Quercetin induces endoplasmic reticulum stress to enhance cDDP cytotoxicity in ovarian cancer: involvement of STAT3 signaling Zongyuan Yang1, Yi Liu1,2, Jing Liao3, Cheng Gong1, Chaoyang Sun1, Xiaoshui Zhou1, Xiao Wei1, Taoran Zhang1, Qinglei Gao1, Ding Ma1 and Gang Chen1 1 Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2 Department of Medicinal Chemistry, School of Pharmacy, Hubei University of Chinese Medicine, Wuhan, China 3 Department of Gynecology, Zhongnan Hospital, Medical College of Wuhan University, China

Keywords cisplatin; ERS; ovarian cancer; quercetin; STAT3 Correspondence G. Chen, Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Fax: +86 27 83662681 Tel: +86 13 886113365 E-mail: [email protected] The first two authors contributed equally to this work (Received 30 September 2014, revised 13 December 2014, accepted 16 January 2015) doi:10.1111/febs.13206

There is an urgent need to make cisplatin (cDDP) more effective and less toxic in the treatment of ovarian cancer for its systemic side effects and high resistance rate. In this study, we investigated the effect of quercetin (Qu) pretreatment on the potentiation of cDDP in ovarian cancer. We found that Qu pretreatment significantly enhanced cDDP cytotoxicity in an ovarian cancer cell line and primary cancer cells. In addition, we demonstrated that Qu elicited obvious endoplasmic reticulum stress (ERS) and activated all three branches of ERS in ovarian cancer. Specific inhibitors of each ERS pathway, as well as the general ERS stabilizer tauroursodeoxycholic acid, notably diminished such enhancing effects. Furthermore, Qu notably suppressed STAT3 phosphorylation, leading to downregulation of the BCL-2 gene downstream of STAT3. Moreover, blocking ERS restored the protein levels of phosphorylated STAT3 as well as BCL-2 expression, thus abolishing the chemosensitization potency of Qu; these results revealed that Qu affected the STAT3 pathway to enhance cDDP cytotoxicity, and this effect involved ERS signaling. In a xenograft mouse model of ovarian cancer, Qu enhanced the antitumor effect of cDDP. Tumors from mice treated with cDDP in combination with Qu pretreatment had repressed STAT3 phosphorylation, lower BCL-2 and higher apoptosis levels compared with those from the other groups. Meanwhile, Qu markedly reduced the elevation of blood creatinine during cDDP intervention. These data indicate that Qu pretreatment potentiates the antitumor effects of cDDP in ovarian cancer while protecting the kidneys against damage. Therefore the strategy of Qu pretreatment may be beneficial in enhancing the therapeutic efficacy of cDDP against ovarian cancer.

Introduction Epithelial ovarian cancer remains a highly lethal malignancy and causes more than 140 000 deaths annually in women worldwide. Cytoreductive surgery followed by platinum-based chemotherapy is currently

the standard treatment for ovarian cancer [1]. Platinum agents have been used extensively over the past 30 years for the treatment of various carcinomas, including head and neck, lung, testicular and gyne

Abbreviations cDDP, cisplatin; ER, endoplasmic reticulum; ERS, endoplasmic reticulum stress; Qu, quercetin; TG, thapsigargin; TUDCA, tauroursodeoxycholic acid; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP-digoxigenin nick-end labeling; UPR, unfolded protein response.

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cological cancers, and relapsed lymphomas, but can result in obvious systemic side effects, a high incidence of relapse and the development of resistance in ovarian cancer patients [2–5]. These observations underscore the need to develop modulators of platinum agents to effectively overcome resistance and systemic side effects. Since conventional anticancer drugs can be highly toxic, plant-derived bioactive compounds are being more intensively investigated as alternative or adjunct therapies for various forms of cancer [6]. Flavonoids, one of the most diverse and widespread groups of natural compounds, are the most common natural phenolics [7] and are regarded as efficient antioxidants that scavenge oxygen radicals and possess anticancer, hypolipidemic, anti-aging and anti-inflammatory activities [8]. Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone, Qu) is the most common flavonoid in nature and is present in many fruits and vegetables [9]. Qu has been reported to possess a variety of anticancer effects, including stimulation of the intrinsic and extrinsic apoptosis pathways, inhibition of mutagenesis, transformation, angiogenesis and tumorigenesis [10,11]. Regarding the antitumor effect of Qu, there is some controversy about using Qu in combination with cDDP. Qu might jeopardize the cytotoxicity of cDDP by scavenging reactive oxygen species when given at the same time, but Maciejczyk and Surowiak [12] reported that Qu pretreatment increases the sensitivity of ovarian cancer cells to cDDP in vitro. However, the exact mechanism involved is unclear. In this study, we sought to explore the specific benefit of Qu pretreatment in the platinum-based chemotherapy of ovarian cancer. Endoplasmic reticulum stress (ERS) was found to be a new pathway leading to apoptosis following the discovery of the death receptor signaling and mitochondrial pathways. ERS is provoked by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen. Mammalian cells utilize ERS to activate the unfolded protein response (UPR) to reestablish ER homeostasis [13]. However, prolonged or irreversible ERS switches the adaptive nature of the UPR into a cell death program [14]. Therefore, drugs that induce ERS overload or block the UPR in cancer cells may shed light on anticancer therapy. Recent studies on Qu have revealed that this compound can elicit evident ERS in a variety of tumor cells, thus challenging the traditional opinion of the effects exerted by Qu in cancer cells [15]. Our recent study indicated that Qu evoked ERS in ovarian cancer cells [16]. We asked whether ERS had a measurable effect on the chemosensitization by Qu in the cDDP treatment of ovarian cancer. 1112

