Mammalian target of rapamycin regulates isoliquiritigenin-induced ...

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Sep 29, 2011 - Mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells.

Apoptosis (2012) 17:90–101 DOI 10.1007/s10495-011-0658-1

ORIGINAL PAPER

Mammalian target of rapamycin regulates isoliquiritigenininduced autophagic and apoptotic cell death in adenoid cystic carcinoma cells Gang Chen • Xiang Hu • Wei Zhang • Ning Xu • Feng-Qin Wang • Jun Jia • Wen-Feng Zhang • Zhi-Jun Sun • Yi-Fang Zhao

Published online: 29 September 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Previous studies, including those from our laboratory, have demonstrated that isoliquiritigenin (ISL), a flavonoid isolated from licorice, is a promising cancer chemotherapeutic agent. However the mechanisms underlying its anticancer effects are still far from clear. We now show, for the first time, that ISL triggers the mammalian target of rapamycin (mTOR)-dependent autophagic and apoptotic cell death in adenoid cystic carcinoma (ACC). Exposure of both ACC-2 and ACC-M cells to ISL resulted in several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, punctate pattern of LC3 immunostaining, and an increase in autophagic flux. Moreover, ISL treatment also resulted in significantly increased apoptosis in ACC cells. The ISL-mediated autophagic and apoptotic cell death were obviously attenuated by transfection with dominant negative Atg5 (DN-Atg5K130R) plasmids or treatment with 3-methyladenine(3-MA). In additon, the data also revealed that the autophagic and apoptotic cell

Gang Chen and Xiang Hu contributed equally to this article.

Electronic supplementary material The online version of this article (doi:10.1007/s10495-011-0658-1) contains supplementary material, which is available to authorized users. G. Chen  X. Hu  W. Zhang  N. Xu  F.-Q. Wang  J. Jia  W.-F. Zhang  Z.-J. Sun (&)  Y.-F. Zhao (&) The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory of Oral Biomedicine Ministry of Education, Department of Oral and Maxillofacial Surgery, School & Hospital of Stomatology, Wuhan University, Wuhan, China e-mail: [email protected] Y.-F. Zhao e-mail: [email protected]

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death induced by ISL occurred through a mTOR-dependent pathway. More importantly, the xenograft model using ACC-M cells provided further evidence of the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppresson of mTOR activation as well as up-regulation of Atg5 expression. Taken together, these findings in our study suggest that induction of mTORdependent autophagic and apoptotic cell death may be an important mechanism in cancer chemotherapy by ISL. Keywords Isoliquiritigenin  Adenoid cystic carcinoma  Autophagy  Apoptosis  mTOR

Introduction Adenoid cystic carcinoma (ACC) is one of the most common malignancies of the major and minor salivary glands [1, 2], and accounts for approximately 15–25% of all the carcinomas at these locations [3]. This neoplasm often presents a prolonged clinical course [4], providing a large window of opportunity for intervention to prevent or slow its malignant progression. Thus, identification and preclinical/clinical development of novel agents that are nontoxic to humans but can delay the onset and/or progression of ACC are highly desirable. Epidemiological studies have shown that dietary intake of flavonoid-containing fruits, vegetables and beverages is associated with a low risk of cancer [5, 6]. Isoliquiritigenin (ISL), 20 ,40 ,40 -3-hydroxychalcone [7, 8], is a natural flavonoid that has been shown to be nontoxic to humans but that has various biological properties, such as antiinflammatory, antioxidant, and antiplatelet aggregation, as well as vasorelaxant and estrogenic effects [8, 9]. More importantly, ISL has also been demonstrated to possess

