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Suppression of P-glycoprotein expression by antipsychotics trifluoperazine in adriamycin-resistant L1210 mouse leukemia cells
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Suppression of P-glycoprotein expression by antipsychotics trifluoperazine in adriamycin-resistant L1210 mouse leukemia cells Soon Young Shin a , Byeong Hyeok Choi a , Jae-Ryong Kim b , Jung-Hye Kim b , Young Han Lee a,∗ a
Division of Molecular and Life Science, Department of Bio-Nano Technology, College of Science and Technology, Hanyang University, Ansan 426-791, Republic of Korea b Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, 705-717, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history:
Multidrug resistance (MDR) to unrelated chemotherapeutic drugs can be mediated by over-
Received 1 August 2005
expression of P-glycoprotein (P-gp), the mdr gene product. Trifluoperazine (TFP), a phenoth-
Received in revised form 12 March
iazine derivative antipsychotics, is known to reverse MDR of tumor cell lines by blocking
2006
P-gp efflux function. In the present study, we evaluated the effect of TFP on the expression
Accepted 13 March 2006
of P-gp in multidrug-resistant L1210/Adr mouse leukemic cell lines, which are characterized
Published on line 17 March 2006
by overexpession of P-gp. We found that TFP induced the downregulation of P-gp protein and mdr1b mRNA in a dose- and time-dependent manner in L1210/Adr cells. TFP reduc-
Keywords:
tion of mdr1b mRNA was paralleled by transcriptional suppression of the mdr1b promoter.
Trifluoperazine
Moreover, TFP restored the adriamycin-induced apoptosis in L1210/Adr cells. These results
Multidrug resistance
suggest that TFP may have utility as an adjuvant in the therapy of leukemia for the reversal
P-glycoprotein
of P-gp-dependent MDR as well as for the management of psychological symptoms in the cancer patients. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Multidrug resistance (MDR) of tumor cells represents a development of clinical barrier to a wide range of structurally and functionally unrelated anti-cancer drugs (Gottesman and Pastan, 1993). Many mechanisms for the development of MDR have been described, including the up-regulation of membrane transporter, activation of pathway for detoxification of drugs, changes in DNA repair response and apoptotic responses to toxic drugs (Simon and Schindler, 1994). One of the most common and well-described mechanism in the development of MDR phenotype is the overexpression of Pglycoprotein (P-gp). P-gp is a transmembrane protein that acts
as an ATP-driven efflux pump for a wide variety of structurally and functionally unrelated agents from cytoplasm, thereby reducing the intracellular drug content to a sublethal level (Fardel et al., 1996; Ambudkar et al., 2003). Trifluoperazine (TFP) is a phenothiazine derivative which is widely used clinically to control psychotic disturbances, such as depression, agitation, anxiety, psychosis, and acute confusional state (Bruera and Nemann, 1998; Nordenberg et al., 1999). The therapeutic effects of TFP are attributed to the antagonism of dopamine D2 receptor and the inhibition of calmodulin (Nordenberg et al., 1999; Takano et al., 2003; Lahti et al., 1993). TFP, apart from their clinical use as a neuroleptics, has been shown to exert antiproliferative effects on many
∗ Corresponding author at: 1271 Sa-1-dong, Sangrok-gu, Hanyang University, Division of Molecular and Life Science, College of Science and Technology, Ansan 426-791, Republic of Korea. Tel.: +82 31 400 5517; fax: +82 31 416 9781. E-mail address: [email protected] (Y.H. Lee).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.03.002
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 300–306
tumor cells (Osborn and Weber, 1980; Naftalovich et al., 1991; Bruera and Nemann, 1998; Nordenberg et al., 1999; Shin et al., 2004a). In addition, TFP has been shown to block the function of P-gp by modification of the membrane structure around the P-gp or by direct interaction with P-gp (Hendrich et al., 2001), and sensitizes MDR cells to chemotherapeutic agents (Ford et al., 1989; Ganapathi et al., 1991). It has been demonstrated that P-gp reversing agents, such as reserpine, verapamil and cyclosporine A, or bromocriptine, an ergot alkaloid known as a D2 dopaminergic receptor agonist, regulate P-gp gene expression (Sonneveld and Wiemer, 1997; Furuya et al., 1997). The aim of the present study was to determine whether MDR reversing agent TFP could modulate P-gp gene expression. We found that TFP decreased levels of mdr1b mRNA and its product P-gp protein in adriamycin-resistant mouse leukemia L1210 model system.
2.
Materials and methods
2.1.
Materials
Trifluoperazine was purchased from RBI (Natick, MA). [␣32 P]dCTP and [14 C]chloramphenicol were from NEN Life Science Products, Inc. (Boston, MA). Acetyl coenzyme A was from Sigma (St. Louis, MO). Rabbit polyclonal P-Glycoprotein antibody was obtained from Oncogene (San Diego, CA) and antiPARP, anti-NFB, and anti-IB antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
2.2.
Cell culture
L1210 mouse leukemia cell line was obtained from American Type Culture Collection (Rockville, MD). Multidrug resistant L1210 subline was obtained by long-term adaptation of parental L1210 cells to adriamycin (Kim et al., 1993). Additional details concerning cross-resistance responsible for MDR phenotype were described elsewhere (Kim et al., 1993; Kim and Kim, 1994; Kim, 1997). L1210 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone, Logan, UT). L1210/Adr cells were routinely maintained in a medium containing 0.2 g/ml adriamycin.
2.3.