STAT3 is a member of a family that modulates the transcription of genes involved in the regulation of a variety of critical functions, including cell differentiation, proliferation, survival, angiogenesis, metastasis, drug resistance and immune responses [17]. The activation of STAT3 was shown to promote ovarian cancer cell growth and survival [18]. The activity of the STAT3 pathway has been associated with cDDP resistance in epithelial ovarian cancer [19]. In our previous study, the inhibition of STAT3 activation using small molecule inhibitors reversed the inherent and acquired chemoresistance of human ovarian cancer cells [20]. Recent studies have shown that Qu is a potent STAT3 inhibitor in human glioblastoma and gastric cancer cells [21,22]. Therefore, we set out to explore whether Qu could affect STAT3 signaling to sensitize cDDP treatment in ovarian cancer. To clarify these issues, we examined the therapeutic outcome of treatment with Qu in combination with cDDP. The Qu pretreatment led to the ERS-mediated inhibition of the STAT3 pathway, accompanied by the downregulation of BCL-2, thus lowering the threshold of the mitochondrial apoptosis pathway and thereby sensitizing the ovarian cancer cells in vitro and in vivo to the cDDP intervention. Furthermore, we pleasantly found that this combination strategy could protect against kidney damage caused by cDDP. Therefore, our results provide a novel insight into the mode of action of Qu-mediated chemosensitization in ovarian cancer and, furthermore, point to the rational design of cancer therapeutic regimens by combining Quinduced ERS with conventional chemotherapeutic drugs.

Results Quercetin pretreatment dramatically enhanced the cytotoxicity of cDDP in ovarian cancer cells In this study, we used two pairs of cells named C13* and OV2008, and P-ris and P-sen (paired primary cancer cells). The cancer cells from the ascites before chemotherapy were labeled P-sen, while the cells derived from the ascites after the chemotherapy relapse were labeled P-ris (Fig. 1A,B). Within 1 h after collecting the ascites, we performed magnetic cell sorting of the tumor epithelial cells. Only those cells that displayed an EPCAM+ component > 90% were further cultured and used for experiments. Annexin V/propidium iodide (PI) double staining assay was used to determine the cytotoxicity of cDDP alone in the paired ovarian cell lines and primary cells. Both types of cells were exposed to increasing concentrations of cDDP FEBS Journal 282 (2015) 1111–1125 ª 2015 FEBS

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concentrations > 40 lM (Fig. 1E). We selected 20 lM Qu for further combination studies due to the negligible cytotoxicity of this dose in both cell types. Qu (20 lM) preconditioning significantly enhanced the cytotoxicity of cDDP in both the C13* and P-ris cells (Fig. 1F). The colony-forming assay presented a similar outcome that cDDP treatment with Qu pretreatment had the greatest inhibitory effect on cancer cell growth (Fig. 1G). This result indicated that Qu pretreatment might be a solution to the systemic side effects and resistance of cDDP. Next, we examined whether the chemosensitization effect was caspase dependent using an immunoblot analysis. As shown in Fig. 1H, cDDP following Qu pretreatment rather than cDDP alone significantly increased the levels of cleaved caspase-9, cleaved caspase-3 and PARP proteins compared with the other groups. Qu at a dose higher than 20 lM had a greater impact on cDDP cytotoxicity, and this influence was dose dependent (data not shown). These results indicated that Qu pretreatment markedly potentiated the cytotoxicity of

cDDP in ovarian cancer cells, and the apoptotic behavior was due to the activation of caspase-9 and caspase-3 of the intrinsic pathway. Quercetin caused obvious ERS and activated all three branches of the UPR in ovarian cancer cells Previous studies have demonstrated that the flavonoids have an obvious effect on ERS in multiple human carcinoma cell lines, and we first confirmed that Qu could evoke ERS in ovarian cancer cells in a previous study [23–25]. As shown in Fig. 2A, the expression of the ERS signature markers GRP78 and CHOP were evidently upregulated with Qu treatment. Among the three canonical branches of the UPR, namely PERK, IRE1a and ATF6, we first explored the effect of Qu on the PERK signaling branch of UPR signaling. PERK activation leads to the phosphorylation of eIF2a at serine 51, and global translation is suppressed under conditions of eIF2a phosphorylation, while the transcription factor ATF4 is a downstream

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Fig. 2. Qu caused evident ERS and activated all three branches of UPR in ovarian cancer cells. In cDDP-resistant C13* and P-ris cells, 20 lM Qu was added for a time duration from 0 to 24 h. Lysates were harvested and immunoblotted for diverse recognized markers of ERS and UPR. (A) The marker of ERS GRP78 and CHOP increased in a typical time-dependent manner. (B) The branch of the PERK pathway was activated in both cell types evidenced by the phosphorylation of eIF2a and activation of its downstream molecule ATF4. (C) The branch of IRE1 was characterized by the phosphorylation of IRE1a and JNK. (D) Qu induced IRE1a pathway activation, evidented by the splicing of XBP1 mRNA in ovarian cancer cells. (E) The branch of ATF6 was activated dramatically characterized by the clevage of ATF6 to the form of ATF6 p50.