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significant antitumor activities, including inhibition of proliferation and/or induction of apoptosis [10] as well as prevention of metastasis [11, 12]. Nevertheless, the precise mechanisms underlying the antitumor activities of ISL are yet to be elucidated. Our recent study [8] revealed that ISL treatment suppressed the growth and angiogenesis-induction activity of ACC cells in vitro and in vivo. We found that treatment with ISL (0–20 lM) for 24 h could inhibit the growth of ACC cells in cultures without the occurrence of cell death. Meanwhile, we also observed that when the treatment period was extended to 48 or 72 h, cell viability was significantly reduced by the same concentrations of ISL. However, the mechanisms behind the cytotoxicity exerted by ISL have not been fully elucidated. The only known intracellular responses are the suppression of the mTOR pathway correlating with concurrent activation of c-Jun NH2-terminal kinase (JNK), and the inhibition of extracellular signal-regulated kinase (ERK). Of note, the mTOR signaling pathway has assumed a central role in the regulation of autophagy, which is an evolutionarily conserved and dynamic process for bulk degradation of cellular macromolecules and organelles [13, 14]. The present study is designed to investigate whether ISL-induced mTOR suppression may lead to autophagy in ACC cells, and whether the occurrence of autophagy may contribute to the cytotoxicity exerted by ISL. Here, we initially evaluated the effects of ISL in cultured ACC cells under cytotoxic conditions (0–20 lM for 48 h), and found that under these conditions ISL treatment not only induced autophagy in ACC cells, but also resulted in significantly increased cell apoptosis. This related to the up-regulation of mTOR-dependent Atg5 expression. Further, we assessed the in vivo effects of ISL by establishing a xenograft model using ACC-M cells, and also observed the mTOR-dependent autophagic and apoptotic cell death induced by ISL. Taken together, these findings suggest that ISL can induce mTOR-dependent autophagic and apoptotic cell death in ACC cells, which likely serve as an important mechanism in its cancer chemotherapeutic effects.

against b-actin was purchased from Santa Cruz. The expression vectors encoding constitutively active mTOR (pRK5-CA-mTOR), as well as the corresponding empty vectors (pRK5) were kindly provided by Dr. Fuyuhiko Tamanoi (University of California, USA). The expression vectors encoding dominant negative Atg5 mutant (pCIneo-DN-Atg5K130R), as well as the corresponding empty vectors (pCI-neo) were provided by Prof. Tamotsu Yoshimori (National Institute for Basic Biology, Okazaki, Japan).

Materials and methods

Detection of acidic vesicular organelles

Chemicals and antibodies

The formation of yellow-orange acidic vesicular organelles (AVOs) was detected as we described earlier [15]. Briefly, both ACC-2 and ACC-M cells (2 9 105) were grown on coverslips in 6-cm dishes and allowed to attach by overnight incubation. After treatment with increased concentrations of ISL for 48 h, cells were stained with 1 lg/ml acridine orange (AO) in PBS for 15 min, washed with PBS, and then examined under a Leica fluorescence microscope.

Unless otherwise noted, all chemicals and reagents including ISL were purchased from Sigma–Aldrich. Primary antibodies for phospho-mTOR (Ser2448), mTOR, phospho-S6 (Ser235/236), S6, Atg5, LC3, Bax, Bcl-2, and cleaved-poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology. Primary antibody

Cell culture and transient transfection The low (ACC-2) and high (ACC-M) metastatic cell lines of human salivary ACC were obtained from the China Center for Type Culture Collection, and were grown in DMEM medium supplemented with 10% FBS. The cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For transient transfection, ACC cells were seeded in 6 cm culture dishes at a density of 106 cells/dish, and then transfected with CA-mTOR, DN-Atg5K130R as well as corresponding vector plasmids using the LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions. The expression levels of p-mTOR and Atg5– Atg12 complex after transfection were determined by Western blotting. Transmission electron microscopy The transmission electron microscopy was performed according to our previous procedures [15]. Briefly, both ACC-2 and ACC-M cells were incubated with either DMSO (Control) or 20 lM ISL for 48 h. Cells were then fixed with 2.5% electron microscopy grade glutaraldehyde, postfixed in 1% osmium tetroxide with 0.1% potassium ferricyanide, dehydrated through a graded series of ethanol (30–90%), and embedded in Epon. Ultrathin sections (65 nm) were cut, stained with 2% uranyl acetate and then examined using a Hitachi H-600 Transmission Electron Microscope.

