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Involvement of P-gp in the process of apoptosis in peripheral blood mononuclear cells
International Immunopharmacology 5 (2005) 821 – 828 www.elsevier.com/locate/intimp
Involvement of P-gp in the process of apoptosis in peripheral blood mononuclear cells A. Pawlika,T, M. Baskiewicz-Masiukb, B. Machalinskib, B. Gawronska-Szklarza a
Department of Pharmacokinetics and Therapeutic Drug Monitoring, Pomeranian Medical University, 70-111 Szczecin, al. Powstan˜co´w Wlkp. 72, Poland b Department of Pathology, Pomeranian Medical University, 70-111 Szczecin, al. Powstan˜co´w Wlkp. 72, Poland Received 18 August 2004; received in revised form 12 October 2004; accepted 20 December 2004
Abstract Multidrug resistance mediated by the drug-efflux protein P (P-gp) is one of mechanisms that cells use to escape death induced by drugs and other agents. The aim of the study was to evaluate the effect of P-gp inhibition on apoptosis of PHAactivated peripheral blood mononuclear cells (MNC) as well as apoptosis induced by methotrexate (MTX), dexamethasone (DEX), methylprednisolone (MP) and cortisone (COR). Apoptosis was quantified by flow cytometry using Annexin V/PI and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL). P-gp expression was inhibited using verapamil (VER) and P-gp specific monoclonal antibodies (mAb). VER and mAb enhanced the apoptosis of PHA-activated MNC. Moreover these agents significantly increased the apoptosis induced by MTX, DEX, MP and COR. The results of this study suggest that P-gp is involved in the process of apoptosis in peripheral blood mononuclear cells. D 2005 Elsevier B.V. All rights reserved. Keywords: Apoptosis; P-glycoprotein; Methotrexate; Glucocorticosteroids
1. Introduction Resistance to chemotherapeutic drugs is a frequent problem encountered in the therapy of malignances and autoimmune diseases. Multidrug resistance (MDR) involves cross-resistance to many commonly used cytotoxic drugs, often is associated with re-
T Corresponding author. Tel./fax: +48 91 4661600. E-mail address: [email protected] (A. Pawlik). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2004.12.014
duced intracellular accumulation and altered cellular distribution of these substances [1]. The mechanism of MDR has been associated with alterations in expression of P-glycoprotein (P-gp), which plays the role in absorption of many drugs [2]. The mechanisms by which different immunosuppressive drugs interfere with T cell activation and proliferation are different. The pharmacological effects of glucocorticosteroids (GS) are based on several mechanisms leading to the modulation of cell activation, cytokine expres-
822
A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828
sion, the secretion of inflammatory mediators, leukocyte migration and induction of apoptosis of T cell [3]. At low concentrations of GS T cell apoptosis is mediated by the glucocorticosteroid receptor (GSR) and can be blocked by the GSR antagonists. At higher doses GS can exert proapoptotic effects by other mechanism such as binding to membrane receptors with the consecutive activation of second messenger system and by affecting the physiochemical properties of the cell membrane [4]. The folate antagonist methotrexate (MTX) is extensively used in therapy of malignances, and chronic inflammatory disorders such as rheumatoid arthritis [5]. MTX exerts antiproliferative activity, which is associated with increased release of adenosine. MTX induces also apoptosis of T lymphocytes [6]. Induction of apoptosis is the key mechanism to eliminate the auto reactive T cell in the periphery and attenuate the immune response. Recently, molecular level of understanding of the regulation of apoptosis has expanded considerably. Nevertheless, it is still not understood how extra cellular signals ranging from withdrawal of growth factors to administration of drugs, influence the apoptosis and which mechanism may protect the cells from proapoptotic action of diverse agents [7]. Previously published data have shown that Pglycoprotein can protect the cells from various apoptotic agents [8–10]. The aim of this study was to examine the effect of P-gp inhibition on apoptosis of peripheral blood mononuclear cells (MNC) as well as on apoptosis induced by methotrexate and glucocorticosteroids (GS): dexamethasone (DEX), methylprednisolone (MP) and cortisone (COR).
(concentration 5 Ag/mL) and incubating MNC for 24 h at 37 8C, 5% CO2 in RPMI 1640 medium. One million cells were incubated in each culture. After 24 h of incubation methotrexate (Methotrexate, Rhone-Poulenc, F) MTX or GS dexamethasone (Dexaven, Jelfa, PL), methylprednisolone (SoluMedrol, Upjohn, B) and cortisone (Hydrocortisonum, Jelfa, PL) were added for 24 h. Apoptosis was measured by flow cytometry (FACScan, Becton Dickinson, USA). Verapamil (VER) (Isoptin, Knoll, D) and P-gp specific monoclonal antibodies (mAb), clone 17F9 (Beckton Dickinson, San Jose, USA) were used in the experiments to inhibit P-gp function. The study was approved by the local ethics committee and written informed consent was obtained from all subjects. 2.2. Apoptosis assays—combined Annexin V–propidium iodide staining The apoptosis was assessed using Annexin V/PI (BD Biosciences, USA) following the manufacturer’s specifications. Binding of fluorescein-conjugated Annexin V and propidium iodide (PI) was analyzed by flow-cytometry (FACScan, Becton Dickinson, USA) [11,12]. 2.3. Apoptosis assays—TUNEL method
2. Materials and methods
Apoptosis was also quantified by using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assays (APO-DIRECT, BD Biosciences Pharmingen, USA) following the manufacturerTs instructions. The labeled DNA breaks with FITC-dUTP were visualized on a FACScan flow cytometer (Becton Dickinson, USA) [13].
