Hispidulin Mediates Apoptosis in Human Renal Cell Carcinoma by Inducing Ceramide Accumulation

Original Article

Hispidulin mediates apoptosis in human renal cell carcinoma by inducing ceramide accumulation

Hui GAO1, 2, #, Ming-quan GAO1, #, Jian-jun PENG2, *, Mei HAN1, Kai-li LIU1, Yan-tao HAN3, *

1Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao 266021, China; 2College of Life Sciences, Chongqing Normal University, Chongqing 401331, China; 3Medical College, Qingdao University, Qingdao 266071, China

Abstract Hispidulin, a polyphenolic flavonoid extracted from the traditional Chinese medicinal plant S involucrata, exhibits anti-tumor effects in a wide array of human cancer cells, mainly through growth inhibition, apoptosis induction and cell cycle arrest. However, its precise anticancer mechanisms remain unclear. In this study, we investigated the molecular mechanisms that contribute to hispidulin- induced apoptosis of human clear-cell renal cell carcinoma (ccRCC) lines Caki-2 and ACHN. Hispidulin (10, 20 µmol/L) decreased the viability of ccRCC cells in dose- and time-dependent manners without affecting that of normal tubular epithelial cells. Moreover, hispidulin treatment dose-dependently increased the levels of cleaved caspase-8 and caspase-9, but the inhibitors of caspase-8 and caspase-9 only partly abrogated hispidulin-induced apoptosis, suggesting that hispidulin triggered apoptosis via both extrinsic and intrinsic pathways. Moreover, hispidulin treatment significantly inhibited the activity of sphingosine kinase 1 (SphK1) and consequently promoted ceramide accumulation, thus leading to apoptosis of the cancer cells, whereasARTICLE pretreatment with K6PC-5, an activator of SphK1, or overexpression of SphK1 significantly attenuated the anti-proliferative and pro-apoptotic effects of hispidulin. In addition, hispidulin treatment dose-dependently activated ROS/JNK signaling and led to cell apoptosis. We further demonstrated in Caki-2 xenograft nude mice that injection of hispidulin (20, 40 mg·kg-1·d-1, ip) dose-dependently suppressed tumor growth accompanied by decreased SphK1 activity and increased ceramide accumulation in tumor tissues. Our findings reveal a new explanation for the anti- tumor mechanisms of hispidulin, and suggest that SphK1 and ceramide may serve as potential therapeutic targets for the treatment of ccRCC.

Keywords: hispidulin; SphK1; ceramide; ROS/JNK signaling; apoptosis; clear-cell renal cell carcinoma

Acta Pharmacologica Sinica (2017) 38: 1618–1631; doi: 10.1038/aps.2017.154; published online 9 Nov 2017

Introduction cinogenesis has led to the development of novel therapeutics Renal cell carcinoma (RCC) is the 7th most common cancer in targeting two interacting pathways: the VHL/HIF/VEGF and the developed world and is by far the most lethal urologic can- PI3K/AKT/mTOR pathways; these strategies have improved cer, accounting for 80% to 85% of kidney cancer cases[1]. There the prognosis of patients with ccRCC[4-7]. Despite advances in are approximately 209 000 new RCC cases and 102 000 related diagnostic and therapeutic strategies, including the introduc- deaths per year worldwide[2]. Clear-cell renal cell carcinoma tion of targeted therapy in clinical practice, clinical outcomes (ccRCC), the most common histological subtype of RCC, can have unfortunately not shown a satisfactory improvement in be cured with partial or radical nephrectomy at an early stage. the past decade, because of tumor recurrence and metastasis. However, approximately 20%–30% of patients present with Therefore, a better understanding of the factors involved in metastatic ccRCC at diagnosis. Moreover, up to 30% of newly the tumorigenesis process of ccRCC is imperative to develop diagnosedRETRACTED patients with localized disease develop metastases, more effective therapeutic strategies. and the recurrence rate is 20%–30% in patients after surgery[3]. A number of bioactive lipids, including ceramide (Cer), During the past decade, a better understanding of ccRCC car- sphingosine (Sph), and sphingosine 1-phosphate (S1P), play a crucial role in the development and progression of human cancers by regulating cell proliferation, apoptosis, migration, #These authors contributed equally to this work. senescence and responses to stressful conditions[8]. Among *To whom correspondence should be addressed. E-mail [email protected] (Jian-jun PENG); these lipids, ceramide has been identified as an anti-tumor [email protected] (Yan-tao HAN) effector that induces cell cycle arrest or apoptosis in cancerous Received 2017-05-15 Accepted 2017-07-09 cells[9]. In contrast, S1P functions as a pro-survival effector[10]. www.chinaphar.com Gao H et al 1619

