Tamoxifen enhances the cytotoxic effects of nelfinavir in breast cancer cells
© Brüning et al.; licensee BioMed Central Ltd. 2010
Received: 1 March 2010
Accepted: 1 July 2010
Published: 1 July 2010
The HIV protease inhibitor nelfinavir is currently under investigation as a new anti-cancer drug. Several studies have shown that nelfinavir induces cell cycle arrest, endoplasmic reticulum stress, autophagy, and apoptosis in cancer cells. In the present article, the effect of nelfinavir on human breast cancer cells is examined and potential combination treatments are investigated.
The effects of nelfinavir and tamoxifen on the human breast cancer cell lines MCF7, T47 D, MDA-MB-453, and MDA-MB-435 were tested by analysing their influence on cell viability (via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay), apoptosis (annexin binding, poly(ADP-ribose) polymerase cleavage), autophagy (autophagy marker light chain 3B expression), endoplasmic reticulum stress (binding protein and activating transcription factor 3 expression), and the occurrence of oxidative stress (intracellular glutathione level).
Nelfinavir induced apoptosis in all four breast cancer cell lines tested, although the extent of autophagy and endoplasmic reticulum stress varied among the cell lines. The concentration of nelfinavir needed for an efficient induction of apoptosis in breast cancer cells could be reduced from 15 μg/ml to 6 μg/ml when combined with tamoxifen. At a concentration of 6 μg/ml, tamoxifen substantially enhanced the endoplasmic reticulum stress reaction in those cell lines that responded to nelfinavir with binding protein (BiP) upregulation (MCF7, T47D), and enhanced autophagy in cell lines that responded to nelfinavir treatment with autophagy marker light chain 3B upregulation (MDA-MB-453). Although tamoxifen has been described to be able to induce oxidative stress at concentrations similar to those applied in this study (6 μg/ml), we observed that nelfinavir but not tamoxifen reduced the intracellular glutathione level of breast cancer cells within hours of application by up to 32%, suggesting the induction of oxidative stress was an early event and an additional cause of the apoptosis induced by nelfinavir.
The results demonstrate that nelfinavir may be an effective drug against breast cancer and could be combined with tamoxifen to enhance its efficacy against breast cancer cells. Moreover, the cytotoxic effect of a tamoxifen and nelfinavir combination was independent of the oestrogen receptor status of the analysed breast cancer cells, suggesting a potential benefit of a combination of these two drugs even in patients with no hormone-responsive tumours. We therefore recommend that clinical studies on nelfinavir with breast cancer patients should include this drug combination to analyse the therapeutic efficacy as well as the safety and tolerability of this potential treatment option.
Breast cancer is the most frequent cancer in the female population . Although tremendous progress in the treatment of breast cancer has been achieved during past decades, it is still the principal cause of cancer death for females worldwide [1, 2].
Tamoxifen is a selective oestrogen receptor antagonist, and since its introduction in cancer therapy has become the standard treatment option for hormone-responsive breast cancer patients [2–5]. Not all breast cancer patients, however, benefit from an endocrine therapy with tamoxifen . Interestingly, several hormone receptor-independent effects of tamoxifen have been described, leading to apoptosis when higher concentrations of tamoxifen are applied [6, 7]. A combination therapy of tamoxifen with other drugs that cause synergistic anti-tumour effects might therefore be an interesting option in the therapy of breast carcinomas.
Nelfinavir (Viracept®; Pfizer, Groton, CT, USA) is an HIV protease inhibitor that has long been an essential component of the antiviral combination highly active antiretroviral therapy. Several recent in vitro studies have indicated that nelfinavir has potential anti-tumoral effects [8, 9], and clinical studies on nelfinavir are ongoing to confirm its efficacy against human cancers in vivo [10–13]. Nelfinavir exerts pleiotropic effects on cancer cells, including induction of apoptosis, necrosis, and autophagy [9, 14, 15]. Nelfinavir is believed to either cross-react with a protease of the cytoplasmic proteasomal protein degradation machinery or with endoplasmic reticulum-resident proteases [15, 16]. In both cases, this protease inhibition can lead to the accumulation of misfolded proteins that cause the unfolded protein response or endoplasmic reticulum stress response [17–19]. These pathways are primarily associated with a transient cell cycle arrest and upregulation of molecular chaperones such as binding protein (BiP) and other members of the heat shock family, in order to repair and prevent further cell damage . A prolonged or irreparable stress reaction, however, eventually switches from a repair and survival mechanism to cell death executed by apoptosis [20, 21]. This nonclassical cell death mechanism has recently become of interest because of its ability to act even on otherwise chemoresistant human cancer cells [15, 22].
