Autocrine production of interleukin-6 confers cisplatin and paclitaxel resistance in ovarian cancer cells

Cancer Letters 295 (2010) 110–123

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Autocrine production of interleukin-6 confers cisplatin and paclitaxel resistance in ovarian cancer cells Yue Wang a,b,*, Xiu Long Niu a, Ye Qu c, Jian Wu a, Ya Qin Zhu a, Wei Jia Sun a, Ling Zhi Li b,d a

Department of Immunology, Medical College of Chinese People’s Armed Police Forces, Tianjin, People’s Republic of China Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard, Tianjin, People’s Republic of China c Department of Microbiology, Medical College of Chinese People’s Armed Police Forces, Tianjin, People’s Republic of China d Department of Pharmaceutical Chemistry, Medical College of Chinese People’s Armed Police Forces, Tianjin, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 5 December 2009 Received in revised form 19 February 2010 Accepted 19 February 2010

Keywords: Interleukin-6 (IL-6) Chemoresistance Multidrug resistance-related genes Apoptosis inhibitory proteins Ovarian cancer

a b s t r a c t It has been shown that IL-6 is elevated in the serum and ascites of ovarian cancer patients, and increased IL-6 concentration correlates with poor prognosis and chemoresistance. However, the role of IL-6 expression in the acquisition of the chemoresistance phenotype and the underlining mechanisms of drug resistance in ovarian cancer cells remain unclear. Here we demonstrate that both exogenous (a relatively short period of treatment with recombination IL-6) and endogenous IL-6 (by transfecting with plasmid encoding for sense IL-6) induce cisplatin and paclitaxel resistance in non-IL-6-expressing A2780 cells, while deleting of endogenous IL-6 expression in IL-6-overexpressing SKOV3 cells (by transfecting with plasmid encoding for antisense IL-6) promotes the sensitivity of these cells to anticancer drugs. IL-6-mediated resistance of ovarian cancer cells exhibits decreased proteolytic activation of caspase-3. Meanwhile, the further study demonstrates that the chemoresistance caused by IL-6 is associated with increased expression of both multidrug resistance-related genes (MDR1 and GSTpi) and apoptosis inhibitory proteins (Bcl-2, Bcl-xL and XIAP), as well as activation of Ras/MEK/ERK and PI3K/Akt signaling. Therefore, modulation of IL-6 expression or its related signaling pathway may be a promising strategy of treatment for drug-resistant ovarian cancer. Ó 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The currently most frequently used therapy for the treatment of ovarian cancer is a combination of carboplatin and paclitaxel. Although up to 80% of patients initially respond well to therapy, the majority of patients suffer recurrent disease [1]. In some cases, patients respond well to repeated treatment with the same chemotherapeutic regimen but they will inevitably succumb to the disease following the eventual emergence of drug resistance. As a

* Corresponding author. Address: Department of Immunology, Medical College of Chinese People’s Armed Police Forces, Hedong District Chenlin Road No. 211, Tianjin 300162, People’s Republic of China. Tel.: +86 22 60578099; fax: +86 22 022 24799262. E-mail address: [email protected] (Y. Wang). 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.02.019

consequence, the overall 5-year survival is only 30% [1]. Thus, there is a pressing need to either identify novel therapies for ovarian cancer or to discover drugs which (re)sensitize tumor cells to existing chemotherapy. Several factors have previously been implicated in drug resistance, including genes which regulate drug influx and efflux, drug metabolism, damage repair, and the apoptotic response to drug-induced damage. Indeed, it is possible that numerous resistance mechanisms could contribute to a drugresistant phenotype and these mechanisms might be coordinately regulated [2]. Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a major role in the response to injury or infection and is involved in the immune response, inflammation, and hematopoiesis [3]. IL-6 is a vital regulator of physiologic functions in diverse organ systems, including the central

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nervous system, the cardiovascular system, the immune system, the hepatic system, and others [3]. In addition, its dysregulation impacts numerous disease states, including many types of cancer [4]. Several studies have addressed the role of IL-6 in promoting the chemoresistance of multiple myeloma [5,6], renal cell carcinoma [7], cholangiocarcinoma [8], prostate cancer [9–11], and breast cancer cells [12]. IL-6 was elevated in the serum and peritoneal fluid from patients with ovarian cancer [13–15], and high levels of IL6 in body fluids were associated with poor prognosis and resistance to chemotherapy [16,17]. It has been demonstrated that ovarian cancer cell lines NOM1 and SKOV cultured with IL-6 have increased chemotactic and/or chemokinetic activity and increased overall invasiveness [18]. Moreover, IL-6 has been reported to be is a potent proangiogenic cytokine in ovarian cancer cells [19]. More recent studies have suggested that IL-6 secreted by ovarian cancer cell lines could be involved in their tumorigenic potential, particularly potentiating their capacity to secrete matrix metalloproteinase-9 [20]. Our previous study demonstrated that IL-6 may contribute to ovarian cancer cell growth partly through the activation of androgen receptor (AR) and estrogen receptor (ER) pathways [21,22]. In vitro studies with ovarian cancer cell lines show that generation of paclitaxel-resistant sublines is often associated with increased IL-6 mRNA expression and protein secretion using cDNA array technology [23]. However, the role of IL-6 expression in the acquisition of the chemoresistance phenotype and the underlining mechanisms of drug resistance in ovarian cancer cells are not yet fully understood. IL-6 acts through a hexametric receptor, which contains the ligand-binding IL-6a chain and the common cytokine receptor signal-transducing subunit gp130. The binding of IL-6 to gp130 activates multiple signal transduction pathways such as signal transducers and activators of transcription (JAK/STATs) pathway, Ras/MEK (mitogen-activated protein or extracellular signal-regulated kinase kinase)/ERK (extracellular signal-regulated kinase) pathway, and PI3K (phosphotidylinositol 3 kinase)/Akt pathway [24]. Recently, growing evidence suggests activation of Ras/MEK/ERK [25–27] and PI3K/Akt [28–31] signaling pathways play an important role in chemoresistance of ovarian cancer. Therefore, we hypothesized that one potential mechanism that IL-6 induces chemoresistance of ovarian cancer cells by triggering activation of Ras/MEK/ ERK and PI3K/Akt signaling. In this study, we investigated the role of IL-6 expression in modulating cellular sensitivity to chemotherapeutic drugs in ovarian cancer cells. Furthermore, we also explored possible underlying mechanisms involved in drug resistance induced by IL-6. Our data suggest that the autocrine production of IL-6 by ovarian cancer cells promotes resistance of these cells to chemotherapy through decrease of proteolytic activation of caspase-3. The further study demonstrates that IL-6-induced resistance of ovarian cancer cells may be associated with up-regulation of both multidrug resistance-related genes [multidrug resistance gene 1 (MDR1) and glutathione S transferase pi (GSTpi)] and apoptosis inhibitory proteins [Bcl-2, Bcl-xL and X-