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concentrations > 40 lM (Fig. 1E). We selected 20 lM Qu for further combination studies due to the negligible cytotoxicity of this dose in both cell types. Qu (20 lM) preconditioning significantly enhanced the cytotoxicity of cDDP in both the C13* and P-ris cells (Fig. 1F). The colony-forming assay presented a similar outcome that cDDP treatment with Qu pretreatment had the greatest inhibitory effect on cancer cell growth (Fig. 1G). This result indicated that Qu pretreatment might be a solution to the systemic side effects and resistance of cDDP. Next, we examined whether the chemosensitization effect was caspase dependent using an immunoblot analysis. As shown in Fig. 1H, cDDP following Qu pretreatment rather than cDDP alone significantly increased the levels of cleaved caspase-9, cleaved caspase-3 and PARP proteins compared with the other groups. Qu at a dose higher than 20 lM had a greater impact on cDDP cytotoxicity, and this influence was dose dependent (data not shown). These results indicated that Qu pretreatment markedly potentiated the cytotoxicity of

cDDP in ovarian cancer cells, and the apoptotic behavior was due to the activation of caspase-9 and caspase-3 of the intrinsic pathway. Quercetin caused obvious ERS and activated all three branches of the UPR in ovarian cancer cells Previous studies have demonstrated that the flavonoids have an obvious effect on ERS in multiple human carcinoma cell lines, and we first confirmed that Qu could evoke ERS in ovarian cancer cells in a previous study [23–25]. As shown in Fig. 2A, the expression of the ERS signature markers GRP78 and CHOP were evidently upregulated with Qu treatment. Among the three canonical branches of the UPR, namely PERK, IRE1a and ATF6, we first explored the effect of Qu on the PERK signaling branch of UPR signaling. PERK activation leads to the phosphorylation of eIF2a at serine 51, and global translation is suppressed under conditions of eIF2a phosphorylation, while the transcription factor ATF4 is a downstream

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Fig. 2. Qu caused evident ERS and activated all three branches of UPR in ovarian cancer cells. In cDDP-resistant C13* and P-ris cells, 20 lM Qu was added for a time duration from 0 to 24 h. Lysates were harvested and immunoblotted for diverse recognized markers of ERS and UPR. (A) The marker of ERS GRP78 and CHOP increased in a typical time-dependent manner. (B) The branch of the PERK pathway was activated in both cell types evidenced by the phosphorylation of eIF2a and activation of its downstream molecule ATF4. (C) The branch of IRE1 was characterized by the phosphorylation of IRE1a and JNK. (D) Qu induced IRE1a pathway activation, evidented by the splicing of XBP1 mRNA in ovarian cancer cells. (E) The branch of ATF6 was activated dramatically characterized by the clevage of ATF6 to the form of ATF6 p50.

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protein that is upregulated to promote the folding capacity of the ER and the adaptation to stress [26]. It was apparent that 20 lM Qu induced eIF2a phosphorylation and ATF4 expression in both cell types as early as 3 h, illustrating the activation of the PERK–eIF2a– ATF4 pathway upon Qu treatment (Fig. 2B). We next investigated the influence of Qu on the IRE1 branch. It was evident that Qu led to a time-dependent elevation of S724-phosphorylated IRE1 expression, as well as JNK phosphorylation at Thr183/Tyr185, a molecular event known to be downstream of IRE1 activation. As shown in Fig. 2C, JNK phosphorylation was clearly induced upon Qu treatment, and the kinetics of the JNK phosphorylation were parallel to those of IRE1. Meanwhile, we found that Qu induced splicing of XBP1 mRNA, which delineated the activation of IRE1 signaling (Fig. 2D). Lastly, the effect of Qu on the ATF6 branch of the UPR was examined. Under conditions of ERS, ATF6 translocates to the Golgi where it is processed to its cytoplasmic domain ATF6f (a fragment of ATF6), which operates as a transcriptional activator that upregulates many UPR genes related to protein folding [27], as shown in Fig. 2E. Qu led to the dramatic induction of ATF6 p50 compared with the control, indicating that ATF6 was processed to its active form upon Qu stimulation. Collectively, these data revealed that Qu (20 lM) can evoke the activation of all three canonical branches of the UPR, reaching a plateau at 12 h; these results provided evidence for the choice of a 12 h Qu pretreatment.

study showed that salubrinal, 3-E-5,6-D and sp600125 all alleviated the potentiation by Qu of the cDDP-mediated inhibition of ovarian cancer cell growth (Fig. 3D). In line with this observation, Qu’s potentiation of cDDP cytotoxicity diminished in the presence of salubrinal, 3-E-5,6-D and sp600125, respectively, as demonstrated by the annexin V–FITC–PI assay (Fig. 3E). Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid derivative which has been effectively used for the treatment of primary biliary cirrhosis and ulcerative colitis, has recently become recognized as a chemical chaperone directed against ERS; this agent was utilized to relieve ERS [31]. As shown in Fig. 4A, B, our data showed that TUDCA remarkably diminished Qu-elicited extensive UPR in ovarian cancer cells. In accordance with the overall silence of Quinduced ERS in the presence of TUDCA, TUDCA addition notably alleviated Qu’s potentiation of the cytotoxicity of cDDP and relieved apoptotic cell death (Fig. 4C,D). To confirm this observation, DNA fragmentation was examined with Hoechst staining. The result demonstrated the protective role of TUDCA in the chemosensitization by Qu (Fig. 4E). Next, we examined the effect of TUDCA on intrinsic apoptosis pathway related proteins, such as caspase-3, caspase-9 and PARP. As shown in Fig. 4F, cleaved caspase 9, cleaved caspase 3 and cleaved PARP recovered notably after treatment with TUDCA. Collectively, our studies suggest that Qu-induced ERS plays a significant role in the potentiation of cDDP cytotoxicity.