Since the orally available drugs tamoxifen and nelfinavir have anti-tumoral properties, a combination of these medications might be an intriguing option in the therapy of breast cancer patients. However, no data regarding their potential synergistic effects are yet available. We therefore tested the effect of nelfinavir and tamoxifen with regard to the influence on apoptosis, endoplasmic reticulum stress, autophagy, and oxidative stress in breast cancer cells with different oestrogen receptor status.
Materials and methods
Cells and cell culture
The breast cancer cell lines T47 D (ATCC HTB 133; oestrogen receptor-positive), MCF7 (ATCC HTB 22; oestrogen receptor-positive), MDA-MB-453 (ATCC HTB 131; oestrogen receptor-negative), and MDA-MB-435 S (ATCC HTB 129; oestrogen receptor-negative) - all kindly provided by G Saretzki (Newcastle, UK) - were cultured in RPMI-1640 medium supplemented with 10% foetal calf serum and antibiotics at 37°C in a humidified atmosphere with 5% CO2. All cell culture reagents were from PAA (Pasching, Austria).
Drugs and drug treatment
Nelfinavir (Viracept®) was generously provided by Pfizer. Nelfinavir was dissolved in ethanol and kept at -20°C as a 100 mg/ml stock solution. Tamoxifen (Sigma, Munich, Germany) was dissolved in dimethylsulfoxide at a concentration of 100 mg/ml. In control experiments, equal amounts of dimethylsulfoxide or ethanol were added.
Cell proliferation analysis
A total of 2 × 104 cells per well were seeded in quadruplicate in 24-well cell culture plates and were incubated with nelfinavir for up to 4 days. The number of viable, trypan blue-excluding cells was determined by a haemocytometer.
For the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay analysis, 20 μl MTT (Sigma) stock solution (5 mg/ml PBS) was added to viable cells in 200 μl cell culture medium for 1 hour under cell culture conditions. The water-insoluble precipitate was dissolved in 100 μl dimethylsulfoxide and analysed by an ELISA reader at 595 nm.
Annexin binding assay
FITC-labelled annexin V (Biocat, Heidelberg, Germany) was applied to viable cells as recommended by the supplier in combination with propidium iodide, and was analysed by FACScan with an FL-1 setting (propidium iodide) at 575 nm and an FL-2 setting (FITC) at 530 nm. FACScan analysis was performed using a Becton Dickinson FACScan analyser (Becton Dickinson, Heidelberg, Germany).
Western blot analysis
Cell extracts of cancer cells cultured in cell culture plates were prepared with radio-immunoprecipitation buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP40, 0.5% doxycholine, 0.1% SDS) and 20 μg protein (BioRad Bradford Assay; BioRad, Munich, Germany) were subjected to SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes in a BioRad Mini Protean II Cell (BioRad) at 1 mA/cm2 membrane in 10% methanol, 192 mM glycine, 25 mM Tris, pH 8.2. Membranes were blocked with 4% nonfat milk powder in PBS-0.05% Tween for 4 hours. Primary antibodies were applied in blocking buffer and incubated at room temperature overnight.
Antibodies against poly(ADP-ribose) polymerase, phospho-ERK1/2 (pp44/pp42), AKT, phospho-AKT, mcl-1, IκB, and autophagy marker light chain 3B were all purchased from Cell Signaling Technology (NEB, Frankfurt, Germany). Antibodies against BiP (H-129), activating transcription factor 3 (C-19) and β-actin (C4) were from SantaCruz Biotech (Heidelberg, Germany). Secondary, alkaline phosphatase-coupled antibodies against the corresponding primary antibodies were from Dianova (Hamburg, Germany). Alkaline phosphatase detection was performed either by the chromogenic BCIP/NBT assay (Promega, Mannheim, Germany) or by the chemiluminescent alkaline phosphatase detection assay (Millipore, Schwalbach, Germany), and the results were analysed and documented using a BioRad QuantityOne Image Analyzer and documentation software (BioRad).