Western blot analysis
Cells were lysed and protein samples (20 g of each) were separated and transferred to nitrocellulose filters as described previously (Shin et al., 2004a). The blots were incubated with anti-P-glycoprotein antibody (Oncogene, La Jolla, CA) and developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech., Piscataway, NJ). For PARP cleavage assay, an anti-PARP antibody that detects both the native 113-kDa and the cleaved 89-kDa fragment was utilized (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The same blot was stripped and reprobed with anti-Erk1/2 or Erk2 antibody for use as an internal control. For detection of NFB and IB, L1210 cells were separated into cytosol and nuclear-enriched fractions as described previously (Shin et al., 2004b).
2.4.
301
Northern blot analysis
Total RNA was prepared using STAT-60 reagent (TEL-TEST, Friendswood, TX) according to the manufacturer’s protocol. Ten micrograms of total RNA was separated on 1.2% agarose gel, transferred to Hybond N+ nylon membrane (Amersham Pharmacia Biotech., Piscataway, NJ), and hybridized with [␣-32 P]dCTP-labeled mdr1 cDNA probe, an insert of pCHP1 (Riordan et al., 1985). The membranes were then washed with 2× SSC/0.1% SDS for 20 min at room temperature, 2× SSC/0.1% SDS at 42 ◦ C for 30 min, and 0.5× SSC/0.1% SDS for 30 min at 65 ◦ C. [␣-32 P]dCTP-labeled gapdh cDNA was used as an internal control.
2.5. Generation of deletions of the rat mdr1b promoter constructs Deletions of the rat mdr1b promoter fragments spanning from the −1237 to +42, −740 to +42, −370 to +42, and −205 to +42 were synthesized by PCR in a reaction containing the 1288 RMICAT plasmid, a rat mdr1b-CAT reporter (Zhou et al., 1996) which was a kind gift of Dr. M. Tien Kuo (University of Texas M.D. Anderson Cancer Institute, Houston, TX;), as a template with the following upstream primers: −1237; 5 caGGTACCtacaaagggaagctgaagag-3 , −740; 5 -caGGTACCggactctgtctcaggttgac-3 , −370; 5 -caGGTACCgctgtcagaagggaacttta3 , and −205; 5 -caGGTACCagagttacctgaacatgtagagac-3 . A KpnI restriction site is indicated by uppercase letters. A single downstream primer (5 -cgTCTAGActcagcctcttacagcttca-3 ) containing a BglII site, which is indicated by uppercase letters, was used in each PCR amplification. The PCR fragments were then digested with the restriction enzymes KpnI and BglII. The fragments were extracted from the agarose gel and inserted into the KpnI and BglII sites of the pGL3-basic luciferaseencoding reporter vector (Promega, Madison, WI, USA). The resultant constructs were verified by DNA sequencing and by restriction enzyme digests.
2.6.
Transient transfection and reporter gene assay
For mdr1b promoter analysis, NIH3T3 cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum. One day after seeding cells into 35-mm dishes (6 × 105 cells), the cells were co-transfected with 0.5 g of 5 deletion constructs of the mdr1b promoter and 50 ng of pRL/null plasmid, which expresses Renilla luciferase (Promega) using the Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s instructions. Twenty-four hours post-transfection, cells were treated with TFP. After 6–8 h, the cell lysates were assayed for luciferase activity as described in the manufacturer’s protocol (Promega) using the TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Renilla luciferase activity was used to normalize the activity of the firefly luciferase.
2.7.
Determination of DNA fragmentation
Cells (5 × 106 ) were lysed in a solution containing 5 mM Tris, pH 7.4, 20 mM EDTA, 0.5% Triton X-100. The lysate was extracted twice with phenol/chloroform and chloroform. Cytosolic frag-
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mented DNA was precipitated with ethanol and subjected to gel electrophoresis on a 1.8% agarose gels. The gel was stained with ethidium bromide and photographed on a UV transilluminator.
3.
Results and discussion
Previously, adriamycin-resistant L1210 subline (L1210/Adr), which confers resistance to a variety of conventional anticancer drugs, was utilized and characterized (Kim and Kim, 1994). In L1210/Adr cells, P-gp protein was highly expressed, but not in parental L1210 cells, C6 glioma and NIH3T3 fibroblasts (Fig. 1A). MRP overexpression also confers an MDR phenotype similar to that of P-gp-dependent MDR (Grant et al., 1994), however, in L1210/Adr cells MRP expression was not detected as revealed by RT-PCR (data not shown) (Kim et al., 1993).
Fig. 1 – Adriamycin-resistant L1210/Adr cells are resistant to adriamycin-induced IB degradation. (A) Expression of P-gp in L1210/Ade cells. Total cell lysates (25 g) were separated on 6% SDS-PAGE gels and analyzed by immunoblot analysis with rabbit anti-P-Glycoprotein antibody (1:500). ␣-tubulin antibody was used as an internal loading control. (B) Resistance of L1210/Adr cells to adriamycin-induced IB degradation. Cells were treated with adriamycin (2 g/ml) for 6 h, and then cytosolic extracts and nuclear extracts were prepared. IB degradation or NFB accumulation was assessed by immunoblot analysis in the cytosolic (B) or in the nuclear (C) fractions, respectively. Results are representative of two independent experiments.
