SGI-1776, an imidazo pyridazine compound, inhibits the proliferation of ovarian cancer cells by inactivating Pim-1

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1 中南大学学报 ( 医学版 ) J Cent South Univ (Med Sci) 214, 39(7) articles 论著 DOI: /j.issn SGI-1776, an imidazo pyridazine compound, inhibits the proliferation of ovarian cancer cells by inactivating Pim-1 XIE Jing 1, BAI Jun 2 (1. Deparetment of Obstetrics and Gynecology, the First Affiliated Hospital of University of South China, Hengyang Hunan 4211; 2. Deparetment of Obstetrics and Gynecology, Hangzhou Red Cross Hospital, Hangzhou 313, China) ABSTRACT KEY WORDS Objective: To investigate the antitumor effect of SGI-1776 on human ovarian cancer HO-891 cells and its molecular mechanism. Methods: HO-891 cells were cultured in vitro, and the proliferation inhibitory effects of SGI were determined by MTT assay and colony formation assay. The effect of SGI-1776 on the distribution of cell cycle phase was observed by flow cytometry with propidium iodide (PI) staining. The inhibition rate of migration and invasion were valued by transwell cell assay. Multiple molecular techniques, such as ELISA, Western blot, sirna and cdna transfection were used to explore the molecular mechanism. Results: SGI-1776 presented dramatic anti-tumor activity against HO-891 cells in vitro, inhibited the cells proliferation and colony formation, and attenuated the migration and invasion in a dosedependent manner, accompanied by cell cycle arrest in G 1 phase. SGI-1776 caused the proliferation inhibition with concomitant decrease in Pim-1 kinase activity, down-regulated the expression of Pim-1 protein and and its downstream genes, such as CDK6, pcdk6, CDK4, pcdk4, CDK2 and pcdk2, and increased the expression of P21 and P27. Down-regulation expression of Pim-1 by sirna followed SGI-1776 treatment resulted in enhanced cell proliferation inhibition rate and attenuated migration/invasion. Up-regulation of Pim-1 by cdna transfection attenuated SGI induced cell proliferation inhibition and its migration/invasion. Conclusion: Pim-1 mediates the biological effect of SGI-1776 in human ovarian cancer HO-891 cells, suggesting Pim-1 might be a novel target for human ovarian cancer. ovarian cancer; SGI-1776; proliferation; migration; invasion; Pim-1 Date of reception: Biography: XIE Jing, master, attending physician, mainly engaged in the research of tumor chemotherapy. Corresponding author: BAI Jun, nhfyxj213@126.com

2 65 中南大学学报 ( 医学版 ), 214, 39(7) SGI-1776 钝化 Pim-1 对卵巢癌细胞增殖和侵袭的抑制作用及其机制 谢晶 1 2, 白军 (1. 南华大学附属第一医院妇产科, 湖南衡阳 4211;2. 杭州市红十字会医院妇产科, 杭州 313) [ 摘要 ] 目的 : 检测 SGI-1776 对卵巢癌 HO-891 细胞增殖 迁移 侵袭的抑制作用及其可能机制 方法 : 体外培养卵巢癌 HO-891 细胞 ;MTT 法和克隆形成法检测 SGI-1776 对 HO-891 细胞的增殖抑制作用 ;PI 染色流式细胞术 (flow cytometry,fcm) 检测 SGI-1776 对 HO-891 细胞周期分布的影响 ;Transwell 法检测 SGI-1776 对 HO-891 细胞迁移及侵袭的抑制作用 ;ELISA,Western 印迹,siRNA/cDNA 转染检测 SGI-1776 对 HO-891 细胞增殖 迁移 侵袭抑制作用的可能分子机制 结果 :SGI-1776 在体外对 HO-891 细胞增殖 迁移 侵袭呈现出浓度依赖性的抑制作用, 阻滞细胞周期于 G 1 期 随 Pim-1 激酶活性降低,Pim-1 蛋白表达下调, 同时 Pim-1 下游蛋白 CDK6,pCDK6,CDK4, pcdk4,cdk2 和 pcdk2 表达下调, 而 P21 和 P27 蛋白表达上调 用 sirna 干扰沉默 Pim-1 基因, 则 SGI-1776 对 HO-891 细胞增殖 迁移 侵袭的抑制作用明显加强 ; 用 Pim-1-cDNA 转染 HO-891 细胞, 则能部分抵消 SGI-1776 对细胞增殖 迁移 侵袭的抑制作用 结论 :SGI-1776 通过钝化 Pim-1 而发挥其抑制卵巢癌 HO-891 细胞增殖 迁移和侵袭的作用, 提示 Pim-1 是卵巢癌治疗的新的分子靶 [ 关键词 ] 卵巢癌 ;SGI-1776; 增殖 ; 迁移 ; 侵袭 ;Pim-1 Ovarian cancer is one of the most common causes of death from all cancers among women and the leading cause of death from malignancies [1]. This high mortality rate is associated with difficulties in diagnosis of the early stages of the disease and because of a high rate of recurrence. Although 8% of cancers initially respond to chemotherapy, the majority ultimately reoccurs with less than 15% remaining in remission [2]. Therefore, the need for new drugs for the prevention and treatment of ovarian cancer is urgent. Ovarian cancer has been shown to have an