- Email: [email protected]
Down-regulation of ICBP90 contributes to doxorubicin resistance
European Journal of Pharmacology 656 (2011) 33–38
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Down-regulation of ICBP90 contributes to doxorubicin resistance Jingxuan Wang 1, Ying Song 1, Shanqi Xu, Qingyuan Zhang ⁎, Yulian Li, Dabei Tang, Shi Jin Department of Medical Oncology, The Third Hospital of Harbin Medical University, Harbin, China
a r t i c l e
i n f o
Article history: Received 19 August 2010 Received in revised form 30 December 2010 Accepted 17 January 2011 Available online 4 February 2011 Keywords: ICBP90 Doxorubicin Topo IIα Breast cancer Cell cycle arrest
a b s t r a c t Acquired resistance to doxorubicin has become a serious obstacle in breast cancer treatment. The underlying mechanism responsible for this has not been completely elucidated. In this study, a doxorubicin-resistant MCF-7/Dox cell was developed to mimic the occurrence of acquired doxorubicin resistance. We next contrasted the expression profiles of ICBP90 and Topo IIα and tumor cell growth of different breast cancer cell lines to doxorubicin. Decreased expression levels of ICBP90 and Topo IIα were found in doxorubicin-resistant cells. To examine its function in chemoresistance, RNA interference (RNAi) and forskolin stimulation experiments further demonstrated that ICBP90 and Topo IIα were involved in the proliferation of cells that had acquired doxorubicin resistance. In MCF-7/Dox and ICBP90-siRNA cells, the cell growth wasn't inhibited by doxorubicin and preferentially arrested in G1 phase. However, after forskolin increased the Topo IIα expression, these breast cancer cells were again found to be inhibited by doxorubicin. Further, immunohistochemical assay breast cancer patients accepted EFC regimen showed ICBP90 was significantly associated with tumor cell proliferation, locally advanced disease and Topo IIα expression. In conclusion, down-regulation of ICBP90 induced the descended expression of Topo IIα protein which is the target enzyme of doxorubicin. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The morbidity of breast cancer has increased dramatically during the past two decades. Doxorubicin (adriamycin) is a potent anthracycline chemotherapeutic agent and is widely used for the treatment of breast cancer, this is attributed to being highly effective and well tolerated. However, despite patients carefully receive adjuvant doxorubicin based chemotherapy regimens after surgical removal of the primary breast tumor, many of these patients recur. This poor prognosis may be attributed to a low incidence of complete response and change in the biologic characteristics of tumor after tumor eradication. The anthracycline drugs is topoisomerase poisons specifically targeting Topo IIα (Withoff et al., 1996). Topo II reduces DNA twisting and super-coiling, allowing selected regions of DNA to untangle and thus engage in transcription, replication, or repair processes (Kellner et al., 2002; Chen and Liu, 1994). Previous studies have reported that deletion or mutation of Topo IIα in breast cancer are associated with drug resistance, whereas high concentrations of Topo IIα indicate anthracycline sensitivity, poor nuclear differentiation and high proliferation (Fry et al., 1991; Sullivan et al., 1987).
⁎ Corresponding author at: Department of Medical Oncology, The Third Hospital of Harbin Medical University, Harbin, 150040, China. Tel./fax: +86 451 86298276. E-mail address: [email protected] (Q. Zhang). 1 Wang Jingxuan and Song Ying contributed equally to this work. 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.01.042
Deregulation of genes that are involved in the progression from G1 to S phase of the cell cycle is a frequent event in numerous tumor types, including lung cancer (Ye et al., 2010), melanoma (Scaini et al., 2009) and esophageal carcinoma (Li et al., 2010). Recently ICBP90 (Inverted CCAAT box Binding Protein, 90 kD) is identified and characterized by a RING finger domain, shares structural homology with the nuclear phosphoprotein Np95 (nuclear protein 95) and the NIRF (Np95/ICBP90-like Ring finger protein), both involved in cellcycle regulation. Functional studies have demonstrated that Np95 is required for the G1/S transition, overexpression of Np95 and cycE/ cdk2 complexes can force reentry of terminally differentiated cells into the cell cycle (Bonapace et al., 2002). NIRF expression is high in proliferating phase but significantly low in G0/G1 phase in normal TIG-7 and WI-38 cells (Mori et al., 2002), while consistently high in tumoral HT-1080 and HepG2 cells. Likewise, ICBP90 also has function in cell-cycle regulation, the expression of ICBP90 is observed throughout cell cycle but peaks at late G1 and G2/M phases in noncancerous human cells. Down-regulation of ICBP90 in HCT116 and Jurkat cells is a critical event in G1-arrested cells (Arima et al., 2004; Abbady et al., 2003). In addition, ICBP90 inhibitors could inhibit the expression of ICBP90 after 24 h treatment, arrested the cell cycle at G1/G0 stage and blocked its entrance from S phase to G2/M phase. Consequently, one proposed function of ICBP90 is that it plays a role in tumorogenesis by deregulating the expression of genes leading to loss of control of G1/S transition and therefore involved in cancerogenesis (Bronner et al., 2007). Among these genes, we can find topoisomerase IIα (Hopfner et al., 2001), RB1 (Jeanblanc et al.,
34
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
2005), putatively p16INK4A, p14ARF and RARb (Unoki et al., 2004). Considering that ICBP90 could bind to one of the inverted CCAAT boxes (called ICB2) of the Topo IIα gene promoter, which is of extreme importance for anthracycline drugs. Therefore, we blocked the expression of ICBP90 in order to observe its effect to doxorubicin resistance. 2. Materials and Methods 2.1. Cell Line For our studies, we used human breast cancer cell line MCF-7 and its sublines resistant to cytotoxic effects of doxorubicin (MCF-7/Dox). They were obtained from Heilongjiang Tumor Research Institute (Heilongjiang, China). MCF-7/Dox cells were derived from the drug sensitive MCF-7 cells by stepwise selection with doxorubicin (adriamycin) (Mehta, 1994). The cells were grown in RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum and 100 units/ml penicillin and 2 mg/ml streptomycin (Sigma Aldrich Co.). To maintain their resistance, this cell line was cultured in the presence of 2 μM doxorubicin (Pfizer Italia S.r.l., Italy) and passaged for 1 week in a drug-free medium before each experiment. 2.2. Western Blot Analysis Cells were rinsed in PBS, and cells were lysed by loading buffer (2% SDS, 10% glycerol, and 50 mm Tris pH 6.8). Samples were subsequently sonicated for 15 s using a microtip. Protein concentration was quantified using Biorad protein assay for all samples lysed in SDS. Cell lysates were denatured at 95 °C for 5 min after the addition of dithiothreitol (DTT) and bromophenol blue. Approximately 25 μg of protein was loaded per lane and separated using a 5–20% gradient polyacrylamide gel (PAGEL, ATTO), and transferred to nitrocellulose membrane (Hybond, Amersham pharmacia). The membranes were probed with primary antibodies. The following antibodies were used: mouse monoclonal antibody for ICBP90 (1RC1C-10, 100 ng/mL), Topo IIα (Ki-S1, 1:1000 dilution; Sigma) and β-actin (1:10 000 dilution; Sigma). Then the membranes were incubated with horseradish peroxidase conjugated secondary antibodies and visualized by chemiluminescense detection system.
cell was treated with a control medium as control. The medium was replaced every 2 days. Then cell proliferation was measured using the MTT assay. The number of viable cells remaining after treatment with various agents was determined by 3-(4,5-di-methylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma Aldrich Co.) reduction assay. Cells were seeded in 96 well microtiter plates at a concentration of 5 × 104 cells/well. Following 48 h of treatment with various concentrations of doxorubicin, MTT was added to each well at a final concentration of 0.5 mg/ml. Four hours later, 100 μl DMSO was added to each well to dissolve the resulting formazan crystals. Absorbance was read at 490 nm using an enzyme-linked immunosorbent assay reader (SpectraMax; Molecular Devices, Sunnyvale, CA). Data were collected from three separate experiments and the percentage of doxorubicininduced cell growth inhibition was determined by comparison to DMSO-treated control cells. MCF-7/Dox and ICBP90-siRNA transfected cells were treated with 0 (control), 25, 50, or 100 μM of forskolin (sigma) and 1 μM doxorubicin for 72 h. Then western blot analysis and MTT assay were then performed. 2.5. Flowcytometric Cell Cycle Analysis Cells plated at a density of 6 106 cells/ml were incubated with doxorubicin (1 μM). The cells were harvested after 48 h, washed then fixed with 70%(v/v) methanol. Staining of cellular DNA was performed using DNA staining solution, composed of 50 μg/l propidium iodide and 500 mg/l protease-free RNase. Cell cycle analysis was performed with the FACSanto software package, BD biosciences corp. The percentage of cells with DNA content corresponding to G0/G1, S and G2/M phases, respectively was computed by the planimetry of the histogram and then compared for different treatment groups. 2.6. Validation on Clinical Samples
ICBP90 small interfering RNA (siRNA) oligonucleotides targeting different regions of ICBP90 mRNA were designed according to a previously reported method, and were synthesized by Qiagen. The control siRNA is a scrambled sequence and does not target any known mammalian mRNA. The oligoribonucleotide used were purchased from Dharmacon, Inc. (Lafayette, CO, U.S.A.) and are as follows: ICBP90siRNA-sense: 5′-AAGATCCAGGAGCTGTTCCAC-3′; anti-sense: 5′- AACGACTGTCGGATCATCTTC-3′. Control siRNA-sense: 5′-UCCGUUUCGGUCCACAUUC-3′; anti-sense: 5′-GAAUGUGGACCGAAACGGA-3′. For siRNA transfection, 60 nM siRNAs were diluted in Opti-MEM with Lipofectamine 2000 (Invitrogen) according to the manufacturers' instructions.
