硫氧还蛋白还原酶抑制剂乙烷硒啉逆转顺铂耐药及其机制研究

Ethaselen: a novel organoselenium anticancer agent targeting thioredoxin reductase 1 reverses cisplatin resistance in drug-resistant K562 cells by inducing apoptosis 硫氧还蛋白还原酶抑制剂乙烷硒啉逆转顺铂耐药及其机制研究 1 2 first-author †E-mail:pkuyesuofu@163.comzenghh@bjmu.edu.cn †E-mail:pkuyesuofu@163.comzenghh@bjmu.edu.cn pkuyesuofu@163.com 3 3 1 2 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:pkuyesuofu@163.comzenghh@bjmu.edu.cn †E-mail:pkuyesuofu@163.comzenghh@bjmu.edu.cn pkuyesuofu@163.com State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China School of Pharmaceutical Sciences, Peking University, Beijing 100191, China School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Keaise Clinical Examination Lab, Wuhan 430000, China Keaise Clinical Examination Lab, Wuhan 430000, China It has been reported that Ethaselen shows inhibitory effects on thioredoxin reductase (TrxR) activity and human tumor cell growth. In order to find an efficient way to reverse cisplatin resistance, we investigated the reversal effects of Ethaselen on cisplatin resistance in K562/cisplatin (CDDP) cells that were established by pulse-inducing human erythrocyte leukemic cell line K562, which are fivefold more resistant to cisplatin compared to K562 cells. The morphology and growth showed that the adhesion of K562/CDDP further decreased while the cell volume increased. The proliferation of K562/CDDP is strengthened. The antitumor activities in vitro were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and combination index (CI), showing the significant synergic effects of cisplatin and Ethaselen. Focusing on apoptosis, a series of comparisons was made between K562 and K562/CDDP. Cisplatin induced higher reactive oxygen species (ROS) generation in K562 and subsequently induced the formation of mitochondrial permeability transition pores (PTPs). In addition, cisplatin increased the ratio of Bax to Bcl-2 in K562, which can influence the mitochondrial membrane permeability. PTP formation and mitochondrial membrane permeabilization eventually resulted in the release of cytochrome c and activation of the Caspase pathway. However, these effects were not clearly seen in K562/CDDP, which may be the reason for the acquired CDDP resistance. However, Ethaselen can induce a high level of ROS in K562/CDDP by TrxR activity inhibition and increased ratio of Bax to Bcl-2 in K562/CDDP by nuclear factor κB (NF-κB) suppression, which subsequently induces the release of cytochrome c in K562/CDDP. This response is partly responsible for the reversal of the cisplatin resistance in K562/CDDP cells. 目的 2 研究凋亡调控相关蛋白来了解顺铂耐药成因，同时考察乙烷硒啉（Ethaselen）在K562耐药细胞中逆转顺铂耐药的作用，并初步探讨其作用机制。 创新点 2 首次研究乙烷硒啉在逆转顺铂耐药中的作用，且此作用与乙烷硒啉诱导细胞凋亡相关。 方法 2 通过长时间脉冲诱导得到顺铂耐药K562细胞，并观察耐药细胞形态及倍增时间。采用MTT法考察乙烷硒啉、顺铂及其联用组在不同细胞株间的生长抑制作用。流式细胞术分析细胞凋亡情况以及细胞内活性氧（ROS）水平。最后，通过蛋白质免疫印迹（Western blot）考察凋亡调控相关蛋白水平的变化。 结论 2 脉冲诱导得到的K562耐药细胞对顺铂的耐受性是原K562细胞的5.34倍。形态学观察发现，耐药细胞体积增大，粘附性进一步降低。乙烷硒啉与顺铂联用表现出协同效应。当加入少量的乙烷硒啉（顺铂与乙烷硒啉的摩尔比率为10:1），顺铂作用K562耐药细胞的半抑制浓度（IC 50 ）值可以减少21倍。流式细胞术及Western blot表明，乙烷硒啉能够诱导耐药细胞凋亡。其逆转顺铂耐药主要是通过调控Bcl-2及Bax蛋白比例以及通过提高细胞内活性氧水平引起线粒体通透转运孔道（PTP）蛋白孔道的形成来促使释放细胞色素 c ，进而引起Caspase凋亡途径。 Cisplatin resistance Cisplatin Bcl-2 Cytochrome c Ethaselen 乙烷硒啉 顺铂耐药 K562 凋亡 Bcl-2 Introduction 1 Cisplatin, a potent antitumor alkylating agent, is widely used for the clinical treatment of many malignancies, such as testicular, ovarian, head and neck, bladder, esophageal, and small cell lung cancers. Cisplatin exerts its anticancer activity by forming 1,2-intrastrand cisplatin–DNA cross-links (Mantri et al., 2007 2007 ; Yaneva et al., 2007 2007 ; Harrington et al., 2010 2010 ). However, a serious limitation in cancer chemotherapy with cisplatin is the development of drug resistance. Furthermore, the dose escalation required to overcome