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Psychological stress induces chemoresistance in breast cancer by upregulating mdr1
BBRC Biochemical and Biophysical Research Communications 329 (2005) 888–897 www.elsevier.com/locate/ybbrc
Psychological stress induces chemoresistance in breast cancer by upregulating mdr1 Fengxi Su a,1, Nengyong Ouyang b,1, Pengcheng Zhu c,d,1, Nengtai Ouyang e, Weijuan Jia a, Chang Gong a, Xuexia Ma a, Huanbin Xu c, Erwei Song a,* a
Department of Breast Surgery, Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University, Guangzhou 510120, PR China b Department of Gynecology, Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University, Guangzhou 510120, PR China c Institute of Genetics, Fudan University, Shanghai 200433, PR China d CBR Institute of Biomedical Research, Harvard Medical School, Boston, MA 02115, USA e Department of Pathology, Guangzhou Medical Institute, Guangzhou 510120, PR China Received 7 February 2005
Abstract Psychological distress reduces the efficacy of chemotherapy in breast cancer patients. The mechanism may be related to the altered neuronal or hormonal secretions during stress. Here, we reported that adrenaline, a hormone mediating the biological activities of stress, upregulates mdr1 gene expression in MCF-7 breast cancer cells via a2-adrenerdric receptors in a dose-dependent manner. Mdr1 upregulation can be specifically inhibited by pretreatment with mdr1-siRNA. Consequently, adrenergic stimulation enhances the pump function of P-glycoprotein and confers resistance of MCF-7 cells to paclitaxel. In vivo, restraint stress increases mdr1 gene expression in the MCF-7 cancers that are inoculated subcutaneously into the SCID mice and provokes resistance to doxorubicin in the implanted tumors. The effect can be blocked by injection of yohimbine, an a2-adrenergic inhibitor, but not by metyrapone, a corticosterone synthesis blocker. Therefore, we conclude that breast cancers may develop resistance against chemotherapeutic drugs under psychological distress by over-expressing mdr1 via adrenergic stimulation. 2005 Elsevier Inc. All rights reserved. Keywords: Psychological stress; Restraint stress; Breast cancer; Chemotherapy; Multi-drug resistance; Adrenaline; RNA interference
Chemotherapy generates heightened psychosocial sequelae, manifested as psychological distress, in a significant number of advanced breast cancer patients [1]. Randomized clinical studies have demonstrated that the psychological distress in these patients predicts poor pathological response of the tumors to chemotherapy and is therefore an independent prognostic factor for survival [2]. Psychological interventions that reduce distress can help one to improve the clinical response of breast cancers to chemotherapy [3]. These findings are further supported by recent animal studies showing that exposure *
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Corresponding author. Fax: +86 20 81332853. E-mail address: [email protected] (E. Song). These authors contributed equally to this work.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.056
of mice bearing Shionogi (SC115) breast cancer or TLX5 lymphoma to rotational stress decreases the anti-tumor effects of the administered chemotherapeutic drugs [4]. Although the mechanisms by which psychological distress regulates chemotherapeutic efficacy in breast cancers remain poorly understood, plenty of evidence has suggested that the altered hormonal and neuronal secretions during stress have a strong impact on the biological activities of breast cancer cells [5]. Among these secretions that function in concert, adrenaline and noradrenaline play an important role in mediating the effect of stress on target cells via adrenergic receptors [6]. Chronic elevation of adrenaline and noradrenaline has been reported in the plasma and urine of breast cancer patients [7]. In addition, highly differentiated
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neuroendocrine cells and their catecholamine products have been found in breast cancer tissues [8]. Furthermore, b-adrenergic receptors were characterized in both human breast cancer cells and experimental mammary tumors, and a2-adrenergic receptors are present in the bovine mammary gland purging system [9]. Recently, it has become clear that catecholamines are not only acute regulators of cellular functions, but also exert longer lasting effects, some of which are related to alterations of gene transcription and expression [10]. Norepinephrine at low concentrations has been shown to stimulate proliferation and provoke migration of breast carcinoma cell lines in vitro [9,11,12]. These findings insinuate the possibility that psychological distress may influence the chemotherapy sensitivity of breast cancer cells by modulating the expression of chemotherapeutic drug resistant genes. Therefore, in the present study, we investigated the effect of adrenergic stimulation on the expression, as well as the function, of a multi-drug resistance (mdr1) gene, and its impact on chemosensitivity in a human breast adenocarcinoma cell line, MCF-7. Furthermore, we employed a mouse model of repetitive restraint stress with elevation of plasma catecholamines and glucocorticoid, which was used to mimic chronic psychological stress in humans [13], to study the influence of stress on MDR phenotype of human tumors implanted in immunodeficient mice. Materials and methods Cell culture and stimulation. Human breast cancer MCF-7 cells (American Type Culture Collection [ATCC]) were cultured in DulbeccoÕs modified EagleÕs medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum at 37 C and 5% CO2. For catecholamine stimulation, adrenaline (()-adrenaline bitartrate, Sigma, St. Louis, MS) was added to the medium at various final concentrations for indicated periods of time, in the absence and presence of 10 lM phentolamine (phentolamine HCl, Sigma), 10 lM prazosin (prazosin HCl, RBI, Natick, MA, USA), 1 lM yohimbine (yohimbine HCl, Sigma), or 10 lM propranolol ((±)-propranolol HCl, Sigma). In parallel, 10 lM UK14,304 (5-bromo-N-(4,5-dihydro-1Himidazol-2-yl)-6-quinoxalinamine, RBI) was added independently to the medium to stimulate MCF-7 cells. siRNA transfection. All siRNA duplexes were synthesized at Dharmacon Research (Lafayette, Colorado). The targeting sequences are as follows: human mdr1 [14], AAGAAGGAAAAGAAACCAA CU (503–523 relative to the start codon); GFP, GGCUACGUCC AGGAGCGCACC. Unmodified siRNA duplexes were used for in vitro experiments, and stability-enhanced siRNA duplexes, siSTABLE, were applied for in vivo injection. In vitro, siRNA duplex (100 nM) was transfected into MCF-7 cells with Oligofectamine (Invitrogen, Carlsbad, CA) following our previous protocol. GFP siRNA was used in every experiment as a control siRNA. About 24 h post-transfection, the medium was changed and the culture was further incubated for an additional 24 h before stimulation with 10 lM adrenaline. TaqMan quantitative RT-PCR for MDR1 mRNA expression. Twenty-four hours after catecholamine stimulation, mdr1 mRNA in MCF-7 cells was quantified using a TaqMan real-time RT-PCR assay. Briefly, total RNA was isolated from the cells using Trizol reagent
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(Life Technologies, Gaithersburg, MD). Primers for mdr1 and GAPDH were designed according to published sequences [15]. A onestep real-time RT-PCR was performed using SYBR green reagent (Applied Biosystems, Foster City, CA). All reactions were done in triplicate in a 25 ll reaction volume following manufacturerÕs instruction. Following reverse transcription for 30 min at 48 C and Taq activation for 10 min at 95 C, 40 cycles of PCR at 95 C for 20 s (melt), 55 C for 30 s (anneal), and 72 C for 30 s (extension) were performed. Real-time expression of mdr1 and GAPDH mRNA was measured on an ABI Prism 5700 Sequence Detection System (Applied Biosystem, Foster City, CA) by calculating the threshold cycles. Relative amount of mdr1 mRNA was standardized to the expression of GAPDH mRNA. A positive control with cDNA from MCF-7/Adr cells and negative controls with no template and genomic DNA as templates were added in every experiment. Paclitaxel accumulation. To evaluate the pump function of P-glycoprotein, we monitored [3H]paclitaxel accumulation in MCF-7 cells. After catecholamine stimulation, the cells were seeded in 6-well plates at 2 · 105 cells per well, and the culture medium was replaced with 1 ml ice-cold fresh medium containing 10 nM [3H]paclitaxel (Moravek, Brea, CA) to incubate at 37 C for indicated periods of time. The medium was then removed and the cells were harvested by trypsinization for scintillation counting. The radioactivity associated with the cells was measured using a liquid scintillation counter. Clonogenic assay for chemosensitivity. The influence of catecholaminergic stimulation on the chemosensitivity of MCF-7 cells was investigated by treating the stimulated tumor cells with Taxol (paclitaxel) (Bristol-Myers Squibb, Evansville, IN), followed by evaluation of the ability of tumor cells to form large colonies. Briefly, the cells were seeded in 