Inhibitors of the UPR alleviated quercetin’s potentiation of cDDP cytotoxicity in ovarian cancer cells

Quercetin selectively suppressed constitutive STAT3 phosphorylation and downregulated the expression levels of STAT3-targeted genes in ovarian cancer cells

To determine whether the potentiation of cDDP cytotoxicity by 20 lM Qu, which had no apparent toxicity in the absence of DNA-damaging agents, was dependent on the evoked ERS response, UPR inhibitors of each ERS pathway were utilized. Salubrinal is a selective inhibitor of the phosphatase complexes that dephosphorylate eIF2a in the PERK pathway [28]. 3-Ethoxy-5,6-dibromosalicylaldehyde (3-E-5,6-D) selectively inhibits XBP1 splicing [29]. sp600125 downregulates the expression of IRE1 and dephosphorylates JNK [30]. As validated by our results, salubrinal addition prevented eIF2a dephosphorylation and further influence of the expression of ATF4, CHOP, STAT3 phosphorylation as well as BCL-2 following Qu treatment (Fig. 3A). 3-E-5,6-D intervention inhibited Quinduced IRE1 pathway activation mediated splicing of XBP1 mRNA (Fig. 3B). sp600125 supplement reduced Qu-induced phosphorylation of IRE1 (Fig. 3C). Our FEBS Journal 282 (2015) 1111–1125 ª 2015 FEBS

It is well recognized that constitutive phosphorylation/ activation of STAT3 contributes to the development of cancers by favoring cancer cell differentiation, proliferation, survival, angiogenesis, metastasis, drug resistance and immune evasion [28], and Qu was reported to be a potent STAT3 inhibitor in some cancer cells [21,22]. Therefore, we investigated whether Qu could modulate the STAT3 phosphorylation status in ovarian cancer cells. It was found that the levels of STAT3 phosphorylated at the tyrosine 705 (Tyr705) site were clearly reduced after 24 h of treatment with 20 lM Qu in both C13* and P-ris cells, and this inhibitory effect was time dependent (Fig. 5A). It has been reported that constitutive STAT3 phosphorylation is usually mediated by upstream tyrosine kinases such as JAK2 and Src, which have been found to contribute to the development of various cancers

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Fig. 3. Inhibitors of UPR alleviated Qu’s potentiation on cDDP cytotoxicity in ovarian cancer cells. The effects of salubrinal (eIF2a phosphatase inhibitor), 3-E-5,6-D (XBP1 splicing inhibitor) and sp600125 (JNK inhibitor) on alleviating Qu’s potentiation on cDDP cytotoxicity were examined. (A) Western blots to demonstrate salubrinal addition prevented eIF2a dephosphorylation and further influence of the expression of ATF4, CHOP, STAT3 phosphorylation as well as Bcl-2 following Qu treatment. (B) 3-E-5,6-D addition inhibited Qu-induced IRE1 RNase activation mediated splicing of XBP1 mRNA. (C) sp600125 addition reduced Qu-induced phosphorylation of IRE1. (D), (E) C13* and P-ris cells were incubated with Qu (20 lM) for 12 h, cDDP (20 lM) for 48 h or pretreatment with Qu (20 lM) in the absence or presence of salubrinal, 3-E-5,6-D and sp600125 respectively for 12 h followed by 48 h treatment of cDDP (20 lM), and then were analyzed by CCK8 assay to determine the growth inhibition, as well as flow cytometry to detect the relative apoptosis rate. The results were similar in at least three independent experiments. *P < 0.05; **P < 0.01.

[32,33]. Our data showed that Qu (20 lM) suppressed the constitutive phosphorylation of JAK2(Y1007/1008) and Src(Tyr416) in both C13* and P-ris cells in a timedependent manner (Fig. 5B). These findings led to the conclusion that Qu effectively and selectively suppresses the constitutive activation of STAT3 in ovarian cancer cells, and this suppressive action can be attributed to the reduction of phosphorylation by JAK2 and Src. STAT3 acts as a transcription factor, the target genes of which, such as MCL-1, BCL-2 and BCL-XL, play important roles in cancer cell growth and survival [34]. We found that in both C13* and P-ris cells Qu decreased the expression of BCL-2, while Qu did not affect the expression levels of MCL-1 or BCL-XL at a concentration of 20 lM (Fig. 5C). Interestingly, the data showed that Qu could sustainably suppress 1116

BCL-2 to a low level even after the withdrawal of Qu for 48 h (Fig. 5D). To further elucidate the connection between STAT3 signaling and Bcl-2 expression in ovarian cancer cells, we used a specific STAT3 inhibitor stattic to inactivate the STAT3 pathway, as a consequent BCL-2 level was notably reduced accordingly (Fig. 5E). Meanwhile, similar results were obtained from the sensitive counterparts OV2008 and P-sen under Qu treatment, which meant that Qu mediated suppression of the STAT3 pathway and downstream signaling was not restricted to the resistant ovarian cancer cells (Fig. 5F). Moreover, the detection of basal level BCL-2 in these two paired cell lines revealed that BCL-2 was vigorously expressed in the resistant cells (C13* and P-ris) (Fig. 5G). Consistently, knocking down of BCL-2 increased the sensitivity of these cells FEBS Journal 282 (2015) 1111–1125 ª 2015 FEBS