The balance between ceramide and S1P is a key signal that Table 1. Clinicopathological features of 3 patients with primary ccRCC. determines cell fate, and sphingosine kinase 1 (SphK1) is an enzyme that plays a key role in the ceramide-S1P balance[10]. Patient No Gender Age TNM stage Lymph node metastasis Aberrant overexpression of SphK1 has been observed in a 1 Male 54 II N0 wide variety of human cancers, and the association between 1 Female 61 II N0 SphK1 expression and prognosis has been established[11]. 1 Male 51 I N0 Regarding ccRCC, SphK1 is up-regulated in ccRCC patients and is correlated with clinical outcomes[12]. Our previous work has also shown that SphK1 is involved in acquired resistance to sunitinib in renal cell carcinoma cells[13]. These data sug- (bFGF) and 10 ng/mL epidermal growth factor (EGF)[32]. After gest that SphK1 is a potential therapeutic target for treating 3 to 6 passages, primary ccRCC cells were used in our study. ccRCC[14]. Hispidulin (4’,5,7-trihydroxy-6-methoxyflavone), The study protocol was reviewed and approved by the Medi- a polyphenolic flavonoid, has been extracted from the tra- cal Ethics Committee of Qingdao University. Consent forms ditional Chinese medicinal plant S involucrata[15, 16] and has were signed by all participating patients. antifungal, anti-inflammatory, antioxidant, anti-thrombosis, antiepileptic, neuroprotective and antiosteoporotic activi- Cell counting kit-8 (CCK-8) assay ties[17-24]. Furthermore, hispidulin has also been identified to The cell viability was determined using a CCK-8 kit (Beyotime, have an anti-proliferative effect on a wide variety of cancer Shanghai, China) according to the manufacturer’s instructions. cells, including pancreatic, gastric, ovarian and glioblastoma The optical density of viable cells was measured using a spec- cells[25-28]. We have previously verified the pro-apoptotic trophotometer (Tecan Group Ltd, Männedorf, Switzerland). effects of hispidulin in hepatocellular carcinoma cells and leukemia cells[29, 30]. However, the underlying mechanisms Flow cytometry analysis of apoptosis through which hispidulin exerts its anti-tumor effects are Cell apoptosis was determined using a FITC Annexin V apop- not fully understood. Therefore, the present study was con- tosis kit (BD Pharmingen, Franklin Lakes, NJ, USA). Briefly, ducted to determine whether hispidulin suppresses tumor cells were harvestedARTICLE at a density of 5×105 cells/mL and incu- growth in ccRCC. Our results showed that hispidulin inhibits bated with Annexin V-FITC and propidium (PI) in the dark SphK1 activity and induces the subsequent accumulation of for 15 min before detection with a flow cytometer (Beckman ceramide in ccRCC cells. Moreover, our findings suggested Coulter Inc, Miami, FL, USA). that increased cellular ceramide levels lead to ROS generation and JNK activation, thus resulting in apoptosis. Measurement of mitochondrial membrane potential (MMP) JC-1 staining was performed to assess changes in the MMP. Materials and methods Briefly, cells were stained with JC-1 and then collected as a cell Cell culture pellet. Excess JC-1 was removed by rinsing the cell pellet with The human ccRCC cell lines Caki-2 and ACHN were pur- PBS. After resuspension, the fluorescence intensity of the cell chased from the ATCC. HK-2 tubule epithelial cells were solution was measured to determine the MMP. purchased from the Cell Bank of the Shanghai Institute of Bio- logical Science (Shanghai, China). Cells were cultured at 37 °C Reactive oxygen species (ROS) assay in a humidified atmosphere with 5% CO2 in DMEM medium The fluorescent probe 2’,7’-dichlorofluorescein-diacetate (HyClone, Logan, UT, USA) with 10% (v/v) heat-inactivated (DCFH-DA) was used to detect the ROS levels in ccRCC cells. FEB (HyClone, Logan, UT, USA), 2 mmol/L glutamine (Sigma, After treatment, ccRCC cells were harvested and resuspended St Louis, MO, USA), 1% nonessential amino acids (Sigma, St in DCFH-DA (10 µmol/L) at 37 °C for 30 min in the dark. Louis, MO, USA) and 100 U/mL streptomycin and penicillin Flow cytometry was used to quantify fluorescence signals, and (Sigma, St Louis, MO, USA). Hispidulin was purchased from the results were analyzed using Cell Quest software. Sigma-Aldrich (St Louis, MO, USA). qRT-PCR analysis of Sphk1 expression Primary culture of human RCC cells Total RNA was extracted from cultured cells by using TRIzol Primary cultures of human RCC cells were obtained from reagent (Thermo Fisher Scientific, Waltham, MA, USA). tissue specimensRETRACTED as previously described[31]. Tumor tissues Briefly, cDNA was acquired using a High Capacity cDNA were collected from 3 patients with ccRCC who received total Archive Kit (Applied Biosystems, Foster City, CA, USA). The nephroureterectomy at the Affiliated Hospital of Qingdao Uni- primers for SphK1 were synthesized on the basis of the pub- versity (Qingdao, China). The baseline information of the 3 lished sequence[33]. First-strand cDNA was obtained using patients is listed in Table 1. The ccRCC tissues were minced Super M-MLV Reverse transcriptase (BioTeke Co, Beijing, and then digested with collagenase I (Sigma, St Louis, MO, China). PCR reactions were performed using SYBR GREEN USA). Cells were obtained by rinsing and filtering. Primary master mix (Solarbio Co, Beijing, China). GAPDH was used ccRCC cells were cultured in FBS-DMEM/F12 medium for normalizing SphK1 mRNA expression. The comparative supplemented with 10 ng/mL basic fibroblast growth factor ΔCt method (ABPrism software, Applied Biosystems, Foster