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linked inhibitor of apoptosis (XIAP)], as well as activation of Ras/MEK/ERK and PI3K/Akt signaling. 2. Materials and methods 2.1. Cell lines and cell culture Human ovarian cancer cell lines A2780, CAOV-3, SKOV3 and ES-2 were obtained from the American Type Culture Collection. A2780, SKOV-3 and ES-2 cells were cultured in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal bovine serum (FBS) (Life Technologies, Inc.), CAOV-3 cells were grown in DMEM (Life Technologies, Inc.) with 15% FBS. Recombinant human IL-6 (R&D Systems, Minneapolis, MN) was used to pretreat A2780 cells. The cells were cultured in the presence of exogenous IL-6 (50 ng/ml) for 10 days. IL-6 was added to the culture every 2 days [12]. After the pretreatment period, the cells (A2780/preIL-6) were harvested, washed, and replated in the presence of IL-6, and their resistance to cisplatin or paclitaxel was determined by the MTT assay. 2.2. Generation and selection of cells stably transfected with pcDNA3.1(+)-ssIl-6 (i.e., sense IL-6 vector) and pcDNA3.1(+)asIL-6 (i.e., antisense IL-6 vector) Transfection was done using Lipofectamine™ 2000 (Invitrogen, San Diego, CA) as recommended by the manufacturer’s instructions. A2780 and SKOV-3 cells (4  105) were plated onto 6-well plates until 90–95% confluence before transfection. A2780 cells were transfected with 4 lg of pcDNA3.1(+)-ssIL-6, and SKOV-3 cells were transfected with 4 lg of pcDNA3.1(+)-asIL-6, kindly provided by Dr. Allen C. Gao (Roswell Park Cancer Institute, Buffalo, NY). Selection for the neomycin gene was initiated 48 h after transfection by adding 500 lg (A2780 cells) or 600 lg (SKOV-3 cells) of G418 (Life Technologies)/ml to the supplemented culture medium. This selection medium was changed every 2 days for 4 weeks, until all non-transfected cells died. Resistant cell clones were isolated and expanded for further characterization. The empty vector pcDNA3.1(+) was also transfected into A2780 or SKOV-3 cells and served as negative controls. 2.3. Semiquantitative RT-PCR Total RNA was isolated from cells with TRIzol (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Primer sequences were designed by Vector NTI 8 software and synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China). The primer sequences were as follow: IL-6, 50 -TTGACAAACAAATTCGGTACA-30 (forward) and 50 GAGG TGCCCATGCTACA-30 (reverse), for MDR1, 50 -TGAC TACCAGGCTCGCCAATGAT-30 (forward) and 50 -TGTGCCACCAAGTAGGCTCCAAA-30 (reverse), for GSTpi, 50 -CAGGAGGG CTCACTCAAAG-30 (forward) and 50 -GATCAGCAGCAAGTCCAGCAG-30 (reverse), for Bcl-2, 50 -TGCACCTGACGCCCTTCAC-30 (forward) and 50 -AGACAGC CAGGAGAAATCA

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AACAG-30 (reverse), for Bcl-xL, 50 -ATGTCTCAGAGCAACCGGGA GC-30 (forward) and 50 -GCGATCCGACTCACCAATACCT-30 (reverse), for XIAP, 50 -ATGATACCATCTTCCAAAATCC-30 (forward) and 50 -TTTCTGTAATGAAGTCTGAC TT-30 (reverse), for b-actin, 50 -TGGAATCCTGTGGCATCCATGAAAC-30 (forward) and 50 -TAAAACGCAGCTCAGTAACAGTCC-30 (reverse). One Step RNA PCR Kit (AMV) (TaKaRa Biotechnology) was used to do RT-PCR. PCR products were fractionated on 1.5% agarose gel and analyzed with Quantity One-4.5.6 software (Bio-Rad, Hercules, CA). The results were normalized against b-actin, and presented as target mRNA: b-actin ratio. 2.4. Enzyme-linked immunosorbent assay (ELISA) The cells were cultured for 48 h in 1 ml of medium containing 5% charcoal-stripped FBS (sFBS) (Life Technologies, Inc.). The supernatants were collected and clarified by centrifugation. The level of IL-6 was measured using ELISA kits (R&D Systems) according to the manufacturer0 s instructions.

2.5. Western blot analysis Cells were lysed in ice-cold RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris–HCl pH 8.0, 1 mM PMSF, 10 lg/ml leupeptin, and 100 lg/ml aprotinin) for 45 min on ice. The lysates were centrifuged to remove cellular debris. Supernatants were analyzed for protein concentration using the bicinchoninic acid assay kit (Pierce Biochemicals, Rockford, IL). Forty microgram of total cell lysates were subjected to 8–12 % SDS–PAGE gels and analyzed by blotting with rabbit polyclonal anti-IL-6Ra, anti-gp130, anti-MDR1, anti-GSTpi, anti-Bcl-2, anti-Bcl-xL, or anti-XIAP antibody (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Membranes were stripped by incubating with stripping buffer, which contains 62.5 mM Tris–HCl (pH 6.8), 2% SDS, and 100 mM b-mercaptoethanol, at 50 °C for 30 min and then blotted with mouse monoclonal anti-b-actin antibody (Sigma). Immunodetection was performed using the corresponding secondary HRP-conjugated antibody, and HRP activity was

Fig. 1. Expression pattern of IL-6 and its receptor (IL-6Ra and gp130) as well as different sensitivity to cisplatin and paclitaxel in four ovarian cancer cell lines. (A and B) Expression of IL-6 protein and mRNA in ovarian cancer cell lines. Secreted IL-6 production in four cell lines were analyzed by ELISA. Data are shown as the mean of three separate experiments with triplicate samples and represent the mean ± SD. The mRNA level of IL-6 was detected by semiquantitative RT-PCR. Target fragment levels were normalized against b-actin. (C) Expression of IL-6Ra and gp130 protein in ovarian cancer cell lines. The same amount of total cell lysate of A2780, CAOV-3, SKOV-3 and ES-2 cells were separated by 8% SDS–PAGE gel, followed by Western blot with anti-IL6Ra antibody, anti-gp130 antibody or anti-b-actin antibody. Target protein levels were normalized against b-actin to control for variance in sample loading and transfer. (D and E) Different responsiveness to cisplatin or paclitaxel in A2780, CAOV-3, SKOV-3 and ES-2 cells was assessed by the MTT assay. The experiment shown is representative of three independent experiments with similar results.