2.1. Cell preparation
2.4. Rhodamine 123 (Rh123) efflux studies
Mononuclear cells (MNC) were isolated from peripheral blood of 10 healthy subjects (7 men, 3 women age 26–47 years, mean 34.7 years, Caucasian origin) using Ficoll Paque. Their viability was assayed by a trypan blue exclusion test. The cells were counted using a haemocytometer and subsequently used for further experiments. PHA stimulation of MNC was performed by adding PHA
The cells were incubated with Rh123 at concentration of 0.5 mg/mL at 37 8C for 30 min for uptake. After washing, Rh123-stained cells were incubated in a dye-free RPMI 1640 medium for an additional 90 min efflux. The percentage of Rh123 positive cells was determined by flow cytometry using FACScan flow cytometer (Becton Dickinson, USA).
A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828
2.5. Statistical analysis The data were expressed as mean valuesFSD and statistically analyzed using two-paired Student’s t-test. Statistica 5.1 software package, Stat Soft, program was used for statistical calculations.
3. Results As a model system to study the effect of P-gp inhibition on apoptosis we used the MNC from peripheral blood. P-gp expression was inhibited
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using verapamil (VER) and P-gp specific monoclonal antibodies (mAb). VER (concentration 1–50 AM) or mAb (concentration 1–10 Ag/mL) were added into PHA-activated MNC cultures for 24 h. The P-gp inhibition by VER and mAb was confirmed using Rh123 efflux test. These two P-gp inhibitors significantly affected the efflux of Rh123 in our preliminary experiments (not shown). The percentage of apoptotic MNC was measured using two methods (Annexin V/PI and TUNEL). As shown in Fig. 1 VER and mAb increased the percentage of apoptotic MNC in PHA-activated cultures, whereas these had no effect on non-activated MNC.
40
Apoptotic cells [%]
35
a b
30
*
25
*
*
20 15 10 5 0 0
1
5
10
50
VER concentration [µM]
40
Apoptotic cells [%]
35
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*
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*
25 20 15 10 5 0 0
1 5 mAb concentration [µg/mL]
10
Fig. 1. The effect of verapamil (VER) and P-gp specific monoclonal antibodies (mAb) on apoptosis of PHA-stimulated (a) and non-stimulated (b) peripheral blood mononuclear cells (MNC), mean valuesFSD, *pb0.05, the results provided using Annexin V/PI method.
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DEX, MP, COR) in PHA-activated MNC cultures VER (concentration 1–50 AM) or mAb (concentration 1–10 Ag/mL), plus MTX, DEX, MP or COR were added for 24 h. The percentage of apoptotic MNC was measured and compared to the results from cultures with MTX, DEX, MP and COR alone. As shown in Fig. 3 VER and mAb increased the apoptosis induced by MTX, DEX, MP and COR. In PHA-activated MNC cultures with VER or mAb plus MTX, DEX, MP or COR we observed the significant increase in apoptotic cells as compared to cultures with MTX, DEX, MP and COR alone (Fig. 3). The increase in apoptotic cells in cultures treated with MTX or GS plus P-gp inhibitors was signifi-
To study the effect of MTX and GS on PHAactivated cells, the MNC were incubated with 5 AM PHA for 24 h and then suppressed by MTX or GS (DEX, MP, COR) for 24 h. MTX concentrations ranging 0.1–10 AM and GS 0.001–1 AM were tested reproducing the concentrations, which may be achieved during clinical administration. As shown in Fig. 2 the increase in MTX and GS concentrations resulted in an increase in percentage of apoptotic cells. Previous reports suggest that P-gp can protect the cells from various apoptotic agents including immunosuppressive drugs. To study the effect of P-gp inhibition on apoptosis induced by drugs (MTX, 60
Apoptotic cells [%]
50
*
*
40
* 30 20 10 0 0
0,1 1 MTX concentration [µM]
10
50 45 Apoptotic cells [%]
40
a b c
35
* * *
30 25
** *
* * *
** *
20 15 10 5 0 0
0,001 0,01 0,1 GS concentration [µM]
1
Fig. 2. The effect of methotrexate (MTX) and glucocorticosteroids (GS): a) methylpredisone, b) dexamethasone, c) cortisone on apoptosis of PHA-stimulated peripheral blood mononuclear cells (MNC), mean valuesFSD, *pb0.05, the results provided using Annexin V/PI method.
A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828
825
70 65
*
Apoptotic cells [%]
60 55
** ** **
* * *
* * *
50
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*
45 40
MTX 10µM MP 1µM DEX 1µM COR 1µM
35 30 0
1
5 10 VER concentration [µM]
50
70 65
* * * *
Apoptotic cells [%]
60 55 50
* * * *
45 40
MTX 10µM MP 1µM DEX 1µM COR 1µM
35 30 0
1 5 mAb concentration [µg/ml]
10
Fig. 3. The effect of verapamil (VER) and P-gp specific monoclonal antibodies (mAb) on apoptosis of PHA-stimulated peripheral blood mononuclear cells (MNC) treated with methotrexate (MTX) and glucocorticosteroids (GS), mean valuesFSD, *pb0.05 vs. cultures without MTX and GS, **pb0.05 vs. cultures with 5 AM VER, the results provided using Annexin V/PI method.
cantly higher than the sum of their individual effects (synergistic effect) (Fig. 4).