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City, CA, USA) was used to quantify PCR results. viously described[37]. Briefly, cells were suspended in assay buffer and incubated at 37 °C for 1 h. The reaction was termi-

Separation of the cytosolic and mitochondrial proteins nated with NaBH4 (5 mg/mL). The activity of SPT was quan- Cytosolic and mitochondrial fractions of proteins were tified by using an HPLC system with a fluorescence detector separated as previously described[34]. After treatment, cells (Agilent, Santa Clara, CA, USA). were resuspended in mitochondrial protein isolation buffer (Amresco, Solon, OH, USA) according to the manufacturer’s Interference vector construction and transfection protocol. The cytosolic and mitochondrial fractions of the pro- The siRNA oligos for SphK1 gene knockdown were designed teins were collected for Western blotting. and synthesized by Sangon (Shanghai, China) as previously described[13]. Two distinct siRNA sequences and one Western blotting scrambled sequence for a control were cloned into the plasmid Proteins were extracted from cells as previously described[30]. vector pGCsi-H1 according to the manufacturer’s instruc- Specific primary antibodies against cleaved caspase-3, cleaved tions. The ccRCC cells in the logarithmic growth phase were caspase-8, cleaved caspase-9, SphK1, cytochrome c and β-actin plated in 6-well plates at a density of 3×105 cells per well, and were purchased from Abcam (Shanghai, China), antibodies transfection was conducted using Lipofectamine 3000 (Invitro- against Ki-67, p-JNK (Thr183/Tyr185), JNK, Fas, Fas-L, and gen, Grand Island, NY, USA) according to the manufacturer's FADD were from Santa Cruz Biotechnology (Santa Cruz, CA, instructions. USA), and the antibody against ceramide was from Sigma- Aldrich (St Louis, MO, USA). The secondary antibodies used SphK1 overexpression in this study were goat anti-rabbit IgG-HRP or anti-mouse IgG- SphK1 overexpressing and control vectors were constructed HRP (Beyotime, Shanghai, China). Signals were monitored as previously described[13]. Transfection of the SphK1 overex- using a chemiluminescent substrate (KPL, Guildford, UK). pressing construct was performed using Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA) according to the manu- Ceramide assay facturer’s instructions. The ceramide level was analyzed as previously described[35], ARTICLE and the results are presented as pmol per nmol of phospho- Xenograft model lipid (PL). Eight-week-old male athymic BALB/c nu/nu mice were maintained under pathogen-free conditions. Caki-2 cells (107 Analysis of sphingosine kinase 1 activity cells) were injected into the left flanks of mice. At 21 d after The activity assay for sphingosine kinase was conducted with injection, mice were randomly allocated into three groups (8 the Sphingosine Kinase Activity Assay Kit (Echelon Research mice per group) to receive intraperitoneal (IP) injections as Laboratories, Inc, Salt Lake City, UT, USA) according to the follows: (A) vehicle (0.9% sodium chloride plus 1% DMSO), manufacturer’s instructions. Briefly, cell lysate (20 μL) was (B) hispidulin (20 mg·kg-1·d-1, dissolved in vehicle), (C) his- incubated with the reaction buffer, 100 μmol/L sphingosine pidulin (40 mg·kg-1·d-1, dissolved in vehicle daily). Mouse body and 10 μmol/L ATP for 1 h at 37 °C, and a luminescence weights and tumor volumes were measured twice per week. attached ATP detector was then added to stop the kinase reac- IHC staining and TUNEL assays were performed on cryostat tion. Kinase activity was measured on the basis of the lumi- sections (4 μm sections) of Caki-2 xenograft tumors; this protocol nescence signals[13]. has been described in detail in our previous work[38]. Animal experiments for this study were approved by the Institutional Analysis of sphingomyelinase (SMase), ceramide synthase, Animal Care and Use Committee at Qingdao University. sphingomyelin synthase (SMS) and glucosylceramide synthase (GCS) activity Statistical analysis The activity of sphingomyelinase, ceramide synthase and Data are expressed as the mean±SD. Statistical comparisons glucosylceramide synthase was determined using NBD- between cell lines were analyzed by one-way ANOVA fol- sphingomyelin from Baijun Biotechnology (Guangzhou, lowed by Dunnett’s t-test. Experimental data were analyzed China) as previously described[36, 37]. Briefly, cells (1×106) were with GraphPad Prism software (GraphPad Software Inc, La lysed and incubated with 15 μmol/L NBD-sphingomyelin. Jolla, CA, USA), and a P value less than 0.05 was considered The reactionRETRACTED was halted with chloroform/methanol (2:1, statistically significant. v:v), and the lipids were obtained by extraction. Lipids were separated with TLC silica gel plates. The fluorescence signal Results of NBD-ceramide (excitation/emission of 460/515 nm) was Hispidulin inhibits cell growth in ccRCC cell lines and primary detected using a Typhoon 9410 variable mode imager (GE, ccRCC cells Shanghai, China). To explore the therapeutic potential of hispidulin in ccRCC cells, the anti-growth effect of hispidulin on cultured ccRCC Enzyme activity assay for serine palmitoyltransferase (SPT) cells was first examined. Figure 1A indicates that hispidulin Serine palmitoyltransferase activity was determined as pre- suppressed the cell growth of both ccRCC cell lines, Caki-2

