRAIL receptors in A2780 cells [18], the same cell line for which we demonstrated a nelfinavir-mediated upregulation of TRAIL receptors by nelfinavir. In cell culture, apoptosis of ovarian cancer cells by nelfinavir as a single agent is not caused by DR5 receptor upregulation because it is generally believed that the extrinsic pathway of apoptosis can only be triggered by ligand activation and TRAIL is no component of the cell culture medium used. However, in vivo, cancer cells are surrounded by activated cells of the immune system that secrete either soluble TRAIL or exhibit membrane-bound TRAIL at their cell membrane [19].Thus, upregulation of DR5 by nelfinavir could enhance apoptosis under in vivo conditions in addition to clinical conditions in which external DR5 antibodies may be applied for therapeutic purposes. In contrast to the apoptosis-inducing effect of nelfinavir in human cancer cells, several reports have shown that HIV protease inhibitors, including nelfinavir, can prevent apoptosis of untransformed cells in certain diseases such as sepsis, hepatitis, pancreatitis, retina degeneration, and stroke [20–22]. These observations could be confirmed by us, showing that nelfinavir did not induce cell death in fibroblasts or peripheral blood mononuclear leukocytes. The exact mechanism and cause of this effect remains unclear, but it appears to be that nelfinavir exerts a mitochondrial membrane-protecting effect, as shown for retina cells [22] and peripheral blood cells [23,24]. Because BJ6 fibroblasts represent an immortalized, proliferating cell line, the difference between the effect of nelfinavir on malignant and non-malignant cells does not appear to be related to the proliferative potential of the cells, probably because nelfinavir does not act as a DNA-damaging or microtubule-interacting drug. Based on our observations on the effect of nelfinavir on ovarian cancer cells and non-malignant cells, nelfinavir could become a highly interesting new drug for the treatment of even chemo-resistant ovarian cancer cells, worthwhile to be tested in further preclinical and clinical studies. In those studies, a combination therapy with TRAIL or a monoclonal TRAIL receptor antibody could be of interest. Using the H157 lung cancer cell lines, Gills et al. [25] have recently shown a growth-inhibiting effect of nelfinavir in a nude mouse model, and Brunner et al. [26] recently presented a phase I clinical study of nelfinavir with promising data on toxicity and activity in patients with pancreatic cancer.
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Fig. 4. Nelfinavir sensitizes to treatment with an apoptosis-inducing TRAIL receptor antibody. (A) A2780 cells were treated for 72 h with the indicated concentrations of nelfinavir and anti-DR5 antibody, and cell survival was analyzed by an ATP assay. (B) The ovarian cancer cell line OVGH5 was treated for 72 h with nelfinavir at the indicated concentration either alone or in combination with carboplatin at 50% peak plasma concentration (PPC), or an apoptosis-inducing TRAIL receptor antibody at 50 ng/ml. (C) The ovarian cancer cell line OVGH5 was treated with 10 lg/ml nelfinavir for the indicated time points (0–72 h) and expression of DR5 was demonstrated by incubation of OVGH5 cells with a monoclonal antibody against TRAIL receptor 2 (DR5), followed by a FITC-conjugated secondary antibody. Dotted line: OVGH5 cells without nelfinavir treatment; solid line: cells treated with nelfinavir for the indicated time points. (E) Expression of DR5 mRNA was analyzed by RT-PCR analysis in a time-dependent manner. (D) OVGH5 cells were treated for 48 h with either 10 lg/ml nelfinavir alone or in combination with 100 ng/ml apoptosis-inducing TRAIL receptor antibody as indicated and analyzed by Western blot analysis. C = control, N = nelfinavir, T = DR5 antibody, T/N = combination of nelfinavir with DR5 antibody. t-bid: truncated bid.
Acknowledgments We greatly appreciate the generous supply with nelfinavir by Pfizer, Groton, CT. This work was supported by the Deutsche Forschungsgemeinschaft (DFG BR 3641/1-1).
