LFS-14265; No of Pages 10 Life Sciences xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
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Sayo Horibe a,b, Akira Matsuda a, Toshihito Tanahashi b,⁎, Jun Inoue b, Shoji Kawauchi b, Shigeto Mizuno b, Masaki Ueno a, Kyohei Takahashi a, Yusaku Maeda a, Tatsuya Maegouchi a, Yoshiki Murakami c, Ryoko Yumoto d, Junya Nagai d, Mikihisa Takano d
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Article history: Received 25 July 2014 Accepted 10 January 2015 Available online xxxx
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Keywords: Cisplatin Drug resistance Cross resistance A549 Cdc2 Cdc25C
Laboratory of Medicinal and Biochemical Analysis, Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Hiroshima 737-0112, Japan Department of Medical Pharmaceutics, Kobe Pharmaceutical University, Kobe 658-8558, Japan c Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan d Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan b
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Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins
a b s t r a c t
Aims: Cisplatin (CDDP) is a platinum-based drug that is widely used in cancer chemotherapy, but the development of resistance in tumor cells is a major weakness of these treatments. Several mechanisms have been proposed to explain cisplatin resistance, and disruption of certain cellular pathways could modulate drug sensitivity to cisplatin. A lower level of cross-resistance to cisplatin leads to better outcomes in clinical use. Main methods: Cross-resistance was assessed using cisplatin resistant lung cancer cell line A549/CDDP. Cell cycle analysis was used to examine the effect of cisplatin on cell signaling pathways regulating G2/M transition in cisplatin resistant cells. Key findings: A549/CDDP cells exhibited cross-resistance to carboplatin, but not oxaliplatin, which is often found in platinum analogues. Flow cytometry showed that nocodazole treatment caused a G2/M block in both A549/ CDDP cells and cisplatin susceptible cells. However, A549/CDDP cells escaped the G2/M block following exposure to cisplatin. Activation of the Cdc2/CyclinB complex is required for transition from G2 to M phase, and the inactive form of phosphorylated Cdc2 is activated by Cdc25C dephosphorylation of Tyr15. In the cisplatin-treated susceptible cells, the levels of phosphorylated Cdc2 and Cdc25C were markedly decreased, leading to a loss of Cdc2 activity and G2/M arrest. In A549/CDDP cells, however, Cdc2 activity was supported by the expression of Cdc2 and Cdc25C after the addition of cisplatin, which resulted in G2/M progression. Significance: The resistance phenotype of G2/M progression has been correlated with dysregulation of Cdc2 in a human lung cancer cell line selected for cisplatin. © 2015 Published by Elsevier Inc.
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addition, cross-resistance to other platinum compounds severely increases the difficulty of chemotherapy [13]. However, the molecular action underlying cisplatin resistance and the acquisition of crossresistance remain unclear. Upon entering a cell, cisplatin becomes positively charged, enabling it to interact with nucleophilic molecules including DNA, RNA and proteins [12]. Cytotoxicity is primarily due to the interaction with DNA forming inter- and intra-strand adducts that hinder both RNA transcription and DNA replication [16,17]. DNA damage resulting from cisplatin treatment induces p53 expression and cell cycle arrest at G2/M phase due to regulation of the expressions of cyclins and cyclin-dependent kinases [9]. It is thought that dysregulation of the cell cycle allows proliferation in the presence of cisplatin-induced DNA damage in the cisplatin-resistant cell line A549/CDDP, but scientific data are lacking [5]. In this study, we evaluated the cross-resistance of A549/CDDP cells to other platinum compounds, which are generally shared in platinum
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Introduction
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Cisplatin (CDDP) has been used as an effective and successful chemotherapeutic agent for various solid tumors, including testicular, ovarian, and non-small and small lung cancers since 1978 [1]. However, the high rate of tumor relapse and the failure of subsequent treatment with platinum agents due to drug resistance have become a major clinical problem. Resistance to cisplatin is generally characterized by a number of mechanisms, including reduced drug accumulation [6], increased detoxification by glutathione [8], and increased repair of cisplatin DNA adducts [20]. An understanding of the mechanism of drug resistance is critical for continued successful treatment of the resistant tumors. In
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⁎ Corresponding author at: Department of Medical Pharmaceutics, Kobe Pharmaceutical University, 4-19-1 Motoyama-kita, Higashinada-ku, Kobe 658-8558, Japan. Tel./fax: +81 78 441 7579. E-mail address: [email protected] (T. Tanahashi).
http://dx.doi.org/10.1016/j.lfs.2015.01.011 0024-3205/© 2015 Published by Elsevier Inc.
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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Cisplatin (CDDP), carboplatin (CBDCA), oxaliplatin (L-OHP), and nocodazole were purchased from Wako Pure Chemical Industries Ltd. (Osaka Japan). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate were purchased from Dojin Laboratories (Kumamoto, Japan).
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Cells and culture
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A549 human lung carcinoma cells were obtained from Riken Cell Bank (Ibaraki, Japan). A549 cells were cultured as previously described [5]. Briefly, the cells were maintained in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2 and 95% air.
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Cisplatin-resistant subline
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A cisplatin-resistant A549 cell subline (A549/CDDP) was established by culturing cells in DMEM containing 4 μM CDDP for 3 months [5]. Cisplatin-resistant A549 cells were grown exponentially in the presence of 4 μM CDDP.
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Cell viability
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Cell viability was assessed using the WST-1 assay [5]. In brief, cells (4 × 103 cells/well) suspended in DMEM (100 μl) were seeded into 96-well plates. At 24 h after seeding, the culture medium was aspirated and exchanged for fresh culture medium containing 0.06, 1, 4, 16, 64, and 256 μM of CDDP. After further incubation for 72 h, the medium was exchanged for 110 μL of medium containing WST-l reagent (10 μl WST-1 reagent and 100 μl DMEM). The cells were further incubated for 90 min, before absorbance was determined at 450 nm with a reference wavelength of 620 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The percentage of cell viability was calculated as follows:
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Viabilityð%Þ ¼ ðA450−A620Þtreated =ðA450−A620Þcontrol 100: 109
The half-maximal inhibitory concentration (IC50) value of CDDP was determined using the following equation:
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n V ¼ 100= 1 þ ð½I =IC50 Þ 112
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where [I] is the concentration of CDDP, V is the viability in the presence of CDDP, and n is the Hill coefficient. The IC50 value was assessed from a curve fitting of the equation using KaleidaGraph™ version 3.08 (Synergy Software Inc., Reading, PA). Relative resistance was indicated by the ratio of the IC50 value of A549/CDDP cells to that of A549 cells.
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Flow cytometry analysis
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Cell cycles were analyzed by flow cytometry as previously described [7]. In brief, cells (1 × 106 cells/well) suspended in DMEM with 10% FBS were seeded into 6-well plates and treated with 5.8 and 20 μM CDDP for up to 72 h. Following treatments of 24, 48, and 72 h, the cells were washed with PBS, fixed with 80% ethanol, and incubated overnight at
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Cell protein extracts
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Cells (5 × 106 cells/dish) suspended in DMEM with 10% FBS were seeded into 60 mm dishes for 24 h and then treated with 20 μM CDDP for up to 72 h. Following treatments of 24, 48, and 72 h, the cells were lysed with RIPA buffer (Wako), composed of 50 mM Tris–HCl (pH 7.4), 1% NP-40, 0.5% SDC, 0.1% SDS, 150 mM NaCl, and protease inhibitor cocktail (Nacalai tesque, Kyoto, Japan). In addition, cells (5 × 106 cells/dish) suspended in DMEM with 10% FBS and nocodazole (100 ng/ml) were seeded into 60 mm dishes, incubated for 24 h, and analyzed. The supernatants obtained after centrifugation at 15,000 g for 10 min at 4 °C were used as whole-cell extracts.