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Figure 1. Effects of hispidulin on cell survival. (A) Hispidulin inhibits the growth of both ccRCC cell lines, Caki-2 and ACHN. Cells were treated with the indicated concentration of hispidulin for 24 h, 48 h, and 72 h. (B) Viability of the primaryARTICLE ccRCC cells and the normal tubular epithelial cells after hispidulin treatment was measured. Cell viability was analyzed by CCK-8 assay. **P<0.01. and ACHN, in a time- and concentration-dependent manner. the levels of both cleaved caspase-8 and cleaved caspase-9 The effects of hispidulin on the cell growth of primary ccRCC (Figure 2C). Given the roles of caspase-8 and caspase-9 in cells were also examined. As shown in Figure 1B, hispidulin extrinsic and intrinsic apoptotic signaling pathways, respec- treatment also dose-dependently decreased the viability of tively, our results suggested that hispidulin triggers apoptosis primary ccRCC cells. Notably, hispidulin did not decrease via both pathways. To further demonstrate the effects of his- survival of HK-2 cells, the normal tubular epithelial cells (Fig- pidulin on the extrinsic pathway, the effects of hispidulin on ure 1B). Taken together, our results suggested that hispidulin the expression of proteins relevant to the Fas death receptor selectively exerted anti-growth effect against ccRCC cells with- pathway were also examined. Although hispidulin did not out harming healthy kidney cells. affect the expression level of TNFR1 in both types of ccRCC cells, dose-dependent activation of Fas/FasL signaling and Hispidulin induces apoptosis in ccRCC cells DR5 were found after hispidulin treatment (Figure 3A). Intrin- To investigate the underlying mechanisms of the growth-inhi- sic apoptosis is characterized by the translocation of cyto- bition by hispidulin, cell apoptosis was analyzed. Following chrome c from mitochondria to the cytosol and by disruption treatment of hispidulin, both Caki-2 and ACHN cells exhib- of the MMP. As shown in Figure 3B and 3C, hispidulin treat- ited dose-dependent increases in the number of apoptotic cells ment led to disruption of the MMP and the loss of cytochrome (Figure 2A). Because activation of caspases plays a crucial role c from mitochondria. Our findings confirmed that both extrin- in apoptotic cell death, specific inhibitors of caspase-3 (z-VAD- sic and intrinsic pathways are involved in hispidulin-induced FMK), caspase-8 (z-LEHD-FMK) and caspase-9 (z-IETD-FMK) apoptosis in Caki-2 and ACHN cells. were used to explore the involvement of caspase activation in hispidulin-induced apoptosis. As shown in Figure 2B and 2C, Hispidulin-induced apoptosis is associated with ceramide hispidulinRETRACTED treatment resulted in marked elevation in level of accumulation and inhibition of SphK1 activity cleaved caspase-3 and caspase-3 inhibitors completely abol- Ceramide is a pro-apoptotic bioactive sphingolipid, and ished the hispidulin-induced apoptosis, thus suggesting that accumulating evidence has shown that the accumulation of hispidulin