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Nelfinavir induces TRAIL receptor upregulation in ovarian cancer cells Ansgar Brüning *, Marianne Vogel, Petra Burger, Martina Rahmeh, Andrea Gingelmaier, Klaus Friese, Miriam Lenhard 1, Alexander Burges 1 University Hospital Munich, Department of Obstetrics/Gynecology, Maistrasse 11, 80337 Munich, Germany
a r t i c l e
i n f o
Article history: Received 29 October 2008 Available online 8 November 2008
Keywords: Ovarian cancer Nelfinavir TRAIL
a b s t r a c t HIV protease inhibitors are currently being discussed to be useful as new and alternative anti-cancer agents, especially as second line treatments for chemo-resistant human cancer types. Among three clinically applied HIV protease inhibitors tested, we found a high efficacy of nelfinavir on ovarian cancer cells, accompanied by apoptosis (annexin binding) and necrosis (propidium iodide permeability). In vitro, at concentrations used to induce cell death in ovarian cancer cells, nelfinavir had no effect on the cellular viability of fibroblasts or peripheral blood mononuclear leukocytes. Nelfinavir sensitized ovarian cancer cells to treatment with an apoptosis-inducing TRAIL receptor antibody due to upregulation of the TRAIL receptor DR5 as shown by RT-PCR and FACScan analysis. We conclude that nelfinavir, an already approved drug, is a highly efficient agent against ovarian cancer cells and could sensitize ovarian cancer cells to TRAIL treatment, either therapeutically applied or endogenously produced by cells of the immune system. Ó 2008 Elsevier Inc. All rights reserved.
HIV protease inhibitors, originally designed to inhibit cleavage of HIV envelope precursor proteins [1] and in current use for HIV patients, have recently been discussed to be of potential use in human cancer treatment [2]. The exact action mechanism of these drugs on human cancer cells remains unclear, but it seems to be a combination of proteasome inhibition, cell stress induction, influence on cell signalling cascades, and autophagy [2–4]. This new action mechanism of cancer cell death could be of special interest for the treatment of ovarian cancer, because treatment of this cancer entity is often limited by a rapidly acquired chemoresistance, resulting in cancer recurrence and high mortality of the disease [5,6]. Ovarian cancer is primarily treated by a combination of carboplatin and taxol, drugs which cause intracellular cell damage and thus activation of the so called intrinsic pathway of apoptosis [7]. This pathway is often accompanied by a destabilization of the mitochondrial membrane potential and subsequent activation of cell-degrading caspases [7]. The involvement of caspases is further common to the extrinsic pathway of apoptosis, in which caspases are activated after binding of extracellular ligands such as FasL, TNF (tumor necrosis factor), or TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) to their corresponding receptors [7]. The identification of HIV protease inhibitors as potential agents against human cancer cells could be of special interest for the * Corresponding author. Fax: +49 89 5160 4916. E-mail address: [email protected] (A. Brüning). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.10.167
treatment of highly refractory ovarian cancer. Therefore, we tested the effect of ViraceptÒ (nelfinavir), KaletraÒ (lopinavir and ritonavir), and InviraseÒ (saquinavir) on ovarian cancer cells with different chemo-resistance and p53 mutation status.
Experimental procedures Cells and cell culture. The established ovarian carcinoma cell lines SKOV-3 (ATCC HTB 77; p53 null), OVCAR-3 (ATCC HTB 161; p53 mutant), A2780 (ECACC 93112519; p53 wild-type), and the BJ6-hTERT human foreskin fibroblast cell line were kindly provided by G. Saretzki, Newcastle, UK. The ovarian cancer cell line OV-GH-5 was established at the Women’s Hospital Großhadern (GH), LMU Munich as a cancer cell line from the ascites of an untreated ovarian cancer patient (OV-GH-5) with informed consent and approval by the local ethics committee. Cells were used at passage numbers between 22 and 33. Cells were cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum and antibiotics at 37 °C in a humidified atmosphere with 5% CO2. All cell culture reagents were from Invitrogen, Karlsruhe, Germany. Cell counting was performed with a hemocytometer. Isolation of peripheral blood mononuclear leukocytes (PBML). Ten milliliter heparinised blood of a healthy donor was centrifuged for 10 min at 996g. The buffy coat was layered on a Biocoll separating solution (1.077 g/ml; Biochrom, Berlin, Germany) and centrifuged for 20 min at 823g. Recovered cells were washed 3-times with PBS at 463 g and resuspended in cell culture medium.