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Western blotting
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Materials and methods
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Total protein (4 or 20 μg) was boiled in a quarter-volume of sample buffer (1 M Tris–HCl [pH 7.4], 640 mM 2-mercaptoethanol, 0.2% bromophenol blue, 4% sodium dodecyl sulfate (SDS), and 20% glycerol) and separated on 7.5% to 12.5% SDS polyacrylamide gels. Proteins on the gels were transferred to polyvinylidene difluoride membranes, which were blocked with Blocking One (Nacalai tesque) overnight at 4 °C. Anti-Cdc2 rabbit polyclonal antibody (1:3000–5000; Cell Signaling, Beverly, MA), anti-phospho-Cdc2 (Tyr15) rabbit polyclonal antibody (1:3000–5000; Cell Signaling), anti-Cdc25C rabbit monoclonal antibody 5H9 (1:1000–3000; Cell Signaling), anti-phospho-Cdc25C (Ser216) rabbit monoclonal antibody 63F9 (1:1000–3000; Cell Signaling), and antiβ-actin mouse monoclonal antibody (1:2000–5000; Sigma-Aldrich, St. Louis, MO) were used as primary antibodies. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The secondary anti-rabbit IgG (Cell Signaling) and anti-mouse IgG antibodies (SigmaAldrich) were diluted to 1:5000–20,000. Protein/antibody complexes were visualized with Chemi-Lumi One Super (Nacalai tesque) and detected using an Image Quant LAS 4000 (GE Healthcare, Piscataway, NJ). The intensities of the blots were quantified using Image Quant TL software (GE Healthcare).
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Results
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80 °C. Cells were stained in PBS containing 1 mg/ml propidium iodide (PI) for 30 min at room temperature. Cell counts were analyzed with a Flow Cytometer (FACSCalibur; BD). In addition, cells (1 × 106 cells/ well) suspended in DMEM with 10% FBS and nocodazole (100 ng/ml) were seeded into 6-well plates, incubated for 24 h, and analyzed. Data were analyzed using ModFit LT for Mac VER 3.0 (Verity Software House).
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analogues. Furthermore, we assessed the effect of cisplatin on the cell cycle and the expressions of cell cycle associated proteins in susceptible and resistant A549 cells, and analyzed their functional roles in the G2/M transition.
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Effect of cisplatin (CDDP), carboplatin (CBDCA), and oxaliplatin (L-OHP) 164 on the viability of A549 and A549/CDDP cells 165 We examined whether cisplatin-selected A549/CDDP cells had also acquired cross-resistance to the other chemotherapeutic drugs, especially platinum. Fig. 1 shows the dose–response curve of A549 and A549/CDDP cells to CDDP, CBDCA, and L-OHP determined by WST-1 assay. Following treatment with 0.06, 1, 4, 16, 64, and 256 μM CDDP for 72 h, IC50 of A549/CDDP cells was 18.6 ± 1.2 μM (Table 1), which was significantly higher (3.67-fold) than that of untreated A549 cells (5.8 ± 0.6 μM). These values were consistent with those described in our previous report (16.8 μM for resistant and 4.2 μM for susceptible cells). A549/CDDP cells also exhibited low level resistance to CBDCA, for which the IC50 value was significantly higher (1.90-fold) than that of A549 cells alone (240 ± 45.7 μM versus 126 ± 5.0 μM for susceptible cells). However, exposure to L-OHP did not produce a significant difference in IC50 values between A549 and its resistant cell line (19.8 ± 1.8 and 28.3 ± 5.8 μM). Also, treatment of L-OHP did not result in a
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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viability (% of control)
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Fig. 1. Effect of cisplatin (CDDP), carboplatin (CBDCA), and oxaliplatin (L-OHP) on the viability of A549 and A549/CDDP cells. At 24 h after the plating of A549 (open circle) and A549/CDDP (closed triangle) cells at a density of 4 × 103 cells/well into 96-well plates, the cells were treated with various concentrations of CDDP (0.06, 1, 4, 16, 64, and 256 μM), CBDCA (0.2, 15.6, 62.5, 250, 1000, and 4000 μM), and L-OHP (0.06, 0.2, 3.9, 15.6, 62.5, 250, and 1000 μM). Cell viability was determined by WST-1 assay as described in the Materials and methods. Each symbol represents the mean ± SE of three or four experiments.
significant difference in the cell viability of A549 and its resistant cell line. These results indicate that cisplatin-resistant A549 cells are crossresistant to CBDCA, but not L-OHP.
Effect of cisplatin (CDDP) on the cell cycle of A549/CDDP cells
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Cell cycle distribution was examined with flow cytometry (Fig. 2). Based on the IC50 of A549 and A549/CDDP cells, CDDP concentrations of 5.8 and 20 μM were chosen for further experiments. After treatment
t1:1 t1:2
Table 1 IC50 values for A549 and A549/CDDP cells.
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Drug Cisplatin Carboplatin Oxaliplatin
A549 IC50 (μM)
A549/CDDP IC50 (μM)
RR
5.8 ± 0.6 126 ± 5.0 19.8 ± 1.8
18.6 ± 1.2⁎ 240 ± 45.7⁎ 28.3 ± 5.8
3.67 1.90 1.43
RR: Relative resistance is indicated by the ratio of the IC50 value of A549/CDDP cells to that of A549 cells. ⁎ Significant difference between A549 and A549/CDDP cells (P b 0.05).
with 5.8 μM CDDP, the percentage of susceptible A549 cells in G2/M was 23.3% at 24 h, and this level persisted for 72 h (31.0%) (Fig. 2A). Similarly, following the treatment with 20 μM CDDP up to 24, 48, and 72 h, the percentage of G2/M phase had remarkably increased to 71.1% at 48 h. The cell shift to G2/M phase had reached 76.2% at 72 h. However, the percentage of G2/M phase cells was lower among A549/CDDP cells (30.7%) than in susceptible cells at 48 h (71.1%) following treatment with 20 μM CDDP. After 72 h, the percentage was further decreased to be 16.5% (Fig. 2B). These results suggest that A549/CDDP cells have an escape response from G2/M arrest after CDDP-induced DNA damage.
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Effect of nocodazole on the cell cycle of A549/CDDP cells
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Nocodazole, a typical microtubule inhibitor, induced G2/M arrest. The cell cycle shift was assessed for 6, 12, and 24 h following nocodazole treatment (Fig. 3). Susceptible A549 cells were blocked at G2/M phase by nocodazole at 24 h (90.5%) (Fig. 3A). Although they progressed through G2/M phase after CDDP treatment, nocodazole-treated A549/ CDDP cells were arrested in G2/M at 24 h (79.7%) (Fig. 3B). These results suggest that A549/CDDP cells have a function in the G2/M transition, which was blocked by treatment with nocodazole, but not CDDP.