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Fig. 4. TUDCA attenuated Qu’s potentiation on cDDP cytotoxicity in ovarian cancer cells. (A), (B) TUDCA addition prevented the overall UPR activation of the three branches including the inhibition of XBP1 mRNA splicing. (C), (D) C13* and P-ris cells were incubated with Qu (20 lM) for 12 h, cDDP (20 lM) for 48 h, or pretreatment with Qu (20 lM) in the absence or presence of TUDCA for 12 h followed by 48 h treatment of cDDP (20 lM), and then were analyzed by CCK8 assay to determine the growth inhibition, as well as flow cytometry to detect the relative apoptosis rate. (E) C13* and P-ris cells were treated with the indicated measures for 48 h; cells were stained with Hoechst and observed under a Leica microscope. The yellow arrows indicate apoptotic cells. (F) C13* and P-ris cells were treated with the indicated measures for 48 h; lysates were harvested and immunoblotted with caspase-9, cleaved caspase-3 and PARP. The results were similar in at least three independent experiments. *P < 0.05; **P < 0.01.

to cDDP (Fig. 5H). This further supports that loss of BCL-2 exerted by Qu is actually responsible for its potentiation towards cDDP. Quercetin suppression of constitutive STAT3 phosphorylation mediated by ERS in ovarian cancer cells Next, we set out to further assess the possible connection between the ERS evoked by Qu and the sustained STAT3 repression. Since we had proved the capacity of TUDCA in attenuating the ERS induced by Qu in ovarian cancer cells, TUDCA was added to stabilize the ERS to examine whether ERS was involved in the suppression of constitutive STAT3 phosphorylation by Qu. In the presence of TUDCA, the protein expression of CHOP, STAT3, phosphorylated STAT3 and BCL-2 was examined. As shown in Fig. 6A, TUDCA restored Qu-induced elevated expression levels of CHOP in a FEBS Journal 282 (2015) 1111–1125 ª 2015 FEBS

concentration-dependent manner,accompanied by the recovering of STAT3 phosphorylation and BCL-2 expression in both cell types. In addition, ovarian cancer cells were transfected with siRNAs targeting CHOP to clarify whether CHOP contributes to constitutive STAT3 suppression. As expected, the Qu-induced upregulation of CHOP was abolished in cells expressing CHOP siRNAs, and notably preventing CHOP elevation restored the phosphorylated STAT3 and BCL-2 protein levels (Fig. 6B). Accordingly, in Fig. 6C, the chemosensitization effect of Qu declined significantly after knocking down of CHOP. Following this, we particularly introduced an ERS inducer thapsigargin (TG), which caused dramatic UPR and increased CHOP expression, thus lowering STAT3 phosphorylation and subsequent BCL-2 level in C13* (Fig. 6D). Coincidently, CHOP siRNA intervention abrogated the chemosensitization effect of TG in C13* to cDDP (Fig. 6E).

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Fig. 5. Qu selectively suppressed constitutive STAT3 phosphorylation and downregulated STAT3 target gene expression levels in ovarian cancer cells. (A), (B) C13* and P-ris cells were treated with Qu (20 lM) for 24 h or a fixed concentration (20 lM) for various durations, and then total cell lysates were subjected to western blot analysis of p-STAT3(Tyr705) and STAT3, as well as molecules downstream of STAT3 signaling such as p-Src(Tyr416), Src, p-JAK2(Y1007/1008) and JAK2. (C) After 12 h treatment of Qu (20 lM), the alterations of BCL-XL, MCL1 and BCL-2 were evaluated by western blot analysis. (D) The levels of BCL-2 were evaluated by western blot analysis: a, C13* cells were treated with DMSO (< 0.1%) for 12 h; b, cells were treated with Qu (20 lM) for 12 h; c, cells were treated with Qu (20 lM) for 12 h, and then it was withdrawn for 24 h; d, cells were treated with Qu (20 lM) for 12 h and then it was withdrawn for 48 h. (E) Specific STAT3 inhibitor stattic was added to C13* cells for 12 h, followed by western blot analysis of p-STAT3(Tyr705), STAT3 and BCL-2. (F) The indicated molecules were examined in two chemosensitive cells OV2008 and P-sen under Qu intervention. (G) Basal level of BCL-2 was detected in paired OV2008 and C13*, P-sen and P-ris. (H) BCL-2 specific siRNA was employed to knock down the BCL-2 expression level in two resistant cell lines C13* and P-ris. 24 h after transfection of BCL-2 siRNA, cancer cells was exposed to various dosages of cDDP for another 48 h; CCK8 assay revealed the relative cell viability alteration in the control and siRNA intervention group. **P < 0.01.

Quercetin potentiated the cDDP antitumor effect in vivo, and ERS was involved in the in vivo sensitization In the light of the synergistic effect of Qu and cDDP in vitro, we studied the in vivo antitumor activity of the combination therapy in nude mice bearing C13* 1118

ovarian cancer xenografts. When the tumors had reached approximately 5–6 mm in size, the mice were injected intraperitoneally with vehicle, Qu, cDDP, or cDDP in combination with Qu; 40 mgkg1 Qu was chosen to be used in the combination therapy for its negligible effect in reducing tumor growth (inhibition rate 2.4%). Our study also showed that only Qu at a FEBS Journal 282 (2015) 1111–1125 ª 2015 FEBS

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Fig. 6. Qu-suppressed constitutive STAT3 phosphorylation was mediated by ERS in ovarian cancer cells. (A) C13* and P-ris cells were treated with Qu (20 lM) for 12 h in the presence or absence of TUDCA (250 and 500 lM), and then cells were subjected to immunoblotting of CHOP, p-STAT3(Tyr705),STAT3 and Bcl-2. (B) C13* and P-ris cells transfected with negative control (NC) siRNA or CHOP siRNA were exposed to 20 lM Qu for 12 h; lysates were immunoblotted for CHOP, p-STAT3(Tyr705), STAT3 and BCL-2. (C) Annexin V–FITC assay was used to determine the apoptosis rate in the NC siRNA or CHOP siRNA transfected cells treated with Qu, cDDP or combination for 48 h. (D) ERS inducer TG (100 nM) caused dramatic UPR and increased CHOP expression, thus lowering STAT3 phosphorylation and subsequent BCL-2 level in C13*. (E) Apoptosis assay showed TG enhanced cDDP sensitivity was diminished by CHOP knocking down. The results were similar in at least three independent experiments. **P < 0.01.