4. Discussion Immunosuppressive drugs are widely used in therapy of autoimmune diseases to suppress the autoreactive T cells. Multidrug resistance to chemotherapeutic drugs is frequently mediated by overexpression of P-glycoprotein—the product of MDR1 gene [9]. Several compounds have been introduced for blocking the function of P-gp [14]. P-gp modulators belong to a number of chemical classes including calcium channel blockers, calm-
odulin inhibitors, indole alkaloids, and antibodies. Tsuruo et al. were the first to demonstrate the ability of calcium channel blocker verapamil to reverse MDR [10]. Verapamil enhanced intracellular accumulation of many anticancer drugs in numerous cell lines. Clinically available calcium antagonists were demonstrated to reverse MDR in vitro [15]. In present study verapamil and anti-P-gp specific monoclonal antibodies enhanced the apoptosis of PHA-activated MNC from peripheral blood as well as increased the apoptosis in PHA-activated MNC treated with methotrexate, dexamethasone, methylprednisolone and cortisone. Vilpo et al. showed that verapamil induced apoptosis of chronic lymphocytic leukemia cells [16]. Moreover, verapamil potenti-
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A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828 60
∆ MTX VER
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mAb
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VER 1µM
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VER 50µM
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mAb 1µg/ml
mAb 5µg/ml
mAb 10µg/ml
Fig. 4. The increase in apoptotic cells in cultures treated with verapamil (VER) (concentrations 1, 5, 50 AM) or P-gp specific monoclonal antibodies (mAb) (concentrations 1, 5, 10 Ag/mL) and MTX 1 AM separately; and with VER (concentrations 1, 5 and 50 AM) or P-gp specific monoclonal antibodies (mAb) (concentrations 1, 5, 10 Ag/mL ) plus MTX 1 AM. *p—comparison between the sum of increases in apoptotic cells in cultures treated with VER or mAb and MTX 1 AM separately vs. the increase in apoptotic cells in cultures treated with VER or mAb plus MTX 1 AM.
ated the toxicity of cyclosporine and other anticancer drugs. In our study we examined the effect of P-gp blocking on apoptosis of PHA-activated MNC as well as PHA-activated MNC treated with MTX and GS. Initially we found that blocking P-gp activity by verapamil or antibody resulted in an increase of apoptosis of PHA-activated MNC. This finding is consistent with a previous observation of Gollapudi et al. which demonstrated that anti-P-gp mAb promoted apoptosis in anti-CD3-activated T cells [17]. We investigated also the apoptosis of PHAactivated MNC treated with P-gp inhibitors and MTX or GS. Genstrier et al. showed that methotrexate induced only the apoptosis of activated but not resting T cells [5]. Methotrexate induced apoptosis required progression of activated T cells to S phase of the cell cycle. In our study we also did not find the significant effect of methotrexate as well as glucocorticosteroids on apoptosis of non-activated MNC (data not shown). Moreover we found that verapamil and anti-Pgp antibody increased the rate of apoptosis in PHAactivated MNC treated with MTX and GS and the rate of apoptosis correlated to the concentration of VER and anti-Pgp antibody. The increase in apoptotic cells in PHA-activated MNC cultures treated with MTX or
GS was higher than the sum of increase of apoptosis induced by P-gp inhibitors (VER and anti-Pgp antibody) and drugs (MTX or GS) individually. For example the addition of VER at concentration 5 AM to PHA-activated MNC resulted in an increase of apoptotic cells by 8% vs. control (PHA-activated MNC without drugs), MTX (1 AM) increased the apoptosis of PHA-activated MNC by 25% vs. control (PHA-activated MNC without drugs) whereas in PHA-activated MNC cultures with MTX 1 AM plus VER 5 AM we observed the increase in apoptotic cells by 43% vs. control (PHA-activated MNC without drugs). The increase of apoptotic cells in cultures treated with P-gp inhibitors plus MTX or GS was statistically significantly higher than the sum of individual effects of these agents (Fig. 4). These results indicate the additive effect of MTX or GS plus P-gp inhibitors on apoptosis of PHA-activated MNC that might suggest the involvement of P-gp in both apoptotic stimuli. The intriguing are similar results of P-gp inhibition found for MTX and GS and the low increase in apoptosis rate in cultures with increased concentrations of GS. GS are known P-gp substrates while MTX is not transported by P-gp. Our results might suggest that the apoptosis induced by these drugs
A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828
weakly correlates with intracellular concentration of these drugs. Schinkel et al. found that in MDR1 knockout mice the concentrations of DEX are elevated only in the brain, whereas there were no differences in DEX concentrations in other organs (muscle, kidney, liver, spleen, plasma) [18]. These results suggest that P-gp is involved in transport of DEX only in brain but not in other cells. It is possible that antiapoptotic properties of P-gp are not related to the decrease of intracellular concentration of these drugs but caused by other mechanisms, however this hypothesis requires further studies. Several studies demonstrated that P-gp may play an important role in regulation of programmed cell death [9,15,19]. P-gp confers resistance to apoptosis induced by a range of chemotherapeutic drugs, Fas crosslinking, binding of TNF-a to its cell surface receptor and UV irradiation. These stimuli induce cell death by activating the common cell-death cascade mediated by caspases. Biochemical analyses have shown that upon Fas ligation, functional P-gp can inhibit the activation of downstream caspases 8 and 3 [9]. This inhibitory