6-well plates in DMEM at a density of 2 · 105 per well for overnight adherence. Fresh DMEM containing paclitaxel at indicated concentrations (10 pM to 1 lM) was added to replace the culture medium, and the cells were incubated at 37 C for 24 h. The cells were then harvested by trypsinization and washed twice with PBS. Resuspended in 300 ll DMEM, the cells were then added to the methylcellulose solution at a final volume of 3 ml, mixed by vortexing vigorously for 20 s, and aliquoted into 6-well plates at 1 ml per well. After incubation at 37 C for 4–6 days, the colonies of viable cells (>50 cells per colony) were counted under light microscopy by randomly choosing 10 fields of view, and the ratio of surviving colonies relative to the control without paclitaxel treatment (surviving fraction) was determined. Animals and tumor implantation. Female severe combined immune deficient (SCID) mice (6- to 8-weeks old) were purchased from Vital River Lab Animal (Beijing, China) and housed at the animal facility of Sun-Yat-Sen University in a pathogen-free environment. All experiments were approved by an Academic Committee on Animal Welfare. Two days prior to inoculation of MCF-7 cells, 17b-estradiol pellets (0.72 mg, 60-day release, Innovative Research of America, Sarasota, FL) were implanted s.c. since the growth of MCF-7 cells is estrogendependent. MCF-7 cells (2 · 106 cells) were implanted s.c. on day 0, and tumors (approximately 50 mm3 in size) were detected by day 4. All mice were sacrificed on day 30 after tumor implantation, and tumor tissue was harvested and snap-frozen for Northern blot and immunohistochemistry. Restraint stress paradigm. The mice bearing tumors were subjected to restraint stress [13] 4 h daily on days 1–5, 11–15, and 21–25 after tumor implantation. All studies were performed in a quiet room under conditions made as constant as possible from day to day to avoid variation in the environment. All experiments began at 07:00 everyday. Mice subjected to restraint stress were placed in the sterilized and ventilated 50 ml tubes for 4 h immediately after lights-on. Tubes were designed to be small enough to restrain a mouse so that it was able to breathe but unable to move freely. On day 26 after tumor implantation, some animals were sacrificed and plasma was separated from the collected blood for hormone detection. Corticosterone was measured by radioimmunoassay (RIA) using a [125I] corticosterone kit (Diagnostic Products, Los Angeles, CA), and adrenaline as well as noradrenaline was assayed by
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high-performance liquid chromatography (HPLC) and electrochemical detection. The control mice were left unhandled and were judged to be unstressed by their low, basal plasma corticosterone levels. To block the a-adrenerdric effect during stress, phentolamine (4 mg/kg, i.p.) or yohimbine (1 mg/kg/day, i.p.) was injected into the mice 20 min prior to each episode of restraint stress on days 1–5, 11– 15, and 21–25 after tumor implantation. In parallel, to eliminate the influence of glucocorticoid, the corticosterone synthesis blocker metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone), dissolved in saline containing 5% of Tween 80, was injected i.p. into the mice 20 min prior to each episode of the restraint stress. Furthermore, to silence mdr1 expression in tumor tissues, mdr1-siSTABLE (200 lg/kg) or a control GFP-siSTABLE (200 lg/kg) dissolved in 50 ll PBS was injected i.v. into the mice at indicated time points. Chemotherapy. On days 6, 12, 18, and 24 after tumor implantation, the mice received intraperitoneal injection of doxorubicin (10 mg/kg), dissolved in PBS. The control mice were injected i.p. with PBS. The length and width of the tumors were measured every 3 days after the initial dose of doxorubicin with a caliper until day 30 after tumor implantation. Tumor volume (TV) was calculated according to the formula TV (mm3) = length · width2 · 0.4. Northern blot. Mdr1 mRNA expression in implanted MCF-7 tumors from the mice without chemotherapy was measured by Northern blot analysis as described previously [16]. After RNA extraction using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA), 20 lg of total RNA was separated by denaturing-formaldehyde agarose gel electrophoresis, transferred onto Hybond membrane (Amersham), and hybridized with 32P-labelled probes for mdr1 or GAPDH. The hybridization probe for mdr1 was the 1.4 kb mdr1 cDNA from plasmid pHDR5A. The hybridized membranes were then washed to a final stringency of 65 C in 2· SSPE, 2% SDS. The signal was visualized and quantified on a phosphorimager. To estimate relative expression levels, the ratio between the signals obtained from mdr1 and GAPDH probes was calculated.
Results Adrenergic stimulation increases mdr1 gene expression To study the effect of adrenaline on mdr1 gene expression in MCF-7 cells, we treated the cells with adrenaline for 24 h and measured mdr1 expression. Treatment with adrenaline dramatically increases the level of mdr1 mRNA, quantified by real-time RT-PCR, in a concentration-dependent manner (Fig. 1A). The expression reaches a maximal level of over 80-fold the control (2.43 ± 0.4 vs. 0.03 ± 0.01 times GAPDH; p < 0.0001) at 10 lM adrenaline. The half-maximal stimulation effect is obtained between 0.01 and 0.1 lM adrenaline (Fig. 1A). To confirm the specificity of mdr1 expression, we transfected MCF-7 cells with siRNA 48 h prior to 10 lM adrenaline stimulation. As shown in Fig. 1A, pretreatment of the cells with a specific mdr1-siRNA abrogates the expression of mdr1 induced by adrenaline, while a control GFP-siRNA does not block the adrenerdric effect. Adrenaline induces mdr1 expression via a2-adrenoreceptors To investigate which adrenergic receptors mediate the effect of adrenaline on mdr1 expression in MCF-7
Fig. 1. Stimulation of a2-adrenergic receptors induces mdr1 expression. (A) Treatment with adrenaline increases the expression of mdr1 gene in a dose-dependent manner. Pretreatment of the cells with mdr1-siRNA, but not GFP-siRNA, prevents the adrenaline-induced upregulation of mdr1. *p < 0.05 as compared with a lower concentration level, and # p < 0.001 as compared with the cells treated with GFP-siRNA + adrenaline (10 lM). (B) Addition of a pan-a-adrenergic receptor blocker, phentolamine (10 lM), or an a2-adrenergic receptor blocker, yohimbine (1 lM), instead of a b-adrenergic receptor blocker, propanolol (10 lM), or an a1-adrenergic receptor blocker, prazosin (10 lM), abrogates the adrenaline-induced mdr1 expression. In agreement, treatment with an a2-adrenergic receptor agonist, UK14,304 (10 lM), but not with agonist of b-adrenergic receptor, isoprenaline (10 lM), or of a1-adrenergic receptor, cirazoline (1 lM), induces the expression of mdr1. *p < 0.001 as compared with the untreated controls, and #p < 0.001 as compared with the adrenaline-treated cells.
cells, various adrenoreceptor agonists and antagonists were used (Fig. 1B). The pan b-adrenoreceptor antagonist, propranolol (10 lM), and the a1-adrenoreceptor antagonist, prazosin (10 lM), do not significantly influence adrenaline-induced mdr1 expression (p > 0.05), but the pan a-adrenoreceptor antagonist, phentolamine (10 lM), and the a2-adrenoreceptor antagonist, yohimbine (1 lM), completely abolish the effect of adrenaline (p < 0.01). The reversing effect of phentolamine and yohimbine is specifically mediated through adrenoreceptor blockade, as treatment with phentolamine (10 lM) or yohimbine (1 lM) alone does not alter basal mdr1 levels as compared with the controls (data not shown). Likewise, neither the pan b-adrenoreceptor agonist, isoprenaline (10 lM), nor the a1-adrenoreceptor
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agonist, cirazoline (1 lM), increases mdr1 expression in MCF-7 cells (p > 0.05). In contrast, the potency of the a2-adrenoreceptor agonist UK14,304 on mdr1 expression is comparable to that of adrenaline, since no significant difference is observed in mdr1 levels between MCF-7 cells treated with the same concentration (10 lM) of either UK14,304 or adrenaline (p > 0.05). Thus, a2-adrenoreceptors alone may mediate the adrenerdric induction of mdr1 expression in MCF-7 cells. Adrenaline enhances mdr1 function To evaluate the efflux function of mdr1 expressed on the adrenaline-stimulated cells, we monitored the accu-
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mulation of an mdr1 substrate, paclitaxel, in the cells as described in Materials and methods. As shown in Fig. 2, treatment with adrenaline or UK14,304, instead of isoprenaline or prazosin, reduces the cellular accumulation of [3H]paclitaxel by approximately seven-fold following incubation of the drug for 150 min. The effect of adrenaline can be reversed by phentolamine or yohimbine, but not by propranolol or prazosin. These data suggest that a2-adrenoreceptor stimulation enhances the pump function of Pgp to efflux paclitaxel. As a control, the reversal effect is not observed in the cells pretransfected with an irrelevant GFP-siRNA. Hence, the reduced accumulation of paclitaxel in adrenaline-treated cells is a result of mdr1 activity.