activated Pim-1 signaling pathway, and it belongs to a family of evolutionary conserved transcriptional regulators that are characterized by the presence of an ATP-binding pocket, an active site, a kinase domain, and lack regulatory domains making them constitutively active [3]. The expression of provirus integration site for Moloney murine leukemia virus kinase 1 (Pim-1), is mediated by the Janus-activated kinase/signal transducers and activators of transcription (STAT) signaling pathway [4]. Pim-1 regulates cell-cycle progression by directly phosphorylating cyclin-dependent kinase inhibitor (CDKI), including P21 and P27 and so on [3]. Studies by Mahalingam et al [4] have shown that SGI-1776 may inhibit Pim-1 activation in renal cancer cells. Accordingly, we hypothesized that SGI-1776 may target the inactivation of Pim-1, which could represent a promising strategy for ovarian cancer therapy. In the present study, we investigated whether SGI [5-6] inhibited the proliferation, migration and invasion of ovarian cancer HO-891 cells and could be attributed to the inhibition of Pim-1 expression. We found that SGI-1776 decreased Pim-1 activity and downregulated the Pim-1 protein expression and its downstream genes, including CDK6, pcdk6, CDK2, pcdk2, CDK4, pcdk4, and increased P27 and P21, resulted in the proliferation, migration and invasion inhibition in ovarian cancer HO-891 cells. These results provided supportive evidence that Pim-1 is a legitimate target in ovarian cancer and that the targeted inactivation of Pim-1, SGI-1776 as shown here, would be highly relevant for designing novel strategies for the prevention of tumor progression and/or treatment of ovarian cancer. 1 Materials and methods 1.1 Cell culture and experimental reagents The human ovarian cancer HO-891 cells were purchased from China Centre for Type Culture Collection (CCTCC, Wuhan, China) and were maintained in DMEM medium (Invitrogen Corp., Carlsbad, CA, USA) supplemented with 1% FBS (Boster Wuhan Biological Technology Ltd., Wuhan, China), 4 mmol/l glutamine, 1 U/mL penicillin, and 1 µg/ml streptomycin, and incubated at 37 in a humidified atmosphere of 5% CO 2. SGI-1776 was kindly provided by Super-Gen Inc. (Dublin, CA, USA). Primary antibodies for Pim-1, CDK6, pcdk6,

3 SGI-1776, an imidazo pyridazine compound, inhibits the proliferation of ovarian cancer cells by inactivating Pim-1 XIE Jing, et al. 651 CDK4, pcdk4, CDK2, pcdk2, P21, P27 and GAPDH, as well as horseradish peroxidase-conjugated rabbit antimouse secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HTScan Pim-1 kinase assay kit was bought from Cell Signaling Technology (Zhonglian Friendship Technology Co., Ltd., Beijing, China). 3-(4,5-dimethylthi-azol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Giemsa stain and all other chemicals were obtained from Sigma (St. Louis, MO, USA). SGI-1776 was dissolved to make a 1 µmol/l stock solution and was added directly to the media at different concentrations. 1.2 MTT assay Cells were seeded in a 96-well plate at a density of 5 cells/well as previously described [7]. After incubation for 24 h, different concentrations of SGI-1776 (.625, 1.25, 2.5, 5, 1, 2, 4 µmol/l) were added to each well and cultured for 48 h. The medium was removed and then incubated with 5 mg/l MTT for 4 h. Next, the supernatant was removed after centrifugation. Finally, 1 µl of DMSO was added and an absorbance at 57 nm wavelength (A57) was measured by enzyme-labeling instrument (ELX-8 type; BioTek, Shanghai, China). Relative cell proliferation inhibition rate (IR)=(1 average A57 of the experimental group/average A57 of the control group) 1%. 