effect can be completely reversed by addition of anti-P-gp monoclonal antibodies or verapamil. The mechanisms of chemotherapy induced apoptosis are not clear and there is conflicting evidence regarding the relationship between drug resistance and resistance to Fas-mediated apoptosis. It has been shown that treatment of cells with chemotherapeutic drugs results in upregulated FasL expression causing Fas-mediated apoptosis [20–22]. Recently, it has been shown that the triggering of apoptosis by chemotherapeutic drugs occurs in a Fas/ FasL independent fashion but the downstream effector proteins caspases are the same as those activated by Fas ligation [23]. Moreover classical multidrug resistant cells K562 expressing high levels of P-gp were less sensitive to apoptosis mediated by cytotoxic drugs and ligation to Fas [9]. Inhibition of P-gp function by P-gp mAb or verapamil reversed resistance to these forms of apoptotic signals. P-gp can protect the cells from apoptotic stimuli in a dual manner. This protein may act as efflux pump, which remove the toxins and drugs from cells and additionally can influence caspase 3-activation, a necessary step for apoptosis induced by drugs. Therefore P-gp may play an important role in pro-
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tecting from a variety of pharmaceutical and physiological apoptotic stimuli. In a previous study we demonstrated the involvement of genetically determined P-gp overexpression in RA refractory to MTX and GS [24]. The probability of remission of RA symptoms was significantly lower in patients with MDR1 genotypes associated with high P-gp expression (3435 CT and 3435CC), than with genotype associated with low Pgp activity (3435TT). Although MTX is not known to be a substrate for P-gp, several studies showed the involvement of P-gp in resistance to MTX. Norris et al. examined the expression of P-gp in a series of leukemia sublines resistant to MTX [25]. They demonstrated increased expression of MDR1 messenger RNA and increased P-gp expression in these sublines. The resistance to MTX was reversed by P-gp specific monoclonal antibodies as well as by verapamil or cyclosporine. In a second study Graaf et al. hypothesized that P-gp may mediate MTX resistance in cells with deficient carrier mediate MTX uptake [26]. These authors reported that insertion of recombinant retrovirus expressing the human MDR1 gene resulted in increased survival of resistance cells. These studies demonstrate the potential of P-gp overexpression in conferring resistance to MTX. The results of this study suggest that P-gp is involved in apoptosis of PHA-activated MNC from peripheral blood. We hypothesize also the involvement of P-gp in apoptosis induced by MTX and GS, however this hypothesis requires further studies.
References [1] Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesmann MM, Pastan I. Expression of a multidrug-resistance gene in human tumors and tissues. Proc Natl Acad Sci U S A 1987;84:265 – 9. [2] Klimecki WT, Futscher BW, Grogan TM, Dalton WS. P-glycoprotein expression and function in circulating blood cells from normal volunteers. Blood 1994;83:2451 – 8. [3] Auphan N, Didonato JA, Helmberg A, Rosette C, Karin M. Immunoregulatory genes and immunosuppression by glucocorticoids. Arch Toxicol, Suppl 1997;19:87 – 95. [4] Zettl UK, Gold R, Toyka KV, Hartung HP. Intravenous glucocorticosteroid treatment augments apoptosis of inflammatory T cells in experimental autoimmune neuritis (EAN) of the Lewis rat. J Neuropathol Exp Neurol 1995;54:540 – 7. [5] Genestier L, Paillot R, Fournel S, Ferraro C, Miossec P, Revillard JP. Immunosuppressive properties of methotrexate:
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A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828 Apoptosis and clonal deletion of activated peripheral T cells. J Clin Invest 1998;102:322 – 8. Herman S, Zurgil N, Langevitz P, Ehrenfeld M, Deutsch M. The induction of apoptosis by methotrexate in activated lymphocytes as indicated by fluorescence hyperpolarization: a possible model for predicting methotrexate therapy for rheumatoid arthritis patients. Cell Struct Funct 2003;28: 113 – 22. Nagata S. Apoptosis by death factor. Cell 1997;88:355 – 65. Johnstone RW, Crethney E, Smyth MJ. P-glycoprotein protects leukemia cells against caspase-dependent, but not caspase-independent cell death. Blood 1999;93:1075 – 85. Smyth MJ, Krasovskis E, Sutton VR, Johnstone RW. The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspasedependent apoptosis. Proc Natl Acad Sci U S A 1998;95: 7024 – 9. Tsuruo T, Iida H, Tsukagoshi S, Sakurai Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res 1981;41:1967 – 72. Kieslinger M, Woldman I, Moriggl R, Hofmann J, Marine JC, Ihle JN, et al. Antiapoptotic activity of STAT5 required during terminal stages of myeloid differentiation. Genes Dev 2000; 14:232 – 44. Bas´kiewicz-Masiuk M, Masiuk M, Machalin´ski B. The influence of STAT5 antisense oligonucleotides on the proliferation and apoptosis of selected human leukaemic cell lines. Cell Prolif 2003;36:265 – 78. Moulian N, Truffault F, Gaudry-Talarmain YM, Serraf A, Berrih-Aknin S. In vivo and in vitro apoptosis of human thymocytes are associated with nitrotyrosine formation. Blood 2001;97:3521 – 30. Feng MR, Liebert M, Wedemeyer G, Grossman HB, Mancini M, Williams M, et al. Effect of verapamil on the uptake and efflux of etoposide (VP16) in both sensitive and resistant cancer cells. Sel Cancer Ther 1991;7:75 – 83. Roepe PD. Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug-resistant cells expressing different levels of P-glycoprotein. Biochemistry 1992;31:12555 – 64. Vilpo J, Koski T, Vilpo L. Calcium antagonists potentiate Pglycoprotein-independent anticancer drugs in chronic lymphocytic leukemia cells in vitro. Haematologica 2000;85:806 – 13.