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Drugs and drug treatment. ViraceptÒ and InviraseÒ were from Roche, Basel Switzerland, and KaletraÒ from Abbott, Wiesbaden, Germany. Additional amounts of nelfinavir (ViraceptÒ) were generously provided by Pfizer, Groton, CT. HIV protease inhibitors were dissolved in DMSO and kept at 20 °C as stock solutions. In control experiments, cells received the same DMSO concentration as used to apply the DMSO-dissolved HIV protease inhibitors. Apoptosisinducing anti-TRAIL receptor antibody (anti-h-Trail R2, R&D Systems, Wiesbaden, Germany) was used at concentrations between 12.5–100 ng/ml. Carboplatin, provided by the University Hospital Pharmacy, Clinic Großhadern, Munich, was applied at a concentration below or equal to the maximal serum concentration of 100% PPC (peak plasma concentration, 15.8 lg/ml) as reached in clinical chemotherapy for ovarian cancer. Cell survival assays (ATP-TCA and MTT assay). To test viability of cancer cells, 5000 cells in a total volume of 200 ll were plated in flat bottom 96-well plates (Nunc, Wiesbaden, Germany) and incubated with the indicated cytostatic drugs for 72 h at 37 °C. For cell extraction, 50 ll tumor cell extraction buffer (DCS Innovative Diagnostik-Systems, Hamburg, Germany) was added to each well, mixed thoroughly and incubated for 20 min at room temperature. Using a MicroLumat LB 96P bioluminometer (EG&G Berthold, Bad Wildbad, Germany), Luciferin–Luciferase agent (DCS Innovative Diagnostik-Systems, Hamburg, Germany) was added automatically to each sample and analyzed for bioluminescence. Annexin binding assay. FITC-labelled annexin V (Biocat, Heidelberg, Germany) was applied on viable cells as recommended by the supplier in combination with propidium iodide. FACScan analysis was performed using a BectonDickinson FACS analyzer (BectonDickinson, Heidelberg, Germany). Annexin V recognizes the exposition of phosphatidylserine at the outer cell membrane and is regarded as a specific and early event of apoptosis. Propidium iodide (PI) is impermeable for intact cell membranes and stains necrotic cells or apoptotic cells with secondary necrosis. FACScan analysis was performed using a BectonDickinson FACS analyzer (BectonDickinson, Heidelberg, Germany). Western blot analysis. Western blot analysis was performed as recently described [8]. Anti-bid, caspase 8, and PARP antibodies were from Cellsignal (NEB, Frankfurt, Germany), anti-actin antibody from Santa Cruz Biotechnology (Heidelberg, Germany). RT-PCR analysis. RT-PCR analysis was performed as recently described [8]. Primer pairs for DR5 were DR5F (50 -GATCCCA CTGAGACTCTGAGAC-30 ) and DR5R (50 -GGACATGGCAGAGTCTG CAT-30 ). Results The ovarian cancer cell lines SKOV3 (p53 negative, carboplatinsensitive), OVCAR3 (p53 mutant, carboplatin-resistant), and A2780 (wild-type p53, carboplatin-sensitive) were treated for 72 h with the HIV protease inhibitors nelfinavir, lopinavir, and saquinavir. Assessment of cell viability by a bioluminescent ATP assay revealed nelfinavir to be the most effective cell death-inducing drug among the tested protease inhibitors (Fig. 1). No obvious differences in the reaction of the ovarian cancer cells with respect to p53 mutation status or chemo resistance status could be observed. FACScan analysis of OVCAR3 and A2780 cells treated with annexin-FITC to detect apoptotic cells (FL2 setting; Fig. 2) and propidium iodide to detect the occurrence of necrotic cells (FL1 setting; Fig. 2) revealed that cell death induced by nelfinavir is associated with apoptotic as well as necrotic cell death (Fig. 2). However, the extent of cell death as viewed by phase contrast microscopy (Fig. 2) appeared to be significantly higher than detectable by FACScan analysis, indicating further cell death mechanisms induced by nelfinavir. Autophagy, as indicated by extensive vacuolization of
Fig. 1. HIV protease inhibitors induce cell death in ovarian cancer cells. The ovarian cancer cell lines SKOV-3 (A), OVCAR-3, (B) and A2780 (C) were treated for 72 h with the HIV protease inhibitors nelfinavir (ViraceptÒ), lopinavir (KaletraÒ), and saquinavir (InviraseÒ) at the indicated concentrations and cell viability was analyzed by a chemo-sensitivity assay (ATP-assay).