mediated caspase-dependent apoptosis in ccRCC ceramide triggers apoptosis signaling in cancerous cells[8]. cells. Moreover, our findings demonstrated that pretreatment Therefore, we investigated the effects of hispidulin on the lev- with either the caspase-8 inhibitor or the caspase-9 inhibitor els of ceramide in Caki-2 and ACHN cells. As shown in Figure partly blocked hispidulin-induced apoptosis (Figure 2B). The 4A, hispidulin treatment for 48 h led to a marked increase in Western blot results also confirmed that hispidulin increased ceramide levels in both cell types by promoting ceramide gen-

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Figure 2. Pro-apoptotic effects of hispidulin on ccRCC cell lines. (A) Hispidulin promotes cell apoptosis in Caki-2 and ACHN cells as measured by flow cytometry. (B) Hispidulin-induced cell apoptosis is significantly abrogated by specific inhibitors of caspase-3 (z-VAD-FMK), caspase-8 (z-LEHD-FMK), and caspase-9 (z-IETD-FMK) as measured by flow cytometry. (C) The expression of cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 were increased RETRACTEDby hispidulin as analyzed by Western blotting. **P<0.01. eration or suppressing its metabolism. Therefore, whether his- mediating sphingomyelin hydrolysis, thereby indicating that pidulin can affect the activity of enzymes related to ceramide the ceramide accumulation resulting from hispidulin treat- generation and metabolism was examined. In cancer cells, ment was not due to excessive generation. Interestingly, intracellular ceramide levels can be increased via de novo syn- hispidulin significantly suppressed the activity of SphK1, thesis or sphingomyelin hydrolysis. As shown in Figure 4B, although no significant effects on the activity of SMS and GCS hispidulin did not significantly alter the activity of SPT and were found (Figure 4C). Furthermore, our results showed that ceramide synthase, two enzymes mediating the de novo syn- hispidulin did not affect the mRNA or protein expression of thesis of ceramide, or neutral and acid SMases, two enzymes SphK1 (Figure 4D). Collectively, our findings suggested that

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Figure 3. Hispidulin induces extrinsic and intrinsic apoptosis in Caki-2 and ACHN cells. (A) Hispidulin activates Fas/FasL signaling and DR5 but does not affect the expression of TNFR1 as determined by Western blotting. (B) Hispidulin releases cytochrome c from mitochondria to the cytoplasm as determined by Western blotting. (C) Hispidulin causes the loss of MMP as measured by flow cytometry. **P<0.01.

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Figure 4. Hispidulin induces ceramide accumulation by inhibiting Sphk1 activity. (A) Effects of hispidulin on the accumulation of ceramide. (B) Effects of hispidulin on enzyme activity involved in ceramide generation. (C) Effects of hispidulin on the activity of SMS, GCS, and SphK1. (D) qRT-PCR and Western blots measuring the mRNA and protein expression of SphK1 after hispidulin treatment. **P<0.01.