Fig. 2. Adrenaline enhances P-glycoprotein function to efflux chemotherapeutic drugs. Treatment with adrenaline or UK14,304, instead of isoprenaline or prazosin, reduces the dynamic accumulation of [3H]paclitaxel in MCF-7 cells (A). The effect of adrenaline can be reversed by phentolamine or yohimbine, but not by propranolol or prazosin (B). Prior to adrenaline stimulation, pretreatment of the cells with mdr1, but not GFP-siRNA, restores paclitaxel accumulation to the levels found in the untreated cells (C).
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Adrenaline desensitizes MCF-7 cells to paclitaxel We then investigated the effect of catecholamine stimulation on the sensitivity of MCF-7 cells to chemotherapy by measuring the viability of the cells subjected to 24-h treatment of paclitaxel. As shown in Fig. 3, paclitaxel is approximately 100-fold less effective to kill
adrenaline-stimulated MCF-7 cells (LD50: 95 nM) than the untreated ones (LD50: 0.9 nM) (p < 0.001). Consistent with the results of mdr1 expression and drug uptake, the sensitivity of MCF-7 cells treated with UK14,304 (LD50: 88 nM) is comparable to adrenalinestimulated ones (p > 0.05), while the sensitivity of isoprenaline- (0.9 nM) or cirazoline- (0.8 nM) treated cells
Fig. 3. Adrenaline reduces the sensitivity of breast cancer cells to chemotherapy. Treatment with adrenaline or UK14,304, instead of isoprenaline or prazosin, reduces the sensitivity of MCF-7 cells to paclitaxel (A). The effect of adrenaline can be reversed by phentolamine or yohimbine, but not by propranolol or prazosin (B). Prior to adrenaline stimulation, pretreatment of the cells with mdr1 restores paclitaxel sensitivity to the levels found in the untreated cells (C).
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is not different from the untreated ones (p > 0.05). In addition, adrenaline-induced chemoresistance cannot be reversed by addition of propranolol or prazosin, but phentolamine or yohimbine restores the sensitivity of the adrenaline-treated cells to paclitaxel (Fig. 3). As a control, adrenaline or UK14,304 alone does not influence the clonogencity of MCF-7 cells (data not shown). Furthermore, transfection with mdr1-siRNAs prior to adrenaline stimulation, prevents the desensitizing effect of adrenaline (Fig. 3), thereby suggesting that adrenaline confers chemoresistance to MCF-7 cells by upregulating mdr1 expression. Restraint stress induces mdr1 expression in MCF-7 cells in vivo The above data suggest that adrenaline induces multidrug resistance (MDR) in MCF-7 cells in vitro, but it is more important to know whether catecholamines, in concert with other neuroendocrine secretions during psychological stress, may induce MDR in breast cancer in vivo. For this purpose, we implanted MCF-7 cells subcutaneously into SCID mice that were subsequently subjected to restraint stress, and examined the expression of mdr1 gene in the tumors. After the last session
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of restraint stress, the MCF-7 tumor tissues express constitutive amount of mdr1 mRNA as determined by Northern blot, which is not observed in the tumor tissues from the control mice (Fig. 4). To confirm the specificity of the mdr1 expression, we injected stabilityenhanced siRNA duplex i.v. into the mice subjected to restraint stress 48 hours before harvesting. Treatment with mdr1-siRNA tremendously reduces mdr1 expression (p < 0.01), while a control GFP-siRNA does not change the expression level (p > 0.05). The major humoral factors related to most psychological distress are catecholamines and glucocorticoids [13]. Therefore, we examined the level of two catecholamines, epinephrine and norepinephrine, as well as a glucocorticoid, hydrocortisone, in the plasma of the mice subjected to restraint stress. After the last session of restraint stress, we detected marked elevation of corticosterone (260 ± 25 ng/ml vs. 35 ± 5 ng/ml in controls; n = 5 per group; p < 0.001), epinephrine (2358 ± 142 pg/ml vs. 656 ± 78 pg/ml in controls; n = 5 per group; p < 0.01), and norepinephrine (3264 ± 110 pg/ ml vs. 1028 ± 96 pg/ml in controls; n = 5 per group; p < 0.01) in the plasma of the mice. In this context, we further investigated whether the stress-induced mdr1 expression could be prevented by administration of
Fig. 4. Restraint stress induces mdr1 expression in implanted breast cancer via adrenaline pathway. SCID mice received subcutaneous implantation of MCF-7 cells were subjected to restraint stress for 4 h daily on days 1–5, 11–15, and 21–25 after tumor implantation. To block the effect of neurotransmitter during stress, phentolamine (4 mg/kg, i.p.), yohimbine (1 mg/kg/day, i.p.), or metyrapone, a corticosterone synthesis blocker, was injected i.p. into the mice 20 min prior to each episode of restraint stress. To silence mdr1 expression, mdr1-siSTABLE (200 g/kg) or a control GFPsiSTABLE (200 lg/kg) dissolved in 50 ll PBS was injected i.v. 48 h before harvesting. The mice were sacrificed on day 30. Expression of mdr1 mRNA in tumor tissue was detected by Northern blot (A), quantified by a phosphorimager as the ratio between the signals obtained from mdr1 and GAPDH probes. Additionally, P-glycoprotein in tumor tissue was examined by immunohistochemistry using a mouse anti-human Pgp antibody (200·, B). Restraint stress induces mdr1 expression in the implanted tumors, which can be blocked by phentolamine, yohimbine or mdr1-siRNA, instead of metyrapone or GFP-siRNA.
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adrenerdric receptor blockers, phentolamine and yohimbine, or corticosterone synthesis inhibitor, metyrapone. Pretreatment with phentolamine or yohimbine, but not with metyrapone, eliminates the expression of mdr1 (Fig. 4) in MCF-7 tumors induced by restraint stress, suggesting that the elevation of plasma epinephrine and norepinephrine during restraint stress is responsible for the increase of mdr1 expression in the tumors, and the effect is mediated through a2-adrenerdric receptors. Restraint stress provokes chemotherapeutic resistance in breast cancer in vivo To correlate the stress-induced mdr1 expression in MCF-7 tumors to chemotherapeutic resistance, we treated the mice with doxorubicin i.v. at 10 mg/kg on days 6, 12, 18, and 24 after tumor inoculation. In the control mice not subjected to restraint stress, tumor regression is observed following treatment with doxorubicin
(Fig. 5A), but not noted in mice receiving PBS injection (Fig. 5B). After the third dose of doxorubicin, the tumors regress to undetectable size (Fig. 5A). In contrast, tumor regression does not happen in mice subjected to repetitive restraint stress following the same protocol of chemotherapy, and the tumor size between stressed and non-stressed animals becomes significantly different (p < 0.01) from day 12 after tumor implantation, i.e., 6 days after the initial dose of doxorubicin. On the other hand, when the mice receive PBS injection, the tumors tend to grow more rapidly with restraint stress, but no statistical difference is observed in terms of tumor size between stressed and non-stressed animals. Hence, restraint stress modulates the sensitivity of MCF-7 tumors to chemotherapy, instead of directly enhancing tumor growth. To confirm that the induced chemoresistance is related to Pgp expression, we treated the mice with i.v. injection of mdr1-siSTABLE or a control GFPsiSTABLE 48 h preceding each administration of doxorubicin. As a result, mdr1-siRNA, instead of GFP-siRNA, restores the sensitivity of MCF-7 tumors to doxorubicin in the mice subjected to restraint stress (Fig. 5A), while treatment with either siRNA does not influence tumor growth in the animals receiving PBS injection (Fig. 5B).