NFB is a transcription factor, which functions in the control of immunity, inflammation, cell survival and protection of apoptosis induced by variety of DNA-damaging agents that included adriamycin (Rayet and Gelinas, 1999). Previously, it has been demonstrated that NFB is involved in basal and insulin-induced mdr1b promoter activity in rat hepatoma cells (Zhou and Kuo, 1997). To determine whether aberrant activation of NFB is associated with accumulation of P-gp protein in L1210/Adr cells, we analyzed the activation of NFB induced by adriamycin. Nuclear and cytoplasmic extracts were prepared and examined the levels of NFB and IB, respectively. In most cells, NFB proteins are rendered inactive in the cytoplasm by inhibitory subunits of IB (Grilli et al., 1993). When the cells were treated with adriamycin, the abundance of cytoplasmic IB was reduced in a dose-dependent manner (Fig. 1B), resulting in the increase of the translocation of NFB into the nucleus (Fig. 1C) in parental L1210 cells, but did not in L1210/Adr cells. A possible explanation for the lack of NFB activation in L1210/Adr cells could be that an enhanced efflux of drugs due to overexpression of P-gp prevents intracellular accumulation of drugs, thereby conferring resistance to adriamycin. This result suggests that although NFB plays an essential role in the basal expression of mdr1 genes, maintenance of MDR phenotype in L1210/Adr subline seems to be NFB-independent. It has been demonstrated that P-gp expression can be regulated by some MDR reversing agents, including reserpine (dopamine reuptake inhibitor) and verapamil (calcim channel blocker) in human colon carcinoma cells (Bhat et al., 1995), and by bromocriptine (D2 dopamine receptor agonist) in H35 rat hepatoma cells (Furuya et al., 1997). Antipsychotics TFP is a well-known MDR reversing agent which interferes with efflux pump activity by modification of the membrane structure around the P-gp or by direct interaction with P-gp (Hendrich et al., 2001). To test if TFP could regulate P-gp expression, we treated parental L1210 and L1210/Adr cells with TFP, and measured the level of P-gp expression using Western blot analysis. The amount of P-gp protein was decreased by treatment with TFP in a concentration-dependent manner (Fig. 2A). To better define the effect of TFP on P-gp expression, a detailed time course analysis was performed. The result demonstrates that the expression level of P-gp was time-dependently reduced by treatment of 20 M TFP in L1210/Adr cells (Fig. 2B). The abundance of P-gp was reduced by approximately 40% and 71.3% after 24 h and 48 h, respectively, after TFP incubation. We also examined a low concentration-dependent response for the P-gp expression in L1210/Adr cells. P-gp protein level was decreased by approximately 25% and 50% in response to 5 days treatment of TFP with 1 and 5 M, respectively (Fig. 2C). These results suggest that TFP has the capacity to reduce the abundance of P-gp protein in L1210/Adr cells. To investigate whether the reduction of the P-gp level may result from a suppression of P-gp mRNA, Northern blot analysis was performed. The level of mdr1 mRNA was decreased in a concentration- (Fig. 3A) and time-dependent (Fig. 3B) manners. To determine whether the mdr1 gene is controlled at the transcriptional level by TFP, we generated a set of mdr1b promoter-luciferase constructs progressively truncated at the 5 end (−1237, −740, −370, and −205 bp) containing 42 bp downstream from the transcription start
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Fig. 2 – TFP decreases the accumulation of P-gp protein in adriamycin-resistant L1210/Adr cells. L1210 and L1210/Adr cells were treated with different concentrations of TFP for 24 h (A) or with 20 M TFP for different times (B). Total cell lysates (25 g) were separated on 6% SDS-PAGE gels and analyzed by immunoblot analysis with rabbit anti-P-Glycoprotein antibody (1:500). Erk1/2 antibody was used as an internal loading control. Results are representative of three independent experiments.
site, and transfected into NIH3T3 cells. Twenty-four hours after transfection, cells were treated without or with 10 M TFP. Adriamycin was included as a positive control. As shown in Fig. 4A, deletion of sequences up to −370 bp significantly
reduced the luciferase activity by TFP (p < 0.01 compared with the untreated control). Further deletion to −205 bp makedly reduced the induction of basal activity, suggesting that strong positive cis-element may be present between −370 and −205.
Fig. 3 – TFP suppresses the expression of mdr1b mRNA. Cells were treated with different concentrations of TFP for 24 h (A) or with 20 M TFP for different times (B). Total RNA (10 g) was isolated from cells, electrophoresed on 1% agarose-gel, and capillary transferred to a nylon filter. The blot was then sequentially hybridized with the 32 P-labeled mdr1 of pCHP1 insert and gapdh cDNA probes. Results are representative of two independent experiments. The relative band intensities of mdr1 mRNA were measured by quantitative scanning densitometer and image analysis software, Bio-1D version 97.04, and the ratio of mdr1 to gapdh intensity was determined.
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Fig. 4 – TFP represses mdr1b promoter activity. (A) NIH3T3 cells were transiently transfected for 24 h with 5 deletion constructs of the rat mdr1b promoter fused to the luciferase gene, and luciferase reporter activity was assayed construct using eletrophoration (260 V, 960 F). Transfected cells were treated with different concentrations of TFP (5–20 M) for 24 h. Prepared extracts were incubated with [14 C]chloramphenicol and acetyl-CoA and CAT activity measured by the production of acetylated chloramphenocol using thin layer chromatography (19:1 chloroform/methanol). The significance of each result was compared only with untreated control (CT) using Student’s-t test. *p < 0.01; **p < 0.001.