[17] Gollapudi S, Gupta S. Anti-P-glycoprotein antibody-induced apoptosis of activated peripheral blood lymphocytes: a possible role of P-glycoprotein in lymphocyte survival. J Clin Immunol 2001;21:420 – 30. [18] Schinkel AH, Wagenaar E, van Deemter L, Mol CA, Borst P. Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J Clin Invest 1995;96:1698 – 705. [19] Schwartzman RA, Cidlowski JA. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 1993;14:133 – 51. [20] Robinson LJ, Roberts WK, Ling TT, Lamming D, Sternberg PD, Roepe PD. Human MDR 1 protein overexpression delays the apoptotic cascade in Chinese hamster ovary fibroblasts. Biochemistry 1997;36:11169 – 78. [21] Kasibhatla S, Brunner T, Genestier L, Echeverri F, Mahboubi DR, Green DR. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1. Mol Cell 1998;1:543 – 51. [22] Friesen C, Herr I, Krammer PH, Debatin KM. Involvement of the CD95 (APO-1/FAS) receptor/ligand system in druginduced apoptosis in leukemia cells. Nat Med 1996;2:574 – 7. [23] Eischen CM, Kottke TJ, Martins LM, Basi GS, Tung JS, Earnshaw WC, et al. Comparison of apoptosis in wild-type and fas-resistant cells: Chemotherapy-induced apoptosis is not dependent on fas/fas ligand interactions. Blood 1997;90: 935 – 43. [24] Pawlik A, Wrzesniewska J, Fiedorowicz-Fabrycy I, Gawronska-Szklarz B. The MDR1 3435 polymorphism in patients with rheumatoid arthritis. Int J Clin Pharmacol Ther 2004;42:496 – 503. [25] Norris MD, De Graaf D, Haber M, Kavallaris M, Madafiglio J, Gilbert J, et al. Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate. Int J Cancer 1996;65: 613 – 9. [26] Graaf D, Sharma RC, Mechetner EB, Schimke RT, Roninson IB. P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake. Proc Natl Acad Sci U S A 1996;93:1238 – 42.
Involvement of P-gp in the process of apoptosis in peripheral blood mononuclear cells A. Pawlika,T, M. Baskiewicz-Masiukb, B. Machalinskib, B. Gawronska-Szklarza a
Department of Pharmacokinetics and Therapeutic Drug Monitoring, Pomeranian Medical University, 70-111 Szczecin, al. Powstan˜co´w Wlkp. 72, Poland b Department of Pathology, Pomeranian Medical University, 70-111 Szczecin, al. Powstan˜co´w Wlkp. 72, Poland Received 18 August 2004; received in revised form 12 October 2004; accepted 20 December 2004
Abstract Multidrug resistance mediated by the drug-efflux protein P (P-gp) is one of mechanisms that cells use to escape death induced by drugs and other agents. The aim of the study was to evaluate the effect of P-gp inhibition on apoptosis of PHAactivated peripheral blood mononuclear cells (MNC) as well as apoptosis induced by methotrexate (MTX), dexamethasone (DEX), methylprednisolone (MP) and cortisone (COR). Apoptosis was quantified by flow cytometry using Annexin V/PI and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL). P-gp expression was inhibited using verapamil (VER) and P-gp specific monoclonal antibodies (mAb). VER and mAb enhanced the apoptosis of PHA-activated MNC. Moreover these agents significantly increased the apoptosis induced by MTX, DEX, MP and COR. The results of this study suggest that P-gp is involved in the process of apoptosis in peripheral blood mononuclear cells. D 2005 Elsevier B.V. All rights reserved. Keywords: Apoptosis; P-glycoprotein; Methotrexate; Glucocorticosteroids
1. Introduction Resistance to chemotherapeutic drugs is a frequent problem encountered in the therapy of malignances and autoimmune diseases. Multidrug resistance (MDR) involves cross-resistance to many commonly used cytotoxic drugs, often is associated with re-
T Corresponding author. Tel./fax: +48 91 4661600. E-mail address: [email protected] (A. Pawlik). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2004.12.014
duced intracellular accumulation and altered cellular distribution of these substances [1]. The mechanism of MDR has been associated with alterations in expression of P-glycoprotein (P-gp), which plays the role in absorption of many drugs [2]. The mechanisms by which different immunosuppressive drugs interfere with T cell activation and proliferation are different. The pharmacological effects of glucocorticosteroids (GS) are based on several mechanisms leading to the modulation of cell activation, cytokine expres-
822
A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828
sion, the secretion of inflammatory mediators, leukocyte migration and induction of apoptosis of T cell [3]. At low concentrations of GS T cell apoptosis is mediated by the glucocorticosteroid receptor (GSR) and can be blocked by the GSR antagonists. At higher doses GS can exert proapoptotic effects by other mechanism such as binding to membrane receptors with the consecutive activation of second messenger system and by affecting the physiochemical properties of the cell membrane [4]. The folate antagonist methotrexate (MTX) is extensively used in therapy of malignances, and chronic inflammatory disorders such as rheumatoid arthritis [5]. MTX exerts antiproliferative activity, which is associated with increased release of adenosine. MTX induces also apoptosis of T lymphocytes [6]. Induction of apoptosis is the key mechanism to eliminate the auto reactive T cell in the periphery and attenuate the immune response. Recently, molecular level of understanding of the regulation of apoptosis has expanded considerably. Nevertheless, it is still not understood how extra cellular signals ranging from withdrawal of growth factors to administration of drugs, influence the apoptosis and which mechanism may protect the cells from proapoptotic action of diverse agents [7]. Previously published data have shown that Pglycoprotein can protect the cells from various apoptotic agents [8–10]. The aim of this study was to examine the effect of P-gp inhibition on apoptosis of peripheral blood mononuclear cells (MNC) as well as on apoptosis induced by methotrexate and glucocorticosteroids (GS): dexamethasone (DEX), methylprednisolone (MP) and cortisone (COR).