even a small increase in resistance of a tumor to cisplatin can cause severe toxicities in patients (Yeh and Cheng, 1997 1997 ; Miyata et al., 2012 2012 ). Therefore, understanding the molecular basis of cisplatin resistance and finding an efficient method to overcome this resistance can improve the clinical effectiveness of this anticancer drug. Unfortunately, the mechanism of cisplatin resistance is a complex biological process involving multiple factors (Chao et al., 1991 1991 ; Torigoe et al., 2005 2005 ; Haslehurst et al., 2012 2012 ; Oiso et al., 2014 2014 ) and still remains largely unclear. The induction of apoptosis in cancer cells is thought to be fundamental to the success of the treatment of the disease. The Bcl-2 family members are intimately involved in the apoptosis, but the role of these proteins in drug-induced death is controversial, including anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Bcl-W, and pro-apoptotic proteins such as Bax, Bad, Bid, and Bim (Chu, 1994 1994 ; Bargou et al., 1995 1995 ; Tilly et al., 1995 1995 ; Tao et al., 1997 1997 ; Hahn et al., 2003 2003 ; Dumay et al., 2006 2006 ; Heath-Engel et al., 2008 2008 ; Tanaka et al., 2010 2010 ; Chen et al., 2014 2014 ; Zheng et al., 2015 2015 ). It is certain that the Bcl-2 family of proteins is closely related to the regulation of cell death by inducing apoptosis, which is initiated by the release of cytochrome c from mitochondria. However, the relationship between the altered expression of apoptosis-regulating proteins and subsequent resistance to apoptosis still needs to be tested and studied. As a novel potent antitumor candidate targeting thioredoxin reductase (TrxR), Ethaselen (1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]ethane; BBSKE) has shown good antitumor effects in many cancer models (Wang et al., 2012 2012 ). It was first prepared by our group and now has entered clinical trial Phase II. Ethaselen was designed to inhibit mammalian TrxR activity by selectively targeting SeCys498/Cys497, the C-terminal active sites of mammalian TrxR, with high binding potential (Shi et al., 2003 2003 ; Zhao et al., 2006 2006 ; Xing et al., 2008 2008 ). Ethaselen was found to inhibit TrxR activity in several human carcinoma cell lines and tumor-bearing mouse models (Shi et al., 2003 2003 ; Zhao et al., 2006 2006 ). Furthermore, TrxR inactivation by Ethaselen correlates with cell death/apoptosis, including A549 (human lung cancer), HeLa (human cervical cancer), BGC823 (human stomach adenocarcinoma), HL60 and K562 (human leukemia), LoVo (human colon cancer), Bel-7402 (human epithelial hepatoma), and Tca8113 (human tongue cancer) cells (Shi et al., 2003 2003 ; Zhao et al., 2006 2006 ; Xing et al., 2008 2008 ; Fu et al., 2011 2011 ; Wang et al., 2011 2011 ; 2012 2012 ). Moreover, a synergistic effect has been observed in the study of the combination of Ethaselen and cisplatin (CDDP) in vivo. Studies have shown that the antitumor efficacy of cisplatin on the stomach cancer cell line BGC-823, the human lung cancer cell line PG-BE1, the subcutaneous transplanted lung cancer cell line A459 in the nude mice, and the human colon adenocarcinoma cell line LoVo would be further enhanced through synergetic combination with Ethaselen (Tan et al., 2010 2010 ). These previous studies have indicated that there is a close link of the antitumor mechanism between Ethaselen and CDDP, suggesting that Ethaselen may be a potential and efficient method for reversing cisplatin resistance. In the present study, we established cisplatin-resistant K562 cells and attempted to clarify the relationship between apoptosis and drug resistance in K562 cells. The expression patterns of the apoptosis-regulating proteins were analyzed to elucidate the mechanism of cisplatin-induced resistance. Furthermore, a