When −205/+42 construct was transfected, TFP treatment also yield a decreased promoter activity, albeit weakly but significantly (p < 0.001 compared with the untreated control). In addition, TFP significantly decreased the luciferase activities in a dose-dependent manner (5 and 10 M; p < 0.01, 20 M; p < 0.001, compared with the untreated control) in −205/+42 construct-ransfected cells (Fig. 4B). These results suggest that the proximal region of mdr1b gene between −205 and +42 sequences is sufficient to confer the TFP-mediated suppression of mdr1b promoter activity. Previous studies have demonstrated that the processing of poly(ADP-ribose)polymerase (PARP) from its native 113-kDa to an inactive 89-kDa fragment is a result of cleavage by caspase3 (Tewari et al., 1995). Thus, the profile of PARP cleavage has been used as a biochemical marker to assess the activation of
caspases and to confirm the induction of apoptosis (Kaufmann et al., 1993; Shah et al., 1996). To address whether the reduction of P-gp expression by TFP has the potential to sensitize L1210/Adr cells to chemotherapy, PARP cleavage was assessed by Western blotting, utilizing antibodies that detect both the native 113-kDa and the cleaved 89-kDa fragment. Typical processing of PARP into the 89-kDa fragment was observed by 24 h in adriamycin-treated parental L1210 cells, but not in L1210/Adr cells (Fig. 5A, lane 2 versus lane 7). However, when L1210/Adr cells were treated with adriamycin in the presence of TFP, PARP cleavage was restored in a TFP dose-dependent manner (ED50 = 8.5 ± 0.2 M). This TFP response was probably not due to cytotoxic effect for L1210/Adr cells because TFP treatment alone had no effect on the induction of PARP cleavage (Fig. 5A, lanes 3–6). DNA agarose electrophoresis
Fig. 5 – TFP reverses the resistance of L1210/Adr cells to adriamycin. (A) Cells were treated with adriamycin for 6 h and the cleavage of PARP was assessed by immunoblot analysis using anti-PARP antibody (1:1000). (B) L1210/Adr cells were treated with adriamycin in the absence or presence of TFP (20 M) for the indicated times, and the cleavage of PARP was assessed by immunoblot analysis. A 113-kDa PARP and 89-kDa of cleaved form are indicated with the arrow and arrow head, respectively. Results are representative of two independent experiments.
european journal of pharmaceutical sciences
also showed that TFP partially restored adriamycin-induced generation of DNA fragmentation in a dose-dependent manner in L1210/Adr cells (Fig. 5B). These results suggest that TFP-mediated suppression of P-gp expression could contribute to reverse the drug resistance phenotype of L1210/Adr cells. The development of MDR is a major limitation to successful anticancer chemotherapy. Although multiple mechanisms contribute to acquire MDR phenotype, overexpression of mdr1 gene and its product P-gp remains the most common mechanism underlying development of MDR (Gottesman et al., 2002). As the common strategy to reverse the MDR phenotype of tumor cells is associated with the inhibition of P-gp transporter function, prevention of mdr expression would also be necessary to prevent MDR. In the present study, we have found that antipsychotic agent TFP reverses the resistance of L1210/Adr cells to adriamycin. Given that TFP decreased the basal promoter activity of the mdr1b gene, it is likely that decreases in the amount of P-gp protein and mdr1 mRNA by TFP is most probably due to the suppression of mdr1 gene transcription. The promoter region of mdr1b contains several transcription factor binding sites for upregulation of mdr1b expression (Cohen et al., 1991; Zhou et al., 1996). The results presented in this study suggest that the promoter function and endogenous expression of the mdr1b gene can be modulated by TFP. Using a serial deletion construct of the mdr1b promoter, we suggest that the region between 205 bp upstream and 42 bp downstream of the transcription start site may contain cis-acting element, which is responsible for TFP-mediated transcriptional repression. The rat mdr1b promoter contains several regulatory elements, such as p53, NFB, C/EBP (also known as NF-IL6), and NF-Y, between −205 and +42. Among them, NFB may be not involved at least in TFP-mediated suppression of mdr1b gene expression, because IB degradation and NFB activation were not induced by adriamycin in L1210/Adr cells. It will be interesting to identify which of cis-acting element is an authentic TFP responsive element and to investigate the upstream signaling mechanism that modulates TFP-mediated suppression of mdr1b gene expression. In summary, we demonstrate here that antipsychotic agent TFP reduces the abundance of P-gp level probably through the negative regulation of mdr1b gene transcription. TFPmediated suppression of P-gp expression may be involved in an alternative mechanism of the reversion of the MDR phenotype in multidrug resistant variant L1210/Adr mouse leukemia cells. Although the detailed mechanism underlying how TFP regulates mdr1 gene expression has been remained, the present report provides the possibility that a combination of anticancer agents with TFP can efficiently control the P-gpmediated MDR via a mechanism involving the downregulation of mdr1 gene transcription, in addition to the inhibition of P-gp transporter activity. In humans, however, it has been shown that at the maximal tolerated doses (60–80 mg daily) the peak plasma concentrations of TFP are of 39.46–129.83 ng/ml, i.e. 0.08–0.27 M (Miller et al., 1988). We would like to stress that, although our present results restricted to the reduction of Pgp level in L1210 mouse leukemia cell line, the determination of the TFP effect in human MDR cell lines or in vivo remains to be critically evaluated.
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Acknowledgements We are grateful to Dr. M. Tien Kuo (University of Texas M.D. Anderson Cancer Center, Houston) for kindly providing -1288 RMICAT mdr1b promoter construct. This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG3-20900-0049). B.H. Choi was awarded by the 2nd stage Brain Korea 21 Project Scholarship (“Human Resources Development for Industrialization of Bionano Fusion Technology”) in 2006.