(concentration 5 Ag/mL) and incubating MNC for 24 h at 37 8C, 5% CO2 in RPMI 1640 medium. One million cells were incubated in each culture. After 24 h of incubation methotrexate (Methotrexate, Rhone-Poulenc, F) MTX or GS dexamethasone (Dexaven, Jelfa, PL), methylprednisolone (SoluMedrol, Upjohn, B) and cortisone (Hydrocortisonum, Jelfa, PL) were added for 24 h. Apoptosis was measured by flow cytometry (FACScan, Becton Dickinson, USA). Verapamil (VER) (Isoptin, Knoll, D) and P-gp specific monoclonal antibodies (mAb), clone 17F9 (Beckton Dickinson, San Jose, USA) were used in the experiments to inhibit P-gp function. The study was approved by the local ethics committee and written informed consent was obtained from all subjects. 2.2. Apoptosis assays—combined Annexin V–propidium iodide staining The apoptosis was assessed using Annexin V/PI (BD Biosciences, USA) following the manufacturer’s specifications. Binding of fluorescein-conjugated Annexin V and propidium iodide (PI) was analyzed by flow-cytometry (FACScan, Becton Dickinson, USA) [11,12]. 2.3. Apoptosis assays—TUNEL method
2. Materials and methods
Apoptosis was also quantified by using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assays (APO-DIRECT, BD Biosciences Pharmingen, USA) following the manufacturerTs instructions. The labeled DNA breaks with FITC-dUTP were visualized on a FACScan flow cytometer (Becton Dickinson, USA) [13].
2.1. Cell preparation
2.4. Rhodamine 123 (Rh123) efflux studies
Mononuclear cells (MNC) were isolated from peripheral blood of 10 healthy subjects (7 men, 3 women age 26–47 years, mean 34.7 years, Caucasian origin) using Ficoll Paque. Their viability was assayed by a trypan blue exclusion test. The cells were counted using a haemocytometer and subsequently used for further experiments. PHA stimulation of MNC was performed by adding PHA
The cells were incubated with Rh123 at concentration of 0.5 mg/mL at 37 8C for 30 min for uptake. After washing, Rh123-stained cells were incubated in a dye-free RPMI 1640 medium for an additional 90 min efflux. The percentage of Rh123 positive cells was determined by flow cytometry using FACScan flow cytometer (Becton Dickinson, USA).
A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828
2.5. Statistical analysis The data were expressed as mean valuesFSD and statistically analyzed using two-paired Student’s t-test. Statistica 5.1 software package, Stat Soft, program was used for statistical calculations.
3. Results As a model system to study the effect of P-gp inhibition on apoptosis we used the MNC from peripheral blood. P-gp expression was inhibited
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using verapamil (VER) and P-gp specific monoclonal antibodies (mAb). VER (concentration 1–50 AM) or mAb (concentration 1–10 Ag/mL) were added into PHA-activated MNC cultures for 24 h. The P-gp inhibition by VER and mAb was confirmed using Rh123 efflux test. These two P-gp inhibitors significantly affected the efflux of Rh123 in our preliminary experiments (not shown). The percentage of apoptotic MNC was measured using two methods (Annexin V/PI and TUNEL). As shown in Fig. 1 VER and mAb increased the percentage of apoptotic MNC in PHA-activated cultures, whereas these had no effect on non-activated MNC.
40
Apoptotic cells [%]
35
a b
30
*
25
*
*
20 15 10 5 0 0
1
5
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50
VER concentration [µM]
40
Apoptotic cells [%]
35
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*
30
*
25 20 15 10 5 0 0
1 5 mAb concentration [µg/mL]
10
Fig. 1. The effect of verapamil (VER) and P-gp specific monoclonal antibodies (mAb) on apoptosis of PHA-stimulated (a) and non-stimulated (b) peripheral blood mononuclear cells (MNC), mean valuesFSD, *pb0.05, the results provided using Annexin V/PI method.