2.6.1. Patient Selection and Immunohistochemical Assay For this study, we randomly selected 220 breast cancer patients presented at the third Hospital of Harbin Medical University, Harbin, China, during the period from February 2003 to January 2006. Informed consent was obtained from all the patients or their relatives prior to analysis, and the project was approved by the Institutional Ethics Committee of Harbin Medical University. These 220 patients had received preoperative EFC chemotherapy before surgery. EFC regimen containing epirubicin 60 (or 70) mg/m2 i.v. on days 1 and 8, 5-fluorouracil 500 mg/m2 i.v. on day 1 and cyclophosphamide 500 mg/m2 i.v. on day 1 every three weeks for 2 cycles. The median age was 48 years (range, 30–81 years). Thirty-eight patients (30.9%) were postmenopausal. The distribution of cases according to clinical stage, histologic type, and menopausal status is shown in Table 1. ICBP90 and Topo IIα were carried out for immunohistochemistry. Tissues from human primary breast cancer were embedded in paraffin and fixed in 10% buffered formalin (Sigma). Serial histological sections (3 mm) were incubated overnight at room temperature with the ICBP90 mAb or with an anti-Topo IIα mAb (Neo Markers, Union City, CA), and specifically bound antibodies were visualized by a Streptavidin–Biotin complex (LAB/LSAB method, Dako LSAB2 System Kit, DAKO, Carpinteria, CA).
2.4. MTT Assay of Cell Viability
2.7. Statistical Analysis
MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells were seeded into 96-well plates at a density of 5 ⁎ 105 cells/well in a RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum and 100 units/ml penicillin and 2 mg/ml streptomycin. Then MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells were respectively treated with doxorubicin (1 μM) for 7 days. The MCF-7
Direct comparisons between MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells or between control and treatment effects were assessed using a Student's t-test analysis. One-way ANOVA was used to assess the significant differences between the treatment groups. Covariates included in multiple logistic regression analysis were ICBP90 status, breast density, histologic grade (1 versus 2/3), locally advanced disease
2.3. Gene Silencing with Small Interfering RNA
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
35
Table 1 Clinical characteristics of the 220 eligible patients. Characteristics
Total (n%)
Age years Median Range Menopausal status Premenopausal Postmenopausal Histologic type Ductal carcinoma Lobular carcinoma Unclassified Clinical stage I II III IV
48 30–81 152 (69.1) 68 (30.9) 168 (76.4) 50 (22.7) 2 (0.9) 52 (23.6) 98 (44.5) 64 (29.1) 6 (2.7)
and Topo IIα status. The statistical calculations were done using SPSS 12.0 with the exception of conditional logistic regression analysis for which LogXact 5.0 was used, and the confidence limits for odds ratios (OR) for which special programming in Maple 8 was applied (Breslow and Day, 1980). In all statistical comparisons, P b 0.05 was considered to be statistically significant. 3. Result 3.1. Different Expression of ICBP90 and Topo IIα in MCF-7 and MCF-7/Dox Cells We investigated the levels of ICBP90 and Topo IIα in these cell lines. ICBP90 is known to play a critical role in regulating the expression of Topo IIα. Decreased basal levels of ICBP90 and Topo IIα were observed in the MCF-7/Dox cells, as compared with the MCF-7 cells. Densitometry readings revealed that, in MCF-7 cells, the levels of ICBP90 protein was 2fold greater (Pb 0.05), respectively, and the levels of Topo IIα protein was 3.5-fold than those of MCF-7/Dox cells (Pb 0.05), respectively (Fig. 1). 3.2. ICBP90 Knockdown Reduced Topo IIα Expression and Inhibited the Anti-proliferative Effect of Doxorubicin in ICBP90-siRNA Transfected Cells To investigate a possible role for ICBP90 in doxorubicin resistance, ICBP90 was specifically silenced with short interfering RNA (siRNA) in MCF-7 cells. Immunoblot analysis performed at 48 h after the transfection of ICBP90 siRNAs revealed a marked reduction of ICBP90 expression compared to control siRNA, it inhibited the expression of Topo IIα (Fig. 1). Later, we tested the anti-proliferative effect of doxorubicin (1 μM) by MTT. However, ICBP90-siRNA transfected cells were the same as MCF-7/Dox cells, significantly decreased the sensitivity to doxorubicin (Fig. 2). Later, the resistant variants ICBP90-siRNA transfected and MCF-7/Dox cells were originated by growing initial MCF-7 cells with raising concentrations of or doxorubicin (from 0.1 to 64 μM), respectively. Doxorubicin was added twice a week after reseeding. Every two months, cell survival was analyzed by MTT assay. IC 50 values for MCF-7, MCF-7/ Dox and ICBP90-siRNA transfected cells were 1, 24 and 32 μM of doxorubicin, respectively. Therefore, MCF-7/Dox and ICBP90-siRNA transfected cells were 24 and 32 times as much resistant to the cytotoxic effect doxorubicin as compared with the MCF-7 cells, respectively. 3.3. Forskolin Positively Modulated Topo IIα and Restored the Anti-Proliferative Effect of Doxorubicin in ICBP90-siRNA Transfected and MCF-7/Dox Cells In order to study the role of ICBP90 in anthracycline resistance, furthermore, we treated the MCF-7/Dox and ICBP90-siRNA trans-
Fig. 1. Western blotting analysis of ICBP90 and Topo IIα in MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells, using β-actin gene as an endogenous control (A). Relative protein levels for ICBP90 and Topo IIα in the three breast cancer cell lines, MCF7, MCF-7/Dox and ICBP90-siRNA transfected cells (B). The results are expressed as the means (S.D.) of triplicate wells and are representative of three separate experiments. *significant differences from MCF-7 at P b 0.05.
fected cells with Forskolin. Stimulation of MCF-7/Dox and ICBP90siRNA transfected cells with increased concentrations of forskolin both resulted in a significant dose dependent increase in Topo IIα between 25 and 100 μM (Fig. 3A). Up to 3-fold of Topo IIα increase was reached at 100 μM of forskolin when compared to the basal level (Fig. 3A and B). Forskolin significantly increased Topo IIα expression level after 24 h of treatment, but had no effect on ICBP90 expression level. Results from MTT assays indicated that doxorubicin could inhibited the viability of MCF-7/Dox and ICBP90-siRNA transfected cells with forskolin at concentrations ≥ 25 μM (Fig. 3C and D). The treatment of MCF-7/Dox and ICBP90-siRNA transfected cells with 0 (control), 25, 50, or 100 μM of forskolin and 1 μM doxorubicin resulted in a dose and time-dependent inhibition of cell growth. For MCF-7/Dox cells, with inhibition rates of 8.6 ± 0.8%,11.4 ± 1.0%, and 20 ± 1.22%(P b 0.05), respectively, at 24 h, compared with control, 15.7 ± 1.20%, 25.5 ± 1.80%, and 37.2 ± 2.33%, respectively, at 48 h (all P values b 0.05) and 20 ± 1.28%,30.7 ± 2.08%, and 38.7 ± 2.86%, respectively, at 72 h (all P values b 0.05). For ICBP90-siRNA transfected cells, with inhibition rates of 17.6 ± 1.88%, 25.5 ± 2.05%, and 31.8 ± 2.60%, respectively, at 24 h (all P values b0.05), compared with control, 13.6 ± 1.68%, 16.8 ± 2.02%, and 27.1 ± 2.38%, respectively, at 48 h (all P values b0.05) and 18.1 ± 1.96%, 27.7 ± 2.06, and 33.7 ± 2.82%, respectively, at 72 h (all P values b 0.05).
36
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
(ICBP90-siRNA transfected cells: 20.46 ± 5.18% in S phase and 13.42 ± 3.18% in G2/M phase; and MCF-7/Dox cells: 25.21 ± 4.89% in S phase and 12.76 ± 3.57% in G2/M phase). The increase of cell population at the S and G2/M phases was accompanied by a decrease of cell population in the G1 phase of the cell cycle. Furthermore, a significant fraction of cells transfected with ICBP90-siRNA arrested at G1 phase (P b 0.05, Table 2), suggested that reduction of ICBP90 contributes to G1 arrest induced by DNA-damage activated checkpoint signals. 3.5. Biomarker Potential of ICBP90 in Clinical Samples To assess the prognostic power of ICBP90 gene in resistance, clinical samples were used to validate the results of cell culture model, we analyzed the expression of ICBP90 by immunohistochemical assay in 220 breast cancer patients. A significant negative correlation was seen between ICBP90 and histologic grade or locally advanced disease (P b 0.05). Patients with ICBP90-negative tumors after preoperative EFC chemotherapy were more likely to present with locally advanced disease, and 25 of 30 patients had ICBP90-negative tumors. The relationship with lymph node spread and breast density was not significant (P = 0.08 and P = 0.55; Table 3). However, a highly significant relationship was found between tumors expressing ICBP90 and Topo II α (OR, 2.8; 95% CI, 1.4–6.5). 4. Discussion
Fig. 2. Growth curves of MCF-7 (A), MCF-7/Dox (B) and ICBP90-siRNA transfected (C) cells in the presence of doxorubicin. MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells were respectively treated with doxorubicin (1 μM) for 7 days. MCF-7 (A), MCF-7/ Dox (B) and ICBP90-siRNA transfected (C) cells were treated with control medium as control, respectively. The medium was replaced every 2 days. Cell proliferation was measured using the MTT assay. The results are expressed as the means (S.D.) of triplicate wells and are representative of three separate experiments. *significant differences from control at P b 0.05.