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Immunofluorescence microscopy for LC3 localization The immunofluorescence for LC3 localization was performed as previously described [15]. Briefly, both ACC-2 and ACC-M cells (2 9 105) were plated on coverslips in 6-cm dishes and allowed to attach by overnight incubation. Cells were then treated with increased concentrations of ISL for 48 h. After that, the cells were washed with PBS and fixed in 4% paraformaldehyde overnight at 4°C. They were then permeabilized with 0.1% Triton X-100 for 10 min and blocked with bovine serum albumin (BSA) buffer for 1 h. Cells were then incubated with the anti-LC3 antibody (1:50) overnight at 4°C, then treated with fluorescein isothiocyanate-conjugated (FITC) donkey antirabbit antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. The cells with a punctate pattern of LC3 localization were then visualized and captured using a Leica fluorescence microscope. Determination of apoptosis ISL-induced apoptosis in ACC-2 and ACC-M cells was detected according to our previous procedures [16, 17] as follows: (a) morphological evaluation by Hoechst staining; (b) quantification of cytoplasmic histone-associated DNA fragments with Cell Death Detection ELISAPLUS assay; (c) caspase-3 activity measurement by Colorimetric Caspase Activity Assay kit; and (d) Western blot analysis for Bax/Bcl-2 ratio and PARP cleavage. Western blot analysis Western blot analysis was performed according to our previous procedures [8, 16, 17]. Briefly, ACC cells were lysed, and the total protein was separated using 10% SDS– polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). The blots were then blocked with 5% non-fat dry milk at room temperature for 1 h, and incubated overnight at 4°C with the corresponding primary antibodies at dilutions recommended by the suppliers, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Pierce) for 1 h. Blots were then developed by a SuperEnhanced chemiluminescence detection kit (Applygen Technologies Inc.). Nude mice xenografts Female BALB/c nude mice (18–20 g; 6–8 weeks of age) were purchased from the Experimental Animal Center of Wuhan University in a pressurized ventilated cage according to institutional regulations. All studies were approved and supervised by the Animal Care and Use

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Committee of Wuhan University. Exponentially growing ACC-M cells (2 9 106 in 0.2 ml medium) were inoculated subcutaneously into the flank of the mice. After 7 days, tumor-bearing mice were randomly divided into three groups, which were treated with ISL (0.5 g/kg or 1 g/kg p.o. daily; n = 8) or corn oil (Control, 100 ll, p.o., daily; n = 8) for 30 consecutive days. Tumor growth was determined by measuring the size of the tumors daily. Tumor volumes were calculated according to the formula (width2 9 length)/2. The mice were euthanized at Day 30, and the tumors were captured, photographed and embedded in paraffin or frozen at -80°C for the following immunohistochemical analysis as well as for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Statistical analysis All data represent mean ± SEM of three independent experiments. Statistical analysis was performed by Student’s t-test at a significance level of P \ 0.05.

Results ISL treatment activates autophagy in ACC cells As seen in Fig. 1a, treatment of both ACC-2 and ACC-M cells with ISL (20 lM) resulted in typical appearance of membranous vacuoles resembling autophagosomes (double arrowheads). On the other hand, the DMSO-treated control cells exhibited normal healthy looking mitochondria (single arrowhead). Moreover, we also examined the autophagic response of ACC cells to ISL by analysis of LC3 processing and recruitment as well as formation of yelloworange AVOs, which are hallmarks of autophagy. We found that exposure to ISL led to an obvious punctate pattern of LC3 immunostaining in both ACC-2 and ACCM cells, which was rare in the control cells (Fig. 1b). In addition, the data also revealed that ACC cells treated with DMSO primarily exhibited green fluorescence. However, treatment with ISL resulted in formation of yellow-orange AVOs in both ACC-2 and ACC-M cells in a concentrationdependent manner (Fig. 1c). Furthermore, ISL treatment also induced the cleavage of LC3 (an increase in the LC3II/LC3-I ratio) in both ACC-2 and ACC-M cells, and meanwhile up-regulated the expression level of Atg5, an essential protein in autophagosome formation (Fig. 1d). To further distinguish whether ISL-mediated autophagosome accumulation is due to autophagy induction or rather to a block in downstream steps, we performed a LC3 turnover assay by Western blot analysis using the lysosomal inhibitor Bafilomycin A1 to measure the effect of ISL on

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autophagic flux. The results demonstrated that the difference in LC3-II levels in the presence and absence of Bafilomycin A1 is much larger in case of treatment with ISL when compared to the untreated controls, indicating that autophagic flux is indeed increased by ISL treatment (Fig. 1e). Collectively, these observations strongly suggest that ISL could cause autophagy in ACC cells. ISL treatment also induces apoptosis in ACC cells As shown in Fig. 2a, after treatment with 20 lM ISL for 48 h, several apoptotic morphologic features such as