1.3 Clonogenic assay Cells were plated in 24-well plates at a density of 3 cells/well for 24 h prior to the addition of various concentrations of SGI-1776 (2.5, 5 and 1 µmol/l). After 48 h of treatment, the drug-containing medium was removed and replaced with complete proliferation medium. Medium was changed every 3 days for 7 to 1 days until visible colonies formed. Colonies were simultaneously fixed and stained with Wright-Giemsa solution in methanol and manually counted. Individual stained colonies in each well were counted and the colony formation fraction was calculated as follows: colony number/(number of cells seeded plating efficiency), where plating efficiency is equivalent to the colony number divided by the number of cells seeded in the drug-free medium as previously described [8]. 1.4 Flow cytometry (FCM) analysis using propidium iodide staining Cells were seeded at a density of cells/well in 1 ml culture flasks for 24 h and then treated with the medium containing various concentrations of the testing agents and 1% FBS for 24 h. Then the cells were harvested and washed by cold PBS twice, fixed in 7% ethanol at 4 and stained in propidium iodide (PI) in darkness for distribution of cell cycle phase was performed by flow cytometry as previously described [9]. 1.5 Transwell cell migration and invasion assay Cells were seeded at a density of in the upper well of each transwell chamber. Conditioned culture medium and 3 µl DMEM containing 1% FBS were placed in the lower compartment of the chemotaxis chamber as a source of chemoattractants. The cells were incubated for 24 h at 37 with 5% CO 2. The cells that had migrated and invaded were fixed with methanol and stained with hexamethylpararosaniline (GenMed). Using light microscopy, at least 4 random fields were selected and the cells in each field were counted. Subsequently, the cells were eluted in 6 µl 33% acetic acid for 1 min and the optical density (OD) of the final cells through the matrigel was determined at 57 nm. HO-891 cells proliferation in standard medium were set as the control group [1]. 1.6 ELISA Anti-Pim-1 protein was diluted by carbonate buffer (.5 mol/l, ph=9.6) into 1 mg/l concentration, and.1 ml solution was added into each reacting hole of ploystyrene board, then incubated in 4 overnight, and then removed the solution and washed in PBS three times, at last blocked using 1% BSA/PBST at room temperature, and the Pim-1 activity of HO-891 cells exposed to different concentration of SGI-1776 (2.5, 5 and 1 µmol/l) was detected using HTScan Pim-1 kinase assay kit (Cell Signaling Technology) that peptide substrate of Pim-1 was biotinylated recombined utilizing colorimetric method to detect Pim-1 kinase activity. Pim-1 kinase activity of the normal saline () group was regarded as 1%, and the relative activity of Pim-1 kinase of different concentration SGI-1776 groups were got through comparing with group. All operations were according to the instruction manual [11]. 1.7 Plasmids and transfections Pim-1 sirna and sirna controls were obtained from Santa Cruz Biotechnology. The Pim-1 cdna plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, USA). Human ovarian cancer HO-891 cells were cultured at a density of cells/well in a 6-well plate, and transfected with 2 µg Pim-1 sirna, and cdna respectively using Lipofectamine 2 (Invitrogen) as described by Yang et al [12]. At 24 h post-transfection, G418 was added to the culture medium (4 µg/ml). The cells

4 652 中南大学学报 ( 医学版 ), 214, 39(7) were collected at 1 days post-transfection, when the majority of the cultured cells had died. The remaining cells were diluted to 1 cell/1 µl and 1 µl cells was added into the 96-well plate with G418. The cells were identified by fluorescence and the positive clones were transferred into a 6 well plate. Following amplifcation for 3 days, the cells were collected for Western blot. 1.8 Western blot Western blot was carried out as previously described [13]. Anti-Pim-1, anti-cdk6, anti-pcdk6, anti-cdk4, antipcdk4, anti-cdk2, anti-pcdk2, Anti-P27, anti-p21 and GAPDH were used as primary antibodies. Cells were lysed in a lysis buffer by incubating for 2 min at 4. The protein concentration was determined by using the Bio- Rad assay system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Total proteins were fractionated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) and transferred onto Polyvinylidene fluoride membrane (PVDF) (Millipore, Billerica, MA, USA). The signals were detected using an ECL advance Western blot analysis system (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). 