concentration >80 mgkg1 had detectable treatment efficacy as a solitary measure (Fig. 7A). The tumor weight was measured once a week, as indicated in Fig. 7B. The weekly intraperitoneal administration of 3 mgkg1 cDDP for 4 weeks only modestly reduced the tumor growth by 44.2%, while the weekly treatment with 3 mgkg1 cDDP 1 day after the 40 mgkg1 Qu treatment remarkably decreased tumor growth by 89.6%. At the end of the experiment, the animals were anesthetized and sacrificed to expose the tumor tissues. It was obvious that the smallest tumor size occurred in the combination treatment group, as shown in Fig. 7C. Furthermore, compared with the cDDP treatment group, the combination group presented a reduced elevation of blood creatinine levels. Thus the combination of Qu and cDDP produced a much more potent tumor growth inhibition effect, while attenuating the toxicity FEBS Journal 282 (2015) 1111–1125 ª 2015 FEBS

of cDDP (Fig. 7D). To evaluate the abilities of the Qu and/or cDDP treatments to induce apoptosis, a terminal deoxynucleotidyl transferase mediated dUTPdigoxigenin nick-end labeling (TUNEL) assay and caspase-3 immunostaining were performed. As shown in Fig. 7E, the percent of TUNEL-positive cells and the expression of active caspase-3 were significantly higher in the tumor tissues of the combination-treated mice. With regard to the ERS marker proteins, the co-treated tumors exhibited increased GRP78 and CHOP levels compared with the control group. The co-treated tumors showed a significant suppression of phosphorylated STAT3 and BCL-2 protein expression compared with the other groups, which was highly inversely correlated with the expression of CHOP. Consistent with the aforementioned in vitro results, these data provide evidence that links the pretreatment of Qu with the Qu-induced activation of ERS,

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Fig. 7. Qu-potentiated cisplatin antitumor effect and ERS involved in the in vivo sensitization. (A) After the C13* xenograft tumor reached a mean volume of 50 mm3, mice in each group (six mice per group) were given intraperitoneally various dosages of Qu (20–100 mgkg1) once a week for 4 weeks. One week after the last Qu intervention procedure, mice were euthanized and the tumors were weighed for treatment efficacy. (B) Mice bearing C13* cell-derived tumors were treated with saline, Qu (40 mgkg1), cDDP (3 mgkg1), and Qu (40 mgkg1) + cDDP (3 mgkg1). Tumor growth was monitored by measuring the tumor volume for 5 weeks (n = 6 mice per group). (C) At the end of the experiment, the tumors were collected and weighed in each group. (D) The blood creatinine level was monitored by analyzing the blood sample taken from each mouse once a week for 4 weeks. (E) Tumor samples were subjected to TUNEL assay and immunohistochemical analysis of active caspase-3 at the end of the animal experiment. (F) The representative immunohistochemical staining of ERS markers GRP78 and CHOP as well as p-STAT3 and BCL-2 in a tumor section from each group. *P < 0.05; **P < 0.01.

followed by STAT3 pathway mediated downregulation of the BCL-2 protein level, and finally the sensitization of cDDP cytotoxicity (Fig. 7F).

Discussion Although cDDP remains a vital component of chemotherapy after tumor debulking surgery, the incidences of recurrence and platinum resistance lead to poor survival rates for ovarian cancer patients [35]. Meanwhile, the clinical use of cDDP is hampered by the dosedependent toxicity of this agent to normal tissues, such as the liver and kidney. Therefore, there is an urgent need to find a way to enhance the specific killing activity while relieving the side effects of cDDP. ERS is a new pathway to apoptosis that has been proved to mediate the cytotoxicity of cDDP in cancer cells in recent studies [36], and was described as a promising way to enhance the outcome of chemotherapy in clinical studies. Qu, a naturally existing flavonoid pres1120

ent in nearly all plants and plant food sources, has many biological and pharmacological activities in a variety of cancer models [15,36] and evoked ERS in ovarian cancer cells in our previous study [16]. In this study, we demonstrated that Qu potentiates cDDP cytotoxicity in drug-resistant ovarian cancer cells both in vitro and in vivo by using a drug combination scheme that involves the pre-administration of a low dose of Qu followed by intervention with cDDP. Further functional analysis showed that the inhibition of ERS markedly alleviated this potentiation, suggesting that the Qu-induced ERS plays a ‘priming’ role for cDDP cytotoxicity in drug-resistant ovarian cancer cells. For the mechanism study, we focused on STAT3, a member of the STAT family of transcription factors, as a key player involved in ovarian cancer [18–20]. Previous studies revealed that Qu could hinder the activation of STAT3 in human glioblastoma and gastric cancer cells [21,22]. Consistent with these reports, our present study first revealed a remarkable inhibition of STAT3 FEBS Journal 282 (2015) 1111–1125 ª 2015 FEBS