3.4. Down-regulation of ICPB90 Contributes to Doxorubicin Resistance by Mediating G1 Arrest After determining that down-regulation of ICPB90 exhibited resistance to doxorubicin, we next investigated the effects of ICPB90 on cell cycle progression. In contrast to MCF-7 cells, analysis of cell cycle distribution after doxorubicin treatment presented evidence of a preferential block of ICBP90-siRNA transfected and MCF-7/Dox breast cancer cells in G0/G1 phase. However, a shift of cell distribution into S and G2/M phases was noticed upon with 100 μM forskolin addition in ICBP90-siRNA transfected(31.92 ± 6.56% in S phase and 15.07 ± 4.78% in G2/M phase) and MCF-7/Dox (32.56 ± 7.08% in S phase and 16.82 ± 5.16% in G2/M phase) breast cancer cells, compared to Dox alone
Acquired resistance to doxorubicin has become a serious obstacle in current breast cancer treatment. Here, it was revealed that abnormal cell cycle regulation was a crucial factor in this crosstalk, the silencing of ICBP90 made cell cycle arrest at G1/S transition and resulted doxorubicin resistance in MCF-7 cell. Cells contain numerous pathways designed to protect them from the genomic instability or toxicity. The cell division phases can be divided into two functional phases (S and M phases) and two preparatory phases (G1 and G2). Cell cycle checkpoints are important control mechanisms that ensure the proper growth. Chemotherapy activates cell cycle checkpoint signaling pathways arresting the cell cycle and inducing apoptosis. Checkpoint defects are frequent in human cancer and can confer resistance toward anticancer chemotherapy. For instance, chemotherapeutic agents cause double-strand DNA breaks involving the function of BRCA1, so losing it contributes to G2/M-phase checkpoint defect in BRCA1-mutant HCC1937 breast cancer cell line. However, transfecting a wild-type BRCA1 gene in these cells could correct the function of this checkpoint (Quinn et al., 2003). A p53independent branch of the DNA damage checkpoint is activated that involves the Chk1 kinase and arrests cells solely before mitosis. While, defects in the function of p53 might eliminate G1 arrest, thereby allowing cells with damaged DNA to enter S phase, which may cause tumor resistance to several different cytotoxic compounds (Geisler et al., 2003). In addition, overexpression of LATS2 inhibited cyclinE/CDK2 kinase activity and also resulted in G1/S cell cycle arrest (Li et al., 2003), which leads to breast cancer in patients who were sensitive to EC (Cyclophosphamide + Epirubicin) treatment. In summary, interfering cell cycle checkpoints could suppress tumor growth (Takahashi et al., 2005). Topo IIα has been regarded as one of the target enzymes for drugresistance cancer cells. Low level expression of Topo IIα protein leads to drug resistance to anthracycline (Harris and Hochhauser, 1992). The relationship between the down-regulated expressive level of Topo IIα protein and drug resistance has been proved by researches on the clinical efficacy of drugs targeted on Topo IIα (Arpino et al., 2005). ICBP90 is first isolated as a protein that binds to a CCAAT box in the promoter region of Topo IIα gene (Hopfner et al., 2002), which localizes in nuclei and contains an ubiquitin-like (UbL) domain (Hopfner et al., 2001). Many cancer cell lines such as HeLa cell line,
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
37
Fig. 3. Effects of forskolin on the expression of ICBP90 and Topo IIα (A and B). Effects of forskolin on the proliferation of MCF-7/Dox (C) and ICBP90-siRNA transfected cells (D). MCF7/Dox and ICBP90-siRNA transfected cells were treated with 0 (control), 25, 50, or 100 μM of forskolin and 1 μM doxorubicin. They were then incubated in a medium containing forskolin and doxorubicin for 72 h. Western blot analysis and MTT assay were then performed. The data are representative of at least three separate experiments. *significant differences from control at P b 0.05.
Jurkat cell line and A549 cell line show ICBP90 expressed throughout the entire cell cycle. Through cAMP signaling pathway, ICBP90 exit escape of forskolin-treated cells from G1 phase and increases binding of ICBP90 to ICB2 element of Topo IIα gene promoter with subsequently up-regulated expression of Topo IIα protein(Trotzier et al., 2004). Thus, it was of interest to compare the expression of ICBP90 and Topo IIα in ICBP90-siRNA transfected, MCF-7/Dox and MCF-7 cells. Compared to MCF-7 cells, ICBP90 and Topo IIα expressions were lower in ICBP90-siRNA transfected cells, which were consistent with our finding of little ICBP90 and Topo IIα in MCF7/Dox cell line. These data showed the decreased expression of ICBP90 subsequently down-regulated the expression of Topo IIα both in ICBP90-siRNA transfected and MCF-7/Dox cells. ICBP90 is likely to play a role in regulating S phase entry, depletion of ICBP90 expression by siRNA transfection induced G1 arrest in a significant population of HeLa cells when the cells were treated with doxorubicin to induce DNA damage. In addition, doxorubicin is a phasespecific drug, affecting predominantly S and G2 phases of the cell cycle, so we next compared cell cycle traverse in the ICBP90-siRNA transfected, MCF-7/Dox and MCF-7cells upon cell exposure to doxorubicin. We have Table 2 Cell cycle distribution of MCF-7 cells and sublines with induced resistance to doxorubicin and/or forskolin. Cell line
Treatment
MCF-7
Dox Dox + For Con Dox Dox + For Con Dox Dox + For Con
MCF-7/Dox
ICBP90-siRNA
Percentage of cells in a phase of the cell cycle G0/G1
S
G2/M (%)
61.11 ± 12.03 56.16 ± 11.33 45.42 ± 9.18 60.17 ± 12.33 50.62 ± 11.29 62.03 ± 13.42 62.86 ± 13.16 53.01 ± 5.46 66.12 ± 14.72
25.12 ± 5.35 28.01 ± 5.11 34.18 ± 6.27 26.31 ± 4.98 32.56 ± 7.08 25.21 ± 4.89 23.15 ± 5.03 31.92 ± 6.56 20.46 ± 5.18
13.77 ± 4.12 15.83 ± 4.05 20.40 ± 5.87 13.52 ± 3.85 16.82 ± 5.16 12.76 ± 3.57 13.99 ± 4.53 15.07 ± 4.78 13.42 ± 3.18
shown that in the MCF-7 cells, incubation with doxorubicin resulted in the significant decrease of S (from 34.18 ± 6.27% to 25.12 ± 5.35%) and G2/M (from 20.40 ± 5.87% to 13.77 ± 4.12%) phase percentage. Meanwhile, doxorubicin treatment resulted in the accumulation of MCF-7 cells in G0/G1 phase with the G0/G1 cell percentage increasing from 45.42 ± 9.18% to 61.11 ± 12.03%. In contrast, exposure to doxorubicin in ICBP90-siRNA transfected cells and MCF-7/Dox cells had no effect on cell cycle traverse (Table 2). Up-regulation of several genes involved in cell cycle regulation, DNA replication, and modification including the Topo IIα gene have been described in Schwann cells as response to forskolin (Schworer et al., 2003). In this study, we found that forskolin plus doxorubicin
Table 3 The number and proportion of cases (%) for different variables and molecular markers according to ICBP90 expression. Characteristics Breast density Low (b 30%) Mod (30–70%) High (N70%) Histologic grade 1–2 3 Locally advanced disease No Yes Nodal status Neg Pos TopoII α Neg Pos a
CI.