1.9 Statistical analysis The database was set up with the SPSS 15. software package (SPSS Inc., Chicago, IL, USA) for analysis. Data are presented as the mean±standard deviation (x±s). The means of multiple groups were compared with oneway ANOVA, after the equal check of variance, and the comparisons among the means were performed using the LSD method. Statistical comparison was also performed with two-tailed t-test when appropriate. P<.5 was considered as statistically significant. 2 Results 2.1 Effects of SGI-1776 on cell proliferation inhibition and cell cycle arrest of ovarian cancer cells First, we examined the effect of SGI-1776 on cell proliferation viability of HO-891 cells using MTT assay, SGI-1776 showed the significant activity against HO-891 cells in a dose-dependent manner in vitro, the inhibiting effect of SGI-1776 (5 µmol/l) was roughly equivalent to DDP (5 µmol/l), and IC 5 of SGI-1776 treated for 48 h was (5.2±.6) µmol/l for HO-891 cells (Figure 1A). Furthermore, the inhibiting effect of SGI-1776 was sharply increased from 1.25 µmol/l to 2 µmol/l in vitro (Figure 1B). In order to confirm our results, we tested the effects of SGI-1776 on cell proliferation viability by clonogenic assay, HO-891 cells exposed to different concentration of SGI-1776 showed in a significant inhibition of colony formation in a dose-dependent manner compared to control (Figure 1C and 1D). Overall, the results from MTT assay and clonogenic assay suggest that SGI-1776 inhibited the proliferation of HO-891 cells in vitro. Additionally, to assess whether the loss of cell viability could be due to the induction of cell cycle arrest partly, we evaluated the effects of SGI-1776 treatment on the distribution in the cell cycle phase using FCM after PI staining. As shown in Figure 1E and 1F, in HO-891 cell line, SGI-1776 treatment caused a significant accumulation of cells in the G 1 phase and a marked decrease in the S/G 2 /M phase when compared with control cells. These results provided convincing data by showing that SGI-1776 induced the proliferation inhibition and accumulation of cell cycle in G 1 phase in HO-891 cells. 2.2 Effects of SGI-1776 on cell migration and invasion ability of ovarian cancer cells Transwell cell assay was performed to evaluate the migration and invasion activity of HO-891 cells, morphological migration and invasive features of HO- 891 cells in the differently conditioned media are shown in Figure 2A and 2C. SGI-1776 inhibited the migration and invasion of HO-891 cells in a dose-dependent manner compared with control groups, and the inhibiting migration and invasion rate of 5 µmol/l SGI-1776 was approximately equal to 5 µmol/l DDP (Figure 2B and 2D). These results suggested that SGI-1776 inhibited the migratory and invasive activity of HO-891 cells in a dosedependent manner in vitro. 2.3 Effect of SGI-1776 on the activity of Pim-1 kinase in ovarian cancer cells The effect of SGI-1776 on the activity of Pim-1 kinase in ovarian cancer cells was determined using HTScan Pim-1 kinase assay kit. The results showed that Pim- 1 kinase activity of HO-891 cells exposed to different concentration SGI-1776 (2.5, 5 and 1 µmol/l) was decreased in a dose-dependent manner, and the Pim- 1 kinase activity were (45.8±3.42)%, (3.7±6.9)% and (23.6±2.96)% compared with the groups (assume the activity is 1%) respectively, and there was significantly difference among groups (P<.5).