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detected using chemiluminescent substrate kit (SuperSignalÒ Westpico Trial Kit, Pierce Biochemicals). The intensity of relative band was assessed by densitometric analysis (Scion Image Analysis software program). A2780 cells were plated in 100-mm culture dishes with the density of 4  106 cells for 24 h, and then incubated in 5% sFBS with vehicle DMSO, or PD98059 (25 or 50 lM), or wortmannin (100 or 200 nM) for 30 min prior to IL-6 (50 ng/ml) for 6 h. Total cell lysates were isolated and quantified. The phosphorylation status of ERK and Akt was analyzed by Western blot as described above, except that the filters were probed with anti-phospho-ERK or anti-phospho-Akt antibodies (Cell Signaling Technology, Beverly, MA) to detect phosphorylated ERK or phosphorylated Akt, visualized by chemiluminescent substrate kit. The filters were subsequently stripped and then reprobed with anti-ERK (BD Biosciences, San Diego, CA) or anti-Akt antibodies (Cell Signaling Technology, Beverly, MA) to detect both the phosphorylated and unphosphorylated forms of ERK or Akt. 2.6. Cytotoxicity assay In vitro cytotoxicity assays were performed by MTT assay as previously described [21,22]. MTT was obtained from Sigma (St. Louis, MO, USA). Briefly, 4  104 cells per well were plated in 96-well plates. Culture medium was RPMI 1640 containing increasing concentrations of cisplatin or paclitaxel (all obtained from commercial sources). After culture for 48 h, MTT solution (0.5 mg/ml PBS) was added to each well and incubated for 4 h. After dissolving the resulting formazan product with acid-isopropanol, the absorbance was measured at 490 nm using ELISA microplate reader. Data represents the average absorbance of six wells in one experiment. The percentage of surviving cells was estimated by dividing the A490 nm of treated cells by the A490 nm of control cells. The IC50 is defined as the drug concentration required to inhibit A490 to 50% of the control value. IC50 values were estimated from the dose– response curve. Data were derived from at least three independent experiments. A2780 cells pretreated with IL-6 (A2780/preIL-6) were plated in 96-well plates at 4  103 cells per well for 24 h, and then incubated in 5% sFBS with vehicle DMSO, or PD98059 (25 or 50 lM), or wortmannin (100 or 200 nM) for 30 min prior to IL-6 (50 ng/ml) and cisplatin (1 or 10 lM) or paclitaxel (0.01 or 0.1 lM) or control for 48 h. MTT assay was performed as described above. Data are shown as the mean ± SD of two separate experiments with sextuple samples.

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measured at a wavelength of 405 nm on a plate reader. Blank values were subtracted, and fold increase in activity was calculated based on activity measured from untreated cells. Each sample was measured in triplicates. 2.8. Statistical analysis Data are expressed as the mean of three experiments, each in triplicate or sextuple samples for individual treatments or dosage regimens. Statistical analysis was carried out using a one-way ANOVA, followed by Tukey’s post hoc test. Values are presented as the mean ± SD. All statistical tests were two-sided and were considered to be statistically significant at P < 0.05. 3. Results 3.1. Comparing expression levels of IL-6 and its receptor (IL-6Ra and gp130) as well as sensitivity to cisplatin and paclitaxel in four ovarian cancer cell lines In order to investigate the role of IL-6 expression in the acquisition of the chemoresistance phenotype in ovarian cancer cells, we first analyzed the expression of IL-6 and its receptor (IL-6Ra and gp130) as well as the response to cisplatin and paclitaxel in four ovarian cancer cell lines. The

2.7. Caspase-3 activation assay Caspase-3 activity was measured using caspase-3 colorimetric assay kit (R&D Systems) according to the manufacturer’s instruction. Briefly, cells left untreated or treated with 10 lM cisplatin or 0.1 lM paclitaxel for 24 h and then lysed in lysis buffer for 10 min on ice. The lysed cells were centrifuged at 14,000 rpm for 5 min, and 100 lg of protein was incubated with 20 ll of reaction buffer and 10 ll of caspase-3 substrate at 37 °C for 1 h, and absorbance was

Fig. 2. Treatment with IL-6 increased the resistance of A2780 cells. A2780 cells were cultured in the presence (A2780/preIL-6) or absence (A2780) of exogenous IL-6 for 10 days. After the pretreatment period, the cells were replated in the presence or absence of IL-6, and their resistance to cisplatin (A) or paclitaxel (B) was determined by the MTT assay. The experiment shown is representative of three independent experiments with similar results.

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Fig. 3. Representative ELISA and RT-PCR demonstrating levels of IL-6 expression in A2780 and SKOV-3 cells and their transfectants. A2780 cells were stably transfected with empty vector or with vector encoding for sense IL-6, and SKOV-3 cells were stably transfected with empty vector or with vector encoding for antisense IL-6. Three stable sense IL-6-transfected A2780 clones that produced low (A2780/ssIL-6L), middle (A2780/ssIL-6M) and high (A2780/ssIL-6H) levels of IL-6 (A and B), two stable antisense IL-6-transfected SKOV-3 clones that were middle (SKOV-3/asIL-6Mi) and high (SKOV-3/asIL-6Hi) inhibition of IL-6 production (C and D), and the corresponding control vector-transfected A2780 and SKOV-3 cells were isolated for further studies.