ICBP90 pos (n = 85), n%
ICBP90 neg (n = 135), n%
OR (95%CI)
P
11 (12.9) 60 (70.6) 14 (16.5)
19 (14.1) 95 (70.4) 21 (15.6)
1.5 (1.4–1.6)a 1.0
0.55
78 (91.8) 7 (8.2)
90 (66.7) 45 (33.3)
1.0 5.9 (2.6–13.5)
b0.01
80 (94.1) 5 (5.9)
110 (81.5) 25 (18.5)
1.0 3.4 (1.3–9.3)
b0.05
60 (70.6) 25 (29.4)
78 (57.8) 57 (42.2)
1.0 1.8 (0.9–3.3)
0.08
19 (22.3) 66 (77.7)
75 (55.5) 60 (44.4)
2.8 (1.4–6.5) 1.0
b0.05
The mean difference between ICBP90-negative and ICBP90-positive cases with 95%
38
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
resulted in a dose and time-dependent growth inhibitions of ICBP90siRNA transfected and MCF-7/Dox cells, forskolin can increase the expression of Topo IIα but could not affect the expression of ICBP90, while down-regulation of ICBP90 would result in significantly decrease of Topo IIα. It seemed that, other proteins other than ICBP90 could affect the expression of Topo IIα in response to forskolin treatment. So in other words, except for ICBP90 protein, there are possible that other proteins regulating Topo IIα are also responsible to anthracycline resistance, such as CRM1 (Turner et al., 2009), R16 (Zhu et al., 2007), HMGB1 and HMGB2 (Stros et al., 2009). Induction of the clinical investigation was done in order to test our preliminary data, the clinical study demonstrated that strong and consistent associations occurred between the negative ICBP90 protein expression and features of aggressive breast cancer, for example anthracycline resistance, such as high tumor cell proliferation, locally advanced disease and reduced Topo IIα expression (Table 3). In conclusion, our study indicates that down-regulation of ICBP90 can be associated with the acquisition of anthracycline resistance in breast cancer. Therefore, the proper control of ICBP90 activity might be a potential strategy to prevent the development of acquired resistance to doxorubicin based anticancer agents. Funding This study was supported by Special Science Funding for Young Scholars in the Heilongjiang Province, China (QC2009C08). Disclosure The authors declare no conflict of interest. Acknowledgments This study was supported, in part, by the Department of Pathology, the third Hospital of Harbin Medical University (Harbin, China). This experiment was finished in the oncobiology key lab of Heilongjiang common institution of higher learning. References Abbady, A.Q., Bronner, C., Trotzier, M.A., Hopfner, R., Bathami, K., Muller, C.D., Jeanblanc, M., Mousli, M., 2003. ICBP90 expression is down-regulated in apoptosis-induced Jurkat cells. Ann. NY Acad. Sci. 1010, 300–303. Arima, Y., Hirota, T., Bronner, C., Mousli, M., Fujiwara, T., Niwa, S., Ishikawa, H., Saya, H., 2004. Downregulation of nuclear protein ICBP90 by p53/p21Cip1/WAF1 dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition. Genes Cells 9, 131–142. Arpino, G., Ciocca, D.R., Weiss, H., Allred, D.C., Daguerre, P., Vargas-Roig, L., Leuzzi, M., Gago, F., Elledge, R., Mohsin, S.K., 2005. Predictive value of apoptosis, proliferation, HER-2, and topoisomerase II alpha for anthracycline chemotherapy in locally advanced breast cancer. Breast Cancer Res. Treat. 92, 69–75. Bonapace, I.M., Latella, L., Papait, R., Nicassio, F., Sacco, A., Muto, M., Crescenzi, M., Di Fiore, P.P., 2002. Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry. J. Cell Biol. 157, 909–914. Breslow, N.E., Day, N.E., 1980. Statistical methods in cancer research. Volume I — the analysis of case–control studies. IARC Sci. Publ. 32, 5–338. Bronner, C., Achour, M., Arima, Y., Chataigneau, T., Saya, H., Schini-Kerth, V.B., 2007. The UHRF family: oncogenes that are drugable targets for cancer therapy in the near future? Pharmacol. Ther. 115, 419–434. Chen, A.Y., Liu, L.F., 1994. DNA topoisomerases: essential enzymes and lethal drugs. Annu. Rev. Pharmacol. Toxicol. 34, 191–218.
Fry, A.M., Chresta, C.M., Davies, S.M., Walker, M.C., Harris, A.L., Hartley, J.A., Masters, J.R., Hickson, I.D., 1991. Relationship between topoisomerase II level and chemosensitivity in human tumor cell lines. Cancer Res. 51, 6592–6595. Geisler, S., Børresen-Dale, A.L., Johnsen, H., Aas, T., Geisler, J., Akslen, L.A., Anker, G., Lønning, P.E., 2003. TP53 gene mutations predict the response to neoadjuvant treatment with 5-fluorouracil and mitomycin in locally advanced breast cancer. Clin. Cancer Res. 9, 5582–5588. Harris, A.L., Hochhauser, D., 1992. Mechanisms of multidrug resistance in cancer treatment. Acta Oncol. 31, 205–213. Hopfner, R., Mousli, M., Garnier, J.M., Redon, R., du Manoir, S., Chatton, B., Ghyselinck, N., Oudet, P., Bronner, C., 2001. Genomic structure and chromosomal mapping of the gene coding for ICBP90, a protein involved in the regulation of the topoisomerase II alpha gene expression. Gene 266, 15–23. Hopfner, R., Mousli, M., Oudet, P., Bronner, C., 2002. Overexpression of ICBP90, a novel CCAAT-binding protein, overcomes cell contact inhibition by forcing topoisomerase II alpha expression. Anticancer Res. 22, 3165–3170. Jeanblanc, M., Mousli, M., Hopfner, R., Bathami, K., Martinet, N., Abbady, A.Q., Siffert, J.C., Mathieu, E., Muller, C.D., Bronner, C., 2005. The retinoblastoma gene and its product are targeted by ICBP90: a key mechanism in the G1/S transition during the cell cycle. Oncogene 24, 7337–7345. Kellner, U., Sehested, M., Jensen, P.B., Gieseler, F., Rudolph, P., 2002. Culprit and victim — DNA topoisomerase II. Lancet Oncol. 3, 235–243. Li, Y., Pei, J., Xia, H., Ke, H., Wang, H., Tao, W., 2003. Lats2, a putative tumor suppressor, inhibits G1/S transition. Oncogene 22, 4398–4405. Li, Q., Kawamura, K., Ma, G., Iwata, F., Numasaki, M., Suzuki, N., Shimada, H., Tagawa, M., 2010. Interferon-lambda induces G1 phase arrest or apoptosis in oesophageal carcinoma cells and produces anti-tumour effects in combination with anti-cancer agents. Eur. J. Cancer 46, 180–190. Mehta, K., 1994. High levels of transglutaminase expression in doxorubicin resistant human breast carcinoma cells. Int. J. Cancer 58, 400–406. Mori, T., Li, Y., Hata, H., Ono, K., Kochi, H., 2002. NIRF, a novel RING finger protein, is involved in cell-cycle regulation. Biochem. Biophys. Res. Commun. 296, 530–536. Quinn, J.E., Kennedy, R.D., Mullan, P.B., Gilmore, P.M., Carty, M., Johnston, P.G., Harkin, D.P., 2003. BRCA1 functions as a differential modulator of chemotherapy-induced apoptosis. Cancer Res. 63, 6221–6228. Scaini, M.C., Rossi, E., de Siqueira Torres, P.L., Zullato, D., Callegaro, M., Casella, C., Quaggio, M., Agata, S., Malacrida, S., Chiarion-Sileni, V., Vecchiato, A., Alaibac, M., Montagna, M., Mann, G.J., Menin, C., D'Andrea, E., 2009. Functional impairment of p16(INK4A) due to CDKN2A p.Gly23Asp missense mutation. Mutat. Res. 671, 26–32. Schworer, C.M., Masker, K.K., Wood, G.C., Carey, D.J., 2003. Microarray analysis of gene expression in proliferating Schwann cells: synergistic response of a specific subset of genes to the mitogenic action of heregulin plus forskolin. J. Neurosci. Res. 73, 456–464. Stros, M., Polanská, E., Struncová, S., Pospísilová, S., 2009. HMGB1 and HMGB2 proteins up-regulate cellular expression of human topoisomerase II alpha. Nucleic Acids Res. 37, 2070–2086. Sullivan, D.M., Latham, M.D., Ross, W.E., 1987. Proliferation dependent topoisomerase II content as a determinant of antineoplastic drug action in human, mouse, and Chinese hamster ovary cells. Cancer Res. 47, 3973–3979. Takahashi, Y., Miyoshi, Y., Takahata, C., Irahara, N., Taguchi, T., Tamaki, Y., Noguchi, S., 2005. Down-regulation of LATS1 and LATS2 mRNA expression by promoter hypermethylation and its association with biologically aggressive phenotype in human breast cancers. Clin. Cancer Res. 11, 1380–1385. Trotzier, M.A., Bronner, C., Bathami, K., Mathieu, E., Abbady, A.Q., Jeanblanc, M., Muller, C.D., Rochette-Egly, C., Mousli, M., 2004. Phosphorylation of ICBP90 by protein kinase A enhances topoisomerase II alpha expression. Biochem. Biophys. Res. Commun. 319, 590–595. Turner, J.G., Marchion, D.C., Dawson, J.L., Emmons, M.F., Hazlehurst, L.A., Washausen, P., Sullivan, D.M., 2009. Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition. Cancer Res. 69, 6899–6905. Unoki, M., Nishidate, T., Nakamura, Y., 2004. ICBP90, an E2F-1 target, recruits HDAC1 and binds to methyl-CpG through its SRA domain. Oncogene 23, 7601–7610. Withoff, S., De Jong, S., De Vries, E.G., Mulder, N.H., 1996. Human DNA topoisomerase II: biochemistry and role in chemotherapy resistance. Anticancer Res. 16, 1867–1880. Ye, X., Zhou, W., Li, Y., Sun, Y., Zhang, Y., Ji, H., Lai, Y., 2010. Darbufelone, a novel antiinflammatory drug, induces growth inhibition of lung cancer cells both in vitro and in vivo. Cancer Chemother.Pharmacol 6, 277–285. Zhu, H., Huang, M., Yang, F., Chen, Y., Miao, Z.H., Qian, X.H., Xu, Y.F., Qin, Y.X., Luo, H.B., Shen, X., Geng, M.Y., Cai, Y.J., Ding, J., 2007. R16, a novel amonafide analogue, induces apoptosis and G2-M arrest via poisoning topoisomerase II. Mol. Cancer Ther. 6, 484–495.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
Down-regulation of ICBP90 contributes to doxorubicin resistance Jingxuan Wang 1, Ying Song 1, Shanqi Xu, Qingyuan Zhang ⁎, Yulian Li, Dabei Tang, Shi Jin Department of Medical Oncology, The Third Hospital of Harbin Medical University, Harbin, China
a r t i c l e
i n f o
Article history: Received 19 August 2010 Received in revised form 30 December 2010 Accepted 17 January 2011 Available online 4 February 2011 Keywords: ICBP90 Doxorubicin Topo IIα Breast cancer Cell cycle arrest