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Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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A A549 72 h
Percentage of cell cycle phase
NT
G0/G1 S G2/M
CDDP (5.8 M) 24 h 48 h 72 h 31.4 ± 2.0 49.9 ± 0.1 62.6± 0.5 45.3 ± 2.1 10.6 ± 0.1 6.4 ± 0.2 23.3 ± 1.9 39.6 ± 0.1 31.0 ± 0.6
G0/G1 S G2/M
CDDP (20 M) 24 h 48 h 72 h 34.5 ± 0.0 11.0 ± 0.8 12.7 ± 1.0 65.2 ± 0.2 17.9 ± 0.7 11.1 ± 0.8 0.3 ± 0.3 71.1 ± 0.7 76.2 ± 1.1
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Percentage of cell cycle phase
G0/G1 S G2/M
NT 24 h 48 h 63.0 ± 0.8 65.8 ± 0.2 22.8 ± 1.5 27.3 ± 0.3 14.2 ± 0.8 7.5 ± 0.4
G0/G1 S G2/M
CDDP (5.8 M) 24 h 48 h 72 h 48.3 ± 0.8 65.3 ± 0.4 81.5 ± 1.0 34.7 ± 1.1 21.5 ± 0.6 10.8 ± 0.4 17.0 ± 0.5 13.2 ± 0.6 7.7 ± 0.6
G0/G1 S G2/M
CDDP (20 M) 24 h 48 h 72 h 34.9 ± 1.0 46.7 ± 0.2 71.8 ± 0.7 45.6 ± 1.5 22.6 ± 0.7 11.7 ± 0.7 19.5 ± 1.0 30.7 ± 0.9 16.5 ± 0.7
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CDDP (20 μM)
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G0/G1 S G2/M
NT 24 h 48 h 56.2 ± 0.2 64.9 ± 2.8 32.7 ± 0.4 33.5 ± 3.5 11.1 ± 0.2 1.6 ± 1.6
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72 h 78.9 ± 1.3 13.4 ± 1.3 7.2 ± 0.1
Fig. 2. Effect of cisplatin on the cell cycles of A549 and A549/CDDP cells. After seeding A549 (A) and A549/CDDP (B) cells at a density of 4 × 106 cells/well, the cells were pre-cultured for 24 h. Cells were then treated with 5.8 and 20 μM CDDP for up to 72 h. After treatments of 24, 48, and 72 h, DNA content was assessed by flow cytometry, and cell cycle distribution was analyzed using ModFit LT software. In the left panel, a representative cell cycle distribution is shown. In the right panel, the percentage of each cell cycle is given as the mean ± standard deviation of three experiments.
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Effect of nocodazole on the expression of cell cycle-associated proteins in A549/CDDP cells
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The expression of cell cycle-associated proteins was examined to characterize the molecular mechanism of G2/M arrest in A549 and A549/
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CDDP cells (Fig. 4). Cdc2 cyclin dependent kinase (also known as Cdk1) is essential for entry into mitosis. Cells remain arrested in G2/M phase when CyclinB1 forms a complex with Cdc2 that is inactive upon Tyr15 and Thr14 phosphorylation [15]. In contrast, progression through G2/M occurs after dephosphorylation of Cdc2 by Cdc25C phosphatase [4].
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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57.9 ± 0.4 35.2 ± 0.4 6.7 ± 0.1
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24 h 58.0 ± 0.4 32.0 ± 0.2 10.0 ± 0.2
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43.4 ± 1.6 39.9 ± 1.3 16.8 ± 0.3
22.1 ± 0.8 35.0 ± 0.8 42.9 ± 0.4
3.6 ± 0.3 5.9 ± 0.2 90.5 ± 0.4
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G2/M
A549
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60.7 ± 1.8 29.6 ± 0.1 9.7 ± 0.7
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A549/CDDP
NT
0h
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12 h
24 h
G0/G1
62.3 ± 0.7 28.9 ± 0.3 8.9 ± 0.6
56.2 ± 0.3 28.6 ± 0.3 15.2 ± 0.1
62.1 ± 0.3 25.1 ± 0.3 12.8 ± 0.1
61.0 ± 0.3 27.1 ± 0.1 11.9 ± 0.2
S G2/M Noc
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G0/G1 S
43.0 ± 0.5 25.9 ± 0.2 31.0 ± 0.5
29.6 ± 0.3 22.1 ± 0.5 48.3 ± 0.7
15.2 ± 0.1 5.1 ± 0.6
G2/M
79.7 ± 0.6
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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Fig. 4. Effect of nocodazole on the expression of cell cycle-associated proteins in A549 and A549/CDDP cells. At 24 h after seeding A549 and A549/CDDP cells at a density of 5 × 106 cells/dish into 60 mm dishes, the cells were treated with nocodazole (100 ng/ml). Whole cell extracts were then prepared from samples at 3, 6, 12, and 24 h. The expression levels of Cdc2, p-Cdc2, Cdc25C, and p-Cdc25C in these cells were measured by Western blotting using β-actin as a loading control (A). Fold changes in protein expressions between A549 (white bar) and A549/ CDDP cells (black bar) are shown (B).
Fig. 3. Effect of nocodazole on cell cycles of A549 and A549/CDDP cells. After A549 (A) and A549/CDDP (B) cells were seeded at a density of 4 × 106 cells/well, the cells were incubated up to 24 h and treated with nocodazole (100 ng/ml). After treatments of 6, 12, and 24 h, DNA content was assessed by flow cytometry, and cell cycle distribution was analyzed by ModFit LT software. Each panel (A and B) contains a representative cell cycle distribution, and a table listing the percentage of each cell cycle, shown as the mean ± standard deviation of three experiments.
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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CDDP (20 μM)
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Fig. 5. Effect of CDDP on the expression of cell cycle-associated proteins in A549/CDDP cells. At 24 h after seeding A549 and A549/CDDP cells at a density of 5 × 106 cells/dish into 60 mm dishes, the cells were treated with 20 μM CDDP for up to 72 h. Whole cell extracts were then prepared at 24, 48, and 72 h. The expression levels of Cdc2, p-Cdc2, Cdc25C, and p-Cdc25C proteins in these cells were measured by Western blotting using β-actin as a loading control. Two different blotting membranes were used. Results of Cdc2 and p-Cdc2 blots are shown in the upper frame and Cdc25C and p-Cdc25C blots are in the lower panel (A). Fold changes in protein expressions between A549 (white bar) and A549/CDDP cells (black bar) are shown (B).
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
8
Cdc2 was consistent with a previous report for MCF-7, a human breast cancer cell line [2]. Similarly, in A549/CDDP cells, Cdc2 expression was promoted for up to 12 h but was reduced 24 h following exposure to nocodazole. In both A549 and A549/CDDP cells, the expression of phosphorylated Cdc2 was induced within 12 h, and repressed at 24 h. These results indicated that nocodazole lowered Cdc2 expression, although
A CBDCA (240 μM) A549 48
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We assessed the effect of nocodazole on the expression of cell cycleassociated proteins in susceptible and resistant A549 cells (Fig. 4A and B). Before nocodazole treatment (0 h), susceptible A549 cells have a lower level of Cdc2 expression. After treatment with nocodazole, Cdc2 expression increased to maximum levels at 12 h and had slightly decreased at 24 h. In susceptible A549 cells, the induction level of
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Fig. 6. Effect of CBDCA on the expression of cell cycle-associated proteins in A549/CDDP cells. At 24 h after seeding A549 and A549/CDDP cells at a density of 5 × 106 cells/dish into 60 mm dishes, the cells were treated with 240 μM CBDCA up to 72 h. Whole cell extracts were then prepared at 24, 48, and 72 h. The expression levels of Cdc2, p-Cdc2, Cdc25C, and p-Cdc25C proteins in these cells were measured by Western blotting using β-actin as a loading control (A). Fold changes in protein expressions between A549 (white bar) and A549/CDDP cells (black bar) are shown (B).
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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Since A549/CDP cells showed cross-resistance to CBDCA, the expression of Cdc2, Cdc25C, and their phosphorylated forms was assessed after treatment with 240 μM CBDCA (Fig. 6). In A549 cells, the treatment increased Cdc2 expression up to 48 h and was decreased at 72 h. However, Cdc2 expression in A549/CDDP cells peaked at two-fold higher than A549 cells at 48 h and remained higher for up to 72 h. In addition, Cdc25C and its phosphorylated form were induced in A549/CDDP cells after exposure to CBDCA, and peaked at 48 h. As with CDDP treatment, the active form of Cdc2 was induced by Cdc25C phosphatase for up to 72 h following CBDCA treatment. In addition, CBDCA treatment led to a weak expression pattern of cell cycle-associated proteins, which was similar to that observed after treatment with CDDP.