Discussion
Fig. 5. Restraint stress inhibits tumor regression under chemotherapy. The tumor-bearing SCID mice subjected to restraint stress stress according to the paradigm as described in Materials and methods received doxorubicin i.v. at 10 mg/kg (A) or PBS i.v. (B) on the indicated days (denoted by arrows). The length and width of the tumors were measured every 3 days after the initial dose of doxorubicin with a caliper until day 30 after tumor implantation. Tumor volume (TV) was calculated as follows: TV (mm3) = length · width2 · 0.4. Restraint stress abolishes tumor regression induced by doxorubicin injection, which can be reversed by injection of mdr1-siRNA (200 g/kg), but not GFP-siRNA (200 lg/kg), 48 h before each doxorubicin treatment (A). Restraint stress or siRNA injection does not interfere with tumor growth without doxorubicin treatment (B).
In a mouse model mimicking chronic psychological stress for humans, we demonstrate that restraint stress is a potent factor to trigger chemotherapeutic resistance in human MCF-7 breast cancer cells implanted into SCID mice by inducing the expression of mdr1 gene. The dramatic effect is mediated by catecholamine hormones secreted into the bloodstream during stress, which are mainly composed of adrenaline and noradrenaline. In vitro, adrenaline induces abundant expression of mdr1 gene and its product P-glycoprotein in MCF-7 cells via a2-adrenergic receptors. The induced P-glycoprotein on MCF-7 cells is capable of expelling chemotherapeutic drugs and contributes to the chemotherapy resistance of the cells. A primary mechanism of MDR in breast cancer patients involves decreased accumulation of chemotherapeutic drugs in the cancer cells, which is the consequence of drug efflux mediated by a number of ATP-binding cassette (ABC) transporter proteins [17], such as P-glycoprotein (Pgp), multi-drug resistant proteins (MRPs), and breast cancer resistance protein (BCRP/ABCG2). Pgp encoded by mdr1 gene is the most common among these proteins [17]. A number of factors related to chemotherapy, such as the dosing and protocol of chemotherapy and additional application of other cytotoxins and alkaloids, may increase the expression of mdr1 gene in breast cancer cells, and ultimately induce
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acquired MDR [17,18]. Our present data demonstrate that psychological stress is also an independent factor resulting in MDR by upregulation of Pgp in human breast cancer cells growing in SCID mice. In our study, we employed a chronic stress paradigm that exerted repetitively restraint stimulation to the mice, and the stress manipulation was mingled with consecutive dosing of doxorubicin to investigate the response of the implanted tumors to chemotherapy. This model imitates the clinical situation that patients with breast cancers suffer from chronic psychological stress that is further heightened by chemotherapy [13]. The analogy is marked since stress-related hormones are elevated in the plasma of the stressed animals, as was observed in patients suffering from chronic distress [7]. Therefore, our finding of stress-induced mdr1 expression may provide mechanistic explanation to the clinical phenomenon that chronic psychosocial stressors reduce the efficacy of cancer therapies in breast cancer [2]. However, further studies to correlate the stress level in patients with mdr1 expression in their cancer tissues are needed to confirm our theory. It has been well established that psychological stress affects the outcome of chemotherapy in cancer patients. Studies in a mouse mammary carcinoma model showed that exposure of the animals to rotational stress decreased the anti-tumor effects of chemotherapeutic drugs in terms of tumor burden, extent of metastasis, and survival time [4,19]. Additionally, restraint stress was also demonstrated to reduce the therapeutic efficacy of a cytotoxic anti-tumor drug in a mouse lung cancer model [20]. However, these reports did not clarify whether stress induces chemotherapeutic resistance by directly regulating the biology of tumor cells or by indirectly modulating the anti-tumor immunity in the hosts [21,22]. Since suppression of anti-tumor immunity may also reduce the efficacy of chemotherapy and psychological stress inhibits the cytotoxic effect of CTLs and NK cells [21–23], it is possible that restraint stress induces MDR by inhibiting host anti-tumor immunity. In our study, we used SCID mice with deficient immune function so that the influence of anti-tumor immunity suppression on MDR is minimized. Furthermore, mdr1 expression in the tumors is dramatically increased in the animals subjected to restraint stress, and silencing mdr1 with siRNA restores the sensitivity of the tumors to doxorubicin, confirming that mdr1 upregulation in tumor tissue is a direct consequence of restraint stress and contributes to stress-induced MDR. The effect of psychological stress is mediated by two major classes of hormones that are released into the peripheral blood, catecholamines, and glucocorticoids [24]. Although both types of hormones may influence the biology of breast cancer [5,9], our data indicate that the stress-induced mdr1 expression in our model is primarily a result of adrenaline or noradrenaline stimula-
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tion, as the effect can be abolished by blocking the adrenerdric receptors, but is not altered if glucocorticoid synthesis is inhibited. This is supported by recent findings that catecholamines not only trigger acute response in mammalian cells, but also modulate longer lasting cellular events by regulating gene expression [10]. For example, adrenaline and noradrenaline regulate gene expression related to cardiac hypertrophy or hyperplasia [25], and promote the expression of various cytokines as well as growth factors [26,27]. Aligned with these reports, our study further demonstrates that treating the MCF-7 cells in vitro dose-dependently increases the expression of mdr1 mRNA, which consequently enhances drug efflux function and induces chemotherapeutic resistance. The effect is specific as it can be reversed by mdr1-siRNA. Therefore, we propose that in patients with psychological stress, the heightened adrenergic stimulation, either from the profuse catecholamines in the bloodstream or from the activated NE-releasing cells distributing in cancer tissues, triggers mdr1 upregulation and the ensuing chemotherapy resistance. We further dissected which adrenergic receptor and intracellular signalling pathway mediate the adrenaline-induced mdr1 expression. Various a- and b-adrenergic receptors have been characterized in many different types of tumors, especially endocrine related cancers, and their activity is directly related to the proliferation [9,28], apoptosis [29], angiogenesis [30], migration [31], and differentiation [32] of the tumor cells. Our study demonstrates that stimulating a2-adrenergic receptors enhances mdr1 expression, while blocking the receptors inhibits it. The activity of a2-adrenergic receptors is also directly related to increased drug efflux and chemotherapy resistance in breast cancer cells. In agreement with our findings, agonizing a2-adrenergic receptors has been stated elsewhere to stimulate tritiated thymidine incorporation in MCF-7 cells [28], indicating that breast cancer cells express a2-adrenergic receptors. In addition to breast cancer cells, other tumors, such as HT29 colon cancers, also express a2-adrenergic receptors, and activating these receptors by catecholamines will result in changes of gene expression in cancer cells [33]. Our present findings not only highlight the mechanism for stress-induced chemoresistance in breast cancer, but also insinuate possible therapeutic approaches to reverse MDR for breast cancer patients subjected to psychological distress. First, reducing the level of stress by psychological therapy, such as relaxation training and guided imagery that allows patients to visualize host defences destroy tumor cells, can be applied to lower plasma catecholamine levels, and thus prevent the upregulation of Pgp on cancer cells. Such therapies have already been used in clinical trials to improve the effect of chemotherapy with encouraging results [34]. Second, blocking a2-adrenergic receptors with highly specific antagonists may also serve as an alternative therapeutic
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approach to reverse MDR by inhibiting Pgp upregulation. yohimbine has been used by urologists to treat male impotence of vascular or diabetic origins[35], and based on our data, its indication can also be extended to treat breast cancer patients with psychological distress. Finally, specifically silencing the expression of mdr1 gene with RNA interference (RNAi) also emerges to be a novel therapeutic approach for stress-induced MDR. RNAi is a sequence specific post-transcriptional gene silencing mechanism, induced by double-stranded RNA (dsRNA) molecules. The potential of RNAi as a new tool in disease therapy lies in its specificity and potency of gene silencing. Although the silencing effect of chemically synthetic siRNA is not prolonged, our data suggest that administering mdr-siRNA prior to each dosing of chemotherapy is effective enough to sensitize tumor cells to chemotherapeutic drugs for growth inhibition. In summary, we demonstrate that mdr1 upregulation via a2-adrenoceptor stimulation is a molecular mechanism for stress-induced insensitivity of breast cancer cells to chemotherapy, and blocking this pathway can help to reverse MDR in the tumors, which can be developed into a promising therapeutic approach for breast cancer patients with psychological stress. To support our findings, further studies are needed to correlate the level of psychological stress in breast cancer patients and mdr1 expression in their cancer samples.
Acknowledgments This work was supported by research grants from the National Natural Science Foundation of China (30440021 and 30471977), the Provincial Natural Science Foundation of Guangdong Province (04009402 and 19980098), and the Department of Medical Science of Sun-Yat-Sen University.