possible effective way of reversing cisplatin resistance by Ethaselen was studied in order to provide a potential clinical application of Ethaselen-cisplatin combined chemotherapy. Materials and methods 1 Chemicals and antibodies 2 Ethaselen (Fig. 1 1 ) was previously synthesized in our lab and verified by high-performance liquid chromatography (HPLC) (Shi et al., 2003 2003 ). Ethaselen was dissolved in 20 mmol/L in dimethyl sulfoxide (DMSO). Cyclosporin A (CsA) and 2',7'-dichloro-dihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (Shanghai, China), and were dissolved in 5 and 20 mmol/L in HPLC-grade DMSO, respectively. The mammalian protease inhibitor cocktail was bought from Amresco Inc. (Solon, USA). Antibodies against Caspase-3 were purchased from Cell Signaling Technology (Shanghai, China). Antibodies against Bax, Bcl-2, and cytochrome c were bought from Santa Cruz Biotechnology Inc. (Shanghai, China), and β-actin was purchased from OriGene Technologies (Beijing, China). Cell culture 2 Human erythrocyte leukemic cell line K562 and K562/CDDP were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in 5% CO 2 atmosphere at 37 °C. K562/CDDP cells were established in our laboratory by pulsed induction (Du et al., 2007 2007 ) and exposed to the final concentration of 30 μmol/ml cisplatin for six months. Cell growth curves and population doubling time 2 K562, K562/CDDP (without CDDP), and K562/CDDP (exposed to 30 μmol/ml cisplatin) were obtained in the logarithmic phase with a cell density of 2000 cells/ml and seeded into 96-well plates (200 μl/well). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Amresco Inc., Solon) assay was conducted each day for the next eight days. The cell growth curves were drawn with time as X -axis and the average absorbance of 16 replicates as Y -axis: the population doubling time (DT)=( t – t 0 )×log2/(log N –log N 0 ), where t and t 0 , respectively, represent the time of culture termination and beginning, and N and N 0 represent the numbers of cells at t and t 0 , respectively. Cell morphology 2 K562 and K562/CDDP, taken from logarithmic phase cells, were used to observe the cell morphology under a 10×40-fold inverted fluorescence microscope. Cell growth inhibition assays 2 Cells in the exponential growth phase were plated into 96-well plates at 5000 cells/well. After exposure to various concentrations of CDDP or Ethaselen separately and in combination (the molar ratio of CDDP to Ethaselen was 10: 1) for 24, 48, and 72 h, the growth inhibition activities of different drug treatments on K562 and K562/CDDP cells were determined using MTT assay. The data were recorded on a microplate reader DNM-9602 (Beijing Perlong New Technology Co., Ltd., Beijing, China) at 570 nm. The inhibitory rate was calculated using the software Origin 7.5 (OriginLab, USA). Median effect analysis 2 The combination index (CI) was used to explore the drug interaction in combinations including synergy (CI<0.95), additivity (0.95≤CI≤1.05), or antagonism (CI>1.05). The CI value was calculated as CI= D 1 / D x 1 + D 2 / D x 2 , where D 1 and D 2 are the dosages of drugs used in combination to get a certain antitumor effect, respectively, and D x 1 and D x 2 are the dosages of drugs used separately to get same antitumor effect, respectively. Flow cytometric analysis of apoptosis and intracellular ROS levels 2 Cells undergoing apoptosis under various treatment conditions were prepared by the Annexin V-propidium iodide (PI) apoptosis detection kit according to the manufacturer’s instructions. After exposure to CDDP alone or to the combination of CDDP and Ethaselen, cells were harvested, washed once with ice-cold phosphate buffer saline (PBS), and incubated with DCFH-DA (100 μmol/L final concentration) at 37 °C for 15 min in the dark (Sainz