Determination of intracellular glutathione levels
To detect intracellular glutathione levels, cells were seeded in 96-well cell culture dishes and allowed to grow for 24 hours under cell culture conditions. Cells were then incubated with the cytotoxic drugs for up to 5 hours. Intracellular glutathione levels were quantified using the bioluminescent Promega GSH-Glo™ glutathione assay (Promega), essentially as recommended by the supplier. In brief, adherent cells were directly dissolved in 100 μl GSH-Glo™ lysis and reaction buffer. After addition of 100 μl GSH-Glo™ Luciferin detection reagent, luminescence was detected using a MicroLumat LB 96P bioluminometer (EG&G Berthold, Bad Wildbad, Germany).
Determination of proteasomal activity
For the determination of cellular proteasome activity, cells were seeded in 96-well cell culture dishes, allowed to grow for 24 hours under cell culture conditions, and then incubated with cytotoxic drugs for up to 5 hours. Proteasomal activity was analysed using the bioluminescent Promega Proteasome-Glo™ assay (Promega) as recommended by the supplier. Adherent cells were directly dissolved in 50 μl Proteasome-Glo™ lysis and reagent buffer, containing Suc-LLVY-aminoluciferin as a substrate. Leukaemia cells were collected by centrifugation before lysis. Bioluminescence was detected using a MicroLumat LB 96P bioluminometer (EG&G Berthold).
All experiments, except western blots and FACScan analysis, were performed in quadruplicate. The results were evaluated using the nonparametric Wilcoxon sum test and the Mann-Whitney U rank-sum test where applicable (PASW version 17.0; SPSS Inc., Chigaco, IL, USA). Values were plotted as the mean ± standard deviation, and significance was assumed at P < 0.05 using the two-tailed test. Significant relations are indicated in the figures and the statistical test used is mentioned in the corresponding figure legend.
All experiments were performed on established cancer cell lines. No ethical approval or informed consent was thus needed.
Nelfinavir reduces cell proliferation of breast cancer cells and is able to induce apoptosis in breast cancer cells when applied at higher concentrations
Tamoxifen enhances the cytotoxic effect of nelfinavir
The mean plasma concentration of nelfinavir in HIV-infected persons taking oral doses of nelfinavir was determined to be 2.2 μg/ml, reaching maximal plasma concentrations of up to 4 μg/ml [23, 24]. These concentrations can achieve only a partial reduction of breast cancer cell proliferation (Figure 1), and are not efficient in inducing apoptosis in breast cancer cells. The plasma concentration of nelfinavir, however, can be significantly increased by administering higher oral doses of nelfinavir or by intravenous application of nelfinavir . Still, a possible combination strategy of nelfinavir with other chemotherapeutic drugs that would allow a reduction of single drug concentrations would be advantageous.
Quantitative analysis of apoptotic and necrotic cells after tamoxifen and nelfinavir treatment
TAM 6 μg/ml
NFV 6 μg/ml
TAM 6 μg/ml
NFV 6 μg/ml
Nelfinavir exerts pleiotropic proapoptotic and anti-apoptotic effects in breast cancer cells
In all four cell lines, no change in the expression of the proteasome-regulated NF-κB inhibitor IκB could be observed, suggesting no influence of nelfinavir on the proteasome activity in breast cancer cells. We have previously described an upregulation of the anti-apoptotic mcl-1 protein by nelfinavir in ovarian cancer cells , but this could only be observed in one (MDA-MB-435) of the four tested breast cancer cell lines. Nelfinavir has further been described as reducing AKT phosphorylation, resulting in enhanced radiosensitivity [11, 16] - which could be of special importance for breast cancer treatment, for which radiotherapy can be applied. We observed no inhibition of AKT signalling in MCF7 and T47 D cells by nelfinavir, however, and we even observed a markedly enhanced AKT phosphorylation level in nelfinavir-treated MDA-MB-453 and MDA-MB-435 cells (Figure 4).