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Psychological stress induces chemoresistance in breast cancer by upregulating mdr1 Fengxi Su a,1, Nengyong Ouyang b,1, Pengcheng Zhu c,d,1, Nengtai Ouyang e, Weijuan Jia a, Chang Gong a, Xuexia Ma a, Huanbin Xu c, Erwei Song a,* a
Department of Breast Surgery, Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University, Guangzhou 510120, PR China b Department of Gynecology, Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University, Guangzhou 510120, PR China c Institute of Genetics, Fudan University, Shanghai 200433, PR China d CBR Institute of Biomedical Research, Harvard Medical School, Boston, MA 02115, USA e Department of Pathology, Guangzhou Medical Institute, Guangzhou 510120, PR China Received 7 February 2005
Abstract Psychological distress reduces the efficacy of chemotherapy in breast cancer patients. The mechanism may be related to the altered neuronal or hormonal secretions during stress. Here, we reported that adrenaline, a hormone mediating the biological activities of stress, upregulates mdr1 gene expression in MCF-7 breast cancer cells via a2-adrenerdric receptors in a dose-dependent manner. Mdr1 upregulation can be specifically inhibited by pretreatment with mdr1-siRNA. Consequently, adrenergic stimulation enhances the pump function of P-glycoprotein and confers resistance of MCF-7 cells to paclitaxel. In vivo, restraint stress increases mdr1 gene expression in the MCF-7 cancers that are inoculated subcutaneously into the SCID mice and provokes resistance to doxorubicin in the implanted tumors. The effect can be blocked by injection of yohimbine, an a2-adrenergic inhibitor, but not by metyrapone, a corticosterone synthesis blocker. Therefore, we conclude that breast cancers may develop resistance against chemotherapeutic drugs under psychological distress by over-expressing mdr1 via adrenergic stimulation. 2005 Elsevier Inc. All rights reserved. Keywords: Psychological stress; Restraint stress; Breast cancer; Chemotherapy; Multi-drug resistance; Adrenaline; RNA interference
Chemotherapy generates heightened psychosocial sequelae, manifested as psychological distress, in a significant number of advanced breast cancer patients [1]. Randomized clinical studies have demonstrated that the psychological distress in these patients predicts poor pathological response of the tumors to chemotherapy and is therefore an independent prognostic factor for survival [2]. Psychological interventions that reduce distress can help one to improve the clinical response of breast cancers to chemotherapy [3]. These findings are further supported by recent animal studies showing that exposure *
1
Corresponding author. Fax: +86 20 81332853. E-mail address: [email protected] (E. Song). These authors contributed equally to this work.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.056
of mice bearing Shionogi (SC115) breast cancer or TLX5 lymphoma to rotational stress decreases the anti-tumor effects of the administered chemotherapeutic drugs [4]. Although the mechanisms by which psychological distress regulates chemotherapeutic efficacy in breast cancers remain poorly understood, plenty of evidence has suggested that the altered hormonal and neuronal secretions during stress have a strong impact on the biological activities of breast cancer cells [5]. Among these secretions that function in concert, adrenaline and noradrenaline play an important role in mediating the effect of stress on target cells via adrenergic receptors [6]. Chronic elevation of adrenaline and noradrenaline has been reported in the plasma and urine of breast cancer patients [7]. In addition, highly differentiated
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neuroendocrine cells and their catecholamine products have been found in breast cancer tissues [8]. Furthermore, b-adrenergic receptors were characterized in both human breast cancer cells and experimental mammary tumors, and a2-adrenergic receptors are present in the bovine mammary gland purging system [9]. Recently, it has become clear that catecholamines are not only acute regulators of cellular functions, but also exert longer lasting effects, some of which are related to alterations of gene transcription and expression [10]. Norepinephrine at low concentrations has been shown to stimulate proliferation and provoke migration of breast carcinoma cell lines in vitro [9,11,12]. These findings insinuate the possibility that psychological distress may influence the chemotherapy sensitivity of breast cancer cells by modulating the expression of chemotherapeutic drug resistant genes. Therefore, in the present study, we investigated the effect of adrenergic stimulation on the expression, as well as the function, of a multi-drug resistance (mdr1) gene, and its impact on chemosensitivity in a human breast adenocarcinoma cell line, MCF-7. Furthermore, we employed a mouse model of repetitive restraint stress with elevation of plasma catecholamines and glucocorticoid, which was used to mimic chronic psychological stress in humans [13], to study the influence of stress on MDR phenotype of human tumors implanted in immunodeficient mice. Materials and methods Cell culture and stimulation. Human breast cancer MCF-7 cells (American Type Culture Collection [ATCC]) were cultured in DulbeccoÕs modified EagleÕs medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum at 37 C and 5% CO2. For catecholamine stimulation, adrenaline (()-adrenaline bitartrate, Sigma, St. Louis, MS) was added to the medium at various final concentrations for indicated periods of time, in the absence and presence of 10 lM phentolamine (phentolamine HCl, Sigma), 10 lM prazosin (prazosin HCl, RBI, Natick, MA, USA), 1 lM yohimbine (yohimbine HCl, Sigma), or 10 lM propranolol ((±)-propranolol HCl, Sigma). In parallel, 10 lM UK14,304 (5-bromo-N-(4,5-dihydro-1Himidazol-2-yl)-6-quinoxalinamine, RBI) was added independently to the medium to stimulate MCF-7 cells. siRNA transfection. All siRNA duplexes were synthesized at Dharmacon Research (Lafayette, Colorado). The targeting sequences are as follows: human mdr1 [14], AAGAAGGAAAAGAAACCAA CU (503–523 relative to the start codon); GFP, GGCUACGUCC AGGAGCGCACC. Unmodified siRNA duplexes were used for in vitro experiments, and stability-enhanced siRNA duplexes, siSTABLE, were applied for in vivo injection. In vitro, siRNA duplex (100 nM) was transfected into MCF-7 cells with Oligofectamine (Invitrogen, Carlsbad, CA) following our previous protocol. GFP siRNA was used in every experiment as a control siRNA. About 24 h post-transfection, the medium was changed and the culture was further incubated for an additional 24 h before stimulation with 10 lM adrenaline. TaqMan quantitative RT-PCR for MDR1 mRNA expression. Twenty-four hours after catecholamine stimulation, mdr1 mRNA in MCF-7 cells was quantified using a TaqMan real-time RT-PCR assay. Briefly, total RNA was isolated from the cells using Trizol reagent
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(Life Technologies, Gaithersburg, MD). Primers for mdr1 and GAPDH were designed according to published sequences [15]. A onestep real-time RT-PCR was performed using SYBR green reagent (Applied Biosystems, Foster City, CA). All reactions were done in triplicate in a 25 ll reaction volume following manufacturerÕs instruction. Following reverse transcription for 30 min at 48 C and Taq activation for 10 min at 95 C, 40 cycles of PCR at 95 C for 20 s (melt), 55 C for 30 s (anneal), and 72 C for 30 s (extension) were performed. Real-time expression of mdr1 and GAPDH mRNA was measured on an ABI Prism 5700 Sequence Detection System (Applied Biosystem, Foster City, CA) by calculating the threshold cycles. Relative amount of mdr1 mRNA was standardized to the expression of GAPDH mRNA. A positive control with cDNA from MCF-7/Adr cells and negative controls with no template and genomic DNA as templates were added in every experiment. Paclitaxel accumulation. To evaluate the pump function of P-glycoprotein, we monitored [3H]paclitaxel accumulation in MCF-7 cells. After catecholamine stimulation, the cells were seeded in 6-well plates at 2 · 105 cells per well, and the culture medium was replaced with 1 ml ice-cold fresh medium containing 10 nM [3H]paclitaxel (Moravek, Brea, CA) to incubate at 37 C for indicated periods of time. The medium was then removed and the cells were harvested by trypsinization for scintillation counting. The radioactivity associated with the cells was measured using a liquid scintillation counter. Clonogenic assay for chemosensitivity. The influence of catecholaminergic stimulation on the chemosensitivity of MCF-7 cells was investigated by treating the stimulated tumor cells with Taxol (paclitaxel) (Bristol-Myers Squibb, Evansville, IN), followed by evaluation of the ability of tumor cells to form large colonies. Briefly, the cells were seeded in 6-well plates in DMEM at a density of 2 · 105 per well for overnight adherence. Fresh DMEM containing paclitaxel at