OVCAR3 cells (Fig. 2) might represent one of these mechanisms. We further extended our study on non-malignant control cells, including peripheral blood mononuclear leukocytes (PBML) and BJ6 fibroblasts, an immortalized human fibroblast cell line. Fig. 2 shows that nelfinavir, used at the same concentrations to induce cell death in ovarian cancer cells, exerts no detectable apoptotic or necrotic effects on PBML or fibroblasts. To get a better insight into the cell death mechanism induced by nelfinavir, we applied a commercially available antibody array (RnD Systems) to screen for the expression of apoptosis-related proteins in cell extracts of A2780 cells treated with (+) or without () nelfinavir (Fig. 3). The antibody array is based on membranebound capture antibodies, spotted in duplicate on carrier membranes (Fig. 3). Unexpectedly, most apoptosis-related proteins, including those of the bcl family, appeared to be uninfluenced by nelfinavir, although a significant increase in the occurrence of the cleaved form of caspase 3 clearly indicated the occurrence of apoptosis (Fig. 3). This reveals a non-classical, but yet unidentified mechanism of apoptosis induced by nelfinavir. Besides activation of caspase 3, the most prominently regulated protein appeared to be the TRAIL receptor 2 (DR5, death receptor 5), indicating a potential TRAIL-sensitizing effect of nelfinavir. Fig. 4a shows that incubation of A2780 cells with nelfinvar enhanced sensitivity of this cell line to treatment with an apoptosis-inducing TRAIL receptor antibody (RnD Systems, DR5-AB, Clone 71903). The combination effect
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Fig. 2. Nelfinavir induces cell death in ovarian cancer cells but not in non-malignant fibroblasts or PBML. OVCAR3 cells (A), A2780 cells (B), BJ6 cells (C), and PBML (D) were treated for 48 h either with or without 10 lg/ml nelfinavir and analyzed by FACScan analysis with FITC-conjugated annexin to reveal apoptosis, and propidium iodide to reveal necrotic cell death. FL-1 setting (propidium iodide): 575 nm; FL-2 setting (FITC): 530 nm. Corresponding figures of OVCAR3 cells (20 lens), A2780 cells (20 lens), BJ6 cells (20 lens), and PBML (40 lens) treated with or without nelfinavir are shown by phase contrast microphotographs.