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apoptotic bodies, cell shrinkage and chromatin condensation were observed in ACC-2 and ACC-M cells by Hoechst 33258 staining assay. We also quantitatively assessed the DNA fragmentation in ISL-treated ACC cells using the Cell Death Detection ELISAPLUS kit. Likewise, the results revealed that treatment with ISL resulted in formation of DNA fragments in both ACC-2 and ACC-M cells in a concentration-dependent manner (Fig. 2b). To further verify the ISL-induced apoptosis in ACC cells, we detected the expression levels of some key apoptosis-related proteins in ACC cells treated with ISL. As the results show, the activity of caspase-3 was found to increase significantly

Fig. 1 ISL treatment causes autophagy in ACC cells. Both ACC-2 and ACC-M cells were grown on coverslips in 6-cm dishes and allowed to attach by overnight incubation. Cells were then treated with indicated concentrations of ISL for 48 h. a Detection and quantification for ISL-induced autophagosomes by transmission electron microscopy. b Detection for ISL-induced punctate pattern of LC3 immunostaining. c Detection for ISL-induced formation of yellow-orange AVOs. d The expression levels of Atg5–Atg12 complex and LC3 were detected by Western blotting. e The difference in LC3 expression levels between control and ISL-treated samples with or without Bafilomycin A1 was compared by Western blotting. All data are presented as mean ± SEM from three different experiments with duplicate. ** P \ 0.01 versus the control group

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in ISL-treated ACC cells (Fig. 2c). On the other hand, both the ratio of Bax/Bcl-2 and the cleavage of PARP in both ACC-2 and ACC-M cells were significantly increased by ISL in a concentration-dependent manner (Fig. 2d), thus confirming the apoptosis-induction by ISL. Inactivation of Atg5 prevented ISL-induced autophagy in ACC cells As described above, Atg5 is an essential protein for autophagosome formation, and Atg5-deficience can significantly diminish the number of autophagic vesicles [18, 19]. Here we evaluated the role of Atg5 in ISL-induced autophagy in ACC cells by transient transfection of DN-Atg5K130R prior to ISL treatment. DN-Atg5K130R is a dominant negative mutant of Atg5, in which Lys130 of Atg5 was replaced with Arg, and has been previously shown to inhibit its conjugation with Atg12 and suppress vacuole formation [20]. Under normal conditions, the conjugation between Atg5 and Atg12 was formed via an isopeptide bond between the C-terminal glycine of Atg12 and Lys130 of Atg5. While when the Lys130 of Atg5 was changed to Arg, the conjugation between Atg5 and Atg12

Fig. 2 ISL induces apoptosis in ACC cells. Both ACC-2 and ACC-M cells were treated with indicated concentrations of ISL for 48 h. a The morphologic changes were captured using fluorescence microscopy with Hoechst 33258 staining. b DNA fragmentation was quantified using Cell Death Detection ELISAPLUS assay. c Caspase-3 activity was measured by Colorimetric Caspase Activity Assay. d The expression levels of cleaved-PARP, Bcl-2 and Bax were determined by Western blotting. All values were presented as means ± SEM from three different experiments with duplicate. ** P \ 0.01 versus the control group

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was destroyed, and therefore the formation of Atg5–Atg12 complex was blocked [21]. As our results show, the punctate pattern of LC3 immunostaining that resulted from ISL treatment for 48 h was significantly reduced by transient transfection of DN-Atg5K130R (Fig. 3a), correlating with the obviously decreased expression of the Atg5– Atg12 complex (Fig. 3b). Consistent with these finding, the autophagic flux induced by ISL in both ACC-2 and ACC-M cells was effectively prevented by inhibition of Atg5 as well (Fig. 3b). Moreover, 3-MA, a well known autophagy inhibitor which could decrease the expression of Atg5 [22, 23], also significantly attenuated the ISL-mediated punctate pattern of LC3 immunostaining (Fig. 3a). All of the above findings indicate that ISL-induced autophagy in ACC cells was dependent on Atg5. ISL-mediated autophagy in ACC cells was a form of cell death but not a protective mechanism The role of autophagy in cancer development and therapy is still unclear. Some studies have suggested that autophagy may serve as a protective mechanism against therapyinduced cell death in various cancer cells [24, 25]. On the