indicated concentrations (10 pM to 1 lM) was added to replace the culture medium, and the cells were incubated at 37 C for 24 h. The cells were then harvested by trypsinization and washed twice with PBS. Resuspended in 300 ll DMEM, the cells were then added to the methylcellulose solution at a final volume of 3 ml, mixed by vortexing vigorously for 20 s, and aliquoted into 6-well plates at 1 ml per well. After incubation at 37 C for 4–6 days, the colonies of viable cells (>50 cells per colony) were counted under light microscopy by randomly choosing 10 fields of view, and the ratio of surviving colonies relative to the control without paclitaxel treatment (surviving fraction) was determined. Animals and tumor implantation. Female severe combined immune deficient (SCID) mice (6- to 8-weeks old) were purchased from Vital River Lab Animal (Beijing, China) and housed at the animal facility of Sun-Yat-Sen University in a pathogen-free environment. All experiments were approved by an Academic Committee on Animal Welfare. Two days prior to inoculation of MCF-7 cells, 17b-estradiol pellets (0.72 mg, 60-day release, Innovative Research of America, Sarasota, FL) were implanted s.c. since the growth of MCF-7 cells is estrogendependent. MCF-7 cells (2 · 106 cells) were implanted s.c. on day 0, and tumors (approximately 50 mm3 in size) were detected by day 4. All mice were sacrificed on day 30 after tumor implantation, and tumor tissue was harvested and snap-frozen for Northern blot and immunohistochemistry. Restraint stress paradigm. The mice bearing tumors were subjected to restraint stress [13] 4 h daily on days 1–5, 11–15, and 21–25 after tumor implantation. All studies were performed in a quiet room under conditions made as constant as possible from day to day to avoid variation in the environment. All experiments began at 07:00 everyday. Mice subjected to restraint stress were placed in the sterilized and ventilated 50 ml tubes for 4 h immediately after lights-on. Tubes were designed to be small enough to restrain a mouse so that it was able to breathe but unable to move freely. On day 26 after tumor implantation, some animals were sacrificed and plasma was separated from the collected blood for hormone detection. Corticosterone was measured by radioimmunoassay (RIA) using a [125I] corticosterone kit (Diagnostic Products, Los Angeles, CA), and adrenaline as well as noradrenaline was assayed by
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high-performance liquid chromatography (HPLC) and electrochemical detection. The control mice were left unhandled and were judged to be unstressed by their low, basal plasma corticosterone levels. To block the a-adrenerdric effect during stress, phentolamine (4 mg/kg, i.p.) or yohimbine (1 mg/kg/day, i.p.) was injected into the mice 20 min prior to each episode of restraint stress on days 1–5, 11– 15, and 21–25 after tumor implantation. In parallel, to eliminate the influence of glucocorticoid, the corticosterone synthesis blocker metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone), dissolved in saline containing 5% of Tween 80, was injected i.p. into the mice 20 min prior to each episode of the restraint stress. Furthermore, to silence mdr1 expression in tumor tissues, mdr1-siSTABLE (200 lg/kg) or a control GFP-siSTABLE (200 lg/kg) dissolved in 50 ll PBS was injected i.v. into the mice at indicated time points. Chemotherapy. On days 6, 12, 18, and 24 after tumor implantation, the mice received intraperitoneal injection of doxorubicin (10 mg/kg), dissolved in PBS. The control mice were injected i.p. with PBS. The length and width of the tumors were measured every 3 days after the initial dose of doxorubicin with a caliper until day 30 after tumor implantation. Tumor volume (TV) was calculated according to the formula TV (mm3) = length · width2 · 0.4. Northern blot. Mdr1 mRNA expression in implanted MCF-7 tumors from the mice without chemotherapy was measured by Northern blot analysis as described previously [16]. After RNA extraction using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA), 20 lg of total RNA was separated by denaturing-formaldehyde agarose gel electrophoresis, transferred onto Hybond membrane (Amersham), and hybridized with 32P-labelled probes for mdr1 or GAPDH. The hybridization probe for mdr1 was the 1.4 kb mdr1 cDNA from plasmid pHDR5A. The hybridized membranes were then washed to a final stringency of 65 C in 2· SSPE, 2% SDS. The signal was visualized and quantified on a phosphorimager. To estimate relative expression levels, the ratio between the signals obtained from mdr1 and GAPDH probes was calculated.
Results Adrenergic stimulation increases mdr1 gene expression To study the effect of adrenaline on mdr1 gene expression in MCF-7 cells, we treated the cells with adrenaline for 24 h and measured mdr1 expression. Treatment with adrenaline dramatically increases the level of mdr1 mRNA, quantified by real-time RT-PCR, in a concentration-dependent manner (Fig. 1A). The expression reaches a maximal level of over 80-fold the control (2.43 ± 0.4 vs. 0.03 ± 0.01 times GAPDH; p < 0.0001) at 10 lM adrenaline. The half-maximal stimulation effect is obtained between 0.01 and 0.1 lM adrenaline (Fig. 1A). To confirm the specificity of mdr1 expression, we transfected MCF-7 cells with siRNA 48 h prior to 10 lM adrenaline stimulation. As shown in Fig. 1A, pretreatment of the cells with a specific mdr1-siRNA abrogates the expression of mdr1 induced by adrenaline, while a control GFP-siRNA does not block the adrenerdric effect. Adrenaline induces mdr1 expression via a2-adrenoreceptors To investigate which adrenergic receptors mediate the effect of adrenaline on mdr1 expression in MCF-7
Fig. 1. Stimulation of a2-adrenergic receptors induces mdr1 expression. (A) Treatment with adrenaline increases the expression of mdr1 gene in a dose-dependent manner. Pretreatment of the cells with mdr1-siRNA, but not GFP-siRNA, prevents the adrenaline-induced upregulation of mdr1. *p < 0.05 as compared with a lower concentration level, and # p < 0.001 as compared with the cells treated with GFP-siRNA + adrenaline (10 lM). (B) Addition of a pan-a-adrenergic receptor blocker, phentolamine (10 lM), or an a2-adrenergic receptor blocker, yohimbine (1 lM), instead of a b-adrenergic receptor blocker, propanolol (10 lM), or an a1-adrenergic receptor blocker, prazosin (10 lM), abrogates the adrenaline-induced mdr1 expression. In agreement, treatment with an a2-adrenergic receptor agonist, UK14,304 (10 lM), but not with agonist of b-adrenergic receptor, isoprenaline (10 lM), or of a1-adrenergic receptor, cirazoline (1 lM), induces the expression of mdr1. *p < 0.001 as compared with the untreated controls, and #p < 0.001 as compared with the adrenaline-treated cells.
cells, various adrenoreceptor agonists and antagonists were used (Fig. 1B). The pan b-adrenoreceptor antagonist, propranolol (10 lM), and the a1-adrenoreceptor antagonist, prazosin (10 lM), do not significantly influence adrenaline-induced mdr1 expression (p > 0.05), but the pan a-adrenoreceptor antagonist, phentolamine (10 lM), and the a2-adrenoreceptor antagonist, yohimbine (1 lM), completely abolish the effect of adrenaline (p < 0.01). The reversing effect of phentolamine and yohimbine is specifically mediated through adrenoreceptor blockade, as treatment with phentolamine (10 lM) or yohimbine (1 lM) alone does not alter basal mdr1 levels as compared with the controls (data not shown). Likewise, neither the pan b-adrenoreceptor agonist, isoprenaline (10 lM), nor the a1-adrenoreceptor
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agonist, cirazoline (1 lM), increases mdr1 expression in MCF-7 cells (p > 0.05). In contrast, the potency of the a2-adrenoreceptor agonist UK14,304 on mdr1 expression is comparable to that of adrenaline, since no significant difference is observed in mdr1 levels between MCF-7 cells treated with the same concentration (10 lM) of either UK14,304 or adrenaline (p > 0.05). Thus, a2-adrenoreceptors alone may mediate the adrenerdric induction of mdr1 expression in MCF-7 cells. Adrenaline enhances mdr1 function To evaluate the efflux function of mdr1 expressed on the adrenaline-stimulated cells, we monitored the accu-
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mulation of an mdr1 substrate, paclitaxel, in the cells as described in Materials and methods. As shown in Fig. 2, treatment with adrenaline or UK14,304, instead of isoprenaline or prazosin, reduces the cellular accumulation of [3H]paclitaxel by approximately seven-fold following incubation of the drug for 150 min. The effect of adrenaline can be reversed by phentolamine or yohimbine, but not by propranolol or prazosin. These data suggest that a2-adrenoreceptor stimulation enhances the pump function of Pgp to efflux paclitaxel. As a control, the reversal effect is not observed in the cells pretransfected with an irrelevant GFP-siRNA. Hence, the reduced accumulation of paclitaxel in adrenaline-treated cells is a result of mdr1 activity.