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hispidulin induces apoptosis through ceramide accumulation off-target effect of hispidulin was demonstrated by SphK1 via inhibiting SphK1 activity. silencing. As shown in Figure 5A, the expression of SphK1 was decreased by more than 70% by RNA interference in both Inhibition of SphK1 activity mediates the pro-apoptotic effect of cell types. Then, the effects of hispidulin on cell growth and hispidulin apoptosis were examined. Figure 5B and 5C show that his- Our results showed that hispidulin promoted ceramide accu- pidulin further augment SphK1 knockdown-induced growth mulation via inhibiting SphK1 activity, leading to apoptosis. inhibition and apoptosis. Moreover, an established pharma- Next, we investigated whether inhibition of SphK1 activity cological inhibitor of SphK1 exerted an additive effect with mediated hispidulin-induced apoptosis in ccRCC cells. The hispidulin in suppressing cell growth and inducing apoptosis

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Figure 5. Repressing SphK1 expression or inhibiting SphK1 activity enhances the anti-growth and proapoptotic effects of hispidulin. (A) Western blots showing the protein expression levels of SphK1 in both Caki-2 and ACHN cells after siRNA transfection. (B) Effects of hispidulin on cell survival after siRNA transfection or pretreatment with a SphK1 inhibitor. (C) Effect of hispidulin on cell apoptosis after SphK1 knockdown or pretreatment with a SphK1 inhibitor as examined by flow cytometry. (D) Effect of hispidulin on the expression of cleaved caspase-3 after SphK1 knockdown or pretreatment with a Sphk1 inhibitor. **P<0.01.

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Figure 6. Overexpression of SphK1 or pretreatment with a SphK1 activator abolishes the anti-growth and proapoptotic effects of hispidulin A. Hispidulin exhibits anti-growth and pro-apoptotic effects by inhibiting SphK1 activity. Transfection of the SphK1-overexpressing plasmid and confirmation of the overexpression of SphK1 in Caki-2 and ACHN cells. The anti-proliferative effects of hispidulin were measured with a CCK-8 assay after Sphk1 overexpression or pretreatment with K6PC-5, an activator of Sphk1. (C) The pro-apoptotic effects of hispidulin with SphK1 overexpression or pretreatment with a SphK1 activator were assessed by flow cytometry. (D) The effects of hispidulin on the expression of cleaved caspase-3 after SphK1 overexpression or pretreatment with a SphK1 activator were detected by Western blotting. **P<0.01.

(Figure 5B, 5C and 5D). Moreover, to further verify the cru- also revealed that pretreatment with K6PC-5, a SphK1 acti- cial contributionRETRACTED of inhibition of SphK1 activity in the pro- vator, significantly reversed the anti-growth and apoptosis apoptotic effect of hispidulin, SphK1 was overexpressed in induction effects of hispidulin. Our findings indicated that Caki-2 and ACHN cells (Figure 6A). CCK-8 assay showed hispidulin-induced apoptosis is mediated by its inhibitory that the anti-proliferative effect of hispidulin was significantly effects on SphK1 activity. compromised in SphK1 overexpressing ccRCC cells (Figure 6B). Correspondingly, flow cytometric analysis and Western Accumulation of ceramide activates ROS/JNK signaling and blot also indicated that hispidulin-induced apoptosis and induces apoptosis caspase-3 activation were significantly attenuated by ectopic A recent study has shown that ROS/JNK signaling is associ- overexpression of SphK1 (Figure 6C and 6D). Our findings ated with ceramide-induced apoptosis in cancer cells[39]. Our

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Figure 7. Hispidulin promotes ROS generation and JNK activation. (A) ROS generation after treatment of Caki-2 and ACHN cells with hispidulin or hispidulin combined with K6PC-5, a Sphk1 activator. (B) The phosphorylation levels of JKN1/2 were detected by Western blotting after treatment with hispidulin or hispidulin combined with the ROS inhibitor NAC. (C) Cell apoptosis was examined by flow cytometry after treatment with hispidulin or hispidulin combined with NAC or the JNK inhibitor SP600125. (D) The expression of cleaved caspase-3 following treatment with hispidulin or hispidulin combined with NAC or the JNK inhibitor SP600125 weas detected by Western blotting. **P<0.01. findings also verified that hispidulin administration was 7D, neither NAC nor SP600125 abolished hispidulin-induced associatedRETRACTED with a dose-dependent increase in intracellular apoptosis in Caki-2 and ACHN cells. Our findings collectively ROS (Figure 7A), and this effect was markedly abrogated by indicated that hispidulin inhibits SphK1 activity and subse- K6PC-5, thus suggesting that the increases in the ROS level quently induces the accumulation of ceramide, which then depended on SphK1 inhibition and subsequent ceramide accu- activates ROS/JNK signaling and leads to apoptosis. mulation. Western blots also showed that hispidulin induced JNK activation in a ROS-dependent manner (Figure 7B). Hispidulin induces apoptosis in vivo Therefore, we next explored the role of ROS/JNK signaling in On the basis of our in vitro results, a xenograft mouse model hispidulin-induced apoptosis by using the ROS inhibitor NAC was used to test the in vivo therapeutic effects of hispidu- and the JNK inhibitor SP600125. As shown in Figure 7C and lin. The dosages of hispidulin were 40 mg·kg-1·d-1 and 20