et al., 2003 2003 ). Then the cells were washed again and maintained in 1 ml PBS. The reactive oxygen species (ROS) levels were determined using the Guava EasyCyte Plus flow cytometry system (Merck Millipore). Western blot analysis 2 After drug treatment, the whole cell lysates of K562 and K562/CDDP cells were prepared by lysing cells in the radioimmunoprecipitation assay (RIPA) buffer (137 mmol/L NaCl, 1 mmol/L ethylene diamine tetraacetic acid (EDTA), 10 mmol/L Tris-HCl (pH 7.4), 0.1% sodium dodecyl sulfate (SDS), 1% (0.01 g/ml) sodium deoxycholate, 1% Triton X-100). Mammalian protease inhibitor cocktail was added to the buffer according to the manufacturer’s instructions. In order to get the cytosolic and mitochondrial fractions, K562 and K562/CDDP cells were suspended in 400 μl of the isotonic buffer (250 mmol/L sucrose, 10 mmol/L Tris-HCl, 1 mmol/L EDTA) including protease inhibitors, and lysed by a tight-fitting Dounce homogenizer. The supernatant was obtained by centrifuging the lysate at 1000 g for 10 min, and subsequently centrifuged at 17 000 g for 15 min to separate the cytosolic fraction and mitochondrial sediment which was further lysed by 150 μl RIPA. Protein concentration was determined by the BCA (bicinchoninic acid) method. The cellular protein sample (50 µg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE), and the separated proteins were subsequently electrically transferred to a polyvinylidene (PVDF) membrane. The membrane was blocked by nonfat milk and incubated with suitable primary and secondary antibodies. The electrochemiluminescence (ECL) signals were recorded on X -ray films. Statistical analysis 2 Data of independent experiments were expressed as mean±standard deviation (SD) and analyzed using a two-tailed t -test. P -values less than 0.05 were considered statistically significant. Results and discussion 1 Resistant cells and parental cell growth curves and population doubling time 2 The growth curves of the parental cell line K562 and resistant cells K562/CDDP without drugs (Fig. 2 2 ) indicated that the proliferative capacities of resistant cells K562/CDDP were higher than those of the parental cells K562. When cultured with the drug in the medium to maintain drug resistance, the proliferative capacities of the resistant cells K562/CDDP were lower than those of the parental cells K562 (Fig. 2 2 and Table 1 1 ), but there was no significant difference ( P > 0.05). P > 0.05, vs. K562, suggesting no significant difference Cells Doubling time (h) K562 30.459±7.722 K562/CDDP 29.399±8.487 a a K562/CDDP (with cisplatin) 36.252±7.993 a a Data are expressed as mean±SD ( n =3) Cell morphology 2 Under a 10×40-fold inverted microscope (Fig. 3 3 ), K562 and K562/CDDP cells were the translucent circular suspension cells. The adhesion of K562 cells was weak, and the cells were dispersed in the medium and less attached to the cell culture flask. The cell volume was small. Compared with K562, the adhesion of K562/CDDP was further decreased while the cell volume increased. Drug sensitivity testing by MTT method in vitro and median effect analysis 2 From Fig. 4 4 , it can be seen that with the increase of drug action time, the effects of CDDP, Ethaselen, and their combinations on the K562 and K562/CDDP cells gradually increased, especially obviously in Figs. 4d–4f 4d–4f . The shapes of dose-response curves of the parental and resistant K562 cells to the same drug or drug combination had some differences, which showed the resistance characteristics of the drug-resistant cells. (a) Dose-response curves of K562 after treatment with CDDP for 24, 48, and 72 h. (b) Dose-response curves of K562/CDDP after treatment with CDDP for 24, 48, and 72 h. (c) Dose-response curves of K562 after treatment with Ethaselen for 24, 48, and 72 h. (d) Dose-response