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Cisplatin-based chemotherapy is effective in the treatment of advanced lung cancer with a survival benefit of 5 years [1]. Unfortunately, some patients gradually have showed the tumor relapse and become resistant to the drug [19]. Two major strategies have been undertaken to improve the efficacy of cisplatin-based therapy. One is the development of platinum analogues with better therapeutic effects. However, cisplatin resistant cells are often cross-resistant to other platinum compounds. Carboplatin (CBDCA) has fewer side effects with an anti-tumor range similar to that of cisplatin, but shows cross-resistance to cisplatin [14]. In contrast, oxaliplatin (L-OHP) exhibits anti-tumor activity against cisplatin-resistant cells and lacks cross-resistance [13,18]. The results of this study using A549/CDDP cells were consistent with the previous findings that oxaliplatin is active, lacks cross-resistance and had a better toxicity profile. These results have made L-OHP as one of the leading treatment options for unresponsive cisplatin-based chemotherapy in human lung cancer. The other strategy is to understand the abnormal mechanisms of cisplatin resistance. Previous examinations of the effects of cisplatin on the cell cycle progression showed that the drug reduces the rates of DNA synthesis [16,17] with a subsequent slowdown of S-phase [11] followed by G2/M arrest [10,11]. In cisplatin-treated cells, G2/M arrest is induced by the inhibition of Cdc2/CyclinB activity, a key regulator of the G2/M transition [10]. The Cdc2/CyclinB complex is activated by dephosphorylation of Tyr15 of Cdc2 by Cdc25 phosphatase, which induces M-phase
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CBDCA, but not L-OHP exhibited cross-resistance to cisplatin in A549/CDDP cells. Under treatment with cisplatin, A549/CDDP cells showed enhanced modification of cell cycle associated proteins, which resulted in G2/M progression. These findings represent a partial molecular explanation of the development of drug resistance in lung cancer cells and their subsequent survival under treatment with cisplatin.
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Conflict of interest statement
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Next, we examined the effect of CDDP on cell cycle-associated proteins (Fig. 5). Treatment of A549 cells with 20 μM CDDP led to a gradual increase in Cdc2 expression for up to 48 h, before a marked decrease was observed at 72 h (Fig. 5A). However, in A549/CDDP cells, expression of Cdc2 was increased and not inhibited at 72 h. In parallel, expression of inactive Cdc2, which was phosphorylated at Tyr15, was increased in A549/CDDP cells, but markedly decreased in A549 cells. With the treatment of CDDP in susceptible A549 cells, the expression of Cdc25C increased for 48 h, but had remarkably decreased by 72 h (Fig. 5B). In A549/CDDP cells, however, Cdc25C expression persisted 72 h after CDDP exposure, and expression of phosphorylated Cdc25C peaked at 24 h. Taken together, these results suggest that the inhibitory form of tyrosine phosphorylated Cdc2 was dephosphorylated to an active state by Cdc25C phosphatase for up to 72 h following CDDP treatment in A549/CDDP cells, but not in A549 cells.
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entry. This study showed that cisplatin certainly inhibits the activity of Cdc2 in susceptible A549 cells and induces G2/M arrest with the treatment of 5.8 and 20 μM CDDP. In addition, we hypothesize that Cdc2 activity may be involved in late events of the escape from G2/M block in cisplatin-resistant cells, and found that Cdc2 expression and its activation by Cdc25C are maintained in A549/CDDP cells. The observed Cdc2 activity appeared to be a unique cellular response to cisplatin in resistant lung cancer cells. It is now widely accepted that the G2/M transition is governed by the Cdc2/CyclinB complex. G2/M arrest associated with CyclinB repression has also been reported following treatment with adriamycin, etoposide, and irinotecan [3], suggesting that CyclinB repression is involved in the G2/M arrest mechanism induced by anti-cancer drugs. To extend the previous findings, our results indicate that repression of Cdc2 is a factor in G2/M arrest during cisplatin treatment. Cross resistance to cisplatin and CBDCA remains poorly understood, but is thought to be related to intrinsic resistance mechanisms; including non-specific inactivation and efflux at the cytoplasmic level, and specific DNA adduct repair mechanisms at the nuclear level [13]. CBDCA treatment weakened expressions of Cdc2, Cdc25C and their phosphorylated forms, but the pattern was similar to that observed upon CDDP treatment. The results suggest that these two drugs affect cell cycle associated proteins in a similar fashion, but the protein expression patterns were not identical. Further studies are required to clarify the actual regulation of cell cycle associated proteins under CBDCA exposure. There was a significant difference in the response to nocodazole in A549/CDDP cells. Nocodazole treatment certainly caused a G2/M block in both cisplatin resistant and susceptible cells. Although the cellular response to nocodazole was normally regulated in A549/CDDP cells, cisplatin treatment triggered dysregulation of G2/M associated proteins, which proceeded through the G2/M block. This paradoxical change is likely due to aberrant cellular signaling in cisplatin resistant cells. It has been proposed that G2/M arrest is essential to the process of engaging cell death following cisplatin treatment [17]. We suggest that G2/M associated proteins play a role in determining the pharmacological properties of cisplatin resistant cell lines.
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the majority of Cdc2 was in a phosphorylated that was inactive form in both CDDP susceptible and resistant A549 cells. Similarly, there was no distinct difference in the expression of Cdc25C and its phosphorylated form following nocodazole treatment.
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All authors have no competing interest.
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This study was supported by a Grant-in-Aid (no. 26460212) to TT and a Grant-in-Aid for Young Scientists (B) (no. 25860568) to JI from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. TT and YM were financially supported by KOSEI-KAKENHI (H25-B sou-Kan-en-general-018). TT was also supported by a Grantin-Aid from Hyogo Science and Technology Association.
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Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
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Sayo Horibe a,b, Akira Matsuda a, Toshihito Tanahashi b,⁎, Jun Inoue b, Shoji Kawauchi b, Shigeto Mizuno b, Masaki Ueno a, Kyohei Takahashi a, Yusaku Maeda a, Tatsuya Maegouchi a, Yoshiki Murakami c, Ryoko Yumoto d, Junya Nagai d, Mikihisa Takano d
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Article history: Received 25 July 2014 Accepted 10 January 2015 Available online xxxx
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Keywords: Cisplatin Drug resistance Cross resistance A549 Cdc2 Cdc25C
Laboratory of Medicinal and Biochemical Analysis, Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Hiroshima 737-0112, Japan Department of Medical Pharmaceutics, Kobe Pharmaceutical University, Kobe 658-8558, Japan c Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan d Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan b
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Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins
a b s t r a c t
Aims: Cisplatin (CDDP) is a platinum-based drug that is widely used in cancer chemotherapy, but the development of resistance in tumor cells is a major weakness of these treatments. Several mechanisms have been proposed to explain cisplatin resistance, and disruption of certain cellular pathways could modulate drug sensitivity to cisplatin. A lower level of cross-resistance to cisplatin leads to better outcomes in clinical use. Main methods: Cross-resistance was assessed using cisplatin resistant lung cancer cell line A549/CDDP. Cell cycle analysis was used to examine the effect of cisplatin on cell signaling pathways regulating G2/M transition in cisplatin resistant cells. Key findings: A549/CDDP cells exhibited cross-resistance to carboplatin, but not oxaliplatin, which is often found in platinum analogues. Flow cytometry showed that nocodazole treatment caused a G2/M block in both A549/ CDDP cells and cisplatin susceptible cells. However, A549/CDDP cells escaped the G2/M block following exposure to cisplatin. Activation of the Cdc2/CyclinB complex is required for transition from G2 to M phase, and the inactive form of phosphorylated Cdc2 is activated by Cdc25C dephosphorylation of Tyr15. In the cisplatin-treated susceptible cells, the levels of phosphorylated Cdc2 and Cdc25C were markedly decreased, leading to a loss of Cdc2 activity and G2/M arrest. In A549/CDDP cells, however, Cdc2 activity was supported by the expression of Cdc2 and Cdc25C after the addition of cisplatin, which resulted in G2/M progression. Significance: The resistance phenotype of G2/M progression has been correlated with dysregulation of Cdc2 in a human lung cancer cell line selected for cisplatin. © 2015 Published by Elsevier Inc.