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Fig. 3 Inactivation of Atg5 prevents ISL-induced autophagy in ACC cells. Both ACC-2 and ACC-M cells were transfected with DN-Atg5K130R or pretreated with 3-MA (4 mM), followed by exposure to ISL (20 lM) for 48 h. a Detection and quantification for punctate pattern of LC3 immunostaining. b The protein expression level of the Atg5–Atg12 complex in ISL-treated ACC cells after transfection with DN-Atg5K130R was evaluated by Western blotting. In addition, the difference in LC3 expression levels between samples with and without Bafilomycin A1 was compared by Western blotting. All data are presented as mean ± SEM from three different experiments with duplicate. ** P \ 0.01 versus the control group

other hand, there are also some studies stating that autophagy could be a form of cell death for various anticancer agents [26, 27]. In order to determine the precise role of autophagy in ISL-treated ACC cells, the effect of 3-MA on ISL-induced cell viability reduction was initially evaluated. As shown in Fig. 4a, the decreased cell viability that resulted from 48 h exposure to ISL was effectively rescued by pretreatment with 3-MA, suggesting that ISLmediated autophagy in ACC cells, at least at this exposure time (48 h) and these concentrations (5–20 lM), might be a form of cell death but not a protective mechanism. More importantly, genetic inhibition of ISL-mediated autophagy by transfection with DN-Atg5K130R also significantly prevented the cytotoxicity exerted by ISL in both ACC-2 and ACC-M cells (Fig. 4a), which further validated our hypothesis. After confirming the detrimental role of ISL-mediated autophagy in ACC cells, we further proceeded to explore the relationship between ISL-induced autophagy and apoptosis by examining the effects of 3-MA treatment and DN-Atg5K130R transfection on ISL-induced apoptotic cell death. As the results show, both 3-MA treatment and DN-Atg5K130R transfection significantly but not completely reversed ISL-induced apoptosis in ACC cells, as evidenced by the Hoechst staining (Fig. 4b) as well as by DNA

fragmentation detection (Fig. 4c). Furthermore, pretreatment with 3-MA or transfection with DN-Atg5K130R effectively, and also partially, prevented ISL-mediated PARP cleavage in both ACC-2 and ACC-M cells (Fig. 4d). Together, these results not only demonstrate an essential role of Atg5 in regulation of ISL-mediated autophagy and apoptosis in ACC cells, but also provide additional evidence to indicate that ISL-induced autophagy and apoptosis are interrelated. ISL-mediated autophagy and apoptosis correlated with suppression of mTOR activation Our previous study has shown that ISL treatment for 24 h inhibited the activation of mTOR, a protein kinase which emerged as a key negative regulator of autophagy [13]. In addition, previous studies including those from our laboratory have proven that mTOR was also a potential regulator of apoptosis in various cancer cells [28–30], including ACC cells [17]. Here, we again confirm that treatment with ISL for 48 h could more effectively suppress the activation of mTOR in both ACC-2 and ACC-M cells (Fig. 5a). Meanwhile, the expression level of phosphorylated-S6, a key downstream target of mTOR [31], was also decreased by ISL in both ACC-2 and ACC-M cells (Fig. 5a). All of

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Fig. 4 Inhibition of autophagy prevents ISL-mediated viability reduction and apoptosis induction in ACC cells. Both ACC-2 and ACC-M cells were transfected without (Control) or with DN-Atg5K130R or corresponding vector plasmids, or were pretreated with 3-MA (4 mM), followed by exposure to ISL (20 lM) for 48 h. a Cell viability was measured using the Vi-CELL cell viability analyzer. b Then morphologic changes were captured using fluorescence microscopy with Hoechst 33258 staining. c DNA fragmentation was quantified using Cell Death Detection ELISAPLUS assay. d The expression level of cleavedPARP was determined by Western blotting. All values were presented as means ± SEM from three different experiments with duplicates. * P \ 0.05 versus the corresponding group without ISL treatment, and # P \ 0.05 versus the control group with only ISL treatment but without any other pretreatment

this evidence suggests that ISL-mediated autophagy and apoptosis in ACC cells is probably due to the suppression of mTOR. To further determine the functional significance of mTOR suppression in the autophagic and apoptotic responses of ACC cells to ISL, we assessed the effect of CA-mTOR over-expression on ISL-mediated autophagy and apoptosis. As evidenced in Fig. 5a, transient transfection with CA-mTOR nearly blocked ISL-mediated mTOR inactivation, but only partially retrieved the up-regulation of Atg5 and the cleavage of LC3 and PARP by ISL. Consistent with these findings, over-expression of CAmTOR in ACC cells significantly but only partially prevented ISL-mediated recruitment of LC3 immunostaining (Fig. 5b). Similarly, the apoptotic ACC cells induced by