a b s t r a c t Acquired resistance to doxorubicin has become a serious obstacle in breast cancer treatment. The underlying mechanism responsible for this has not been completely elucidated. In this study, a doxorubicin-resistant MCF-7/Dox cell was developed to mimic the occurrence of acquired doxorubicin resistance. We next contrasted the expression profiles of ICBP90 and Topo IIα and tumor cell growth of different breast cancer cell lines to doxorubicin. Decreased expression levels of ICBP90 and Topo IIα were found in doxorubicin-resistant cells. To examine its function in chemoresistance, RNA interference (RNAi) and forskolin stimulation experiments further demonstrated that ICBP90 and Topo IIα were involved in the proliferation of cells that had acquired doxorubicin resistance. In MCF-7/Dox and ICBP90-siRNA cells, the cell growth wasn't inhibited by doxorubicin and preferentially arrested in G1 phase. However, after forskolin increased the Topo IIα expression, these breast cancer cells were again found to be inhibited by doxorubicin. Further, immunohistochemical assay breast cancer patients accepted EFC regimen showed ICBP90 was significantly associated with tumor cell proliferation, locally advanced disease and Topo IIα expression. In conclusion, down-regulation of ICBP90 induced the descended expression of Topo IIα protein which is the target enzyme of doxorubicin. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The morbidity of breast cancer has increased dramatically during the past two decades. Doxorubicin (adriamycin) is a potent anthracycline chemotherapeutic agent and is widely used for the treatment of breast cancer, this is attributed to being highly effective and well tolerated. However, despite patients carefully receive adjuvant doxorubicin based chemotherapy regimens after surgical removal of the primary breast tumor, many of these patients recur. This poor prognosis may be attributed to a low incidence of complete response and change in the biologic characteristics of tumor after tumor eradication. The anthracycline drugs is topoisomerase poisons specifically targeting Topo IIα (Withoff et al., 1996). Topo II reduces DNA twisting and super-coiling, allowing selected regions of DNA to untangle and thus engage in transcription, replication, or repair processes (Kellner et al., 2002; Chen and Liu, 1994). Previous studies have reported that deletion or mutation of Topo IIα in breast cancer are associated with drug resistance, whereas high concentrations of Topo IIα indicate anthracycline sensitivity, poor nuclear differentiation and high proliferation (Fry et al., 1991; Sullivan et al., 1987).
⁎ Corresponding author at: Department of Medical Oncology, The Third Hospital of Harbin Medical University, Harbin, 150040, China. Tel./fax: +86 451 86298276. E-mail address: [email protected] (Q. Zhang). 1 Wang Jingxuan and Song Ying contributed equally to this work. 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.01.042
Deregulation of genes that are involved in the progression from G1 to S phase of the cell cycle is a frequent event in numerous tumor types, including lung cancer (Ye et al., 2010), melanoma (Scaini et al., 2009) and esophageal carcinoma (Li et al., 2010). Recently ICBP90 (Inverted CCAAT box Binding Protein, 90 kD) is identified and characterized by a RING finger domain, shares structural homology with the nuclear phosphoprotein Np95 (nuclear protein 95) and the NIRF (Np95/ICBP90-like Ring finger protein), both involved in cellcycle regulation. Functional studies have demonstrated that Np95 is required for the G1/S transition, overexpression of Np95 and cycE/ cdk2 complexes can force reentry of terminally differentiated cells into the cell cycle (Bonapace et al., 2002). NIRF expression is high in proliferating phase but significantly low in G0/G1 phase in normal TIG-7 and WI-38 cells (Mori et al., 2002), while consistently high in tumoral HT-1080 and HepG2 cells. Likewise, ICBP90 also has function in cell-cycle regulation, the expression of ICBP90 is observed throughout cell cycle but peaks at late G1 and G2/M phases in noncancerous human cells. Down-regulation of ICBP90 in HCT116 and Jurkat cells is a critical event in G1-arrested cells (Arima et al., 2004; Abbady et al., 2003). In addition, ICBP90 inhibitors could inhibit the expression of ICBP90 after 24 h treatment, arrested the cell cycle at G1/G0 stage and blocked its entrance from S phase to G2/M phase. Consequently, one proposed function of ICBP90 is that it plays a role in tumorogenesis by deregulating the expression of genes leading to loss of control of G1/S transition and therefore involved in cancerogenesis (Bronner et al., 2007). Among these genes, we can find topoisomerase IIα (Hopfner et al., 2001), RB1 (Jeanblanc et al.,
34
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
2005), putatively p16INK4A, p14ARF and RARb (Unoki et al., 2004). Considering that ICBP90 could bind to one of the inverted CCAAT boxes (called ICB2) of the Topo IIα gene promoter, which is of extreme importance for anthracycline drugs. Therefore, we blocked the expression of ICBP90 in order to observe its effect to doxorubicin resistance. 2. Materials and Methods 2.1. Cell Line For our studies, we used human breast cancer cell line MCF-7 and its sublines resistant to cytotoxic effects of doxorubicin (MCF-7/Dox). They were obtained from Heilongjiang Tumor Research Institute (Heilongjiang, China). MCF-7/Dox cells were derived from the drug sensitive MCF-7 cells by stepwise selection with doxorubicin (adriamycin) (Mehta, 1994). The cells were grown in RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum and 100 units/ml penicillin and 2 mg/ml streptomycin (Sigma Aldrich Co.). To maintain their resistance, this cell line was cultured in the presence of 2 μM doxorubicin (Pfizer Italia S.r.l., Italy) and passaged for 1 week in a drug-free medium before each experiment. 2.2. Western Blot Analysis Cells were rinsed in PBS, and cells were lysed by loading buffer (2% SDS, 10% glycerol, and 50 mm Tris pH 6.8). Samples were subsequently sonicated for 15 s using a microtip. Protein concentration was quantified using Biorad protein assay for all samples lysed in SDS. Cell lysates were denatured at 95 °C for 5 min after the addition of dithiothreitol (DTT) and bromophenol blue. Approximately 25 μg of protein was loaded per lane and separated using a 5–20% gradient polyacrylamide gel (PAGEL, ATTO), and transferred to nitrocellulose membrane (Hybond, Amersham pharmacia). The membranes were probed with primary antibodies. The following antibodies were used: mouse monoclonal antibody for ICBP90 (1RC1C-10, 100 ng/mL), Topo IIα (Ki-S1, 1:1000 dilution; Sigma) and β-actin (1:10 000 dilution; Sigma). Then the membranes were incubated with horseradish peroxidase conjugated secondary antibodies and visualized by chemiluminescense detection system.
cell was treated with a control medium as control. The medium was replaced every 2 days. Then cell proliferation was measured using the MTT assay. The number of viable cells remaining after treatment with various agents was determined by 3-(4,5-di-methylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma Aldrich Co.) reduction assay. Cells were seeded in 96 well microtiter plates at a concentration of 5 × 104 cells/well. Following 48 h of treatment with various concentrations of doxorubicin, MTT was added to each well at a final concentration of 0.5 mg/ml. Four hours later, 100 μl DMSO was added to each well to dissolve the resulting formazan crystals. Absorbance was read at 490 nm using an enzyme-linked immunosorbent assay reader (SpectraMax; Molecular Devices, Sunnyvale, CA). Data were collected from three separate experiments and the percentage of doxorubicininduced cell growth inhibition was determined by comparison to DMSO-treated control cells. MCF-7/Dox and ICBP90-siRNA transfected cells were treated with 0 (control), 25, 50, or 100 μM of forskolin (sigma) and 1 μM doxorubicin for 72 h. Then western blot analysis and MTT assay were then performed. 2.5. Flowcytometric Cell Cycle Analysis Cells plated at a density of 6 106 cells/ml were incubated with doxorubicin (1 μM). The cells were harvested after 48 h, washed then fixed with 70%(v/v) methanol. Staining of cellular DNA was performed using DNA staining solution, composed of 50 μg/l propidium iodide and 500 mg/l protease-free RNase. Cell cycle analysis was performed with the FACSanto software package, BD biosciences corp. The percentage of cells with DNA content corresponding to G0/G1, S and G2/M phases, respectively was computed by the planimetry of the histogram and then compared for different treatment groups. 2.6. Validation on Clinical Samples
ICBP90 small interfering RNA (siRNA) oligonucleotides targeting different regions of ICBP90 mRNA were designed according to a previously reported method, and were synthesized by Qiagen. The control siRNA is a scrambled sequence and does not target any known mammalian mRNA. The oligoribonucleotide used were purchased from Dharmacon, Inc. (Lafayette, CO, U.S.A.) and are as follows: ICBP90siRNA-sense: 5′-AAGATCCAGGAGCTGTTCCAC-3′; anti-sense: 5′- AACGACTGTCGGATCATCTTC-3′. Control siRNA-sense: 5′-UCCGUUUCGGUCCACAUUC-3′; anti-sense: 5′-GAAUGUGGACCGAAACGGA-3′. For siRNA transfection, 60 nM siRNAs were diluted in Opti-MEM with Lipofectamine 2000 (Invitrogen) according to the manufacturers' instructions.