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DEX, MP, COR) in PHA-activated MNC cultures VER (concentration 1–50 AM) or mAb (concentration 1–10 Ag/mL), plus MTX, DEX, MP or COR were added for 24 h. The percentage of apoptotic MNC was measured and compared to the results from cultures with MTX, DEX, MP and COR alone. As shown in Fig. 3 VER and mAb increased the apoptosis induced by MTX, DEX, MP and COR. In PHA-activated MNC cultures with VER or mAb plus MTX, DEX, MP or COR we observed the significant increase in apoptotic cells as compared to cultures with MTX, DEX, MP and COR alone (Fig. 3). The increase in apoptotic cells in cultures treated with MTX or GS plus P-gp inhibitors was signifi-
To study the effect of MTX and GS on PHAactivated cells, the MNC were incubated with 5 AM PHA for 24 h and then suppressed by MTX or GS (DEX, MP, COR) for 24 h. MTX concentrations ranging 0.1–10 AM and GS 0.001–1 AM were tested reproducing the concentrations, which may be achieved during clinical administration. As shown in Fig. 2 the increase in MTX and GS concentrations resulted in an increase in percentage of apoptotic cells. Previous reports suggest that P-gp can protect the cells from various apoptotic agents including immunosuppressive drugs. To study the effect of P-gp inhibition on apoptosis induced by drugs (MTX, 60
Apoptotic cells [%]
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*
*
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* 30 20 10 0 0
0,1 1 MTX concentration [µM]
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50 45 Apoptotic cells [%]
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a b c
35
* * *
30 25
** *
* * *
** *
20 15 10 5 0 0
0,001 0,01 0,1 GS concentration [µM]
1
Fig. 2. The effect of methotrexate (MTX) and glucocorticosteroids (GS): a) methylpredisone, b) dexamethasone, c) cortisone on apoptosis of PHA-stimulated peripheral blood mononuclear cells (MNC), mean valuesFSD, *pb0.05, the results provided using Annexin V/PI method.
A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828
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70 65
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Apoptotic cells [%]
60 55
** ** **
* * *
* * *
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**
*
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MTX 10µM MP 1µM DEX 1µM COR 1µM
35 30 0
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5 10 VER concentration [µM]
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70 65
* * * *
Apoptotic cells [%]
60 55 50
* * * *
45 40
MTX 10µM MP 1µM DEX 1µM COR 1µM
35 30 0
1 5 mAb concentration [µg/ml]
10
Fig. 3. The effect of verapamil (VER) and P-gp specific monoclonal antibodies (mAb) on apoptosis of PHA-stimulated peripheral blood mononuclear cells (MNC) treated with methotrexate (MTX) and glucocorticosteroids (GS), mean valuesFSD, *pb0.05 vs. cultures without MTX and GS, **pb0.05 vs. cultures with 5 AM VER, the results provided using Annexin V/PI method.
cantly higher than the sum of their individual effects (synergistic effect) (Fig. 4).
4. Discussion Immunosuppressive drugs are widely used in therapy of autoimmune diseases to suppress the autoreactive T cells. Multidrug resistance to chemotherapeutic drugs is frequently mediated by overexpression of P-glycoprotein—the product of MDR1 gene [9]. Several compounds have been introduced for blocking the function of P-gp [14]. P-gp modulators belong to a number of chemical classes including calcium channel blockers, calm-
odulin inhibitors, indole alkaloids, and antibodies. Tsuruo et al. were the first to demonstrate the ability of calcium channel blocker verapamil to reverse MDR [10]. Verapamil enhanced intracellular accumulation of many anticancer drugs in numerous cell lines. Clinically available calcium antagonists were demonstrated to reverse MDR in vitro [15]. In present study verapamil and anti-P-gp specific monoclonal antibodies enhanced the apoptosis of PHA-activated MNC from peripheral blood as well as increased the apoptosis in PHA-activated MNC treated with methotrexate, dexamethasone, methylprednisolone and cortisone. Vilpo et al. showed that verapamil induced apoptosis of chronic lymphocytic leukemia cells [16]. Moreover, verapamil potenti-
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A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828 60
∆ MTX VER
50
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Apoptotic cells [%]
mAb
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*
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*
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13
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2
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VER 1µM
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8 2
VER 5µM
VER 50µM
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8
2
mAb 1µg/ml
mAb 5µg/ml
mAb 10µg/ml
Fig. 4. The increase in apoptotic cells in cultures treated with verapamil (VER) (concentrations 1, 5, 50 AM) or P-gp specific monoclonal antibodies (mAb) (concentrations 1, 5, 10 Ag/mL) and MTX 1 AM separately; and with VER (concentrations 1, 5 and 50 AM) or P-gp specific monoclonal antibodies (mAb) (concentrations 1, 5, 10 Ag/mL ) plus MTX 1 AM. *p—comparison between the sum of increases in apoptotic cells in cultures treated with VER or mAb and MTX 1 AM separately vs. the increase in apoptotic cells in cultures treated with VER or mAb plus MTX 1 AM.