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ISL, demonstrated by Hoechst staining as well as DNA fragment detection, was also significantly but partially reversed by CA-mTOR transfection (Fig. 5c). These results confirmed that suppression of the mTOR signaling pathway is indispensable for ISL-mediated autophagy and apoptosis, and also indicated that there must be other mechanisms involved, although this requires further investigation. ISL inhibited ACC xenograft growth in association with induced autophagy and apoptosis To further authenticate the effects of ISL described above, animal experiment were designed to test whether ISL could induce autophagic and apoptotic cell death in vivo., and ACC xenografts in nude mice were established using

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Fig. 5 mTOR regulates ISLinduced autophagic and apoptotic cell death in ACC cells. Both ACC-2 and ACC-M cells were transfected with or without CA-mTOR, followed by exposure to indicated concentrations of ISL for 48 h. a The expression levels of p-mTOR, mTOR, p-S6, S6, Atg5–Atg12 complex, LC3 and cleaved-PARP were determined by Western blotting. b Detection and quantification for the punctate pattern of LC3 immunostaining. c The morphologic changes were captured using fluorescence microscopy with Hoechst 33258 staining, and meanwhile the DNA fragmentation was quantified using Cell Death Detection ELISAPLUS assay. * P \ 0.05 versus the corresponding group without ISL treatment, and # P \ 0.05 versus the control group with only ISL treatment but without any other pretreatment

ACC-M cells. As the results show, the growth of ACC-M tumors was significantly inhibited by ISL treatment in a dose-dependent manner compared with the vehicle controls, but the body weight was not affected (Fig. 6a). Importantly, the tumors from ISL-treated mice exhibited significantly increased expression of LC3 (Fig. 6b), suggesting activated autophagic responses. Moreover, treatment with ISL significantly induced cell apoptosis in tumor tissues as evidenced by the TUNEL assay, which is related to the suppression of mTOR activation as well as to the up-regulation of Atg5 expression (Fig. 6b). These results

indicated that ISL could induce mTOR-dependent autophagic and apoptotic cell death in ACC cells in vivo, which might be an important mechanism underlying the antitumor activities of ISL.

Discussion Our previous study has shown that the dietary cancer chemotherapeutic agent ISL could suppress tumor-induced angiogenesis in ACC in vitro and in vivo associating with

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Fig. 6 ISL induced mTORdependent autophagic and apoptotic cell death in vivo. a Tumor volume and body weight of animal in vehicletreated control mice and ISL-treated mice. b Immunohistochemical analysis of the indicated biomarkers in both control and ISL-treated ACC-M tumor tissues. * P \ 0.05 versus the control group

inhibition of mTOR [8], a highly conserved serine/threonine kinase plays a central role in regulation of autophagy [13, 32, 33]. Thus, the present study was designed to investigate the effects of ISL on autophagy. We exposed ACC cells to increased concentrations of ISL for 48 h, a time period which was enough to cause cell death in ACC cells, and found several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, recruitment of the microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, and an increase in autophagic flux. At the same time, the induction of apoptosis by ISL was also detected, manifested in the formation of DNA fragments, cleaved-PARP and an increased in the Bax/Bcl-2 ratio. Furthermore, both ISL-mediated autophagy and apoptosis were obviously attenuated by treatment with 3-MA or transfection with DN-Atg5K130R. More importantly, the xenograft model provided further evidence for the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppression of mTOR and up-regulation of Atg5. Taken together, these results strongly suggest that ISL can induce autophagic and apoptotic cell death in ACC cells, which likely serve as an important mechanism in its cancer chemotherapeutic effects. Although the molecular mechanisms for autophagy induction in mammalian cells are still poorly understood, at least 30 autophagy-related genes have been identified in yeast [34], among which mTOR has emerged as a key negative regulator of autophagy in yeast and possibly in mammalian cells [31, 35–37]. To determine the precise functional significance of mTOR suppresson in ISLinduced autophagy, we evaluate the influence of