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Figure 8. Anti-neoplastic activity of hispidulin in Caki-2 xenograft tumors. (A) Measurements of tumor volume at the indicated time point after treatment with hispidulin (20 mg/kg or 40 mg/kg). (B) Effect of hispidulin on SphK1 activity in vivo. (C) TUNEL and immunohischemistry assay performed on cryostat sections were used to detect cell apoptosis and the expression of Ki-67, ceramide, cleaved caspase-3, and p-JNK1/2 after hispidulin treatment. **P<0.01.

mg·kg-1·d-1. As shown in Figure 8A, both dosages significantly pro-apoptotic effects in a panel of cancerous cells, includ- suppressed tumor growth (P<0.01 vs control). Corresponding ing gastric cancer cells, pancreatic cancer cells and hepatoma to our observation of tumor growth, TUNEL and immuno- cells[28, 44, 45]. Moreover, our previous study has also demon- histochemistry assays showed that hispidulin treatment was strated that hispidulin inhibits cell proliferation and induces associated with dose-dependent increases in cell apoptosis, as apoptosis in vitro in hepatocellular carcinoma and gallblad- well as increases in the expression of cleaved caspase-3 (Figure der cancer[29, 41]. Our recent work has revealed that hispidulin 8C). Moreover, our results showed that tumor growth inhibi- induces mitochondrial apoptosis in leukemia cells, in addition tion by hispidulin was correlated with decreased activity of to solid tumors[30]. Here, we explored the role of hispidulin SphK1, ceramide accumulation and increased expression of in ccRCC and elucidated its potential molecular mechanisms. p-JNK in tissues (Figure 8B and 8C), thus supporting our in Our findings suggested that hispidulin inhibits SphK1 activity vitro findings that hispidulin mediates apoptosis in ccRCC by and induces the subsequent accumulation of ceramide, which inhibiting SphK1 and consequently inducing ceramide accu- in turn activates ROS/JNK signaling and eventually leads to mulation, which in turn activates ROS/JNK signaling. apoptosis in ccRCC cells (Figure 9). Apoptosis has been considered as the main pathway Discussion through which chemotherapeutics eradicate cancer cells[46]. HispidulinRETRACTED has been used as an antifungal and anti-inflam- Our findings in this study also showed that the in vitro anti- matory agent in China for centuries[16]. The anti-neoplastic proliferative effects and the in vivo tumor suppressing effects activity of hispidulin, a flavonoid compound, has also been of hispidulin correlated with its pro-apoptotic effects, thus documented[25, 27, 29, 40-42]. The role of hispidulin as a chemopre- suggesting that hispidulin exerts its anti-tumor effects at ventive agent was first reported in 1992[43]. In 2010, Way et al least partly by inducing apoptosis. Apoptosis mainly occurs revealed that hispidulin induces apoptosis in ovarian cancer through two distinct pathways, the extrinsic or death receptor and glioblastoma multiforme cells through activating AMPK pathway and the intrinsic or mitochondrial pathway[47]. Both signaling[25, 26]. Hispidulin also has been found to exerts its pathways involve the activation of initiator caspases, caspase-8