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Suppression of P-glycoprotein expression by antipsychotics trifluoperazine in adriamycin-resistant L1210 mouse leukemia cells Soon Young Shin a , Byeong Hyeok Choi a , Jae-Ryong Kim b , Jung-Hye Kim b , Young Han Lee a,∗ a
Division of Molecular and Life Science, Department of Bio-Nano Technology, College of Science and Technology, Hanyang University, Ansan 426-791, Republic of Korea b Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, 705-717, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history:
Multidrug resistance (MDR) to unrelated chemotherapeutic drugs can be mediated by over-
Received 1 August 2005
expression of P-glycoprotein (P-gp), the mdr gene product. Trifluoperazine (TFP), a phenoth-
Received in revised form 12 March
iazine derivative antipsychotics, is known to reverse MDR of tumor cell lines by blocking
2006
P-gp efflux function. In the present study, we evaluated the effect of TFP on the expression
Accepted 13 March 2006
of P-gp in multidrug-resistant L1210/Adr mouse leukemic cell lines, which are characterized
Published on line 17 March 2006
by overexpession of P-gp. We found that TFP induced the downregulation of P-gp protein and mdr1b mRNA in a dose- and time-dependent manner in L1210/Adr cells. TFP reduc-
Keywords:
tion of mdr1b mRNA was paralleled by transcriptional suppression of the mdr1b promoter.
Trifluoperazine
Moreover, TFP restored the adriamycin-induced apoptosis in L1210/Adr cells. These results
Multidrug resistance
suggest that TFP may have utility as an adjuvant in the therapy of leukemia for the reversal
P-glycoprotein
of P-gp-dependent MDR as well as for the management of psychological symptoms in the cancer patients. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Multidrug resistance (MDR) of tumor cells represents a development of clinical barrier to a wide range of structurally and functionally unrelated anti-cancer drugs (Gottesman and Pastan, 1993). Many mechanisms for the development of MDR have been described, including the up-regulation of membrane transporter, activation of pathway for detoxification of drugs, changes in DNA repair response and apoptotic responses to toxic drugs (Simon and Schindler, 1994). One of the most common and well-described mechanism in the development of MDR phenotype is the overexpression of Pglycoprotein (P-gp). P-gp is a transmembrane protein that acts
as an ATP-driven efflux pump for a wide variety of structurally and functionally unrelated agents from cytoplasm, thereby reducing the intracellular drug content to a sublethal level (Fardel et al., 1996; Ambudkar et al., 2003). Trifluoperazine (TFP) is a phenothiazine derivative which is widely used clinically to control psychotic disturbances, such as depression, agitation, anxiety, psychosis, and acute confusional state (Bruera and Nemann, 1998; Nordenberg et al., 1999). The therapeutic effects of TFP are attributed to the antagonism of dopamine D2 receptor and the inhibition of calmodulin (Nordenberg et al., 1999; Takano et al., 2003; Lahti et al., 1993). TFP, apart from their clinical use as a neuroleptics, has been shown to exert antiproliferative effects on many
∗ Corresponding author at: 1271 Sa-1-dong, Sangrok-gu, Hanyang University, Division of Molecular and Life Science, College of Science and Technology, Ansan 426-791, Republic of Korea. Tel.: +82 31 400 5517; fax: +82 31 416 9781. E-mail address: [email protected] (Y.H. Lee).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.03.002
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tumor cells (Osborn and Weber, 1980; Naftalovich et al., 1991; Bruera and Nemann, 1998; Nordenberg et al., 1999; Shin et al., 2004a). In addition, TFP has been shown to block the function of P-gp by modification of the membrane structure around the P-gp or by direct interaction with P-gp (Hendrich et al., 2001), and sensitizes MDR cells to chemotherapeutic agents (Ford et al., 1989; Ganapathi et al., 1991). It has been demonstrated that P-gp reversing agents, such as reserpine, verapamil and cyclosporine A, or bromocriptine, an ergot alkaloid known as a D2 dopaminergic receptor agonist, regulate P-gp gene expression (Sonneveld and Wiemer, 1997; Furuya et al., 1997). The aim of the present study was to determine whether MDR reversing agent TFP could modulate P-gp gene expression. We found that TFP decreased levels of mdr1b mRNA and its product P-gp protein in adriamycin-resistant mouse leukemia L1210 model system.
2.
Materials and methods
2.1.
Materials
Trifluoperazine was purchased from RBI (Natick, MA). [␣32 P]dCTP and [14 C]chloramphenicol were from NEN Life Science Products, Inc. (Boston, MA). Acetyl coenzyme A was from Sigma (St. Louis, MO). Rabbit polyclonal P-Glycoprotein antibody was obtained from Oncogene (San Diego, CA) and antiPARP, anti-NFB, and anti-IB antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
2.2.
Cell culture
L1210 mouse leukemia cell line was obtained from American Type Culture Collection (Rockville, MD). Multidrug resistant L1210 subline was obtained by long-term adaptation of parental L1210 cells to adriamycin (Kim et al., 1993). Additional details concerning cross-resistance responsible for MDR phenotype were described elsewhere (Kim et al., 1993; Kim and Kim, 1994; Kim, 1997). L1210 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone, Logan, UT). L1210/Adr cells were routinely maintained in a medium containing 0.2 g/ml adriamycin.
2.3.