curves of K562/CDDP after treatment with Ethaselen for 24, 48, and 72 h. (e) Dose-response curves of K562 after treatment with CDDP+Ethaselen for 24, 48, and 72 h. (f) Dose-response curves of K562/CDDP after treatment with CDDP+Ethaselen for 24, 48, and 72 h As can be seen from Table 2 2 , K562/CDDP produced a certain degree of resistance to CDDP, Ethaselen, and their combination. The half-maximum inhibitory concentration (IC 50 ) of CDDP on K562/CDDP was 5.34 times that of CDDP on K562 ( P < 0.01) and the IC 50 of Ethaselen on K562/CDDP was 1.36 times that of Ethaselen on K562 ( P < 0.01). The dosages of the combination of CDDP and Ethaselen added to K562 and K562/CDDP cells to produce a specific inhibitory effect were lower than when they were used separately. The CI of the drug combination group was 0.169 on K562 cells and 0.107 on K562/CDDP cells, showing significant synergism ( P < 0.05) of CDDP and Ethaselen on the two types of cells. By the presence of small amounts of Ethaselen, the IC 50 of CDDP on K562/CDDP cells could reduce from 260.986 to 12.368, which indicated that Ethaselen can reverse cisplatin resistance in drug-resistant K562 cells. IC 50 : half-maximum inhibitory concentration; CI: combination index. Data are expressed as mean±SD ( n =3) 2 Cell line 3 Inhibitory effect IC 50 (%) * * 2 CI CDDP Ethaselen CDDP+Ethaselen (10:1) K562 48.748±0.697 15.270±3.313 6.255±0.664 0.169 (synergism) K562/CDDP 260.986±6.898 20.694±1.814 12.368±1.611 0.107 (synergism) 5 P -value <0.01 <0.01 <0.01 Flow cytometric analysis of apoptosis 2 After treating with the vehicle (control), 50 μmol/L CDDP, and the combination of 1.5 μmol/L Ethaselen and 15 μmol/L CDDP for 48 h, K562/CDDP cells were harvested for apoptosis analysis by an Annexin V-PI detection kit. As can be seen from the results (Figs. 5a and 5b 5a and 5b ), there is only a small increase in the percentage of cells treated with CDDP alone undergoing apoptosis compared to the control (3.96% vs. 3.17%). When cells were treated with Ethaselen and CDDP, a significant increase was observed in the percentage of Annexin V+/PI− cells (Fig. 5c 5c ), indicating an early stage apoptosis induced by the addition of a small amount of Ethaselen. (a) Apoptosis analysis of K562/CDDP cells treated with vehicle (control) for 24 h. (b) Apoptosis analysis of K562/CDDP cells treated with 50 μmol/L CDDP for 24 h. (c) Apoptosis analysis of K562/CDDP cells treated with the combination of 1.5 μmol/L Ethaselen and 15 μmol/L CDDP for 24 h Intracellular ROS content detection 2 As shown in Fig. 6 6 , the ROS level in K562/CDDP cells was twice of that in K562 cells. Exposure to CDDP resulted in an increase of the ROS levels in K562, while this increase was not obvious in K562/CDDP. By using the combination of CDDP and small amounts of Ethaselen, the ROS level further increased in both K562 and K562/CDDP cells, which was significantly different ( P < 0.05) from that with CDDP treatment alone. These results (Fig. 6 6 ) indicate that the degree of tolerance for ROS in K562/CDDP cells and the failure of inducing ROS by CDDP may partly be the cause of cisplatin resistance. In addition, further inducing the increase of ROS will reverse the cisplatin resistance in K562/CDDP cells. Western blot analysis for the expressions of Bax, Bcl-2, Caspase-3, and cytochrome c in K562 and K562/CDDP cells after different treatments 2 Release of cytochrome c is associated with the decrease of Bcl-2 and increase of Bax followed by activating Caspase-9 and Caspase-3 (Rossé et al., 1998 1998 ; Jia et al., 2015 2015 ). Compared to K562 cells, as shown in Figs. 7 7 and 8 8 , a tiny increase in the expression of Bcl-2 and a small decrease in the expression of Bax in K562/CDDP cells were observed. Western blot analysis revealed that Bax protein