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synthesis from palmitoyl-CoA and L-serine by using serine palmitoyl transferase (SPT) and ceramide synthase or through sphingomyelin hydrolysis by using sphingomyelinases (SMase)[9]. However, cellular ceramides are consumed for the production of sphingomyelin, glucosylceramide, or ceramide 1-phosphate through the actions of sphingomyelin synthase (SMS), glucosylceramide synthase (GCS), or SphK1, respec- tively[48]. A number of chemotherapeutic agents, including natural compounds, have been proposed to induce apoptosis in cancerous cells through ceramide accumulation by either promoting the de novo synthesis of ceramide or suppressing the metabolism of ceramide via targeting the above-mentioned enzymes. An early study has reported that resveratrol, a phy- toalexin present in grapes and red wine, inhibits cell proliferation and pro-apoptosis in metastatic breast cancer cells via de novo ceramide signaling via the activation of SPT[49]. Resve- ratrol has been found to increase the intracellular generation and accumulation of apoptotic ceramides through down- regulating GCS and SphK1[50]. Stichoposide C, a marine triter- pene glycoside, has been found to induce apoptosis in leukemia and colorectal cancer cells through ceramide generation via activating SMase[51]. Sobota et al have demonstrated that curcumin induces apoptosis in multidrug-resistant human Figure 9. Proposed signal transduction pathways through which hispidulin leukemia HL60 cells by activating neutral sphingomyelinase induces apoptosis in ccRCC cells. (nSMase), afterARTICLE the inhibition of sphingomyelin synthase[36]. In our study, we found that ceramide accumulation is involved in the extrinsic pathway and caspase-9 in the intrinsic path- in hispidulin-induced apoptosis in ccRCC cells. Moreover, way; this process is followed by the activation of effector cas- our findings indicated that hispidulin does not affect the pases, such as caspase-3, as apoptosis executioners[47]. Herein, generation of ceramide, but it suppresses the consumption of we verified that the apoptosis induced by hispidulin in ccRCC ceramide by inhibiting SphK1. cells occurred via both extrinsic and intrinsic pathways. The The dysregulation of SphK1 has been documented in a vari- activation of the extrinsic apoptosis pathway by hispidulin ety of human malignancies, and both preclinical and clinical was confirmed by caspase-8 activation and the increased evidence has shown that SphK1 is related to various cancer expression of Fas protein levels in cells, whereas activation processes, such as cell oncogenesis, survival, metastasis and [52] of the intrinsic apoptosis pathway by hispidulin was identi- tumor microenvironment neovascuelarization , thus making [11] fied by the activation of caspase-9, disruption of the MMP, SphK1 a promising therapeutic target . In fact, SphK1 inhibi- and cytochrome c release from mitochondria to the cytosol. tors are under evaluation in pre-clinical and clinical studies [11] The results of the present study are consistent with Way’s for their therapeutic effects . Regarding ccRCC, fingolimod, results showing that hispidulin activates both the intrinsic and a functional antagonist of the S1P receptor and an inhibitor of extrinsic apoptosis pathways by inducing p53 expression in SphK1, chemosensitizes and promotes vascular remodeling in [53] human glioblastoma multiforme cells[26]. However, our previ- ccRCC . In line with this recent finding, our results revealed ous studies using leukemia and hepatocellular carcinoma cells that hispidulin induces apoptosis mainly by inhibiting SphK1 have shown that hispidulin induces apoptosis via only the activity, thus suggesting that modulating SphK1 activity may mitochondrial pathway, a result also in line with Yu’s findings provide a novel therapeutic strategy for ccRCC treatment. in gastric cancer cells[28-30]. We postulate that the mechanism In summary, our findings in the current study showed that through which hispidulin induces apoptosis might depend hispidulin exerts a potent anti-neoplastic effect in ccRCC by on the major molecules or signaling pathway that contributes inducing apoptosis. Furthermore, our findings also suggested to the pro-apoptoticRETRACTED effects of hispidulin in specific cancerous that hispidulin inhibits SphK1 activity and consequently cells. However, more data need to be collected to confirm our induces ceramide accumulation, thus resulting in apoptosis postulation. through activation of ROS/JNK signaling. The balance of ceramide-sphingosine-S1P rheostat has been identified to play a crucial role in deciding the fate of cancer Acknowledgements cells[9]. In cancer cells, cellular ceramide accumulation cor- This work was supported by the National Natural Sci- relates with anti-growth pathways, such as senescence and ence Foundation of China (No 31470570 and 81603337), apoptosis[9]. Therefore, ceramide is considered a “tumor sup- the Chongqing Natural Science Foundation (No cstc2014j- pressor lipid”. Cancer cells produce ceramide through de novo cyjA80013), the Science Foundation of Chongqing Educa-

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