Fig. 4. Effect of IL-6 expression on the responsiveness of ovarian cancer cells to cisplatin and paclitaxel. Three stable sense IL-6-transfected A2780 clones and their parental and control vector-transfected cells were plated out, and the sensitivity of the cells to cisplatin (A) and paclitaxel (B) was determined by the MTT assay. Similarly, the responsiveness of two antisense IL-6-transfected SKOV-3 clones and their parental and control vector-transfected cells to cisplatin (C) and paclitaxel (D) was examined by the MTT assay.

Y. Wang et al. / Cancer Letters 295 (2010) 110–123 secretion levels of IL-6 were significant various in four ovarian cancer as shown in Fig. 1A. High, middle and low levels of IL-6 secretion were observed in CAOV-3 (15873.47 ± 620.52 pg/ml), SKOV-3 (2347.76 ± 78.65 pg/ml) and ES-2 cells (211.59 ± 11.98 pg/ml), respectively. However, no IL-6 was detected in the supernatant from A2780 cells. The mRNA levels of IL-6 resembled their respective protein levels in four ovarian cancer cells (Fig. 1B). As determined by Western blot analysis, these four cell lines were demonstrated to express IL-6Ra and gp130 (Fig. 1C). The sensitivity to cisplatin and paclitaxel also varied among these cell lines as shown in Fig. 1D and E. A2780 cells were the most sensitive (IC50 for cisplatin and paclitaxel were 8.06 ± 0.49 lV and 0.74 ± 0.08 lV respectively), then came ES-2 cells (IC50 for cisplatin and paclitaxel were 10.63 ± 0.86 lV and 1.41 ± 0.50 lV respectively), whereas CAOV-3 (IC50 for cisplatin and paclitaxel were 74.40 ± 0.82 lV and 9.65 ± 0.35 lV respectively) and SKOV-3 cells (IC50 for cisplatin and paclitaxel were 66.19 ± 3.42 lV and 6.67 ± 0.17 lV respectively) were drug-resistant, indicating that autocrine production level of IL-6 by ovarian cancer cell lines were inversely associated with their sensitivity to cisplatin and paclitaxel. Taken together, these results suggest that IL-6 receptor-bearing ovarian cancer cell lines, A2780 (non-IL-6-expressing and drug-sensitive) and CAOV-3 or SKOV-3 (IL-6-overexpressing and drug-resistant) are the suitable cell models to investigate the effect of IL-6 on cisplatin- or paclitaxel-mediated cytotoxicity in ovarian cancer cells.

3.2. IL-6 confers cisplatin and paclitaxel resistance in ovarian cancer cells Previous reports have shown that overexpression of IL-6 correlates with poor prognosis and chemoresistance [16,17]. In correlation, our above results showed that autocrine production level of IL-6 by ovarian

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cancer cell lines were inversely associated with their responsiveness to cisplatin and paclitaxel. It suggests that IL-6 could play a role in the resistance of ovarian cancer cells to the cytotoxic activities of anticancer compounds. To address this hypothesis, A2780 cells were cultured in the presence or absence of IL-6 for 10 days. After the pretreatment period, the cells were harvested, washed, and replated in the presence or absence of IL-6, and their resistance to cisplatin or paclitaxel was determined by the MTT assay [21,22]. Pretreatment of A2780 cells with IL-6 caused 6.25-fold and 7.31-fold increase in resistance to cisplatin and paclitaxel, respectively (Fig. 2), indicating that the presence of exogenous IL-6 increased the resistance of ovarian cancer cells to cisplatin or paclitaxel treatment. To determine whether the endogenous production of IL-6 by tumor cells could provide self-protection against drug-induced cell death, we constitutively expressed IL-6 in A2780 cells and inhibited expression of IL-6 in SKOV-3 cells and examined the effect of IL-6 expression on the drug resistance of these cells. A2780 cells were transfected with plasmid encoding for sense IL-6 and SKOV-3 cells were transfected with plasmid encoding for antisense IL-6. Stable A2780 (A2780/ssIL-6) and SKOV-3 (SKOV-3/asIL-6) transfected clones were isolated and screened for their ability to produce IL-6. Three representative clones that produced low (23.53 ± 2.24 pg/ml, A2780/ssIL-6L), middle (58.22 ± 5.78 pg/ml, A2780/ ssIL-6 M) and high (106.18 ± 12.32 pg/ml, A2780/ssIL-6H) levels of IL-6 (Fig. 3A) and two representative clones that were middle (67.66%, SKOV-3/asIL-6 Mi) and high (83.25%, SKOV-3/asIL-6Hi) inhibition of IL-6 production (Fig. 3C) compared with the corresponding parental (i.e., untransfected) and control vector-transfected A2780 and SKOV-3 cells were chosen for subsequent studies. The levels of IL-6 gene expression in the these stable transfected clones were also examined by semiquantitative RT-PCR analysis. The levels of IL-6 mRNA were consistent with the secreted IL-6 levels in these stable transfected clones (Fig. 3B and D).

Fig. 5. Caspase-3 activity in sense IL-6-transfected A2780 cells (A and B), antisense IL-6-transfected SKOV-3 cells (C and D) and the corresponding parental and control vector-transfected cells at baseline and with cisplatin or paclitaxel treatment. The cells were treated with 10 lM cisplatin or 0.1 lM paclitaxel for 24 h. Caspase-3 activity was measured using the caspase-3 colorimetric assay. Data are shown as the mean of three separate experiments with triplicate samples and represent the mean ± SD.