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addition, cross-resistance to other platinum compounds severely increases the difficulty of chemotherapy [13]. However, the molecular action underlying cisplatin resistance and the acquisition of crossresistance remain unclear. Upon entering a cell, cisplatin becomes positively charged, enabling it to interact with nucleophilic molecules including DNA, RNA and proteins [12]. Cytotoxicity is primarily due to the interaction with DNA forming inter- and intra-strand adducts that hinder both RNA transcription and DNA replication [16,17]. DNA damage resulting from cisplatin treatment induces p53 expression and cell cycle arrest at G2/M phase due to regulation of the expressions of cyclins and cyclin-dependent kinases [9]. It is thought that dysregulation of the cell cycle allows proliferation in the presence of cisplatin-induced DNA damage in the cisplatin-resistant cell line A549/CDDP, but scientific data are lacking [5]. In this study, we evaluated the cross-resistance of A549/CDDP cells to other platinum compounds, which are generally shared in platinum
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Introduction
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Cisplatin (CDDP) has been used as an effective and successful chemotherapeutic agent for various solid tumors, including testicular, ovarian, and non-small and small lung cancers since 1978 [1]. However, the high rate of tumor relapse and the failure of subsequent treatment with platinum agents due to drug resistance have become a major clinical problem. Resistance to cisplatin is generally characterized by a number of mechanisms, including reduced drug accumulation [6], increased detoxification by glutathione [8], and increased repair of cisplatin DNA adducts [20]. An understanding of the mechanism of drug resistance is critical for continued successful treatment of the resistant tumors. In
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⁎ Corresponding author at: Department of Medical Pharmaceutics, Kobe Pharmaceutical University, 4-19-1 Motoyama-kita, Higashinada-ku, Kobe 658-8558, Japan. Tel./fax: +81 78 441 7579. E-mail address: [email protected] (T. Tanahashi).
http://dx.doi.org/10.1016/j.lfs.2015.01.011 0024-3205/© 2015 Published by Elsevier Inc.
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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Cisplatin (CDDP), carboplatin (CBDCA), oxaliplatin (L-OHP), and nocodazole were purchased from Wako Pure Chemical Industries Ltd. (Osaka Japan). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate were purchased from Dojin Laboratories (Kumamoto, Japan).
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A549 human lung carcinoma cells were obtained from Riken Cell Bank (Ibaraki, Japan). A549 cells were cultured as previously described [5]. Briefly, the cells were maintained in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2 and 95% air.
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A cisplatin-resistant A549 cell subline (A549/CDDP) was established by culturing cells in DMEM containing 4 μM CDDP for 3 months [5]. Cisplatin-resistant A549 cells were grown exponentially in the presence of 4 μM CDDP.
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Cell viability was assessed using the WST-1 assay [5]. In brief, cells (4 × 103 cells/well) suspended in DMEM (100 μl) were seeded into 96-well plates. At 24 h after seeding, the culture medium was aspirated and exchanged for fresh culture medium containing 0.06, 1, 4, 16, 64, and 256 μM of CDDP. After further incubation for 72 h, the medium was exchanged for 110 μL of medium containing WST-l reagent (10 μl WST-1 reagent and 100 μl DMEM). The cells were further incubated for 90 min, before absorbance was determined at 450 nm with a reference wavelength of 620 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The percentage of cell viability was calculated as follows:
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where [I] is the concentration of CDDP, V is the viability in the presence of CDDP, and n is the Hill coefficient. The IC50 value was assessed from a curve fitting of the equation using KaleidaGraph™ version 3.08 (Synergy Software Inc., Reading, PA). Relative resistance was indicated by the ratio of the IC50 value of A549/CDDP cells to that of A549 cells.
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Flow cytometry analysis
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Cell cycles were analyzed by flow cytometry as previously described [7]. In brief, cells (1 × 106 cells/well) suspended in DMEM with 10% FBS were seeded into 6-well plates and treated with 5.8 and 20 μM CDDP for up to 72 h. Following treatments of 24, 48, and 72 h, the cells were washed with PBS, fixed with 80% ethanol, and incubated overnight at
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Cell protein extracts
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Cells (5 × 106 cells/dish) suspended in DMEM with 10% FBS were seeded into 60 mm dishes for 24 h and then treated with 20 μM CDDP for up to 72 h. Following treatments of 24, 48, and 72 h, the cells were lysed with RIPA buffer (Wako), composed of 50 mM Tris–HCl (pH 7.4), 1% NP-40, 0.5% SDC, 0.1% SDS, 150 mM NaCl, and protease inhibitor cocktail (Nacalai tesque, Kyoto, Japan). In addition, cells (5 × 106 cells/dish) suspended in DMEM with 10% FBS and nocodazole (100 ng/ml) were seeded into 60 mm dishes, incubated for 24 h, and analyzed. The supernatants obtained after centrifugation at 15,000 g for 10 min at 4 °C were used as whole-cell extracts.
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Total protein (4 or 20 μg) was boiled in a quarter-volume of sample buffer (1 M Tris–HCl [pH 7.4], 640 mM 2-mercaptoethanol, 0.2% bromophenol blue, 4% sodium dodecyl sulfate (SDS), and 20% glycerol) and separated on 7.5% to 12.5% SDS polyacrylamide gels. Proteins on the gels were transferred to polyvinylidene difluoride membranes, which were blocked with Blocking One (Nacalai tesque) overnight at 4 °C. Anti-Cdc2 rabbit polyclonal antibody (1:3000–5000; Cell Signaling, Beverly, MA), anti-phospho-Cdc2 (Tyr15) rabbit polyclonal antibody (1:3000–5000; Cell Signaling), anti-Cdc25C rabbit monoclonal antibody 5H9 (1:1000–3000; Cell Signaling), anti-phospho-Cdc25C (Ser216) rabbit monoclonal antibody 63F9 (1:1000–3000; Cell Signaling), and antiβ-actin mouse monoclonal antibody (1:2000–5000; Sigma-Aldrich, St. Louis, MO) were used as primary antibodies. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The secondary anti-rabbit IgG (Cell Signaling) and anti-mouse IgG antibodies (SigmaAldrich) were diluted to 1:5000–20,000. Protein/antibody complexes were visualized with Chemi-Lumi One Super (Nacalai tesque) and detected using an Image Quant LAS 4000 (GE Healthcare, Piscataway, NJ). The intensities of the blots were quantified using Image Quant TL software (GE Healthcare).
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80 °C. Cells were stained in PBS containing 1 mg/ml propidium iodide (PI) for 30 min at room temperature. Cell counts were analyzed with a Flow Cytometer (FACSCalibur; BD). In addition, cells (1 × 106 cells/ well) suspended in DMEM with 10% FBS and nocodazole (100 ng/ml) were seeded into 6-well plates, incubated for 24 h, and analyzed. Data were analyzed using ModFit LT for Mac VER 3.0 (Verity Software House).