Western blot analysis
Cells were lysed and protein samples (20 g of each) were separated and transferred to nitrocellulose filters as described previously (Shin et al., 2004a). The blots were incubated with anti-P-glycoprotein antibody (Oncogene, La Jolla, CA) and developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech., Piscataway, NJ). For PARP cleavage assay, an anti-PARP antibody that detects both the native 113-kDa and the cleaved 89-kDa fragment was utilized (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The same blot was stripped and reprobed with anti-Erk1/2 or Erk2 antibody for use as an internal control. For detection of NFB and IB, L1210 cells were separated into cytosol and nuclear-enriched fractions as described previously (Shin et al., 2004b).
2.4.
301
Northern blot analysis
Total RNA was prepared using STAT-60 reagent (TEL-TEST, Friendswood, TX) according to the manufacturer’s protocol. Ten micrograms of total RNA was separated on 1.2% agarose gel, transferred to Hybond N+ nylon membrane (Amersham Pharmacia Biotech., Piscataway, NJ), and hybridized with [␣-32 P]dCTP-labeled mdr1 cDNA probe, an insert of pCHP1 (Riordan et al., 1985). The membranes were then washed with 2× SSC/0.1% SDS for 20 min at room temperature, 2× SSC/0.1% SDS at 42 ◦ C for 30 min, and 0.5× SSC/0.1% SDS for 30 min at 65 ◦ C. [␣-32 P]dCTP-labeled gapdh cDNA was used as an internal control.
2.5. Generation of deletions of the rat mdr1b promoter constructs Deletions of the rat mdr1b promoter fragments spanning from the −1237 to +42, −740 to +42, −370 to +42, and −205 to +42 were synthesized by PCR in a reaction containing the 1288 RMICAT plasmid, a rat mdr1b-CAT reporter (Zhou et al., 1996) which was a kind gift of Dr. M. Tien Kuo (University of Texas M.D. Anderson Cancer Institute, Houston, TX;), as a template with the following upstream primers: −1237; 5 caGGTACCtacaaagggaagctgaagag-3 , −740; 5 -caGGTACCggactctgtctcaggttgac-3 , −370; 5 -caGGTACCgctgtcagaagggaacttta3 , and −205; 5 -caGGTACCagagttacctgaacatgtagagac-3 . A KpnI restriction site is indicated by uppercase letters. A single downstream primer (5 -cgTCTAGActcagcctcttacagcttca-3 ) containing a BglII site, which is indicated by uppercase letters, was used in each PCR amplification. The PCR fragments were then digested with the restriction enzymes KpnI and BglII. The fragments were extracted from the agarose gel and inserted into the KpnI and BglII sites of the pGL3-basic luciferaseencoding reporter vector (Promega, Madison, WI, USA). The resultant constructs were verified by DNA sequencing and by restriction enzyme digests.
2.6.
Transient transfection and reporter gene assay
For mdr1b promoter analysis, NIH3T3 cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum. One day after seeding cells into 35-mm dishes (6 × 105 cells), the cells were co-transfected with 0.5 g of 5 deletion constructs of the mdr1b promoter and 50 ng of pRL/null plasmid, which expresses Renilla luciferase (Promega) using the Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s instructions. Twenty-four hours post-transfection, cells were treated with TFP. After 6–8 h, the cell lysates were assayed for luciferase activity as described in the manufacturer’s protocol (Promega) using the TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Renilla luciferase activity was used to normalize the activity of the firefly luciferase.
2.7.
Determination of DNA fragmentation
Cells (5 × 106 ) were lysed in a solution containing 5 mM Tris, pH 7.4, 20 mM EDTA, 0.5% Triton X-100. The lysate was extracted twice with phenol/chloroform and chloroform. Cytosolic frag-
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mented DNA was precipitated with ethanol and subjected to gel electrophoresis on a 1.8% agarose gels. The gel was stained with ethidium bromide and photographed on a UV transilluminator.
3.
Results and discussion
Previously, adriamycin-resistant L1210 subline (L1210/Adr), which confers resistance to a variety of conventional anticancer drugs, was utilized and characterized (Kim and Kim, 1994). In L1210/Adr cells, P-gp protein was highly expressed, but not in parental L1210 cells, C6 glioma and NIH3T3 fibroblasts (Fig. 1A). MRP overexpression also confers an MDR phenotype similar to that of P-gp-dependent MDR (Grant et al., 1994), however, in L1210/Adr cells MRP expression was not detected as revealed by RT-PCR (data not shown) (Kim et al., 1993).
Fig. 1 – Adriamycin-resistant L1210/Adr cells are resistant to adriamycin-induced IB degradation. (A) Expression of P-gp in L1210/Ade cells. Total cell lysates (25 g) were separated on 6% SDS-PAGE gels and analyzed by immunoblot analysis with rabbit anti-P-Glycoprotein antibody (1:500). ␣-tubulin antibody was used as an internal loading control. (B) Resistance of L1210/Adr cells to adriamycin-induced IB degradation. Cells were treated with adriamycin (2 g/ml) for 6 h, and then cytosolic extracts and nuclear extracts were prepared. IB degradation or NFB accumulation was assessed by immunoblot analysis in the cytosolic (B) or in the nuclear (C) fractions, respectively. Results are representative of two independent experiments.