of nelfinavir with TRAIL was further analyzed on the OV-GH5 cell line, a carboplatin- and TRAIL-sensitive ovarian cancer cell line of low passage number, derived from the ascites of an untreated ovarian cancer patient established at our laboratory. Nelfinavir did not seem to cooperate or sensitize to carboplatin treatment (Fig. 4b), but a cooperative effect for nelfinavir and the DR5 antibody could be observed (Fig. 4b). FACScan analysis for TRAIL receptor expression with a non-apoptosis-inducing DR5 antibody showed time-dependent upregulation of DR5 by nelfinavir at the cell membrane of OVGH5 cells (Fig. 4c), explaining its sensitizing effect to the apoptosis-inducing DR5 antibody. DR5 was found to be upregulated at the mRNA level, as shown by RT-PCR analysis
(Fig. 4d). However, FACScan as well as RT-PCR analysis revealed that DR5 upregulation was a late effect, obtained after prolonged incubation with nelfinavir. Fig. 4e further shows that combination of nelfinavir with the DR5 antibody significantly enhanced cleavage of PARP, associated with cleavage of caspase 8 and bid. Discussion Among three HIV protease inhibitors tested in our study, nelfinavir proved to be the most effective drug against ovarian cancer cells. Most noticeable, nelfinavir acted on carboplatin-sensitive (A2780, OVGH5, SKOV3) as well as on carboplatin-resistant ovarian cancer
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Fig. 3. Antibody array analysis for apoptosis-relevant proteins influenced by nelfinavir. A Human Apoptosis Proteome ProfilerArray (R&D Systems) comprising 35 apoptosis-relevant protein antibodies was incubated with cell extracts from A2780 cells treated either with (+) or without () nelfinavir.
cells (OVCAR3) and could thus be of interest to be used as a second line treatment in case of recurrent, chemo-resistant ovarian cancer. Cell death was independent of the p53 status and was induced in A2780 p53 wild-type cancer cells as well as in p53 mutant SKOV3 and OVCAR3 cell lines. Further, and of special interest for clinical purposes, nelfinavir had no effect on the cell survival of fibroblasts or peripheral blood mononuclear leukocytes when applied at concentrations used to induce apoptosis in ovarian cancer cells. As already discussed by Gills et al. [4], the cell death mechanism of nelfinavir is not well understood and involves mechanisms of apoptosis, necrosis, and autophagy. In fact, our FACScan data (Fig. 2) confirm induction of apoptosis (annexin binding) and necrosis (PI staining). Western blot analysis further revealed PARP cleavage and caspase 3 activation (antibody array). Extensive vacuolization of OVCAR3 cells by nelfinavir (Fig. 2) further indicated induction of autophagy, although less in A2780 cells. Thus, induction of ovarian cancer cell death by nelfinavir is mediated by a yet not well understood pleiotropic effect and needs further independent studies. Besides its effect as a single agent, a sensitizing effect could be observed for co-treatment of nelfinavir with an apoptosis-inducing
antibody against the TRAIL receptor DR5. Efficacy and potential use of TRAIL treatment against gynecological cancers has recently been shown by us [9] and others [10–12]. However, not all ovarian cancer cells lines respond to TRAIL treatment as shown in our present study, and drugs that could sensitize to TRAIL treatment by e.g. TRAIL receptor upregulation could be rather useful to enhance and broaden efficacy of TRAIL treatment against ovarian cancer cells. TRAIL, either in its membrane-bound form or as a trimeric soluble peptide, is able to induce apoptosis in target cells by causing receptor trimerization [7] and thus activation of either the TRAIL receptor 1 (DR4, death receptor 4) or TRAIL receptor 2 (DR5). In contrast to e.g. the tumor necrosis factor TNF, which is far less effective on cancer cells than TRAIL and causes severe systemic side effects, TRAIL appears to be rather well tolerable, except an often observed negative effect on liver cells [13,14]. Therefore, several human monoclonal antibodies have been developed that can induce trimerization and activation of either DR4 or DR5 to induce TRAIL receptor-mediated apoptosis in human cancer cells [15–17], similar to that induced by us with a commercially available mouse monoclonal antibody. Interestingly, whereas normal human body cells are rather insensitive to TRAIL [13], the expression