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activation in response to Qu treatment of drug-resistant ovarian cancer cells as well as in a drug-resistant ovarian tumor xenograft model. Because STAT3 was reported to be activated by the upstream tyrosine kinases JAK and Src [33], we demonstrated that Qu decreased the phospho-JAK2 and phospho-Src levels in C13* and P-ris ovarian cancer cells in a time-dependent manner, while the expression levels of total JAK2 and Src were not influenced. STAT3 directly regulates the expression of several survival genes. The expression levels of the antiapoptotic genes BCL-2 and BCL-XL appear to be upregulated in cisplatin-resistant ovarian cancer, which positively correlates with the p-STAT3 levels [37]. The present study revealed that Qu specifically inhibited the expression of BCL-2 but not that of MCL-1 or BCL-XL. The data also showed that Qu continually suppressed BCL-2 to a low level, even after the Qu was withdrawn for an extended time. Because cDDP-induced apoptosis is mainly associated with the mitochondrial pathway, this result provides direct evidence for the potentiation of cDDP by Qu. In our current study, we present evidence that TUDCA or an siRNA-mediated knockdown of CHOP could also abrogate the inhibition of STAT3 activation and BCL-2 expression exerted by Qu treatment, thus alleviating the cDDP-sensitizing effect. Taken together, our data demonstrate that ERS plays a pivotal role in the potentiation of cDDP by Qu by repressing STAT3 phosphorylation and the expression of the STAT3 target gene BCL-2. In conclusion, our current study revealed a hitherto undescribed cellular response showing that Qu pretreatment primes drug-resistant ovarian cancer cells for cDDP chemotherapy by involving the ERS process. In addition, we demonstrated the contribution of altered STAT3 signaling in this process as modulated by ERS (Fig. 8). This report provides favorable evidence that Qu pretreatment could be a promising way to solve the systemic side effects and high incidence of resistance to cDDP in ovarian cancer.

Quercetin induces endoplasmic reticulum stress

Fig. 8. Schematic model illustrating the potential pathway associated with Qu’s potentiation of cDDP cytotoxicity in ovarian cancer. Qu induced ERS to inhibit the STAT3 signaling and BCL-2 expression, which lowered the threshold of cDDP cytotoxicity. Disruption of the potentiation effect by using either TUDCA or UPR branch inhibitors (salubrinal, 3-E-5,6-D and sp600125) could markedly attenuate Qu’s potentiation of cDDP cytotoxicity in ovarian cancer cells.

IgG were purchased from Epitomics Inc. (Burlingame, CA, USA). Qu, Hoechst 33258 and 3-E-5,6-D were purchased from Sigma (St Louis, MO, USA). TUDCA, salubrinal and sp600125 were purchased from Millipore (Calbiochem, San Diego, CA, USA). Cisplatin-sensitive ovarian cancer cell line (OV2008) and its resistant variant (C13*) were gifts from B. K. Tsang in the Ottawa Health Research Institute, Ottawa, Canada, and the cells were cultured in McCoy’s 5A (Modified) medium (Invitrogen, Eugene, OR, USA) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2.

Materials and methods Cell Counting Kit 8 (CCK-8) assay Reagents and cells The antibodies against BCL-2, MCL-1, BCL-XL, Caspase-3, Caspase-9, PARP, phospho-STAT3(Tyr705), STAT3, phosphor-JAK2(Y1007/1008), JAK2, phosphor-Src (Tyr416), Src, CHOP, glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and b-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies against GRP78, ATF4, ATF6, p-Eif2a, p-IRE1a, p-JNK, horseradish peroxidase conjugated anti-mouse IgG, and anti-rabbit

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Cell viability was determined using the CCK-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Cells were plated in 96-well plates at a density of 5000 cells per well; after plating (24 h), the cells were subjected to various treatments for the indicated times. The CCK-8 solution (10 lL) was added to each well, and the cells were incubated for another 3 h at 37 °C; absorbance was read at 450 nm on a microplate reader. Cells that stained positively with the CCK-8 solution were considered

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viable and are presented as a percentage compared with control cells. Each assay was performed in triplicate.

Colony-forming assay Cells were seeded into 6-cm dishes (Corning Inc., Corning, NY, USA) in triplicate at a density of 1000 cells per dish. The cells receiving various treatments were cultured for another 14 days in a humidified incubator at 37 °C. Colonies were fixed with 4% paraformaldehyde (Sigma), stained with 0.5% crystal violet (Sigma) and counted.

Annexin V–FITC–PI assay An annexin V–FITC apoptosis kit (KeyGEN Biotech, NanJing, China) was used to determine the number of apoptotic cells according to the manufacturer’s instructions. Briefly, cells were grown in six-well plates; after 80–90% confluence, the cells were treated with various concentrations of Qu, cDDP or cDDP following Qu pretreatment. The cells were harvested after the indicated time, washed twice with ice-cold PBS and resuspended in 500 lL of binding buffer. Then 5 lL annexin V–FITC and 10 lL PI was added and the mixture was incubated in the dark at 4 °C for 15 min. A total of 100 000 events per sample were analyzed. Flow cytometric analysis was performed with a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with WINMDI 2.8 software.

tion at 225 9 g for 15 min, the supernatant was collected. Total protein concentration was determined using a BCA protein assay kit (Beyotime). Briefly, 40 lg of total proteins from each sample were loaded, separated by SDS/PAGE and transferred to polyvinyl difluoride membranes (Millipore). The transferred membranes were blocked for 1 h with NaCl/ Tris containing 0.1% Tween-20 and 5% BSA at room temperature, and then incubated overnight with an appropriate dilution of the primary antibody at 4 °C. After washing three times with NaCl/Tris containing 0.1% Tween-20, membranes were incubated with the corresponding horseradish peroxidase linked secondary antibody. Finally, the immune complexes were visualized via fluorography using an enhanced ECL system (Thermo Fisher Scientific, San Jose, CA, USA).