5 SGI-1776, an imidazo pyridazine compound, inhibits the proliferation of ovarian cancer cells by inactivating Pim-1 XIE Jing, et al. 653 Growth inhibition rate /% SGI μmol/l SGI μmol/l Time/h A Growth inhibition rate/% HO-891 cells Concentration of SGI-1776/(μmol/L) B 12 SGI μmol/l Number of colony/per well HO-891 cells Cell Number Cell Number 6 9/9=1.% /1=1.% G 1 =6.7% 85 G 1 =7.5% S/G 2 /M=39.3% S/G 2 /M=29.5% DNA Content DNA Content SGI μmol/l 69 1/1=1.% 6 1/1=1.% G 1 =66.6% 575 G 1 =75.9% S/G 2 /M=33.4% 5 S/G 2 /M=24.1% DNA Content Cell Number Cell Number DNA Content C E Distibution of cell cycle phase/% μmol/l DDP Concentration of SGI-1776/(μmol/L) G 1 S/G 2 /M 5 μmol/l DDP Concentration of SGI-1776/(μmol/L) D F Figure 1 Proliferation inhibiting rate of SGI-1776 on HO-891 cells (A, B), inhibition rate of colony formation of SGI-1776 on HO- 891 cells (C, D), and increasing accumulation rate of cell cycle G 1 phase of SGI-1776 on HO-891 cells (E, F). P<.5 vs the group; P<.5 vs the 5 µmol/l DDP group 2.4 Effect of SGI-1776 on the expression of Pim-1 and its downstream target genes in ovarian cancer cells To determine the expression of these proteins, we used Western blot analysis and found that SGI-1776 inhibited the expression of Pim-1, CDK6, pcdk6, CDK4, pcdk4, CDK2 and pcdk2, and increased P27 and P21 at the protein levels in HO-891 cells (Figure 3).

6 654 中南大学学报 ( 医学版 ), 214, 39(7) SGI μmol/l SGI μmol/l A Invasion inhibition rate /% Migration inhibition rate/% HO-891 cells HO-891 cells 5 μmol/l DDP Concentration of SGI-1776/(μmol/L) B C 5 μmol/l DDP Concentration of SGI-1776/(μmol/L) D Figure 2 Inhibiting rate of migration (A, B) and invasion (C, D) of SGI-1776 on HO-891 cells. P<.5 vs the group; P<.5 vs the 5 µmol/l DDP group. SGI-1776/(µmol/L) Pim-1 CDK-6 pcdk-6 CDK-4 pcdk-4 CDK-2 pcdk-2 P21 P27 GAPDH Figure 3 Western blot showing the effect of SGI-1776 on the proteins expression of Pim-1 and its downstream target gene in HO-891 cells. Data was the relative gray value. P<.5, P<.1 vs the group 2.5 Effect of down-regulation of Pim-1 expression by sirna on SGI-1776 induced proliferation inhibition and migration and invasion in HO-891 cell Down-regulation of Pim-1 by sirna transfection showed less expression of Pim-1 protein in HO-891 cells, as confirmed by Western blot (Figure 4A). Furthermore, we found that the down-regulation of Pim-1 expression by SGI-1776 significantly inhibited cell viability (Figure 4B), arrested cell in G 1 phase (Figure 4C), and inhibited the migration and invasion (Figure 4D and 4E). In addition, SGI-1776 plus Pim-1 sirna inhibited cell proliferation, migration and invasion, and induced G 1 phase accumulation to a greater degree compared with SGI alone (Figure 4B 4E). These results provide some molecular evidence suggesting the SGI-1776 induced inhibition of cell proliferation, migration and invasion are mediated via the inactivation of Pim-1 in HO-891 cells. 2.6 Effect of over-expression of Pim-1 by cdna on SGI-1776 induced proliferation inhibition and migration and invasion in HO- 891 cells Up-regulation of Pim-1 by cdna transfection showed an over-expression of Pim-1 protein in HO-891 cells,