2.6.1. Patient Selection and Immunohistochemical Assay For this study, we randomly selected 220 breast cancer patients presented at the third Hospital of Harbin Medical University, Harbin, China, during the period from February 2003 to January 2006. Informed consent was obtained from all the patients or their relatives prior to analysis, and the project was approved by the Institutional Ethics Committee of Harbin Medical University. These 220 patients had received preoperative EFC chemotherapy before surgery. EFC regimen containing epirubicin 60 (or 70) mg/m2 i.v. on days 1 and 8, 5-fluorouracil 500 mg/m2 i.v. on day 1 and cyclophosphamide 500 mg/m2 i.v. on day 1 every three weeks for 2 cycles. The median age was 48 years (range, 30–81 years). Thirty-eight patients (30.9%) were postmenopausal. The distribution of cases according to clinical stage, histologic type, and menopausal status is shown in Table 1. ICBP90 and Topo IIα were carried out for immunohistochemistry. Tissues from human primary breast cancer were embedded in paraffin and fixed in 10% buffered formalin (Sigma). Serial histological sections (3 mm) were incubated overnight at room temperature with the ICBP90 mAb or with an anti-Topo IIα mAb (Neo Markers, Union City, CA), and specifically bound antibodies were visualized by a Streptavidin–Biotin complex (LAB/LSAB method, Dako LSAB2 System Kit, DAKO, Carpinteria, CA).
2.4. MTT Assay of Cell Viability
2.7. Statistical Analysis
MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells were seeded into 96-well plates at a density of 5 ⁎ 105 cells/well in a RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum and 100 units/ml penicillin and 2 mg/ml streptomycin. Then MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells were respectively treated with doxorubicin (1 μM) for 7 days. The MCF-7
Direct comparisons between MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells or between control and treatment effects were assessed using a Student's t-test analysis. One-way ANOVA was used to assess the significant differences between the treatment groups. Covariates included in multiple logistic regression analysis were ICBP90 status, breast density, histologic grade (1 versus 2/3), locally advanced disease
2.3. Gene Silencing with Small Interfering RNA
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
35
Table 1 Clinical characteristics of the 220 eligible patients. Characteristics
Total (n%)
Age years Median Range Menopausal status Premenopausal Postmenopausal Histologic type Ductal carcinoma Lobular carcinoma Unclassified Clinical stage I II III IV
48 30–81 152 (69.1) 68 (30.9) 168 (76.4) 50 (22.7) 2 (0.9) 52 (23.6) 98 (44.5) 64 (29.1) 6 (2.7)
and Topo IIα status. The statistical calculations were done using SPSS 12.0 with the exception of conditional logistic regression analysis for which LogXact 5.0 was used, and the confidence limits for odds ratios (OR) for which special programming in Maple 8 was applied (Breslow and Day, 1980). In all statistical comparisons, P b 0.05 was considered to be statistically significant. 3. Result 3.1. Different Expression of ICBP90 and Topo IIα in MCF-7 and MCF-7/Dox Cells We investigated the levels of ICBP90 and Topo IIα in these cell lines. ICBP90 is known to play a critical role in regulating the expression of Topo IIα. Decreased basal levels of ICBP90 and Topo IIα were observed in the MCF-7/Dox cells, as compared with the MCF-7 cells. Densitometry readings revealed that, in MCF-7 cells, the levels of ICBP90 protein was 2fold greater (Pb 0.05), respectively, and the levels of Topo IIα protein was 3.5-fold than those of MCF-7/Dox cells (Pb 0.05), respectively (Fig. 1). 3.2. ICBP90 Knockdown Reduced Topo IIα Expression and Inhibited the Anti-proliferative Effect of Doxorubicin in ICBP90-siRNA Transfected Cells To investigate a possible role for ICBP90 in doxorubicin resistance, ICBP90 was specifically silenced with short interfering RNA (siRNA) in MCF-7 cells. Immunoblot analysis performed at 48 h after the transfection of ICBP90 siRNAs revealed a marked reduction of ICBP90 expression compared to control siRNA, it inhibited the expression of Topo IIα (Fig. 1). Later, we tested the anti-proliferative effect of doxorubicin (1 μM) by MTT. However, ICBP90-siRNA transfected cells were the same as MCF-7/Dox cells, significantly decreased the sensitivity to doxorubicin (Fig. 2). Later, the resistant variants ICBP90-siRNA transfected and MCF-7/Dox cells were originated by growing initial MCF-7 cells with raising concentrations of or doxorubicin (from 0.1 to 64 μM), respectively. Doxorubicin was added twice a week after reseeding. Every two months, cell survival was analyzed by MTT assay. IC 50 values for MCF-7, MCF-7/ Dox and ICBP90-siRNA transfected cells were 1, 24 and 32 μM of doxorubicin, respectively. Therefore, MCF-7/Dox and ICBP90-siRNA transfected cells were 24 and 32 times as much resistant to the cytotoxic effect doxorubicin as compared with the MCF-7 cells, respectively. 3.3. Forskolin Positively Modulated Topo IIα and Restored the Anti-Proliferative Effect of Doxorubicin in ICBP90-siRNA Transfected and MCF-7/Dox Cells In order to study the role of ICBP90 in anthracycline resistance, furthermore, we treated the MCF-7/Dox and ICBP90-siRNA trans-
Fig. 1. Western blotting analysis of ICBP90 and Topo IIα in MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells, using β-actin gene as an endogenous control (A). Relative protein levels for ICBP90 and Topo IIα in the three breast cancer cell lines, MCF7, MCF-7/Dox and ICBP90-siRNA transfected cells (B). The results are expressed as the means (S.D.) of triplicate wells and are representative of three separate experiments. *significant differences from MCF-7 at P b 0.05.
fected cells with Forskolin. Stimulation of MCF-7/Dox and ICBP90siRNA transfected cells with increased concentrations of forskolin both resulted in a significant dose dependent increase in Topo IIα between 25 and 100 μM (Fig. 3A). Up to 3-fold of Topo IIα increase was reached at 100 μM of forskolin when compared to the basal level (Fig. 3A and B). Forskolin significantly increased Topo IIα expression level after 24 h of treatment, but had no effect on ICBP90 expression level. Results from MTT assays indicated that doxorubicin could inhibited the viability of MCF-7/Dox and ICBP90-siRNA transfected cells with forskolin at concentrations ≥ 25 μM (Fig. 3C and D). The treatment of MCF-7/Dox and ICBP90-siRNA transfected cells with 0 (control), 25, 50, or 100 μM of forskolin and 1 μM doxorubicin resulted in a dose and time-dependent inhibition of cell growth. For MCF-7/Dox cells, with inhibition rates of 8.6 ± 0.8%,11.4 ± 1.0%, and 20 ± 1.22%(P b 0.05), respectively, at 24 h, compared with control, 15.7 ± 1.20%, 25.5 ± 1.80%, and 37.2 ± 2.33%, respectively, at 48 h (all P values b 0.05) and 20 ± 1.28%,30.7 ± 2.08%, and 38.7 ± 2.86%, respectively, at 72 h (all P values b 0.05). For ICBP90-siRNA transfected cells, with inhibition rates of 17.6 ± 1.88%, 25.5 ± 2.05%, and 31.8 ± 2.60%, respectively, at 24 h (all P values b0.05), compared with control, 13.6 ± 1.68%, 16.8 ± 2.02%, and 27.1 ± 2.38%, respectively, at 48 h (all P values b0.05) and 18.1 ± 1.96%, 27.7 ± 2.06, and 33.7 ± 2.82%, respectively, at 72 h (all P values b 0.05).
36
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
(ICBP90-siRNA transfected cells: 20.46 ± 5.18% in S phase and 13.42 ± 3.18% in G2/M phase; and MCF-7/Dox cells: 25.21 ± 4.89% in S phase and 12.76 ± 3.57% in G2/M phase). The increase of cell population at the S and G2/M phases was accompanied by a decrease of cell population in the G1 phase of the cell cycle. Furthermore, a significant fraction of cells transfected with ICBP90-siRNA arrested at G1 phase (P b 0.05, Table 2), suggested that reduction of ICBP90 contributes to G1 arrest induced by DNA-damage activated checkpoint signals. 3.5. Biomarker Potential of ICBP90 in Clinical Samples To assess the prognostic power of ICBP90 gene in resistance, clinical samples were used to validate the results of cell culture model, we analyzed the expression of ICBP90 by immunohistochemical assay in 220 breast cancer patients. A significant negative correlation was seen between ICBP90 and histologic grade or locally advanced disease (P b 0.05). Patients with ICBP90-negative tumors after preoperative EFC chemotherapy were more likely to present with locally advanced disease, and 25 of 30 patients had ICBP90-negative tumors. The relationship with lymph node spread and breast density was not significant (P = 0.08 and P = 0.55; Table 3). However, a highly significant relationship was found between tumors expressing ICBP90 and Topo II α (OR, 2.8; 95% CI, 1.4–6.5). 4. Discussion
Fig. 2. Growth curves of MCF-7 (A), MCF-7/Dox (B) and ICBP90-siRNA transfected (C) cells in the presence of doxorubicin. MCF-7, MCF-7/Dox and ICBP90-siRNA transfected cells were respectively treated with doxorubicin (1 μM) for 7 days. MCF-7 (A), MCF-7/ Dox (B) and ICBP90-siRNA transfected (C) cells were treated with control medium as control, respectively. The medium was replaced every 2 days. Cell proliferation was measured using the MTT assay. The results are expressed as the means (S.D.) of triplicate wells and are representative of three separate experiments. *significant differences from control at P b 0.05.