XBP1 splicing assay C13* and P-ris cells were treated with Qu (20 lM) or other agents. Total RNAs from drug-treated cells were extracted thereafter and the status of XBP1 mRNA splicing was then monitored by semi-quantitative RT-PCR analysis using the primer pair 50 -CCTTGTAGTTGAGAACCAGG-30 (forTGG-30 ward) and 50 -GGGGCTTGGTATATATG (reverse). A 442-bp PCR product was derived from the unspliced form of XBP1 mRNA, which contains the 26-bp intron, and a 416-bp PCR product was derived from the spliced form of XBP1 mRNA. GAPDH was used as the control for equal loading using the primer pair 50 -GGAG CGAGATCCCTCCAAAAT-30 (forward) and 50 - GGCTG TTGTCATA CT TCTCATGG-30 (reverse).

Hoechst 33258 staining The nuclear morphologies were analyzed by Hoechst 33258 staining using a fluorescence microscope. Briefly, cells were incubated with or without Qu, cDDP, cDDP following Qu in the presence or absence of TUDCA, after which the cells were stained with Hoechst 33258 (1 mgmL1) for 20 min. The digital images were captured under a fluorescence microscope (Olympus Optical Co. Ltd, Tokyo, Japan).

TUNEL assay To evaluate the cellular apoptosis inside tumor tissues, we applied the TUNEL technique to detect the apoptosis rate in paraffin blocks. Procedures were performed on 6 lm paraffin sections from C13* xenograft tumor tissues with or without Qu, cDDP, cDDP plus Qu intervention according to the instructions of the manufacturer (KeyGEN Biotech).

Immunohistochemical staining After the mice were sacrificed, tumors were excised, stored in 10% formalin for 24 h, and then embedded in paraffin, sectioned, and subjected to immunohistochemical staining for GRP78, CHOP, p-STAT3 and BCL-2 expression using the streptavidin-peroxidase technique as described previously. Briefly, sections were deparaffinized and incubated with 3% H2O2 (Sigma) in distilled water to block endogenous peroxidase activity. After antigen retrieval, the sections were blocked with goat serum for 30 min and then incubated with primary antibody overnight at 4 °C. After washing with PBS, the sections were incubated with horseradish peroxidase linked secondary antibody for 30 min. After washing with PBS, the sections were developed in 2,4-diaminobutyric acid solution until the desired staining intensity was achieved. Finally, the sections were counterstained with hematoxylin.

Western blot analysis Cells were harvested, washed twice with cold PBS, and lysed in lysis buffer (Beyotime, ShangHai, China) containing 1 mM PMSF (Beyotime) and protease inhibitor cocktail (Roche, Mannheim, Germany) for 30 min on ice. After centrifuga-

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Transfection of siRNA Knockdown of CHOP expression in C13* and primary cancer cells was achieved by specific siRNA, and non-

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targeted siRNA was used as a negative control. CHOP and non-targeting control siRNAs were purchased from Santa Cruz Biotechnology (Texas, USA). Cells were transfected with 100 nM concentrations of siRNAs diluted in Opti-Eagle’s minimal essential medium (Invitrogen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions; 48 h after transfection, cells were subsequently exposed to different measures.

In vivo experiments This study was performed with the approval of the Committee on the Ethics of Animal Experiments in Hubei province. All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Tongji Hospital in Hubei. Female nude athymic BALB/c-nu mice 4–6 weeks old were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. Human ovarian cancer cells C13* (5 9 106 in 100 lL volume of PBS) were injected subcutaneously into the right supra scapula region of mice. Tumor volume was estimated by using the formula volume = length 9 width2/ 2. About 1 week after tumor implantation, when the tumor reached a mean group size of 50 mm3, the mice were randomized to four groups with six mice per group and treated intraperitoneally with 40 mgkg1 Qu once a week (group 1), intraperitoneally with 3 mgkg1 cDDP once a week (group 2), a combination of 3 mgkg1 cDDP intraperitoneally 1 day following 40 mgkg1 Qu once a week (group 3) or vehicle control injected with the same volume of saline (group 4) for 4 weeks. The tumor volumes were determined by caliper measurement once a week. The blood was drawn once a week from the retinal venous plexus for biochemical analysis since drug intervention. When control mice started to succumb to their tumors, the mice in all treatment groups were euthanized and the tumors were weighed for treatment efficacy. Tumor tissue samples from mice were isolated for histopathological evaluations.

Biochemical analysis To judge the influence of Qu preconditioning on the side effects caused by cDDP, the blood of mice was collected once a week for creatinine level analysis in the core laboratory of Tongji Hospital (Wuhan, China) since drug intervention.

Statistical analysis All the data were presented as mean  SD of at least three independent experiments. Statistical analysis was performed using the SPSS16.0 software. The Student t test was used to determine statistical differences between treatment and control values, and P < 0.05 was considered significant.

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Acknowledgements This work was supported by the National Basic Research Program of China (973 Program, 2009CB521808) and the National High Technology Research and National Development Program of China (863 program, 2012AA02A507); National Nature and Science Foundation of China (81072135; 81372801; 81230038; 81272859; 81025011; 81090414; 81000979; 81101962); Nature and Science Foundation of Hubei Province (2011CBD542); and the Fundamental Research Funds for the Central Universities (HUST: 2012TS058).

Author contribution The first two authors contribute equally to this paper.

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