level of K562 was significantly increased by CDDP alone compared to K562/CDDP cells, while Bcl-2 protein expression in K562 treated by CDDP alone was significantly suppressed compared to that in K562/CDDP cells (Figs. 7 7 and 8 8 ). Hence, the increased ratio of Bax to Bcl-2 protein after CDDP treatment, following the cytochrome c release and Caspase pathway activation in K562 cells, was not seen in K562/CDDP cells. This result suggests that the ratio of pro-apoptotic drive to anti-apoptotic drive, as revealed by the ratio of Bax to Bcl-2 protein, may determine the difference in CDDP-induced apoptosis between K562 and K562/CDDP cells. By the combined CDDP-Ethaselen treatment, the ratio of Bax/Bcl-2 was obviously increased, subsequently inducing cytochrome c release from mitochondria to cytosol and Caspase-3 activation in both K562 and K562/CDDP cells. These results indicated that Ethaselen could reverse cisplatin resistance in drug-resistant K562 cells via activating the mitochondria-mediated intrinsic pathway. Data are expressed as mean±SD ( n =3). * P < 0.05 Enhanced mitochondrial membrane permeability by the formation of permeability transition pores, following the cytochrome c release 2 As mitochondrial membrane permeability (MMP) is enhanced by the formation of permeability transition pores (PTPs), which are triggered by ROS, and CsA (a PTP inhibitor) was used to block the formation of PTPs in this study. As shown in Figs. 9 9 and 10 10 , whether or not adding CsA, there was no obvious difference in cytochrome c release in K562/CDDP after exposure to CDDP alone, which confirmed that CDDP cannot induce the increase in the ROS level (Fig. 6 6 ), followed by the failure of PTP formation in K562/CDDP cells. The treatment of CDDP and Ethaselen triggered the release of cytochrome c , which was blocked by the treatment with CsA, suggesting that the cytochrome c release depends, at least partially, on the formation of PTPs. This result further confirmed that Ethaselen reversing cisplatin resistance by inducing apoptosis is initiated by the release of cytochrome c from mitochondria, suggesting that the increase in ROS level may directly trigger the formation of PTPs to disrupt mitochondrial membrane integrity in a Bcl-2-independent manner. Data are expressed as mean±SD ( n =3) Conclusions 1 A potent and efficient way to reverse cisplatin resistance in K562/CDDP cells by Ethaselen was observed and confirmed through the 21 times decline of the IC 50 of CDDP in K562/CDDP cells by adding small amounts of Ethaselen. The investigation of apoptosis-related molecules and mitochondrial-function-related factors further indicated two possible ways to reverse cisplatin resistance (Fig. 11 11 ): 1. Ethaselen can specifically inhibit TrxR activities, resulting in the reduction of nuclear factor κB (NF-κB) activities (Lan et al., 2007 2007 ), followed by the upregulation of Bax and downregulation of Bcl-2. The increased ratio of Bax/Bcl-2 subsequently induces cytochrome c release from mitochondria to cytosol and Caspase-3 activation in K562/CDDP cells. 2. The elevated ROS levels in K562/CDDP through the inhibition of TrxR by Ethaselen can trigger the formation of PTPs and then enhance MMP, which further confirms that Ethaselen reversing cisplatin resistance by inducing apoptosis is initiated by the release of cytochrome c from mitochondria in a Bcl-2-independent manner. Our results shed new light on a comprehensive understanding of the mechanism of CDDP resistance in K562 cells and a better knowledge of the relationship between CDDP resistance and Ethaselen-induced changes of TrxR, ROS, and apoptosis-related molecules, providing an efficient way to reverse cisplatin resistance in drug-resistant K562 cells by Ethaselen. 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