Fig. 2. Adrenaline enhances P-glycoprotein function to efflux chemotherapeutic drugs. Treatment with adrenaline or UK14,304, instead of isoprenaline or prazosin, reduces the dynamic accumulation of [3H]paclitaxel in MCF-7 cells (A). The effect of adrenaline can be reversed by phentolamine or yohimbine, but not by propranolol or prazosin (B). Prior to adrenaline stimulation, pretreatment of the cells with mdr1, but not GFP-siRNA, restores paclitaxel accumulation to the levels found in the untreated cells (C).
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Adrenaline desensitizes MCF-7 cells to paclitaxel We then investigated the effect of catecholamine stimulation on the sensitivity of MCF-7 cells to chemotherapy by measuring the viability of the cells subjected to 24-h treatment of paclitaxel. As shown in Fig. 3, paclitaxel is approximately 100-fold less effective to kill
adrenaline-stimulated MCF-7 cells (LD50: 95 nM) than the untreated ones (LD50: 0.9 nM) (p < 0.001). Consistent with the results of mdr1 expression and drug uptake, the sensitivity of MCF-7 cells treated with UK14,304 (LD50: 88 nM) is comparable to adrenalinestimulated ones (p > 0.05), while the sensitivity of isoprenaline- (0.9 nM) or cirazoline- (0.8 nM) treated cells
Fig. 3. Adrenaline reduces the sensitivity of breast cancer cells to chemotherapy. Treatment with adrenaline or UK14,304, instead of isoprenaline or prazosin, reduces the sensitivity of MCF-7 cells to paclitaxel (A). The effect of adrenaline can be reversed by phentolamine or yohimbine, but not by propranolol or prazosin (B). Prior to adrenaline stimulation, pretreatment of the cells with mdr1 restores paclitaxel sensitivity to the levels found in the untreated cells (C).
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is not different from the untreated ones (p > 0.05). In addition, adrenaline-induced chemoresistance cannot be reversed by addition of propranolol or prazosin, but phentolamine or yohimbine restores the sensitivity of the adrenaline-treated cells to paclitaxel (Fig. 3). As a control, adrenaline or UK14,304 alone does not influence the clonogencity of MCF-7 cells (data not shown). Furthermore, transfection with mdr1-siRNAs prior to adrenaline stimulation, prevents the desensitizing effect of adrenaline (Fig. 3), thereby suggesting that adrenaline confers chemoresistance to MCF-7 cells by upregulating mdr1 expression. Restraint stress induces mdr1 expression in MCF-7 cells in vivo The above data suggest that adrenaline induces multidrug resistance (MDR) in MCF-7 cells in vitro, but it is more important to know whether catecholamines, in concert with other neuroendocrine secretions during psychological stress, may induce MDR in breast cancer in vivo. For this purpose, we implanted MCF-7 cells subcutaneously into SCID mice that were subsequently subjected to restraint stress, and examined the expression of mdr1 gene in the tumors. After the last session
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of restraint stress, the MCF-7 tumor tissues express constitutive amount of mdr1 mRNA as determined by Northern blot, which is not observed in the tumor tissues from the control mice (Fig. 4). To confirm the specificity of the mdr1 expression, we injected stabilityenhanced siRNA duplex i.v. into the mice subjected to restraint stress 48 hours before harvesting. Treatment with mdr1-siRNA tremendously reduces mdr1 expression (p < 0.01), while a control GFP-siRNA does not change the expression level (p > 0.05). The major humoral factors related to most psychological distress are catecholamines and glucocorticoids [13]. Therefore, we examined the level of two catecholamines, epinephrine and norepinephrine, as well as a glucocorticoid, hydrocortisone, in the plasma of the mice subjected to restraint stress. After the last session of restraint stress, we detected marked elevation of corticosterone (260 ± 25 ng/ml vs. 35 ± 5 ng/ml in controls; n = 5 per group; p < 0.001), epinephrine (2358 ± 142 pg/ml vs. 656 ± 78 pg/ml in controls; n = 5 per group; p < 0.01), and norepinephrine (3264 ± 110 pg/ ml vs. 1028 ± 96 pg/ml in controls; n = 5 per group; p < 0.01) in the plasma of the mice. In this context, we further investigated whether the stress-induced mdr1 expression could be prevented by administration of
Fig. 4. Restraint stress induces mdr1 expression in implanted breast cancer via adrenaline pathway. SCID mice received subcutaneous implantation of MCF-7 cells were subjected to restraint stress for 4 h daily on days 1–5, 11–15, and 21–25 after tumor implantation. To block the effect of neurotransmitter during stress, phentolamine (4 mg/kg, i.p.), yohimbine (1 mg/kg/day, i.p.), or metyrapone, a corticosterone synthesis blocker, was injected i.p. into the mice 20 min prior to each episode of restraint stress. To silence mdr1 expression, mdr1-siSTABLE (200 g/kg) or a control GFPsiSTABLE (200 lg/kg) dissolved in 50 ll PBS was injected i.v. 48 h before harvesting. The mice were sacrificed on day 30. Expression of mdr1 mRNA in tumor tissue was detected by Northern blot (A), quantified by a phosphorimager as the ratio between the signals obtained from mdr1 and GAPDH probes. Additionally, P-glycoprotein in tumor tissue was examined by immunohistochemistry using a mouse anti-human Pgp antibody (200·, B). Restraint stress induces mdr1 expression in the implanted tumors, which can be blocked by phentolamine, yohimbine or mdr1-siRNA, instead of metyrapone or GFP-siRNA.
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adrenerdric receptor blockers, phentolamine and yohimbine, or corticosterone synthesis inhibitor, metyrapone. Pretreatment with phentolamine or yohimbine, but not with metyrapone, eliminates the expression of mdr1 (Fig. 4) in MCF-7 tumors induced by restraint stress, suggesting that the elevation of plasma epinephrine and norepinephrine during restraint stress is responsible for the increase of mdr1 expression in the tumors, and the effect is mediated through a2-adrenerdric receptors. Restraint stress provokes chemotherapeutic resistance in breast cancer in vivo To correlate the stress-induced mdr1 expression in MCF-7 tumors to chemotherapeutic resistance, we treated the mice with doxorubicin i.v. at 10 mg/kg on days 6, 12, 18, and 24 after tumor inoculation. In the control mice not subjected to restraint stress, tumor regression is observed following treatment with doxorubicin
(Fig. 5A), but not noted in mice receiving PBS injection (Fig. 5B). After the third dose of doxorubicin, the tumors regress to undetectable size (Fig. 5A). In contrast, tumor regression does not happen in mice subjected to repetitive restraint stress following the same protocol of chemotherapy, and the tumor size between stressed and non-stressed animals becomes significantly different (p < 0.01) from day 12 after tumor implantation, i.e., 6 days after the initial dose of doxorubicin. On the other hand, when the mice receive PBS injection, the tumors tend to grow more rapidly with restraint stress, but no statistical difference is observed in terms of tumor size between stressed and non-stressed animals. Hence, restraint stress modulates the sensitivity of MCF-7 tumors to chemotherapy, instead of directly enhancing tumor growth. To confirm that the induced chemoresistance is related to Pgp expression, we treated the mice with i.v. injection of mdr1-siSTABLE or a control GFPsiSTABLE 48 h preceding each administration of doxorubicin. As a result, mdr1-siRNA, instead of GFP-siRNA, restores the sensitivity of MCF-7 tumors to doxorubicin in the mice subjected to restraint stress (Fig. 5A), while treatment with either siRNA does not influence tumor growth in the animals receiving PBS injection (Fig. 5B).