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To determine whether the endogenous production of IL-6 in A2780 cells can confer resistance to drug treatment, we examined the susceptibility or resistance of sense IL-6-transfected A2780 cells to cisplatin or paclitaxel treatment using the MTT assay. As shown in Fig.4A and B, A2780/ssIL-6L, A2780/ssIL-6M and A2780/ssIL-6H cells exhibited increased resistance to both cisplatin (7.13-fold, 7.47-fold and 8.34-fold, respectively) and paclitaxel (7.61-fold, 8.27-fold and 10.70-fold, respectively) as compared with parental A2780 cells (P < 0.001). Control A2780/pcDNA3.1(+) cells that did not produce IL-6 exhibited similar drug sensitivity to parental A2780 cells (P > 0.05). These data suggest that overexpressing of IL-6 confers a moderate level of drug resistance in ovarian cancer cells. To determine whether deleting of endogenous IL-6 in SKOV-3 cells could increase their responsiveness to drug treatment, we also examined the susceptibility or resistance of antisense IL-6-transfected SKOV-3 cells to cisplatin and paclitaxel. In correlation with data from sense IL-6-transfected A2780 cells, SKOV-3/asIL-6Mi and SKOV-3/asIL-6Hi cells exhibited increased responsiveness to both cisplatin (IC50 was 9.82 ± 0.56 lV and 6.71 ± 0.12 lV respectively, Fig. 4C) and paclitaxel (IC50 was 0.71 ± 0.08 lV and 0.55 ± 0.03 lV respectively, Fig. 4D) as compared with parental SKOV-3 (IC50 for cisplatin and paclitaxel were 66.50 ± 3.42 lV and 6.79 ± 0.45 lV respectively) and control SKOV-3/pcDNA3.1(+) cells (IC50 for cisplatin and paclitaxel were 65.32 ± 4.18 lV and 6.74 ± 0.33 lV respectively) (P < 0.001), which did not vary (P > 0.05). These results indicate that deleting of endogenous IL-6 by ovarian cancer cells restores their response to chemotherapy. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. To test whether cisplatin and paclitaxel resistance or susceptibility in sense or antisense IL-6-transfected cells may have affected caspase-3 activity with cisplatin or paclitaxel exposure, we measured caspase-3 activities in these cells after exposure to two drugs. As shown in Fig. 5, there was a significantly reduced level of caspase-3 in sense IL-6-transfected A2780 cells (Fig. 5A

and B), while there was a markedly increased in antisense IL-6-transfected SKOV3 cells (Fig. 5C and D) as compared with the corresponding parental and control vector-transfected cells.

3.3. IL-6 regulates MDR1 and GSTpi expression in ovarian cancer cells Cross-resistance to both cisplatin and paclitaxel suggests a multidrug resistant phenotype possibly explained by drug transport or metabolism, cellular repair or detoxification mechanisms. To evaluate this possibility the expression of several genes already known to be involved in the multidrug resistance phenomenon [MDR1, GSTpi, multidrug resistanceassociated protein (MRP), lung resistance-related protein (LRP) and topoisomerasel (Topol)] in above several ovarian cancer cell lines was first measured by semiquantitative RT-PCR and Western blot analysis. This analysis demonstrates that the mRNA and protein levels of MDR1,GSTpi (Fig. 6A and B), MRP, LRP and Topol (data not shown) are lower in A2780 and ES-2 cells, while those are higher in CAOV-3 and SKOV-3 cells, indicating that autocrine production levels of IL-6 by ovarian cancer cell lines were consistent with the expression levels of above several putative resistance factors in these cells. To determine the effects of exogenous and endogenous IL-6 on mutidrug resistance-related genes, we further studied IL-6-induced above several putative resistance factors expression of mRNA and protein in A2780 cells treated with IL-6, sense or antisense IL-6-transfected cells and the corresponding untransfected and control vector-transfected cells by semiquantitative RT-PCR and Western blot analysis. IL-6 significantly up-regulated the mRNA and protein levels of MDR1 and GSTpi (Fig. 6C and D) but not MRP, LRP and Topol (data not shown) in a dose-dependent manner in A2780 cells. The mRNA and protein levels of MDR1 and GSTpi (Fig. 7) but not MRP, LRP and Topol (data not shown) enhanced in sense IL-6-transfected A2780 cells (Fig. 7A and B), and reduced in antisense IL-6-transfected SKOV3 cells (Fig. 7C and D) compared with the corresponding parental and control vector-trans-

Fig. 6. Different MDR1 and GSTpi expression of mRNA and protein in four ovarian cancer cell lines (A and B). Exogenous IL-6 increases MDR1 and GSTpi expression in both mRNA and protein levels in A2780 cells (C and D). The mRNA and protein levels of MDR1 and GSTpi were measured with semiquantitative RT-PCR and Western blot, respectively, as described above. The experiment shown is representative of three independent experiments with similar results.

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Fig. 7. Expression of MDR1 and GSTpi mRNA and protein in three stable ssIL-6-transfected A2780 clones, vector control A2780/pcDNA3.1(+) and parental A2780 cells (A and B). The mRNA and protein levels of MDR1 and GSTpi in two asIL-6-transfected SKOV-3 clones, vector control SKOV-3/pcDNA3.1(+) and parental SKOV-3 cells (C and D). The mRNA and protein levels of MDR1 and GSTpi was detected by semiquantitative RT-PCR and Western blot, respectively, as described above. The experiment shown is representative of three independent experiments with similar results.

fected cells, which had no difference. Therefore, these data suggest that IL-6 may confer cisplatin and paclitaxel resistance in ovarian cancer cells by increasing MDR1 and GSTpi expression.

3.4. IL-6 regulates expression of Bcl-2, Bcl-xL and XIAP in ovarian cancer cells Previous study demonstrated that the expression of apoptosis inhibitory proteins [32–35] may be an important mechanism responsible for chemotherapy resistance in ovarian cancer. To determine the another potential mechanism through which IL-6 causes chemotherapy resistance in ovarian cancer cells, we also examined the expression levels of several apoptosis inhibitory proteins in above several ovarian cancer cell lines. The mRNA and protein levels of Bcl-2, Bcl-xL, XIAP (Fig. 8A and B) and myeloid cell leukemia 1 (Mcl-1) (data not shown), are lower in A2780 and ES-2 cells, where those are higher in CAOV-3 and SKOV-3 cells, suggesting that autocrine production levels of IL-6 by ovarian cancer cell lines were also in agreement with the expression levels of four apoptosis inhibitory proteins studied in these cells. To determine the effects of IL-6 on these apoptosis inhibitory proteins, we further studied IL-6-mediated mRNA and protein expression of Bcl-2, Bcl-xL, Mcl-1 and XIAP, in A2780 cells treated with IL-6, sense or antisense IL-6-transfected cells and the corresponding untransfected and control vector-transfected cells by semiquantitative RT-PCR and Western blot analysis. IL-6 significantly increased the mRNA and protein levels of Bcl-2, Bcl-xL and XIAP (Fig. 8C and D) in a dose-dependent manner, but had no effect on the mRNA and protein levels of Mcl-1 (data not shown) in A2780 cells. The mRNA and protein levels of Bcl-2, Bcl-xL and XIAP (Fig. 9) but not Mcl-1 (data not shown) up-regulated in sense IL-6-transfected A2780 cells (Fig. 9A and B), and down-regulated in antisense IL-6-transfected SKOV3 cells (Fig. 9C and D) compared with the corresponding untransfected and control vector-transfected cells, which did not vary. Taken together, these results suggest that IL-6 may cause chemoresistance in ovarian cancer cells by enhancing Bcl-2, Bcl-xL and XIAP expression.