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analogues. Furthermore, we assessed the effect of cisplatin on the cell cycle and the expressions of cell cycle associated proteins in susceptible and resistant A549 cells, and analyzed their functional roles in the G2/M transition.
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Effect of cisplatin (CDDP), carboplatin (CBDCA), and oxaliplatin (L-OHP) 164 on the viability of A549 and A549/CDDP cells 165 We examined whether cisplatin-selected A549/CDDP cells had also acquired cross-resistance to the other chemotherapeutic drugs, especially platinum. Fig. 1 shows the dose–response curve of A549 and A549/CDDP cells to CDDP, CBDCA, and L-OHP determined by WST-1 assay. Following treatment with 0.06, 1, 4, 16, 64, and 256 μM CDDP for 72 h, IC50 of A549/CDDP cells was 18.6 ± 1.2 μM (Table 1), which was significantly higher (3.67-fold) than that of untreated A549 cells (5.8 ± 0.6 μM). These values were consistent with those described in our previous report (16.8 μM for resistant and 4.2 μM for susceptible cells). A549/CDDP cells also exhibited low level resistance to CBDCA, for which the IC50 value was significantly higher (1.90-fold) than that of A549 cells alone (240 ± 45.7 μM versus 126 ± 5.0 μM for susceptible cells). However, exposure to L-OHP did not produce a significant difference in IC50 values between A549 and its resistant cell line (19.8 ± 1.8 and 28.3 ± 5.8 μM). Also, treatment of L-OHP did not result in a
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Fig. 1. Effect of cisplatin (CDDP), carboplatin (CBDCA), and oxaliplatin (L-OHP) on the viability of A549 and A549/CDDP cells. At 24 h after the plating of A549 (open circle) and A549/CDDP (closed triangle) cells at a density of 4 × 103 cells/well into 96-well plates, the cells were treated with various concentrations of CDDP (0.06, 1, 4, 16, 64, and 256 μM), CBDCA (0.2, 15.6, 62.5, 250, 1000, and 4000 μM), and L-OHP (0.06, 0.2, 3.9, 15.6, 62.5, 250, and 1000 μM). Cell viability was determined by WST-1 assay as described in the Materials and methods. Each symbol represents the mean ± SE of three or four experiments.
significant difference in the cell viability of A549 and its resistant cell line. These results indicate that cisplatin-resistant A549 cells are crossresistant to CBDCA, but not L-OHP.
Effect of cisplatin (CDDP) on the cell cycle of A549/CDDP cells
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Cell cycle distribution was examined with flow cytometry (Fig. 2). Based on the IC50 of A549 and A549/CDDP cells, CDDP concentrations of 5.8 and 20 μM were chosen for further experiments. After treatment
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3.67 1.90 1.43
RR: Relative resistance is indicated by the ratio of the IC50 value of A549/CDDP cells to that of A549 cells. ⁎ Significant difference between A549 and A549/CDDP cells (P b 0.05).
with 5.8 μM CDDP, the percentage of susceptible A549 cells in G2/M was 23.3% at 24 h, and this level persisted for 72 h (31.0%) (Fig. 2A). Similarly, following the treatment with 20 μM CDDP up to 24, 48, and 72 h, the percentage of G2/M phase had remarkably increased to 71.1% at 48 h. The cell shift to G2/M phase had reached 76.2% at 72 h. However, the percentage of G2/M phase cells was lower among A549/CDDP cells (30.7%) than in susceptible cells at 48 h (71.1%) following treatment with 20 μM CDDP. After 72 h, the percentage was further decreased to be 16.5% (Fig. 2B). These results suggest that A549/CDDP cells have an escape response from G2/M arrest after CDDP-induced DNA damage.
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Effect of nocodazole on the cell cycle of A549/CDDP cells
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Nocodazole, a typical microtubule inhibitor, induced G2/M arrest. The cell cycle shift was assessed for 6, 12, and 24 h following nocodazole treatment (Fig. 3). Susceptible A549 cells were blocked at G2/M phase by nocodazole at 24 h (90.5%) (Fig. 3A). Although they progressed through G2/M phase after CDDP treatment, nocodazole-treated A549/ CDDP cells were arrested in G2/M at 24 h (79.7%) (Fig. 3B). These results suggest that A549/CDDP cells have a function in the G2/M transition, which was blocked by treatment with nocodazole, but not CDDP.
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Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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A A549 72 h
Percentage of cell cycle phase
NT
G0/G1 S G2/M
CDDP (5.8 M) 24 h 48 h 72 h 31.4 ± 2.0 49.9 ± 0.1 62.6± 0.5 45.3 ± 2.1 10.6 ± 0.1 6.4 ± 0.2 23.3 ± 1.9 39.6 ± 0.1 31.0 ± 0.6
G0/G1 S G2/M
CDDP (20 M) 24 h 48 h 72 h 34.5 ± 0.0 11.0 ± 0.8 12.7 ± 1.0 65.2 ± 0.2 17.9 ± 0.7 11.1 ± 0.8 0.3 ± 0.3 71.1 ± 0.7 76.2 ± 1.1
D
P
CDDP (20 μM)
B
72 h
T
48 h
C
24 h
E
A549/CDDP
Percentage of cell cycle phase
G0/G1 S G2/M
NT 24 h 48 h 63.0 ± 0.8 65.8 ± 0.2 22.8 ± 1.5 27.3 ± 0.3 14.2 ± 0.8 7.5 ± 0.4
G0/G1 S G2/M
CDDP (5.8 M) 24 h 48 h 72 h 48.3 ± 0.8 65.3 ± 0.4 81.5 ± 1.0 34.7 ± 1.1 21.5 ± 0.6 10.8 ± 0.4 17.0 ± 0.5 13.2 ± 0.6 7.7 ± 0.6
G0/G1 S G2/M
CDDP (20 M) 24 h 48 h 72 h 34.9 ± 1.0 46.7 ± 0.2 71.8 ± 0.7 45.6 ± 1.5 22.6 ± 0.7 11.7 ± 0.7 19.5 ± 1.0 30.7 ± 0.9 16.5 ± 0.7
R
R
E
NT
U
N
C
O
CDDP (5.8 μM)
CDDP (20 μM)
72 h 87.7 ± 1.4 9.5 ± 0.7 2.8 ± 1.2
R O
CDDP (5.8 μM)
G0/G1 S G2/M
NT 24 h 48 h 56.2 ± 0.2 64.9 ± 2.8 32.7 ± 0.4 33.5 ± 3.5 11.1 ± 0.2 1.6 ± 1.6
F
48 h
O
24 h
72 h 78.9 ± 1.3 13.4 ± 1.3 7.2 ± 0.1
Fig. 2. Effect of cisplatin on the cell cycles of A549 and A549/CDDP cells. After seeding A549 (A) and A549/CDDP (B) cells at a density of 4 × 106 cells/well, the cells were pre-cultured for 24 h. Cells were then treated with 5.8 and 20 μM CDDP for up to 72 h. After treatments of 24, 48, and 72 h, DNA content was assessed by flow cytometry, and cell cycle distribution was analyzed using ModFit LT software. In the left panel, a representative cell cycle distribution is shown. In the right panel, the percentage of each cell cycle is given as the mean ± standard deviation of three experiments.