of TRAIL receptors and sensitivity to TRAIL is found predominantly associated with cancer cells, thus representing a potential Achilles heel of cancer cells. Bortezomib, a specific proteasome inhibitor, has previously been shown to induce upregulation of TRAIL receptors in A2780 cells [18], the same cell line for which we demonstrated a nelfinavir-mediated upregulation of TRAIL receptors by nelfinavir. In cell culture, apoptosis of ovarian cancer cells by nelfinavir as a single agent is not caused by DR5 receptor upregulation because it is generally believed that the extrinsic pathway of apoptosis can only be triggered by ligand activation and TRAIL is no component of the cell culture medium used. However, in vivo, cancer cells are surrounded by activated cells of the immune system that secrete either soluble TRAIL or exhibit membrane-bound TRAIL at their cell membrane [19].Thus, upregulation of DR5 by nelfinavir could enhance apoptosis under in vivo conditions in addition to clinical conditions in which external DR5 antibodies may be applied for therapeutic purposes. In contrast to the apoptosis-inducing effect of nelfinavir in human cancer cells, several reports have shown that HIV protease inhibitors, including nelfinavir, can prevent apoptosis of untransformed cells in certain diseases such as sepsis, hepatitis, pancreatitis, retina degeneration, and stroke [20–22]. These observations could be confirmed by us, showing that nelfinavir did not induce cell death in fibroblasts or peripheral blood mononuclear leukocytes. The exact mechanism and cause of this effect remains unclear, but it appears to be that nelfinavir exerts a mitochondrial membrane-protecting effect, as shown for retina cells [22] and peripheral blood cells [23,24]. Because BJ6 fibroblasts represent an immortalized, proliferating cell line, the difference between the effect of nelfinavir on malignant and non-malignant cells does not appear to be related to the proliferative potential of the cells, probably because nelfinavir does not act as a DNA-damaging or microtubule-interacting drug. Based on our observations on the effect of nelfinavir on ovarian cancer cells and non-malignant cells, nelfinavir could become a highly interesting new drug for the treatment of even chemo-resistant ovarian cancer cells, worthwhile to be tested in further preclinical and clinical studies. In those studies, a combination therapy with TRAIL or a monoclonal TRAIL receptor antibody could be of interest. Using the H157 lung cancer cell lines, Gills et al. [25] have recently shown a growth-inhibiting effect of nelfinavir in a nude mouse model, and Brunner et al. [26] recently presented a phase I clinical study of nelfinavir with promising data on toxicity and activity in patients with pancreatic cancer.
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Fig. 4. Nelfinavir sensitizes to treatment with an apoptosis-inducing TRAIL receptor antibody. (A) A2780 cells were treated for 72 h with the indicated concentrations of nelfinavir and anti-DR5 antibody, and cell survival was analyzed by an ATP assay. (B) The ovarian cancer cell line OVGH5 was treated for 72 h with nelfinavir at the indicated concentration either alone or in combination with carboplatin at 50% peak plasma concentration (PPC), or an apoptosis-inducing TRAIL receptor antibody at 50 ng/ml. (C) The ovarian cancer cell line OVGH5 was treated with 10 lg/ml nelfinavir for the indicated time points (0–72 h) and expression of DR5 was demonstrated by incubation of OVGH5 cells with a monoclonal antibody against TRAIL receptor 2 (DR5), followed by a FITC-conjugated secondary antibody. Dotted line: OVGH5 cells without nelfinavir treatment; solid line: cells treated with nelfinavir for the indicated time points. (E) Expression of DR5 mRNA was analyzed by RT-PCR analysis in a time-dependent manner. (D) OVGH5 cells were treated for 48 h with either 10 lg/ml nelfinavir alone or in combination with 100 ng/ml apoptosis-inducing TRAIL receptor antibody as indicated and analyzed by Western blot analysis. C = control, N = nelfinavir, T = DR5 antibody, T/N = combination of nelfinavir with DR5 antibody. t-bid: truncated bid.
Acknowledgments We greatly appreciate the generous supply with nelfinavir by Pfizer, Groton, CT. This work was supported by the Deutsche Forschungsgemeinschaft (DFG BR 3641/1-1).
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