3.5. IL-6-induced chemoresistance to ovarian cancer cells is through Ras/Raf/ MEK/ERK and PI3K/Akt activation To investigate what role Ras/MEK/ERK and PI3K/Akt pathways play in the signal transduction of IL-6 in ovarian cancer cells, we determined the effects of PD98059, a MEK1/2 specific inhibitor at 25 or 50 lmol/L, and wortmannin, a PI3K specific inhibitor at 100 or 200 nmol/L, on IL-6-induced phosphorylation of ERK and Akt and IL-6-induced cisplatin and paclitaxel resistance of A2780 cells. It was found that PD98059 and wortmannin significantly antagonized IL-6-induced phosphorylation of ERK and Akt, respectively (Fig. 10A and B), and both of them blocked IL-6-induced cisplatin and paclitaxel resistance (Fig. 10C and D) and the inhibitory effects of PD98059 and wortmannin were dependent on its concentration. These data confirm that activation of ERK and Akt are mediated by MEK1/2 and PI3K-dependent mechanism, respectively, and suggest that IL-6-induced cisplatin or paclitaxel resistance to ovarian cancer cells is through activation of Ras/MEK/ERK and PI3K/Akt.

4. Discussion It has been widely reported that IL-6 is overexpressed in the serum and ascites of ovarian cancer patients [13–15], and increased IL-6 concentration correlates with a poor final outcome and chemoresistance [16,17]. Previous work from our group and others has shown that IL-6 promotes ovarian cancer cell growth [21,22,36]. In the present study, we first demonstrated that autocrine production level of IL-6 by ovarian cancer cell lines, including A2780, CAOV3, SKOV-3, and ES-2, is inversely associated with their response to cisplatin and paclitaxel. Some studies have con-

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sistently demonstrated that CAOV3 cells secrete a large amount of IL-6 and are resistant to cisplatin, while A2780 cells do not and are responsive [37,38]. Our data further demonstrated that CAOV3 and A2780 cells were also paclitaxel-resistant and paclitaxel-sensitive respectively. Furthermore, we found that SKOV3 cells also produced higher levels of IL-6 and were cisplatin/paclitaxel-refractory. Notably, we also observed that A2780 cells expressed IL-6 receptor, though they did not secrete IL-6, suggesting that the expression of IL-6 receptor by ovarian cancer cells could be not associated with their IL-6 production status. Therefore, IL-6 receptor-bearing ovarian cancer cell lines, non-IL-6-expressing and cisplatin/paclitaxel-responsive A2780, and IL-6-overexpressing and cisplatin/paclitaxelresistant CAOV-3 or SKOV-3 were used to study the effect

of IL-6 on multidrug resistance, but in our study, A2780 and SKOV-3 cell lines were chosen as suitable cell models. Several recent studies have addressed the role of IL-6 in tumor cell chemoresistance, including in multiple myeloma [5,6], renal cell carcinoma [7], cholangiocarcinoma [8], prostate cancer [9–11], and breast cancer cells [12]. However, the role of IL-6 expression in the acquisition of the multidrug resistance phenotype in ovarian cancer has not been investigated. Here we show that both exogenous (a relatively short period of treatment with recombination IL-6) and endogenous IL-6 (by transfecting with plasmid encoding for sense IL-6) induce cisplatin and paclitaxel resistance in non-IL-6-producing A2780 cells, whereas deleting of endogenous IL-6 expression in IL-6-overexpressing SKOV3 cells (by transfecting with plasmid

Fig. 8. Various Bcl-2, Bcl-xL and XIAP expression of mRNA and protein in four ovarian cancer cell lines (A and B). Exogenous IL-6 increases Bcl-2, Bcl-xL and XIAP expression in both mRNA and protein level in A2780 cells (C and D). The mRNA and protein levels of MDR1 and GSTpi was detected by semiquantitative RT-PCR and Western blot, respectively, as described above. The experiment shown is representative of three independent experiments with similar results.

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encoding for antisense IL-6) promotes the sensitivity of these cells to anticancer drugs. Meanwhile, we confirm that IL-6-mediated resistance of ovarian cancer cells exhibits decreased proteolytic activation of caspase-3. These findings suggest that the production of IL-6 protects the cells from cytotoxic agents through down-regulation of proteolytic activation of caspase-3 and expression level of IL-6 is positively associated with their degree of chemoresistance in ovarian cancer cells. Other studies have shown that transfection of IL-6 into breast cancer [12] or osteosarcoma cell line [39] causes drug resistance and inhibition of IL-6 secretion in prostate cancer cell lines by anti-IL-6 antiserum [9] or antisense IL-6 oligonucleotide phosphorothioates [11] increases the sensitivity of these cells to anticancer drugs. Thus, some tumor cells may acquire the ability to express and produce IL-6 as a protective mechanism against drug induced death. The stimuli responsible

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for the constitutive expression of IL-6 in chemoresistant cells remain to be determined. The first and most widely studied factor known to modulate multidrug resistance is MDR1 (also known as ABCB1, which encodes the P-glycoprotein) and is believed to mediate multidrug resistance by reducing the intracellular accumulation of cytotoxic drugs and compounds. Another putative resistance factor is GSTpi which acts by detoxification of some chemotherapeutic drugs, namely, alkylating agents and Platinum-based drugs. In ovarian cancer, up to two-thirds tumor specimens have been found to overexpress MDR1 on immunohistochemistry [40–42], and this overexpression has been shown in some cases to correlate with poor overall survival and chemotherapy resistance. On the other hand, primary ovarian cancer biopsies [43] and cell lines [44] established from drug-resistant cancer patients have been shown to express high levels of GSTpi.