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Effect of nocodazole on the expression of cell cycle-associated proteins in A549/CDDP cells
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The expression of cell cycle-associated proteins was examined to characterize the molecular mechanism of G2/M arrest in A549 and A549/
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CDDP cells (Fig. 4). Cdc2 cyclin dependent kinase (also known as Cdk1) is essential for entry into mitosis. Cells remain arrested in G2/M phase when CyclinB1 forms a complex with Cdc2 that is inactive upon Tyr15 and Thr14 phosphorylation [15]. In contrast, progression through G2/M occurs after dephosphorylation of Cdc2 by Cdc25C phosphatase [4].
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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A
0h
6h
5
12 h
24 h
O
F
NT
R O
Noc (0.1 μg/mL)
0
0h
6h
G0/G1 S
79.5 ± 1.5 17.3 ± 1.5 3.2 ± 0.1
57.9 ± 0.4 35.2 ± 0.4 6.7 ± 0.1
E
Noc G0/G1 S
0h
24 h 58.0 ± 0.4 32.0 ± 0.2 10.0 ± 0.2
6h
12 h
24 h
43.4 ± 1.6 39.9 ± 1.3 16.8 ± 0.3
22.1 ± 0.8 35.0 ± 0.8 42.9 ± 0.4
3.6 ± 0.3 5.9 ± 0.2 90.5 ± 0.4
E
24 h
R
R
12 h
150
N C O
NT
6h
C
B
T
G2/M
120
12 h
D
G2/M
A549
60 90 Channels (FL2-A)
60.7 ± 1.8 29.6 ± 0.1 9.7 ± 0.7
P
NT
30
U
Noc (0.1 μg/mL)
A549/CDDP
NT
0h
6h
12 h
24 h
G0/G1
62.3 ± 0.7 28.9 ± 0.3 8.9 ± 0.6
56.2 ± 0.3 28.6 ± 0.3 15.2 ± 0.1
62.1 ± 0.3 25.1 ± 0.3 12.8 ± 0.1
61.0 ± 0.3 27.1 ± 0.1 11.9 ± 0.2
S G2/M Noc
6h
12 h
24 h
G0/G1 S
43.0 ± 0.5 25.9 ± 0.2 31.0 ± 0.5
29.6 ± 0.3 22.1 ± 0.5 48.3 ± 0.7
15.2 ± 0.1 5.1 ± 0.6
G2/M
79.7 ± 0.6
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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A
Noc (100 ng/mL) A549/CDDP
A549 0
3
6
12
24
0
3
6
12
MW 24 (h) (kDa) 34
p-Cdc2
34
O
F
Cdc2
p-Cdc25C
p-Cdc2
E
Cdc2 5.0
T
4.0
C
3.0 2.0 1.0
E
Fold change (/ACTB)
42
D
B
5.0 4.0
12
5.0
A549
4.0
A549/CDDP
3.0 2.0 1.0 0.0
24 (h)
0
3
6
12
24 (h)
3
6
12
24 (h)
p-Cdc25C 5.0 4.0 3.0
2.0
2.0
N
C
3.0
6
O
Cdc25C
3
R
0
R
0.0
Fold change (/ACTB)
60
P
β-acn
1.0
1.0
U
60
R O
Cdc25C
0.0
0.0 0
3
6
12
24 (h)
0
Fig. 4. Effect of nocodazole on the expression of cell cycle-associated proteins in A549 and A549/CDDP cells. At 24 h after seeding A549 and A549/CDDP cells at a density of 5 × 106 cells/dish into 60 mm dishes, the cells were treated with nocodazole (100 ng/ml). Whole cell extracts were then prepared from samples at 3, 6, 12, and 24 h. The expression levels of Cdc2, p-Cdc2, Cdc25C, and p-Cdc25C in these cells were measured by Western blotting using β-actin as a loading control (A). Fold changes in protein expressions between A549 (white bar) and A549/ CDDP cells (black bar) are shown (B).
Fig. 3. Effect of nocodazole on cell cycles of A549 and A549/CDDP cells. After A549 (A) and A549/CDDP (B) cells were seeded at a density of 4 × 106 cells/well, the cells were incubated up to 24 h and treated with nocodazole (100 ng/ml). After treatments of 6, 12, and 24 h, DNA content was assessed by flow cytometry, and cell cycle distribution was analyzed by ModFit LT software. Each panel (A and B) contains a representative cell cycle distribution, and a table listing the percentage of each cell cycle, shown as the mean ± standard deviation of three experiments.
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
S. Horibe et al. / Life Sciences xxx (2015) xxx–xxx
7
CDDP (20 μM)
A
A549/CDDP
A549 0
24
48
72
0
24
MW 72 (h) (kDa)
48
34
p-Cdc2
34
F
Cdc2
CDDP (20 μM)
A549/CDDP
A549 24
48
72
0
C
B
E
Cdc2
60
42
N C O
5.0
A549/CDDP
60.0 40.0 20.0
0.0
0
A549
80.0
R
10.0
p-Cdc2 100.0
R
15.0
Fold change (/ACTB)
60
T
β-acn
42
MW 72 (h) (kDa)
E
p-Cdc25C
U
48
D
Cdc25C
Fold change (/ACTB)
24
P
0
R O
O
β-acn
0.0 24
48
0
72 (h)
Cdc25C
p-Cdc25C
25.0
15.0
24
48
72 (h)
24
48
72 (h)
20.0 10.0
15.0 10.0
5.0
5.0 0.0
0.0 0
24
48
72 (h)
0
Fig. 5. Effect of CDDP on the expression of cell cycle-associated proteins in A549/CDDP cells. At 24 h after seeding A549 and A549/CDDP cells at a density of 5 × 106 cells/dish into 60 mm dishes, the cells were treated with 20 μM CDDP for up to 72 h. Whole cell extracts were then prepared at 24, 48, and 72 h. The expression levels of Cdc2, p-Cdc2, Cdc25C, and p-Cdc25C proteins in these cells were measured by Western blotting using β-actin as a loading control. Two different blotting membranes were used. Results of Cdc2 and p-Cdc2 blots are shown in the upper frame and Cdc25C and p-Cdc25C blots are in the lower panel (A). Fold changes in protein expressions between A549 (white bar) and A549/CDDP cells (black bar) are shown (B).
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
8
Cdc2 was consistent with a previous report for MCF-7, a human breast cancer cell line [2]. Similarly, in A549/CDDP cells, Cdc2 expression was promoted for up to 12 h but was reduced 24 h following exposure to nocodazole. In both A549 and A549/CDDP cells, the expression of phosphorylated Cdc2 was induced within 12 h, and repressed at 24 h. These results indicated that nocodazole lowered Cdc2 expression, although
A CBDCA (240 μM) A549 48
72
0
24
MW 72 (h) (kDa)
48
F
24
A549/CDDP
O
0
34
R O
Cdc2
p-Cdc2
34
60
D
P
Cdc25C
60
E
p-Cdc25C
42
C
T
β-acn
B
E
Cdc2
R
15.0
5.0
0
A549/CDDP
2.0 1.0 0.0
24
48
72 (h)
N
C
0.0
A549
5.0
3.0
R
10.0
p-Cdc2 4.0
O
Fold change (/ACTB)
222 223
0
24
48
72 (h)
24
48
72 (h)
p-Cdc25C
Cdc25C
U
220 221
We assessed the effect of nocodazole on the expression of cell cycleassociated proteins in susceptible and resistant A549 cells (Fig. 4A and B). Before nocodazole treatment (0 h), susceptible A549 cells have a lower level of Cdc2 expression. After treatment with nocodazole, Cdc2 expression increased to maximum levels at 12 h and had slightly decreased at 24 h. In susceptible A549 cells, the induction level of
Fold change (/ACTB)
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15.0
25.0 20.0
10.0
15.0 10.0
5.0
5.0 0.0
0.0 0
24
48
72 (h)
0
Fig. 6. Effect of CBDCA on the expression of cell cycle-associated proteins in A549/CDDP cells. At 24 h after seeding A549 and A549/CDDP cells at a density of 5 × 106 cells/dish into 60 mm dishes, the cells were treated with 240 μM CBDCA up to 72 h. Whole cell extracts were then prepared at 24, 48, and 72 h. The expression levels of Cdc2, p-Cdc2, Cdc25C, and p-Cdc25C proteins in these cells were measured by Western blotting using β-actin as a loading control (A). Fold changes in protein expressions between A549 (white bar) and A549/CDDP cells (black bar) are shown (B).