NFB is a transcription factor, which functions in the control of immunity, inflammation, cell survival and protection of apoptosis induced by variety of DNA-damaging agents that included adriamycin (Rayet and Gelinas, 1999). Previously, it has been demonstrated that NFB is involved in basal and insulin-induced mdr1b promoter activity in rat hepatoma cells (Zhou and Kuo, 1997). To determine whether aberrant activation of NFB is associated with accumulation of P-gp protein in L1210/Adr cells, we analyzed the activation of NFB induced by adriamycin. Nuclear and cytoplasmic extracts were prepared and examined the levels of NFB and IB, respectively. In most cells, NFB proteins are rendered inactive in the cytoplasm by inhibitory subunits of IB (Grilli et al., 1993). When the cells were treated with adriamycin, the abundance of cytoplasmic IB was reduced in a dose-dependent manner (Fig. 1B), resulting in the increase of the translocation of NFB into the nucleus (Fig. 1C) in parental L1210 cells, but did not in L1210/Adr cells. A possible explanation for the lack of NFB activation in L1210/Adr cells could be that an enhanced efflux of drugs due to overexpression of P-gp prevents intracellular accumulation of drugs, thereby conferring resistance to adriamycin. This result suggests that although NFB plays an essential role in the basal expression of mdr1 genes, maintenance of MDR phenotype in L1210/Adr subline seems to be NFB-independent. It has been demonstrated that P-gp expression can be regulated by some MDR reversing agents, including reserpine (dopamine reuptake inhibitor) and verapamil (calcim channel blocker) in human colon carcinoma cells (Bhat et al., 1995), and by bromocriptine (D2 dopamine receptor agonist) in H35 rat hepatoma cells (Furuya et al., 1997). Antipsychotics TFP is a well-known MDR reversing agent which interferes with efflux pump activity by modification of the membrane structure around the P-gp or by direct interaction with P-gp (Hendrich et al., 2001). To test if TFP could regulate P-gp expression, we treated parental L1210 and L1210/Adr cells with TFP, and measured the level of P-gp expression using Western blot analysis. The amount of P-gp protein was decreased by treatment with TFP in a concentration-dependent manner (Fig. 2A). To better define the effect of TFP on P-gp expression, a detailed time course analysis was performed. The result demonstrates that the expression level of P-gp was time-dependently reduced by treatment of 20 M TFP in L1210/Adr cells (Fig. 2B). The abundance of P-gp was reduced by approximately 40% and 71.3% after 24 h and 48 h, respectively, after TFP incubation. We also examined a low concentration-dependent response for the P-gp expression in L1210/Adr cells. P-gp protein level was decreased by approximately 25% and 50% in response to 5 days treatment of TFP with 1 and 5 M, respectively (Fig. 2C). These results suggest that TFP has the capacity to reduce the abundance of P-gp protein in L1210/Adr cells. To investigate whether the reduction of the P-gp level may result from a suppression of P-gp mRNA, Northern blot analysis was performed. The level of mdr1 mRNA was decreased in a concentration- (Fig. 3A) and time-dependent (Fig. 3B) manners. To determine whether the mdr1 gene is controlled at the transcriptional level by TFP, we generated a set of mdr1b promoter-luciferase constructs progressively truncated at the 5 end (−1237, −740, −370, and −205 bp) containing 42 bp downstream from the transcription start
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Fig. 2 – TFP decreases the accumulation of P-gp protein in adriamycin-resistant L1210/Adr cells. L1210 and L1210/Adr cells were treated with different concentrations of TFP for 24 h (A) or with 20 M TFP for different times (B). Total cell lysates (25 g) were separated on 6% SDS-PAGE gels and analyzed by immunoblot analysis with rabbit anti-P-Glycoprotein antibody (1:500). Erk1/2 antibody was used as an internal loading control. Results are representative of three independent experiments.
site, and transfected into NIH3T3 cells. Twenty-four hours after transfection, cells were treated without or with 10 M TFP. Adriamycin was included as a positive control. As shown in Fig. 4A, deletion of sequences up to −370 bp significantly
reduced the luciferase activity by TFP (p < 0.01 compared with the untreated control). Further deletion to −205 bp makedly reduced the induction of basal activity, suggesting that strong positive cis-element may be present between −370 and −205.
Fig. 3 – TFP suppresses the expression of mdr1b mRNA. Cells were treated with different concentrations of TFP for 24 h (A) or with 20 M TFP for different times (B). Total RNA (10 g) was isolated from cells, electrophoresed on 1% agarose-gel, and capillary transferred to a nylon filter. The blot was then sequentially hybridized with the 32 P-labeled mdr1 of pCHP1 insert and gapdh cDNA probes. Results are representative of two independent experiments. The relative band intensities of mdr1 mRNA were measured by quantitative scanning densitometer and image analysis software, Bio-1D version 97.04, and the ratio of mdr1 to gapdh intensity was determined.
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Fig. 4 – TFP represses mdr1b promoter activity. (A) NIH3T3 cells were transiently transfected for 24 h with 5 deletion constructs of the rat mdr1b promoter fused to the luciferase gene, and luciferase reporter activity was assayed construct using eletrophoration (260 V, 960 F). Transfected cells were treated with different concentrations of TFP (5–20 M) for 24 h. Prepared extracts were incubated with [14 C]chloramphenicol and acetyl-CoA and CAT activity measured by the production of acetylated chloramphenocol using thin layer chromatography (19:1 chloroform/methanol). The significance of each result was compared only with untreated control (CT) using Student’s-t test. *p < 0.01; **p < 0.001.