Fig. 9. The mRNA and protein levels of Bcl-2, Bcl-xL and XIAP in three stable ssIL-6-transfected A2780 clones (A and B), two asIL-6-transfected SKOV-3 clones (C and D), and the corresponding parental and vector control cells. The mRNA and protein levels of Bcl-2, Bcl-xL and XIAP were detected by semiquantitative RT-PCR and Western blot, respectively, as described above. The experiment shown is representative of three independent experiments with similar results.

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Previous studies have suggested that the overexpression of the GSTpi might be a critical factor in cisplatin resistance [45]. Recently studies have shown that inhibition of MDR1 and GSTpi expression by small interfering doublestranded RNAs (siRNA) in human multidrug resistant ovarian cancer cell lines enhances the intracellular accumulation of and restored sensitivity to cisplatin [46]. The results of the many studies cited above suggests that MDR1 and GSTpi play an important role in the mechanisms responsible for chemoresistance of ovarian cancer cells. Here we demonstrate that IL-6 regulates expression of MDR1 and GSTpi, but not MRP, LRP and Topolin ovarian cancer cells, in correlation, increases the resistance of the cells to cisplatin/paclitaxel treatment. Thus, the regulation of MDR1 and GSTpi gene expression is a potential mechanism by which IL-6 provides drug protection. The enhancement of MDR1 gene expression by IL-6 in ovarian cancer cells is in accordance with the results of Conze et al. [12] in breast cancer cell line, but is different from the results of Duan et al. [39], who have reported that MDR1 levels are not altered in IL-6 transfectants of osteosarcoma cell

line. While the up-modulation of GSTpi gene expression by IL-6 in ovarian cancer cells is in accordance with the results of Mizutani et al. [7] , who have reported that treatment of human renal carcinoma cells with cisplatin in combination with anti-IL-6 antibody or anti-IL-6 receptor can overcome their cisplatin resistance and that the down-regulation of GSTpi expression by anti-IL-6 or antiIL-6 receptor antibody may play a role in the enhanced cytotoxicity. In this study, we first demonstrate that IL-6 up-regulates MDR1 and GSTpi gene expression in a dosedependent manner in ovarian cancer cells. A number of studies have shown that the anti-apoptotic ability of IL-6 was associated with expression of the Bcl-2 family proteins such as Bcl-2 [47], Bcl-xL [48] and Mcl-1 [49]. Alternatively, Yamagiwa et al. [50] have recently demonstrated that IL-6 may increase expression of XIAP, a Inhibitor of Apoptosis (IAP) family member, by translation at an internal ribosome entry site to inhibit apoptosis in cholangiocarcinoma cells. Bcl-2 [32], Bcl-xL [33] and XIAP [34,35] have been shown in ovarian cancer to be associated with resistance to chemotherapy. Thus, we investi-

Fig. 10. Effects of PD98059 or wortmannin on IL-6-induced phosphorylation of ERK or Akt in A2780 cells and IL-6-mediated cisplatin and paclitaxel resistance to A2780 cells pretreated with IL-6 (A2780/preIL-6). The cells were pretreated with PD98059 (25 or 50 lM), wortmannin (100 or 200 nM) or DMSO of equal volume for 30 min at 37 °C before IL-6 was added into the medium. (A and B) A2780 cells were cultured for 6 h in the presence of IL-6. After the cells were collected and washed, whole-cell extracts were prepared and subjected to Western blot assay. (C and D) The cells were seeded into 96-well plate (4  104 cells per well) and cultured in the presence of IL-6 (50 ng/ml) and cisplatin (1 or 10 lM) or paclitaxel (0.01 or 0.1 lM) for 48 h. Afterwards, MTT assay was performed to determine the effect of PD98059 and wortmannin on IL-6-induced cisplatin or paclitaxe resistance to A2780 cells pretreated with IL-6 (A2780/preIL-6). The experiment shown is representative of three independent experiments with similar results. P > 0.05, compared with control; P < 0.001, compared with cisplatin or paclitaxel (no inhibitors).

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gated whether IL-6 alters the expression of apoptosis inhibitory proteins as a mechanism of drug resistance. We found that IL-6 increased expression of Bcl-2, Bcl-xL and XIAP but not Mcl-1 in a dose-dependent manner in ovarian cancer cells as a mechanism of drug resistance. Several lines of evidence implicated that the activation of Ras/MEK/ERK [25–27] and PI3K/Akt [28–31], the most important cell survival signalings, protects ovarian cancer cells from chemotherapy. It has been shown that cisplatin treatment activates ERK in ovarian cancer cells and that activation of ERK protects ovarian cancer cells from cisplatin-induced death [25–27]. Furthermore, inhibition of ERK signaling by the MEK1/2 inhibitor PD98059 blocked ERK activation and increased cisplatin sensitivity in SKOV3 cells [26]. It has reported that Akt inactivation sensitizes human ovarian cancer cells to cisplatin [28] and paclitaxel [29]. Moreover, the inactivation of a downstream targets of the PI3K/Akt pathway, such as BAD [28] and the transcription factors of Forkhead [51] and NFjB [52], also sensitize human ovarian cancer cells to cisplatin in vitro. Finally, Akt inactivation by a PI3K inhibitor also enhances the sensitivity of ovarian cancer to cisplatin [30] and paclitaxel [31] in vivo. In this study, IL-6-induced activition of ERK and Akt in ovarian cancer cells is blocked by their specific inhibitors to signal transducers, which inhibit IL-6-induced cisplatin and paclitaxel resistance of ovarian cancer cells. Taken together, our data suggest that IL-6 promotes chemoresistance of ovarian cancer cells via activation of multiple signal transduction pathways including ERK cascade and PI3K/Akt pathway. In toto, these results provide support for these signal transduction pathways as a strategy for reversing drug resistance. STAT3 is a major downstream component of the IL-6JAK signaling pathway. Recently, Duan et al. [53] has reported that inhibition of STAT3 expression increases the sensitivity of ovarian cancer cell lines to paclitaxel treatment in vitro, suggesting that the STAT3 pathway may also involve in chemoresistance of ovarian cancer cells. They found that IL-6 induced phosphorylation of STAT3 in several, but not all, of the examined ovarian cancer cell lines not including A2780 cells [53]. The lack of enhanced STAT3 phosphorylation in a subset of the cell lines may be due to suboptimization of IL-6 dose, absence of an intact IL-6RJAK-STAT3 axis, or saturation of the IL-6R from autocrine secretion of IL-6, thereby limiting augmentation of pathway activation through the addition of exogenous IL-6. Furthermore, it is possible that STAT3 could be activated through IL-6-independent mechanisms such as Src, epidermal growth factor receptor, or other cytokines like oncostatin in different cancer cells [54–59]. In summary, we conclude that IL-6 secreted by ovarian cancer cells may contribute to the refractoriness of these cells to conventional chemotherapy through down-regulation of proteolytic activation of caspase-3. Furthermore, IL6-induced chemoresistance may be associated with increase of both multidrug resistance-related genes (MDR1 and GSTpi) and apoptosis inhibitory proteins (Bcl-2, BclxL and XIAP), as well as activation of Ras /MEK/ERK and PI3K/Akt. Therefore, modulation of IL-6 expression or its related signaling pathway may be a promising strategy of treatment for drug-resistant ovarian cancer.