Please cite this article as: S. Horibe, et al., Cisplatin resistance in human lung cancer cells is linked with dysregulation of cell cycle associated proteins..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.01.011
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Effect of CBDCA on the expression of cell cycle-associated proteins in A549/ CDDP cells
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264
Since A549/CDP cells showed cross-resistance to CBDCA, the expression of Cdc2, Cdc25C, and their phosphorylated forms was assessed after treatment with 240 μM CBDCA (Fig. 6). In A549 cells, the treatment increased Cdc2 expression up to 48 h and was decreased at 72 h. However, Cdc2 expression in A549/CDDP cells peaked at two-fold higher than A549 cells at 48 h and remained higher for up to 72 h. In addition, Cdc25C and its phosphorylated form were induced in A549/CDDP cells after exposure to CBDCA, and peaked at 48 h. As with CDDP treatment, the active form of Cdc2 was induced by Cdc25C phosphatase for up to 72 h following CBDCA treatment. In addition, CBDCA treatment led to a weak expression pattern of cell cycle-associated proteins, which was similar to that observed after treatment with CDDP.
265
Discussion
266
Cisplatin-based chemotherapy is effective in the treatment of advanced lung cancer with a survival benefit of 5 years [1]. Unfortunately, some patients gradually have showed the tumor relapse and become resistant to the drug [19]. Two major strategies have been undertaken to improve the efficacy of cisplatin-based therapy. One is the development of platinum analogues with better therapeutic effects. However, cisplatin resistant cells are often cross-resistant to other platinum compounds. Carboplatin (CBDCA) has fewer side effects with an anti-tumor range similar to that of cisplatin, but shows cross-resistance to cisplatin [14]. In contrast, oxaliplatin (L-OHP) exhibits anti-tumor activity against cisplatin-resistant cells and lacks cross-resistance [13,18]. The results of this study using A549/CDDP cells were consistent with the previous findings that oxaliplatin is active, lacks cross-resistance and had a better toxicity profile. These results have made L-OHP as one of the leading treatment options for unresponsive cisplatin-based chemotherapy in human lung cancer. The other strategy is to understand the abnormal mechanisms of cisplatin resistance. Previous examinations of the effects of cisplatin on the cell cycle progression showed that the drug reduces the rates of DNA synthesis [16,17] with a subsequent slowdown of S-phase [11] followed by G2/M arrest [10,11]. In cisplatin-treated cells, G2/M arrest is induced by the inhibition of Cdc2/CyclinB activity, a key regulator of the G2/M transition [10]. The Cdc2/CyclinB complex is activated by dephosphorylation of Tyr15 of Cdc2 by Cdc25 phosphatase, which induces M-phase
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C
254 255
E
247 248
R
245 246
R
243 244
N C O
241 242
U
239 240
Conclusion
327
CBDCA, but not L-OHP exhibited cross-resistance to cisplatin in A549/CDDP cells. Under treatment with cisplatin, A549/CDDP cells showed enhanced modification of cell cycle associated proteins, which resulted in G2/M progression. These findings represent a partial molecular explanation of the development of drug resistance in lung cancer cells and their subsequent survival under treatment with cisplatin.
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Conflict of interest statement
334
F
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237 238
O
249 250
Next, we examined the effect of CDDP on cell cycle-associated proteins (Fig. 5). Treatment of A549 cells with 20 μM CDDP led to a gradual increase in Cdc2 expression for up to 48 h, before a marked decrease was observed at 72 h (Fig. 5A). However, in A549/CDDP cells, expression of Cdc2 was increased and not inhibited at 72 h. In parallel, expression of inactive Cdc2, which was phosphorylated at Tyr15, was increased in A549/CDDP cells, but markedly decreased in A549 cells. With the treatment of CDDP in susceptible A549 cells, the expression of Cdc25C increased for 48 h, but had remarkably decreased by 72 h (Fig. 5B). In A549/CDDP cells, however, Cdc25C expression persisted 72 h after CDDP exposure, and expression of phosphorylated Cdc25C peaked at 24 h. Taken together, these results suggest that the inhibitory form of tyrosine phosphorylated Cdc2 was dephosphorylated to an active state by Cdc25C phosphatase for up to 72 h following CDDP treatment in A549/CDDP cells, but not in A549 cells.
R O
236
290 291
P
Effect of CDDP on the expression of cell cycle-associated proteins in A549/ CDDP cells
entry. This study showed that cisplatin certainly inhibits the activity of Cdc2 in susceptible A549 cells and induces G2/M arrest with the treatment of 5.8 and 20 μM CDDP. In addition, we hypothesize that Cdc2 activity may be involved in late events of the escape from G2/M block in cisplatin-resistant cells, and found that Cdc2 expression and its activation by Cdc25C are maintained in A549/CDDP cells. The observed Cdc2 activity appeared to be a unique cellular response to cisplatin in resistant lung cancer cells. It is now widely accepted that the G2/M transition is governed by the Cdc2/CyclinB complex. G2/M arrest associated with CyclinB repression has also been reported following treatment with adriamycin, etoposide, and irinotecan [3], suggesting that CyclinB repression is involved in the G2/M arrest mechanism induced by anti-cancer drugs. To extend the previous findings, our results indicate that repression of Cdc2 is a factor in G2/M arrest during cisplatin treatment. Cross resistance to cisplatin and CBDCA remains poorly understood, but is thought to be related to intrinsic resistance mechanisms; including non-specific inactivation and efflux at the cytoplasmic level, and specific DNA adduct repair mechanisms at the nuclear level [13]. CBDCA treatment weakened expressions of Cdc2, Cdc25C and their phosphorylated forms, but the pattern was similar to that observed upon CDDP treatment. The results suggest that these two drugs affect cell cycle associated proteins in a similar fashion, but the protein expression patterns were not identical. Further studies are required to clarify the actual regulation of cell cycle associated proteins under CBDCA exposure. There was a significant difference in the response to nocodazole in A549/CDDP cells. Nocodazole treatment certainly caused a G2/M block in both cisplatin resistant and susceptible cells. Although the cellular response to nocodazole was normally regulated in A549/CDDP cells, cisplatin treatment triggered dysregulation of G2/M associated proteins, which proceeded through the G2/M block. This paradoxical change is likely due to aberrant cellular signaling in cisplatin resistant cells. It has been proposed that G2/M arrest is essential to the process of engaging cell death following cisplatin treatment [17]. We suggest that G2/M associated proteins play a role in determining the pharmacological properties of cisplatin resistant cell lines.
D
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T
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the majority of Cdc2 was in a phosphorylated that was inactive form in both CDDP susceptible and resistant A549 cells. Similarly, there was no distinct difference in the expression of Cdc25C and its phosphorylated form following nocodazole treatment.
E
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9
All authors have no competing interest.
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Acknowledgments
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This study was supported by a Grant-in-Aid (no. 26460212) to TT and a Grant-in-Aid for Young Scientists (B) (no. 25860568) to JI from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. TT and YM were financially supported by KOSEI-KAKENHI (H25-B sou-Kan-en-general-018). TT was also supported by a Grantin-Aid from Hyogo Science and Technology Association.
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References
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