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CA-mTOR on ISL-mediated autophagic and apoptotic cell death in ACC cells. As the results show, transient transfection of ACC cells with CA-mTOR significantly but only partially reversed ISL-induced autophagy as well as apoptosis, suggesting that there must be other mechanisms involved in ISL-mediated autophagic response, which still needs further investigation. The role of autophagy in cancer development and therapy has been debated with regard to whether autophagy is beneficial for survival or is detrimental for death. There is no doubt that apoptosis, the type I programmed cell death, is a cellular control mechanism for death [16, 38]. However, manipulation of the type II programmed cell death, autophagy, may play dual and opposing roles in cancer therapy [25, 39, 40]. After some anticancer treatments, cancer cells may adapt to survive through activation of autophagy [41], whereas in other cases the cancer cells might undergo autophagic cell death [42, 43]. In the present study, our results indicated that ISL-induced autophagy at 48 h was indeed detrimental for death, since inhibition of autophagy by 3-MA treatment or DNAtg5K130R transfection significantly reversed ISL-induced reduction of ACC cell viability. Additionally, we found it particularly interesting that short exposure to ISL (12–24 h) could also slightly activate autophagy in ACC cells but showed no effect on cell viability (data not shown). These findings may support a more rational belief that autophagy could facilitate the removal of damaged proteins and organelles, and meanwhile maintain energy metabolism and macromolecular synthesis to promote cell survival under unfavorable stresses; however, if excess or prolonged, autophagy may reversely lead to cell death.

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To date, the relationship between autophagic and apoptotic cell death is also poorly understood, especially concerning whether autophagy directly executes cell death or is the secondary effect of apoptosis. Here our data revealed that ISL-induced apoptotic cell death was both autophagy-dependent and autophagy-independent, because autophagy suppression by DN-Atg5K130R transfection significantly but only partially prevented ISL-mediated apoptotic DNA fragmentation in ACC cells. Indeed, previous studies have shown that enhanced expression of Atg5 can not only promote autophagy but can also increase susceptibility toward apoptotic stimuli irrespective of the cell type [43–45]. These results not only demonstrated the essential role of Atg5 in regulation of ISL-mediated autophagy and apoptosis in ACC cells, but also provide additional evidence to indicate that ISL-induced autophagy and apoptosis are interrelated. In addition to Atg5, there several other signal transduction pathways can elicit both autophagy and apoptosis. The transcription factor p53 is one such molecular. Generally, p53 is known as a quintessential tumor suppressor and apoptosis inducer. However, accumulating evidence suggests p53 also has a positive role in autophagy activation. Moreover, previous study has demonstrated that ISL could induce apoptosis in HepG2 cells through a p53dependent pathway [46]. Thus, we also tried to detect the effects of ISL on p53 signaling pathway in ACC cells, however, unfortunately, we found that ISL treatment failed to affect either the total expression or the transcription activity of p53 (data not shown), suggesting that ISLinduced autophagy and apoptosis is independent on p53 signaling pathway. We then proceeded to test the possible involvement of another molecule Bax in the ISL-induced autophagy and apoptosis, using Bax-inhibiting peptide-V5. As the consequence, inhibition of Bax nearly abrogated ISL-mediated apoptosis-induction in ACC cells (Supplementary Fig. 1a), and meanwhile significantly, albeit partially, prevented ISL-induced viability-reduction (Supplementary Fig. 1c), but showed no effect on ISL-induced autophagy (Supplementary Fig. 1b). More important, we also found that when V5 and 3-MA were used in combination, the cell viability-reduced by ISL could be more obviously prevented (Supplementary Fig. 1c), suggesting that ISL-induced autophagy which is independent on Bax may indeed contribute to part of the cell death in ACC cells. These findings indicated that Bax is indispensable for ISL-mediated apoptosis, but not autophagy in ACC cells; in other words, Bax is only partially essential for ISLmediated cell death. Nevertheless, the precise molecular events, especially those cross talk mechanisms behind the autophagic and apoptotic cell death induced by ISL in ACC cells still needs further investigation.

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In conclusion, our results revealed for the first time that ISL could induce both autophagic and apoptotic cell death in ACC cells, relating to the up-regulation of mTORdependent Atg5, which likely serves as an important mechanism in cancer chemotherapy by ISL. Acknowledgments This work was supported by grants from National Natural Science Foundation of China (81072203) to Dr. Z. J. Sun (30801305) to Dr. J. Jia (30973329) to Prof. W. F. Zhang, and (30872894, 30973330) to Prof. Y. F. Zhao. We would like to thank Shelagh Powers in Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research for expert editorial assistance. Conflict of interest disclosed.

No potential conflicts of interest were

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