Tamoxifen enhances the pleiotropic effects of nelfinavir in breast cancer cells
Nelfinavir reduces glutathione levels in breast cancer cells
The intracellular glutathione levels might vary not only because of external drug applications, but likewise due to differences in cell growth, nutrient concentrations (especially that of cysteine), and the redox state within the cells or of the surrounding medium. These factors can vary under cell culture conditions. Exogenously applied glutathione or cysteine derivatives such as N-acetyl-cysteine can enhance or replenish intracellular glutathione levels or support the antioxidative effect of glutathione. To analyse the involvement of glutathione depletion on nelfinavir-induced apoptosis, intracellular glutathione contents were replenished by the addition of externally applied glutathione or N-acetyl-cysteine. Figure 6c demonstrates that the addition of glutathione, and even more so the addition of N-acetyl-cysteine, can attenuate the cytotoxic effect of nelfinavir on breast cancer cells. In the absence of external glutathione or N-acetyl-cysteine, only 5% of the MCF7 cells and 12% of the MDA-MB-435 cells survived the application of 15 μg/ml nelfinavir (Figure 6c). Although the addition of 5 mM glutathione was not without negative effects on the cell viability of MCF7 cells (Figure 6c), the presence of external glutathione enhanced the remaining cell viability of MCF7 cells to 15% and to 17% in the case of MDA-MB-435 cells (Figure 6c). In the presence of 5 mM N-acetyl-cysteine, 18% of the MCF7 cells remained viable, and up to 34% of the MDA-MB-435 cells survived the addition of nelfinavir (Figure 6c).
Nelfinavir has no effect on the chymotrypsin-like proteasome activity of breast cancer cells
Some previous studies have indicated the inhibition of proteasomal activity by HIV protease inhibitors, including nelfinavir [26, 28]. The fact that nelfinavir has no influence on expression of the proteasome-regulated NF-κB inhibitor IκB (Figure 4), however, indirectly suggests that nelfinavir has no effect on the proteasomal activity of breast cancer cells.
The HIV protease inhibitor nelfinavir is a prospective new anti-cancer drug, as shown by several in vitro as well as in vivo studies [8–13]. The concentrations of nelfinavir needed to induce cell death of cancer cells are higher than those applied for HIV-infected individuals for HIV suppression, but this may be achieved by the application of higher oral or intravenous doses of nelfinavir . Still, the prospects of nelfinavir as an anti-cancer drug will rely less on its efficacy as a single drug and more on its ability to cooperate with or sensitise to other chemotherapeutic drugs or cancer treatment options. For example, we have recently demonstrated that nelfinavir cooperates with the multiple kinase inhibitor sorafenib to induce apoptosis in various cancer cell types [26, 29], and enhances TNF-related apoptosis inducing ligand sensitivity in ovarian cancer cells .
The present results show that the cytotoxic effects of nelfinavir on breast cancer cells can be enhanced by combination with tamoxifen, thus allowing the effective concentration of nelfinavir to be reduced. Tamoxifen, although originally designed and applied as a selective oestrogen receptor modulator, also represents a drug with several described pleiotropic anti-tumoral effects [6, 7] - and two recent and independent studies observed that tamoxifen is able to induce the endoplasmic reticulum stress reaction [31, 32], thus explaining the synergistic effect of nelfinavir and tamoxifen on the induction of endoplasmic reticulum stress. The nelfinavir-boosting effect of tamoxifen was obviously independent of its ability to induce oxidative stress . Instead, we observed that nelfinavir itself reduced cellular glutathione levels, indicating the occurrence of oxidative stress after nelfinavir treatment. Induction of oxidative stress occurs within a few hours as an early effect of nelfinavir treatment and has so far been neglected as an additional mechanism of the pleiotropic anti-cancer effects of nelfinavir. The observation that the effect of nelfinavir can be attenuated by the addition of antioxidants (glutathione or N-acetyl-cysteine) could have an impact on the efficacy of nelfinavir in cancer cells, as well as on the nelfinavir-induced adverse effects occurring in HIV-infected persons.