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Conflicts of interest None declared. Acknowledgments We thank Dr. Allen C. Gao for providing pcDNA3.1(+)ssIL-6 and pcDNA3.1(+)-asIL-6 plasmids. This work was supported by grants from Tianjin Municipal Science and Technology Commission 08JCYBJC06900, Key Program for Science and Technology in Medical College of Chinese People’s Armed Police Forces WJZ2007-1 and Postdoctoral Science Foundation of China 200804441177. References [1] R. Agarwal, S.B. Kaye, Ovarian cancer: strategies for overcoming resistance to chemotherapy, Nat. Rev. Cancer 3 (2003) 502–516. [2] A. Richardson, S.B. Kaye, Drug resistance in ovarian cancer: the emerging importance of gene transcription and spatio-temporal regulation of resistance, Drug Resist. Updates 8 (2005) 311–321. [3] P.B. Sehgal, L. Wang, R. Rayanade, H. Pan, L. Margulies, Interleukin-6type cytokines, Ann. NY. Acad. Sci. 762 (1995) 1–14. [4] D.S. Hong, L.S. Angelo, R. Kurzrock, Interleukin-6 and its receptor in cancer: implications for translational therapeutics, Cancer 110 (2007) 1911–1928. [5] J. Hardin, S. MacLeod, I. Grigorieva, R. Chang, B. Barlogie, H. Xiao, J. Epstein, Interleukin-6 prevents dexamethasone-induced myeloma cell death, Blood 84 (1994) 3063–3070. [6] D. Chauhan, S. Kharbanda, A. Ogata, M. Urashima, G. Teoh, M. Robertson, D.W. Kufe, K.C. Anderson, Interleukin-6 inhibits Fasinduced apoptosis and stress-activated protein kinase activation in multiple myeloma cells, Blood 89 (1997) 227–234. [7] Y. Mizutani, B. Bonavida, Y. Koishihara, K. Akamatsu, Y. Ohsugi, O. Yoshida, Sensitization of human renal cell carcinoma cells to cisdiamminedichloroplatinum (II) by anti-interleukin 6 monoclonal antibody or anti-interleukin 6 receptor monoclonal antibody, Cancer Res. 55 (1995) 590–596. [8] G.J. Gores, Cholangiocarcinoma: current concepts and insights, Hepatology 37 (2003) 961–969. [9] N. Borsellino, A. Belldegrun, B. Bonavida, Endogenous interleukin 6 is a resistance factor for cis-diamminedichloroplatinum and etoposidemediated cytotoxicity of human prostate carcinoma cell lines, Cancer Res. 55 (1995) 4633–4639. [10] A. Hobisch, R. Ramoner, D. Fuchs, S. Godoy-Tundidor, G. Bartsch, H. Klocker, Z. Culig, Prostate cancer cells (LNCaP) generated after longterm interleukin 6 (IL-6) treatment express IL-6 and acquire an IL-6 partially resistant phenotype, Clin. Cancer Res. 7 (2001) 2941–2948. [11] Y.S. Pu, T.C. Hour, S.E. Chuang, A.L. Cheng, M.K. Lai, M.L. Kuo, Interleukin-6 is responsible for drug resistance and anti-apoptotic effects in prostatic cancer cells, Prostate 60 (2004) 120–129. [12] D. Conze, L. Weiss, P.S. Regen, A. Bhushan, D. Weaver, P. Johnson, M. Rincón, Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells, Cancer Res. 61 (2001) 8851–8858. [13] M. Plante, S.C. Rubin, G.Y. Wong, M.G. Federici, C.L. Finstad, G.A. Gastl, Interleukin-6 level in serum and ascites as a prognostic factor in patients with epithelial ovarian cancer, Cancer 73 (1994) 1882– 1888. [14] G. Scambia, U. Testa, P.B. Panici, R. Martucci, E. Foti, M. Petrini, M. Amoroso, V. Masciullo, C. Peschle, S. Mancuso, Interleukin-6 serum levels in patients with gynecological tumors, Int. J. Cancer 57 (1994) 318–323. [15] C. Tempfer, H. Zeisler, G. Sliutz, G. Haeusler, E. Hanzal, C. Kainz, Serum evaluation of interleukin 6 in ovarian cancer patients, Gynecol. Oncol. 66 (1997) 27–30. [16] G. Scambia, U. Testa, P. Benedetti, E. Foti, R. Martucci, A. Gadducci, A. Perillo, V. Facchini, C. Peschle, S. Mancuso, Prognostic significance of interleukin 6 serum levels in patients with ovarian cancer, Brit. J. Cancer 71 (1995) 354–356. [17] R.T. Penson, K. Kronish, Z. Duan, A.J. Feller, P. Stark, S.E. Cook, L.R. Duska, A.F. Fuller, A.K. Goodman, N. Nikrui, K.M. MacNeill, U.A. Matulonis, F.I. Preffer, M.V. Seiden, Cytokines IL-1beta, IL-2, IL-6, IL8, MCP-1, GM-CSF and TNFalpha in patients with epithelial ovarian cancer and their relationship to treatment with paclitaxel, Int. J. Gynecol. Cancer 10 (2000) 33–41.

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