Discussion
Fig. 5. Restraint stress inhibits tumor regression under chemotherapy. The tumor-bearing SCID mice subjected to restraint stress stress according to the paradigm as described in Materials and methods received doxorubicin i.v. at 10 mg/kg (A) or PBS i.v. (B) on the indicated days (denoted by arrows). The length and width of the tumors were measured every 3 days after the initial dose of doxorubicin with a caliper until day 30 after tumor implantation. Tumor volume (TV) was calculated as follows: TV (mm3) = length · width2 · 0.4. Restraint stress abolishes tumor regression induced by doxorubicin injection, which can be reversed by injection of mdr1-siRNA (200 g/kg), but not GFP-siRNA (200 lg/kg), 48 h before each doxorubicin treatment (A). Restraint stress or siRNA injection does not interfere with tumor growth without doxorubicin treatment (B).
In a mouse model mimicking chronic psychological stress for humans, we demonstrate that restraint stress is a potent factor to trigger chemotherapeutic resistance in human MCF-7 breast cancer cells implanted into SCID mice by inducing the expression of mdr1 gene. The dramatic effect is mediated by catecholamine hormones secreted into the bloodstream during stress, which are mainly composed of adrenaline and noradrenaline. In vitro, adrenaline induces abundant expression of mdr1 gene and its product P-glycoprotein in MCF-7 cells via a2-adrenergic receptors. The induced P-glycoprotein on MCF-7 cells is capable of expelling chemotherapeutic drugs and contributes to the chemotherapy resistance of the cells. A primary mechanism of MDR in breast cancer patients involves decreased accumulation of chemotherapeutic drugs in the cancer cells, which is the consequence of drug efflux mediated by a number of ATP-binding cassette (ABC) transporter proteins [17], such as P-glycoprotein (Pgp), multi-drug resistant proteins (MRPs), and breast cancer resistance protein (BCRP/ABCG2). Pgp encoded by mdr1 gene is the most common among these proteins [17]. A number of factors related to chemotherapy, such as the dosing and protocol of chemotherapy and additional application of other cytotoxins and alkaloids, may increase the expression of mdr1 gene in breast cancer cells, and ultimately induce
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acquired MDR [17,18]. Our present data demonstrate that psychological stress is also an independent factor resulting in MDR by upregulation of Pgp in human breast cancer cells growing in SCID mice. In our study, we employed a chronic stress paradigm that exerted repetitively restraint stimulation to the mice, and the stress manipulation was mingled with consecutive dosing of doxorubicin to investigate the response of the implanted tumors to chemotherapy. This model imitates the clinical situation that patients with breast cancers suffer from chronic psychological stress that is further heightened by chemotherapy [13]. The analogy is marked since stress-related hormones are elevated in the plasma of the stressed animals, as was observed in patients suffering from chronic distress [7]. Therefore, our finding of stress-induced mdr1 expression may provide mechanistic explanation to the clinical phenomenon that chronic psychosocial stressors reduce the efficacy of cancer therapies in breast cancer [2]. However, further studies to correlate the stress level in patients with mdr1 expression in their cancer tissues are needed to confirm our theory. It has been well established that psychological stress affects the outcome of chemotherapy in cancer patients. Studies in a mouse mammary carcinoma model showed that exposure of the animals to rotational stress decreased the anti-tumor effects of chemotherapeutic drugs in terms of tumor burden, extent of metastasis, and survival time [4,19]. Additionally, restraint stress was also demonstrated to reduce the therapeutic efficacy of a cytotoxic anti-tumor drug in a mouse lung cancer model [20]. However, these reports did not clarify whether stress induces chemotherapeutic resistance by directly regulating the biology of tumor cells or by indirectly modulating the anti-tumor immunity in the hosts [21,22]. Since suppression of anti-tumor immunity may also reduce the efficacy of chemotherapy and psychological stress inhibits the cytotoxic effect of CTLs and NK cells [21–23], it is possible that restraint stress induces MDR by inhibiting host anti-tumor immunity. In our study, we used SCID mice with deficient immune function so that the influence of anti-tumor immunity suppression on MDR is minimized. Furthermore, mdr1 expression in the tumors is dramatically increased in the animals subjected to restraint stress, and silencing mdr1 with siRNA restores the sensitivity of the tumors to doxorubicin, confirming that mdr1 upregulation in tumor tissue is a direct consequence of restraint stress and contributes to stress-induced MDR. The effect of psychological stress is mediated by two major classes of hormones that are released into the peripheral blood, catecholamines, and glucocorticoids [24]. Although both types of hormones may influence the biology of breast cancer [5,9], our data indicate that the stress-induced mdr1 expression in our model is primarily a result of adrenaline or noradrenaline stimula-
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tion, as the effect can be abolished by blocking the adrenerdric receptors, but is not altered if glucocorticoid synthesis is inhibited. This is supported by recent findings that catecholamines not only trigger acute response in mammalian cells, but also modulate longer lasting cellular events by regulating gene expression [10]. For example, adrenaline and noradrenaline regulate gene expression related to cardiac hypertrophy or hyperplasia [25], and promote the expression of various cytokines as well as growth factors [26,27]. Aligned with these reports, our study further demonstrates that treating the MCF-7 cells in vitro dose-dependently increases the expression of mdr1 mRNA, which consequently enhances drug efflux function and induces chemotherapeutic resistance. The effect is specific as it can be reversed by mdr1-siRNA. Therefore, we propose that in patients with psychological stress, the heightened adrenergic stimulation, either from the profuse catecholamines in the bloodstream or from the activated NE-releasing cells distributing in cancer tissues, triggers mdr1 upregulation and the ensuing chemotherapy resistance. We further dissected which adrenergic receptor and intracellular signalling pathway mediate the adrenaline-induced mdr1 expression. Various a- and b-adrenergic receptors have been characterized in many different types of tumors, especially endocrine related cancers, and their activity is directly related to the proliferation [9,28], apoptosis [29], angiogenesis [30], migration [31], and differentiation [32] of the tumor cells. Our study demonstrates that stimulating a2-adrenergic receptors enhances mdr1 expression, while blocking the receptors inhibits it. The activity of a2-adrenergic receptors is also directly related to increased drug efflux and chemotherapy resistance in breast cancer cells. In agreement with our findings, agonizing a2-adrenergic receptors has been stated elsewhere to stimulate tritiated thymidine incorporation in MCF-7 cells [28], indicating that breast cancer cells express a2-adrenergic receptors. In addition to breast cancer cells, other tumors, such as HT29 colon cancers, also express a2-adrenergic receptors, and activating these receptors by catecholamines will result in changes of gene expression in cancer cells [33]. Our present findings not only highlight the mechanism for stress-induced chemoresistance in breast cancer, but also insinuate possible therapeutic approaches to reverse MDR for breast cancer patients subjected to psychological distress. First, reducing the level of stress by psychological therapy, such as relaxation training and guided imagery that allows patients to visualize host defences destroy tumor cells, can be applied to lower plasma catecholamine levels, and thus prevent the upregulation of Pgp on cancer cells. Such therapies have already been used in clinical trials to improve the effect of chemotherapy with encouraging results [34]. Second, blocking a2-adrenergic receptors with highly specific antagonists may also serve as an alternative therapeutic
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approach to reverse MDR by inhibiting Pgp upregulation. yohimbine has been used by urologists to treat male impotence of vascular or diabetic origins[35], and based on our data, its indication can also be extended to treat breast cancer patients with psychological distress. Finally, specifically silencing the expression of mdr1 gene with RNA interference (RNAi) also emerges to be a novel therapeutic approach for stress-induced MDR. RNAi is a sequence specific post-transcriptional gene silencing mechanism, induced by double-stranded RNA (dsRNA) molecules. The potential of RNAi as a new tool in disease therapy lies in its specificity and potency of gene silencing. Although the silencing effect of chemically synthetic siRNA is not prolonged, our data suggest that administering mdr-siRNA prior to each dosing of chemotherapy is effective enough to sensitize tumor cells to chemotherapeutic drugs for growth inhibition. In summary, we demonstrate that mdr1 upregulation via a2-adrenoceptor stimulation is a molecular mechanism for stress-induced insensitivity of breast cancer cells to chemotherapy, and blocking this pathway can help to reverse MDR in the tumors, which can be developed into a promising therapeutic approach for breast cancer patients with psychological stress. To support our findings, further studies are needed to correlate the level of psychological stress in breast cancer patients and mdr1 expression in their cancer samples.
Acknowledgments This work was supported by research grants from the National Natural Science Foundation of China (30440021 and 30471977), the Provincial Natural Science Foundation of Guangdong Province (04009402 and 19980098), and the Department of Medical Science of Sun-Yat-Sen University.
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