When −205/+42 construct was transfected, TFP treatment also yield a decreased promoter activity, albeit weakly but significantly (p < 0.001 compared with the untreated control). In addition, TFP significantly decreased the luciferase activities in a dose-dependent manner (5 and 10 M; p < 0.01, 20 M; p < 0.001, compared with the untreated control) in −205/+42 construct-ransfected cells (Fig. 4B). These results suggest that the proximal region of mdr1b gene between −205 and +42 sequences is sufficient to confer the TFP-mediated suppression of mdr1b promoter activity. Previous studies have demonstrated that the processing of poly(ADP-ribose)polymerase (PARP) from its native 113-kDa to an inactive 89-kDa fragment is a result of cleavage by caspase3 (Tewari et al., 1995). Thus, the profile of PARP cleavage has been used as a biochemical marker to assess the activation of
caspases and to confirm the induction of apoptosis (Kaufmann et al., 1993; Shah et al., 1996). To address whether the reduction of P-gp expression by TFP has the potential to sensitize L1210/Adr cells to chemotherapy, PARP cleavage was assessed by Western blotting, utilizing antibodies that detect both the native 113-kDa and the cleaved 89-kDa fragment. Typical processing of PARP into the 89-kDa fragment was observed by 24 h in adriamycin-treated parental L1210 cells, but not in L1210/Adr cells (Fig. 5A, lane 2 versus lane 7). However, when L1210/Adr cells were treated with adriamycin in the presence of TFP, PARP cleavage was restored in a TFP dose-dependent manner (ED50 = 8.5 ± 0.2 M). This TFP response was probably not due to cytotoxic effect for L1210/Adr cells because TFP treatment alone had no effect on the induction of PARP cleavage (Fig. 5A, lanes 3–6). DNA agarose electrophoresis
Fig. 5 – TFP reverses the resistance of L1210/Adr cells to adriamycin. (A) Cells were treated with adriamycin for 6 h and the cleavage of PARP was assessed by immunoblot analysis using anti-PARP antibody (1:1000). (B) L1210/Adr cells were treated with adriamycin in the absence or presence of TFP (20 M) for the indicated times, and the cleavage of PARP was assessed by immunoblot analysis. A 113-kDa PARP and 89-kDa of cleaved form are indicated with the arrow and arrow head, respectively. Results are representative of two independent experiments.
european journal of pharmaceutical sciences
also showed that TFP partially restored adriamycin-induced generation of DNA fragmentation in a dose-dependent manner in L1210/Adr cells (Fig. 5B). These results suggest that TFP-mediated suppression of P-gp expression could contribute to reverse the drug resistance phenotype of L1210/Adr cells. The development of MDR is a major limitation to successful anticancer chemotherapy. Although multiple mechanisms contribute to acquire MDR phenotype, overexpression of mdr1 gene and its product P-gp remains the most common mechanism underlying development of MDR (Gottesman et al., 2002). As the common strategy to reverse the MDR phenotype of tumor cells is associated with the inhibition of P-gp transporter function, prevention of mdr expression would also be necessary to prevent MDR. In the present study, we have found that antipsychotic agent TFP reverses the resistance of L1210/Adr cells to adriamycin. Given that TFP decreased the basal promoter activity of the mdr1b gene, it is likely that decreases in the amount of P-gp protein and mdr1 mRNA by TFP is most probably due to the suppression of mdr1 gene transcription. The promoter region of mdr1b contains several transcription factor binding sites for upregulation of mdr1b expression (Cohen et al., 1991; Zhou et al., 1996). The results presented in this study suggest that the promoter function and endogenous expression of the mdr1b gene can be modulated by TFP. Using a serial deletion construct of the mdr1b promoter, we suggest that the region between 205 bp upstream and 42 bp downstream of the transcription start site may contain cis-acting element, which is responsible for TFP-mediated transcriptional repression. The rat mdr1b promoter contains several regulatory elements, such as p53, NFB, C/EBP (also known as NF-IL6), and NF-Y, between −205 and +42. Among them, NFB may be not involved at least in TFP-mediated suppression of mdr1b gene expression, because IB degradation and NFB activation were not induced by adriamycin in L1210/Adr cells. It will be interesting to identify which of cis-acting element is an authentic TFP responsive element and to investigate the upstream signaling mechanism that modulates TFP-mediated suppression of mdr1b gene expression. In summary, we demonstrate here that antipsychotic agent TFP reduces the abundance of P-gp level probably through the negative regulation of mdr1b gene transcription. TFPmediated suppression of P-gp expression may be involved in an alternative mechanism of the reversion of the MDR phenotype in multidrug resistant variant L1210/Adr mouse leukemia cells. Although the detailed mechanism underlying how TFP regulates mdr1 gene expression has been remained, the present report provides the possibility that a combination of anticancer agents with TFP can efficiently control the P-gpmediated MDR via a mechanism involving the downregulation of mdr1 gene transcription, in addition to the inhibition of P-gp transporter activity. In humans, however, it has been shown that at the maximal tolerated doses (60–80 mg daily) the peak plasma concentrations of TFP are of 39.46–129.83 ng/ml, i.e. 0.08–0.27 M (Miller et al., 1988). We would like to stress that, although our present results restricted to the reduction of Pgp level in L1210 mouse leukemia cell line, the determination of the TFP effect in human MDR cell lines or in vivo remains to be critically evaluated.
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Acknowledgements We are grateful to Dr. M. Tien Kuo (University of Texas M.D. Anderson Cancer Center, Houston) for kindly providing -1288 RMICAT mdr1b promoter construct. This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG3-20900-0049). B.H. Choi was awarded by the 2nd stage Brain Korea 21 Project Scholarship (“Human Resources Development for Industrialization of Bionano Fusion Technology”) in 2006.
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