7 SGI-1776, an imidazo pyridazine compound, inhibits the proliferation of ovarian cancer cells by inactivating Pim-1 XIE Jing, et al. 655 as confirmed by Western blot (Figure 4A). The results showed that over-expression of Pim-1 rescued SGI-1776 induced cell viability inhibition, migration and invasion, and cell cycle G 1 phase accumulation to a certain degree (Figure 4B 4E). These results provided mechanistic evidence suggesting that SGI-1776 inhibited cell proliferation, migration and invasion were partly due to the inactivation of Pim-1 signaling pathway in HO-891 cells. SGI-1776 sirna+ cdna+ (5 μmol/l) sirna SGI-1776 cdna SGI-1776 Pim GAPDH A Cell of growth rate/% Migration rate/% HO-891 cells SGI-1776 sirna sirna+ cdna cdna+ (5 μmol/l) SGI-1776 SGI-1776 HO-891 cells B Invasion rate/% Distribution of cell cycle/% G 1 S/G 2 /M SGI-1776 sirna sirna+ cdna cdna+ (5 μmol/l) SGI-1776 SGI-1776 HO-891 cells C 2 SGI-1776 sirna sirna+ cdna cdna+ (5 μmol/l) SGI-1776 SGI D SGI-1776 sirna sirna+ cdna cdna+ (5 μmol/l) SGI-1776 SGI-1776 E Figure 4 Effect of Pim-1 sirna, Pim-1 cdna transfection, and Pim-1 sirna/ Pim-1 cdna transfection combining with SGI-1776 on HO- 891 cells. A: Western blot showing the proliferation, migration and invasion; B: MTT assay showing inhibition of cell proliferation; C: Flow cytometry showing induction of cell cycle G 1 accumulation; D: Transwell cell assay showing inhibition rate of migration; E: Transwell cell assay showing inhibition rate of invasion. Data was the relative gray value. P<.5, P<.1 vs the group; P<.5 vs the 5 µmol/l SGI-1776 group 3 Discussion A variety of studies have shown the over-expressed Pim-1 in human cancer cells and tissues, including ovarian cancer cells and so on. Pim-1 gene play an import role in tumor cells formation, proliferation, differentiation, apoptosis, and inhibiting Pim-1 gene could suppress cancer cells proliferation and promote them apoptosis, which suggests that the inactivation of Pim-1 may play an important role in cancer therapy [3]. SGI-1776, an imidazo

8 656 中南大学学报 ( 医学版 ), 214, 39(7) pyridazine compound initially found through virtual screening, which has been shown to have inhibitory activity against Pim-1, Pim-2, Pim-3 kinase, and induced tumor cells apoptosis and inhibited cancer cell proliferation, has already entered phase I of clinical trials [5-6], but the effects and mechanism of SGI-1776 on ovarian cancer has not been reported in public. Thus, in the present study, we investigated whether SGI-1776 induced inhibition of ovarian cancer HO-891 cells viability and its molecular mechanisms. In our study, SGI-1776 elicited a dramatic inhibitory effect on the proliferation of HO-891 cells as shown by MTT assay, clonogenic assay, transwell cell migration and invasion assay, accompanied by decrease of Pim-1 kinase activity and down-regulation of Pim-1 protein expression. Our results suggested that Pim-1 as a target of SGI-1776 in HO-891 cells because Pim-1 is known to induce oncogenesis and its down-regulation causes inhibition of cell proliferation. Undeniably, we found that down-regulation of Pim-1 by sirna together with SGI-1776 treatment inhibited cell proliferation to a greater degree in HO-891 cells compared to SGI treatment alone. Up-regulation of Pim-1 by cdna transfection showed over-expression of Pim-1 protein as confirmed by Western blot, and this over-expression in Pim-1 attenuated SGI-1776 induced cell proliferation inhibition, migration and invasion in HO-891 cells. SGI-1776 has been shown to inhibit Pim-1 activation in kidney cancer cells, leading to cell cycle G 1 phase arrested [14], we investigated whether SGI-1776 could regulate Pim-1 signaling pathways using molecular and mechanistic