ated the toxicity of cyclosporine and other anticancer drugs. In our study we examined the effect of P-gp blocking on apoptosis of PHA-activated MNC as well as PHA-activated MNC treated with MTX and GS. Initially we found that blocking P-gp activity by verapamil or antibody resulted in an increase of apoptosis of PHA-activated MNC. This finding is consistent with a previous observation of Gollapudi et al. which demonstrated that anti-P-gp mAb promoted apoptosis in anti-CD3-activated T cells [17]. We investigated also the apoptosis of PHAactivated MNC treated with P-gp inhibitors and MTX or GS. Genstrier et al. showed that methotrexate induced only the apoptosis of activated but not resting T cells [5]. Methotrexate induced apoptosis required progression of activated T cells to S phase of the cell cycle. In our study we also did not find the significant effect of methotrexate as well as glucocorticosteroids on apoptosis of non-activated MNC (data not shown). Moreover we found that verapamil and anti-Pgp antibody increased the rate of apoptosis in PHAactivated MNC treated with MTX and GS and the rate of apoptosis correlated to the concentration of VER and anti-Pgp antibody. The increase in apoptotic cells in PHA-activated MNC cultures treated with MTX or
GS was higher than the sum of increase of apoptosis induced by P-gp inhibitors (VER and anti-Pgp antibody) and drugs (MTX or GS) individually. For example the addition of VER at concentration 5 AM to PHA-activated MNC resulted in an increase of apoptotic cells by 8% vs. control (PHA-activated MNC without drugs), MTX (1 AM) increased the apoptosis of PHA-activated MNC by 25% vs. control (PHA-activated MNC without drugs) whereas in PHA-activated MNC cultures with MTX 1 AM plus VER 5 AM we observed the increase in apoptotic cells by 43% vs. control (PHA-activated MNC without drugs). The increase of apoptotic cells in cultures treated with P-gp inhibitors plus MTX or GS was statistically significantly higher than the sum of individual effects of these agents (Fig. 4). These results indicate the additive effect of MTX or GS plus P-gp inhibitors on apoptosis of PHA-activated MNC that might suggest the involvement of P-gp in both apoptotic stimuli. The intriguing are similar results of P-gp inhibition found for MTX and GS and the low increase in apoptosis rate in cultures with increased concentrations of GS. GS are known P-gp substrates while MTX is not transported by P-gp. Our results might suggest that the apoptosis induced by these drugs
A. Pawlik et al. / International Immunopharmacology 5 (2005) 821–828
weakly correlates with intracellular concentration of these drugs. Schinkel et al. found that in MDR1 knockout mice the concentrations of DEX are elevated only in the brain, whereas there were no differences in DEX concentrations in other organs (muscle, kidney, liver, spleen, plasma) [18]. These results suggest that P-gp is involved in transport of DEX only in brain but not in other cells. It is possible that antiapoptotic properties of P-gp are not related to the decrease of intracellular concentration of these drugs but caused by other mechanisms, however this hypothesis requires further studies. Several studies demonstrated that P-gp may play an important role in regulation of programmed cell death [9,15,19]. P-gp confers resistance to apoptosis induced by a range of chemotherapeutic drugs, Fas crosslinking, binding of TNF-a to its cell surface receptor and UV irradiation. These stimuli induce cell death by activating the common cell-death cascade mediated by caspases. Biochemical analyses have shown that upon Fas ligation, functional P-gp can inhibit the activation of downstream caspases 8 and 3 [9]. This inhibitory effect can be completely reversed by addition of anti-P-gp monoclonal antibodies or verapamil. The mechanisms of chemotherapy induced apoptosis are not clear and there is conflicting evidence regarding the relationship between drug resistance and resistance to Fas-mediated apoptosis. It has been shown that treatment of cells with chemotherapeutic drugs results in upregulated FasL expression causing Fas-mediated apoptosis [20–22]. Recently, it has been shown that the triggering of apoptosis by chemotherapeutic drugs occurs in a Fas/ FasL independent fashion but the downstream effector proteins caspases are the same as those activated by Fas ligation [23]. Moreover classical multidrug resistant cells K562 expressing high levels of P-gp were less sensitive to apoptosis mediated by cytotoxic drugs and ligation to Fas [9]. Inhibition of P-gp function by P-gp mAb or verapamil reversed resistance to these forms of apoptotic signals. P-gp can protect the cells from apoptotic stimuli in a dual manner. This protein may act as efflux pump, which remove the toxins and drugs from cells and additionally can influence caspase 3-activation, a necessary step for apoptosis induced by drugs. Therefore P-gp may play an important role in pro-
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tecting from a variety of pharmaceutical and physiological apoptotic stimuli. In a previous study we demonstrated the involvement of genetically determined P-gp overexpression in RA refractory to MTX and GS [24]. The probability of remission of RA symptoms was significantly lower in patients with MDR1 genotypes associated with high P-gp expression (3435 CT and 3435CC), than with genotype associated with low Pgp activity (3435TT). Although MTX is not known to be a substrate for P-gp, several studies showed the involvement of P-gp in resistance to MTX. Norris et al. examined the expression of P-gp in a series of leukemia sublines resistant to MTX [25]. They demonstrated increased expression of MDR1 messenger RNA and increased P-gp expression in these sublines. The resistance to MTX was reversed by P-gp specific monoclonal antibodies as well as by verapamil or cyclosporine. In a second study Graaf et al. hypothesized that P-gp may mediate MTX resistance in cells with deficient carrier mediate MTX uptake [26]. These authors reported that insertion of recombinant retrovirus expressing the human MDR1 gene resulted in increased survival of resistance cells. These studies demonstrate the potential of P-gp overexpression in conferring resistance to MTX. The results of this study suggest that P-gp is involved in apoptosis of PHA-activated MNC from peripheral blood. We hypothesize also the involvement of P-gp in apoptosis induced by MTX and GS, however this hypothesis requires further studies.
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