Nelfinavir has been reported to exert a radiosensitising effect by inhibiting proteasome activity and AKT signalling . Inhibition of proteasomal activity or AKT signalling in breast cancer cells, however, was not observed in the present study. In contrast, nelfinavir markedly enhanced AKT phosphorylation in some breast cancer cell lines (MDA-MB-453 cells and MDA-MB-435 cells). This observation is not surprising, however, since we previously demonstrated activation of the cell-protective ERK1/2 signalling pathway by nelfinavir . The endoplasmic stress reaction is primarily a cell-protective mechanism, aiming to rescue cells from transient stress-induced cell damage [21, 33]. Longer exposure to cell stress mechanisms or a cellular inability to cope with the stress-induced cell damage then finally induces a switch from cell protection to autophagy and apoptosis [21, 33]. Endoplasmic reticulum stress has repeatedly been shown to induce activation of both ERK1/2 and AKT signalling [34–37]. Several studies, however, have likewise shown that AKT activation, which can occur directly at the endoplasmic reticulum , primarily represents a short-term effect, and prolonged exposure of cells to endoplasmic reticulum stress indeed induces AKT inactivation [38, 39]. In fact, we observed a reduced AKT phosphorylation when breast cancer cells were treated with nelfinavir for more than 48 hours (data not shown), although this indicates downregulation of AKT phosphorylation as a secondary event. The present data therefore do not exclude the potential use of nelfinavir as a radiosensitiser even for breast cancer patients, but a potential negative interaction between these two treatment options, especially shortly after nelfinavir application, should be kept in mind.
In addition to the data on AKT signalling, the present data revealed some other differences to previous studies performed by us and other workers on different cancer cell types. For example, upregulation of the anti-apoptotic mcl-1 protein by nelfinavir - as we observed in ovarian cancer cells  and leukaemia cells  - could only be observed in a single breast cancer cell line (MDA-MB-435 cells), and only at high concentrations of nelfinavir (Figure 4). Further, we could not demonstrate proteasome inhibition by nelfinavir in breast cancer cells.
Although nelfinavir induced cell death in all four breast cancer cell lines tested, the data presented further indicate that the cell lines respond quite differently to nelfinavir, especially regarding the effect on cell stress, autophagy, and apoptosis. This variation might be due to the different hormone receptor status of the cells, but likewise may be due to the different malignancies of the tumours from which these cell lines have been derived. We therefore tried to include various types of breast cancer cell lines in the present study, ranging from hormone receptor-positive breast cancer cells of a high differentiation grade (T47 D cells) to highly de-differentiated hormone receptor-negative breast cancer cells (MDA-MB-435 cells). Interestingly, especially when low doses of nelfinavir were applied, the de-differentiated hormone receptor-negative breast cancer cell lines (MDA-MB-453 and MDA-MB-435 cells) appeared to react even better to nelfinavir than the T47 D and MCF7 cells (Figure 1).
We observed that the combination of tamoxifen and nelfinavir was able to induce cell death in oestrogen receptor-positive as well as in oestrogen receptor-negative breast cancer cell lines. This indicates that both oestrogen receptor-positive as well as oestrogen receptor-negative breast cancer patients could benefit from a combination of these two drugs.
Since both nelfinavir and tamoxifen have to be used at concentrations higher than those used to inhibit the HIV protease in HIV-infected persons or the oestrogen receptor in hormone receptor-positive breast cancer patients, however, care has to be taken that no unexpected adverse effects occur - especially when both drugs, although displaying moderate and tolerable adverse effects as single agents, are combined. Further, the observed reduction in glutathione levels by nelfinavir might cause an unexpected drug sensitisation in other tissues.
Clinical studies on breast cancer patients testing the described combination of nelfinavir and tamoxifen are thus of high interest in order to assess both efficacy and safety of this drug combination.
The present study demonstrates the efficacy of nelfinavir in breast cancer cells as a single agent, and a possible combination treatment with tamoxifen. Both nelfinavir and tamoxifen are already-approved drugs with known pharmacokinetics, and they generally exhibit relative mild and well tolerable adverse effects even after long-term application. Since the concentrations of both drugs have to be increased for an efficient cancer therapy, and a combination of these two drugs has not yet been tested in humans, however, it is important to first test the safety and tolerability of this combination in phase I studies.
activating transcription factor 3
enzyme-linked immunosorbent assay
extracellular signal-regulated kinase: FACS: fluorescence-activated cell sorting
tumour necrosis factor.
The authors are greatly indebted to Marianne Vogel, Reinhild Joswig, and Petra Burger for their excellent and highly motivated technical assistance. The generous supply of nelfinavir by Pfizer (Groton, CT, USA) is gratefully appreciated. This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft DFG BR 3641/1-1 and DFG BR 3641/3-1).
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