approaches. Cell cycle regulation mainly depends on cyclin, cycle dependent kinase (CDK), cycle dependent kinase inhibitor (CKI), checkpoint, and their synergistic effects. Cells will accumulate in G 1 /S phase, and not pass G 1 /S checkpoint, unless cyclin proteins combine CDK6/CDK4/CDK2 into cyclin/cdk compounds, then CDK6/CDK4/ CDK2 are phosphorylated, and not inhibited by CKI, such as P21, P27 and so on [15-16]. Many studies showed that Pim-1 promotes cells proliferation and stimulates the expression of critical genes for the G 1 / S transition [3]. Because down-regulation of Pim-1 by SGI reduced cell proliferation, we investigated whether cell proliferation inhibition was due to cell cycle arrest in any specific phase of the cell cycle [6]. We found that treatment with SGI-1776 increased cell population in the G 1 phase and decreased cells in the S/G 2 /M phase. Diminished cell population of S/G 2 /M phase and decrease of proliferation rate were associated with increased expression of the CDKI protein, such as P27 and/or P21. In our study, the decreased protein expression level of Pim-1, CDK6, pcdk6, CDK4, pcdk4, CDK2 and pcdk2, and the increased expression of P27 and P21 were strongly correlated with the altered cell cycle distribution phenotype and proliferation suppression. These results suggested that SGI-1776 affected the ovarian cancer cell cycle by regulating the expression levels of Pim-1, CDK6, pcdk6, CDK4, pcdk4, CDK2 and pcdk2, and CDK inhibitors P27 and P21 through down-regulation of Pim-1 expression. In addition, we also found that the down-regulation of Pim-1 by sirna together with SGI-1776 treatment inhibited cells proliferation viability, migration and invasion accompanied to cell cycle G 1 phase accumulation to a greater degree in ovarian cancer cells when compared to SGI-1776 treatment alone. At the same time, upregulation of Pim-1 by cdna transfection showed an over-expression of Pim-1 protein in HO-891 cells, and the over-expression of Pim-1 rescued SGI-1776 induced cell viability inhibition, migration, invasion, and cell cycle G 1 phase to a certain degree compared to SGI-1776 treatment alone. In view of these findings, SGI-1776 not only inhibited proliferation, migration and invasion of the normal ovarian cancer HO-891 cells but also had the same effects on HO-891 cells transfected sirna and cdna, we strongly believe that the inactivation of Pim-1 by SGI-1776 resulted in the down-regulation of its target genes, which are believed to be mechanistically linked with inhibition of tumor cells proliferation, migration and invasion by treatment with SGI In summary, we presented experimental evidence that strongly supported the role of SGI-1776 as an antitumor agent mediated through inactivation of Pim-1 signaling pathway. Further in depth investigations are necessary in order to identify whether SGI-1776 could regulate the expression level of Pim-1 mrna and how to affect the promoter gene transcription. It is also tempting to speculate that the inactivation of Pim-1 combining with the treatment of ovarian cancer cells with conventional agents could be a useful strategy toward better treatment. Acknowledgements We are grateful to Professor CAO Jianguo (The medical college of Hunan Normal University) for his continuous kindly direction and encouragement. 参考文献 1. Rouhana-Toubi A, Wasser SP, Agbarya A, et al. Inhibitory effect of ethyl

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