3.4. Down-regulation of ICPB90 Contributes to Doxorubicin Resistance by Mediating G1 Arrest After determining that down-regulation of ICPB90 exhibited resistance to doxorubicin, we next investigated the effects of ICPB90 on cell cycle progression. In contrast to MCF-7 cells, analysis of cell cycle distribution after doxorubicin treatment presented evidence of a preferential block of ICBP90-siRNA transfected and MCF-7/Dox breast cancer cells in G0/G1 phase. However, a shift of cell distribution into S and G2/M phases was noticed upon with 100 μM forskolin addition in ICBP90-siRNA transfected(31.92 ± 6.56% in S phase and 15.07 ± 4.78% in G2/M phase) and MCF-7/Dox (32.56 ± 7.08% in S phase and 16.82 ± 5.16% in G2/M phase) breast cancer cells, compared to Dox alone
Acquired resistance to doxorubicin has become a serious obstacle in current breast cancer treatment. Here, it was revealed that abnormal cell cycle regulation was a crucial factor in this crosstalk, the silencing of ICBP90 made cell cycle arrest at G1/S transition and resulted doxorubicin resistance in MCF-7 cell. Cells contain numerous pathways designed to protect them from the genomic instability or toxicity. The cell division phases can be divided into two functional phases (S and M phases) and two preparatory phases (G1 and G2). Cell cycle checkpoints are important control mechanisms that ensure the proper growth. Chemotherapy activates cell cycle checkpoint signaling pathways arresting the cell cycle and inducing apoptosis. Checkpoint defects are frequent in human cancer and can confer resistance toward anticancer chemotherapy. For instance, chemotherapeutic agents cause double-strand DNA breaks involving the function of BRCA1, so losing it contributes to G2/M-phase checkpoint defect in BRCA1-mutant HCC1937 breast cancer cell line. However, transfecting a wild-type BRCA1 gene in these cells could correct the function of this checkpoint (Quinn et al., 2003). A p53independent branch of the DNA damage checkpoint is activated that involves the Chk1 kinase and arrests cells solely before mitosis. While, defects in the function of p53 might eliminate G1 arrest, thereby allowing cells with damaged DNA to enter S phase, which may cause tumor resistance to several different cytotoxic compounds (Geisler et al., 2003). In addition, overexpression of LATS2 inhibited cyclinE/CDK2 kinase activity and also resulted in G1/S cell cycle arrest (Li et al., 2003), which leads to breast cancer in patients who were sensitive to EC (Cyclophosphamide + Epirubicin) treatment. In summary, interfering cell cycle checkpoints could suppress tumor growth (Takahashi et al., 2005). Topo IIα has been regarded as one of the target enzymes for drugresistance cancer cells. Low level expression of Topo IIα protein leads to drug resistance to anthracycline (Harris and Hochhauser, 1992). The relationship between the down-regulated expressive level of Topo IIα protein and drug resistance has been proved by researches on the clinical efficacy of drugs targeted on Topo IIα (Arpino et al., 2005). ICBP90 is first isolated as a protein that binds to a CCAAT box in the promoter region of Topo IIα gene (Hopfner et al., 2002), which localizes in nuclei and contains an ubiquitin-like (UbL) domain (Hopfner et al., 2001). Many cancer cell lines such as HeLa cell line,
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
37
Fig. 3. Effects of forskolin on the expression of ICBP90 and Topo IIα (A and B). Effects of forskolin on the proliferation of MCF-7/Dox (C) and ICBP90-siRNA transfected cells (D). MCF7/Dox and ICBP90-siRNA transfected cells were treated with 0 (control), 25, 50, or 100 μM of forskolin and 1 μM doxorubicin. They were then incubated in a medium containing forskolin and doxorubicin for 72 h. Western blot analysis and MTT assay were then performed. The data are representative of at least three separate experiments. *significant differences from control at P b 0.05.
Jurkat cell line and A549 cell line show ICBP90 expressed throughout the entire cell cycle. Through cAMP signaling pathway, ICBP90 exit escape of forskolin-treated cells from G1 phase and increases binding of ICBP90 to ICB2 element of Topo IIα gene promoter with subsequently up-regulated expression of Topo IIα protein(Trotzier et al., 2004). Thus, it was of interest to compare the expression of ICBP90 and Topo IIα in ICBP90-siRNA transfected, MCF-7/Dox and MCF-7 cells. Compared to MCF-7 cells, ICBP90 and Topo IIα expressions were lower in ICBP90-siRNA transfected cells, which were consistent with our finding of little ICBP90 and Topo IIα in MCF7/Dox cell line. These data showed the decreased expression of ICBP90 subsequently down-regulated the expression of Topo IIα both in ICBP90-siRNA transfected and MCF-7/Dox cells. ICBP90 is likely to play a role in regulating S phase entry, depletion of ICBP90 expression by siRNA transfection induced G1 arrest in a significant population of HeLa cells when the cells were treated with doxorubicin to induce DNA damage. In addition, doxorubicin is a phasespecific drug, affecting predominantly S and G2 phases of the cell cycle, so we next compared cell cycle traverse in the ICBP90-siRNA transfected, MCF-7/Dox and MCF-7cells upon cell exposure to doxorubicin. We have Table 2 Cell cycle distribution of MCF-7 cells and sublines with induced resistance to doxorubicin and/or forskolin. Cell line
Treatment
MCF-7
Dox Dox + For Con Dox Dox + For Con Dox Dox + For Con
MCF-7/Dox
ICBP90-siRNA
Percentage of cells in a phase of the cell cycle G0/G1
S
G2/M (%)
61.11 ± 12.03 56.16 ± 11.33 45.42 ± 9.18 60.17 ± 12.33 50.62 ± 11.29 62.03 ± 13.42 62.86 ± 13.16 53.01 ± 5.46 66.12 ± 14.72
25.12 ± 5.35 28.01 ± 5.11 34.18 ± 6.27 26.31 ± 4.98 32.56 ± 7.08 25.21 ± 4.89 23.15 ± 5.03 31.92 ± 6.56 20.46 ± 5.18
13.77 ± 4.12 15.83 ± 4.05 20.40 ± 5.87 13.52 ± 3.85 16.82 ± 5.16 12.76 ± 3.57 13.99 ± 4.53 15.07 ± 4.78 13.42 ± 3.18
shown that in the MCF-7 cells, incubation with doxorubicin resulted in the significant decrease of S (from 34.18 ± 6.27% to 25.12 ± 5.35%) and G2/M (from 20.40 ± 5.87% to 13.77 ± 4.12%) phase percentage. Meanwhile, doxorubicin treatment resulted in the accumulation of MCF-7 cells in G0/G1 phase with the G0/G1 cell percentage increasing from 45.42 ± 9.18% to 61.11 ± 12.03%. In contrast, exposure to doxorubicin in ICBP90-siRNA transfected cells and MCF-7/Dox cells had no effect on cell cycle traverse (Table 2). Up-regulation of several genes involved in cell cycle regulation, DNA replication, and modification including the Topo IIα gene have been described in Schwann cells as response to forskolin (Schworer et al., 2003). In this study, we found that forskolin plus doxorubicin
Table 3 The number and proportion of cases (%) for different variables and molecular markers according to ICBP90 expression. Characteristics Breast density Low (b 30%) Mod (30–70%) High (N70%) Histologic grade 1–2 3 Locally advanced disease No Yes Nodal status Neg Pos TopoII α Neg Pos a
CI.
ICBP90 pos (n = 85), n%
ICBP90 neg (n = 135), n%
OR (95%CI)
P
11 (12.9) 60 (70.6) 14 (16.5)
19 (14.1) 95 (70.4) 21 (15.6)
1.5 (1.4–1.6)a 1.0
0.55
78 (91.8) 7 (8.2)
90 (66.7) 45 (33.3)
1.0 5.9 (2.6–13.5)
b0.01
80 (94.1) 5 (5.9)
110 (81.5) 25 (18.5)
1.0 3.4 (1.3–9.3)
b0.05
60 (70.6) 25 (29.4)
78 (57.8) 57 (42.2)
1.0 1.8 (0.9–3.3)
0.08
19 (22.3) 66 (77.7)
75 (55.5) 60 (44.4)
2.8 (1.4–6.5) 1.0
b0.05
The mean difference between ICBP90-negative and ICBP90-positive cases with 95%
38
J. Wang et al. / European Journal of Pharmacology 656 (2011) 33–38
resulted in a dose and time-dependent growth inhibitions of ICBP90siRNA transfected and MCF-7/Dox cells, forskolin can increase the expression of Topo IIα but could not affect the expression of ICBP90, while down-regulation of ICBP90 would result in significantly decrease of Topo IIα. It seemed that, other proteins other than ICBP90 could affect the expression of Topo IIα in response to forskolin treatment. So in other words, except for ICBP90 protein, there are possible that other proteins regulating Topo IIα are also responsible to anthracycline resistance, such as CRM1 (Turner et al., 2009), R16 (Zhu et al., 2007), HMGB1 and HMGB2 (Stros et al., 2009). Induction of the clinical investigation was done in order to test our preliminary data, the clinical study demonstrated that strong and consistent associations occurred between the negative ICBP90 protein expression and features of aggressive breast cancer, for example anthracycline resistance, such as high tumor cell proliferation, locally advanced disease and reduced Topo IIα expression (Table 3). In conclusion, our study indicates that down-regulation of ICBP90 can be associated with the acquisition of anthracycline resistance in breast cancer. Therefore, the proper control of ICBP90 activity might be a potential strategy to prevent the development of acquired resistance to doxorubicin based anticancer agents. Funding This study was supported by Special Science Funding for Young Scholars in the Heilongjiang Province, China (QC2009C08). Disclosure The authors declare no conflict of interest. Acknowledgments This study was supported, in part, by the Department of Pathology, the third Hospital of Harbin Medical University (Harbin, China). This experiment was finished in the oncobiology key lab of Heilongjiang common institution of higher learning. References Abbady, A.Q., Bronner, C., Trotzier, M.A., Hopfner, R., Bathami, K., Muller, C.D., Jeanblanc, M., Mousli, M., 2003. ICBP90 expression is down-regulated in apoptosis-induced Jurkat cells. Ann. NY Acad. Sci. 1010, 300–303. Arima, Y., Hirota, T., Bronner, C., Mousli, M., Fujiwara, T., Niwa, S., Ishikawa, H., Saya, H., 2004. Downregulation of nuclear protein ICBP90 by p53/p21Cip1/WAF1 dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition. Genes Cells 9, 131–142. Arpino, G., Ciocca, D.R., Weiss, H., Allred, D.C., Daguerre, P., Vargas-Roig, L., Leuzzi, M., Gago, F., Elledge, R., Mohsin, S.K., 2005. Predictive value of apoptosis, proliferation, HER-2, and topoisomerase II alpha for anthracycline chemotherapy in locally advanced breast cancer. Breast Cancer Res. Treat. 92, 69–75. Bonapace, I.M., Latella, L., Papait, R., Nicassio, F., Sacco, A., Muto, M., Crescenzi, M., Di Fiore, P.P., 2002. Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry. J. Cell Biol. 157, 909–914. Breslow, N.E., Day, N.E., 1980. Statistical methods in cancer research. Volume I — the analysis of case–control studies. IARC Sci. Publ. 32, 5–338. Bronner, C., Achour, M., Arima, Y., Chataigneau, T., Saya, H., Schini-Kerth, V.B., 2007. The UHRF family: oncogenes that are drugable targets for cancer therapy in the near future? Pharmacol. Ther. 115, 419–434. Chen, A.Y., Liu, L.F., 1994. DNA topoisomerases: essential enzymes and lethal drugs. Annu. Rev. Pharmacol. Toxicol. 34, 191–218.
Fry, A.M., Chresta, C.M., Davies, S.M., Walker, M.C., Harris, A.L., Hartley, J.A., Masters, J.R., Hickson, I.D., 1991. Relationship between topoisomerase II level and chemosensitivity in human tumor cell lines. Cancer Res. 51, 6592–6595. Geisler, S., Børresen-Dale, A.L., Johnsen, H., Aas, T., Geisler, J., Akslen, L.A., Anker, G., Lønning, P.E., 2003. TP53 gene mutations predict the response to neoadjuvant treatment with 5-fluorouracil and mitomycin in locally advanced breast cancer. Clin. Cancer Res. 9, 5582–5588. Harris, A.L., Hochhauser, D., 1992. Mechanisms of multidrug resistance in cancer treatment. Acta Oncol. 31, 205–213. Hopfner, R., Mousli, M., Garnier, J.M., Redon, R., du Manoir, S., Chatton, B., Ghyselinck, N., Oudet, P., Bronner, C., 2001. Genomic structure and chromosomal mapping of the gene coding for ICBP90, a protein involved in the regulation of the topoisomerase II alpha gene expression. Gene 266, 15–23. Hopfner, R., Mousli, M., Oudet, P., Bronner, C., 2002. Overexpression of ICBP90, a novel CCAAT-binding protein, overcomes cell contact inhibition by forcing topoisomerase II alpha expression. Anticancer Res. 22, 3165–3170. Jeanblanc, M., Mousli, M., Hopfner, R., Bathami, K., Martinet, N., Abbady, A.Q., Siffert, J.C., Mathieu, E., Muller, C.D., Bronner, C., 2005. The retinoblastoma gene and its product are targeted by ICBP90: a key mechanism in the G1/S transition during the cell cycle. Oncogene 24, 7337–7345. Kellner, U., Sehested, M., Jensen, P.B., Gieseler, F., Rudolph, P., 2002. Culprit and victim — DNA topoisomerase II. Lancet Oncol. 3, 235–243. Li, Y., Pei, J., Xia, H., Ke, H., Wang, H., Tao, W., 2003. Lats2, a putative tumor suppressor, inhibits G1/S transition. Oncogene 22, 4398–4405. Li, Q., Kawamura, K., Ma, G., Iwata, F., Numasaki, M., Suzuki, N., Shimada, H., Tagawa, M., 2010. Interferon-lambda induces G1 phase arrest or apoptosis in oesophageal carcinoma cells and produces anti-tumour effects in combination with anti-cancer agents. Eur. J. Cancer 46, 180–190. Mehta, K., 1994. High levels of transglutaminase expression in doxorubicin resistant human breast carcinoma cells. Int. J. Cancer 58, 400–406. Mori, T., Li, Y., Hata, H., Ono, K., Kochi, H., 2002. NIRF, a novel RING finger protein, is involved in cell-cycle regulation. Biochem. Biophys. Res. Commun. 296, 530–536. Quinn, J.E., Kennedy, R.D., Mullan, P.B., Gilmore, P.M., Carty, M., Johnston, P.G., Harkin, D.P., 2003. BRCA1 functions as a differential modulator of chemotherapy-induced apoptosis. Cancer Res. 63, 6221–6228. Scaini, M.C., Rossi, E., de Siqueira Torres, P.L., Zullato, D., Callegaro, M., Casella, C., Quaggio, M., Agata, S., Malacrida, S., Chiarion-Sileni, V., Vecchiato, A., Alaibac, M., Montagna, M., Mann, G.J., Menin, C., D'Andrea, E., 2009. Functional impairment of p16(INK4A) due to CDKN2A p.Gly23Asp missense mutation. Mutat. Res. 671, 26–32. Schworer, C.M., Masker, K.K., Wood, G.C., Carey, D.J., 2003. Microarray analysis of gene expression in proliferating Schwann cells: synergistic response of a specific subset of genes to the mitogenic action of heregulin plus forskolin. J. Neurosci. Res. 73, 456–464. Stros, M., Polanská, E., Struncová, S., Pospísilová, S., 2009. HMGB1 and HMGB2 proteins up-regulate cellular expression of human topoisomerase II alpha. Nucleic Acids Res. 37, 2070–2086. Sullivan, D.M., Latham, M.D., Ross, W.E., 1987. Proliferation dependent topoisomerase II content as a determinant of antineoplastic drug action in human, mouse, and Chinese hamster ovary cells. Cancer Res. 47, 3973–3979. Takahashi, Y., Miyoshi, Y., Takahata, C., Irahara, N., Taguchi, T., Tamaki, Y., Noguchi, S., 2005. Down-regulation of LATS1 and LATS2 mRNA expression by promoter hypermethylation and its association with biologically aggressive phenotype in human breast cancers. Clin. Cancer Res. 11, 1380–1385. Trotzier, M.A., Bronner, C., Bathami, K., Mathieu, E., Abbady, A.Q., Jeanblanc, M., Muller, C.D., Rochette-Egly, C., Mousli, M., 2004. Phosphorylation of ICBP90 by protein kinase A enhances topoisomerase II alpha expression. Biochem. Biophys. Res. Commun. 319, 590–595. Turner, J.G., Marchion, D.C., Dawson, J.L., Emmons, M.F., Hazlehurst, L.A., Washausen, P., Sullivan, D.M., 2009. Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition. Cancer Res. 69, 6899–6905. Unoki, M., Nishidate, T., Nakamura, Y., 2004. ICBP90, an E2F-1 target, recruits HDAC1 and binds to methyl-CpG through its SRA domain. Oncogene 23, 7601–7610. Withoff, S., De Jong, S., De Vries, E.G., Mulder, N.H., 1996. Human DNA topoisomerase II: biochemistry and role in chemotherapy resistance. Anticancer Res. 16, 1867–1880. Ye, X., Zhou, W., Li, Y., Sun, Y., Zhang, Y., Ji, H., Lai, Y., 2010. Darbufelone, a novel antiinflammatory drug, induces growth inhibition of lung cancer cells both in vitro and in vivo. Cancer Chemother.Pharmacol 6, 277–285. Zhu, H., Huang, M., Yang, F., Chen, Y., Miao, Z.H., Qian, X.H., Xu, Y.F., Qin, Y.X., Luo, H.B., Shen, X., Geng, M.Y., Cai, Y.J., Ding, J., 2007. R16, a novel amonafide analogue, induces apoptosis and G2-M arrest via poisoning topoisomerase II. Mol. Cancer Ther. 6, 484–495.