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Animal and In Vitro Models
Temsirolimus, the mTOR inhibitor, induces autophagy in adenoid cystic carcinoma: In vitro and in vivo Wenlei Liu 1 , Shengyun Huang 1 , Zhanwei Chen, Huachun Wang, Haiwei Wu, Dongsheng Zhang ∗ Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China
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Article history: Received 13 November 2013 Received in revised form 2 February 2014 Accepted 10 March 2014 Keywords: Autophagy mTOR Adenoid cystic carcinoma Temsirolimus
a b s t r a c t Temsirolimus acts as a mammalian target of rapamycin (mTOR)-dependent autophagic inhibitor. In order to clarify its effects and mechanisms on human salivary adenoid cystic carcinoma (ACC), we examined whether temsirolimus induced autophagy as the mTOR inhibitor in ACC, both in vitro and in vivo. In this study, MTT assay showed that the inhibition effect of temsirolimus assumed an obvious dose–response relationship on ACC-M cells, and the 50% inhibitory concentration (IC50 ) approached 20 mol/l; numerous autophagosomes were observed by the transmission electron microscopy (TEM) in temsirolimus treatment groups; notably, expression of LC3 and Beclin1 was significantly up-regulated by temsirolimus. More importantly, the xenograft model provided further evidence of temsirolimus-induced autophagy in vivo by inhibiting mTOR activation as well as up-regulation the expression of Beclin1. These results suggest that temsirolimus could act as an mTOR inhibitor to induce autophagy in adenoid cystic carcinoma both in vitro and in vivo. © 2014 Published by Elsevier GmbH.
Introduction Adenoid cystic carcinoma (ACC), a highly aggressive neoplasm mostly occurring in the salivary gland, accounts for approximately 15–25% of all the carcinomas at this location [1,2]. This neoplasm often presents a prolonged clinical course [3]. After curative surgery, radiotherapy, and chemotherapy, the disease-specific survival at 10 years for patients with ACC remains to be 29–40% [4]. Most deaths from salivary ACC are caused by local recurrence and distant metastasis following treatment with those conventional therapies. Therefore, more effective agents with minimal side-effect are still needed for treatment of ACC. Autophagy is a process whereby cellular components such as proteins and organelles are engulfed by autophagosomes, and are delivered to the lysosomes for degradation [5]. Autophagy facilitates cellular survival by maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation; it also functions in many physiopathologic processes (i.e.,
∗ Corresponding author at: Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwu Road, Jinan 250021, China. Tel.: +86 531 68776950; fax: +86 531 87035697. E-mail addresses: [email protected], [email protected] (D. Zhang). 1 These two authors contributed equally to this study.
differentiation and development, antiaging, innate and adaptive immunity) and cancer [6]. According to the recent report, mild and/or slow autophagy may enhance cell survival while more severe and/or rapid autophagy would take part in inducing cell death [7]. Several anticancer agents have been reported to induce autophagy and either protect or further sensitize cells to drug treatment [8]. Furthermore, autophagy is now considered to be a therapeutic target in tumor cells that are resistant to anti-tumor drugs [9]. The autophagy pathway has been conserved among all eukaryotes, and in the last decades, many autophagyspecific genes regulating autophagy (Atg) have been identified, including 16 homologues in human [10]. In these homologues, it has been proved that LC3 (microtubule-associated protein 1 light chain 3) and Beclin1 genes play a pivotal role in the autophagy of mammalian, which are identified as the mammalian orthologue of yeast Atg8 and Atg6, respectively. The regulation and role of autophagy in human carcinoma has been researched, and the expression of Beclin1 and LC3 has been explored in detail in various cancers [11,12]. According to our previous study, we demonstrated that the progression of head and neck ACC was associated with the down-regulated expression of LC3 and Beclin1 [13]. However, the mechanism of autophagy is still far from clear in ACC. Temsirolimus (CCI-779), approved for the treatment of advanced renal cell carcinoma (RCC), is a prodrug as a mammalian
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Please cite this article in press as: W. Liu, et al., Temsirolimus, the mTOR inhibitor, induces autophagy in adenoid cystic carcinoma: In vitro and in vivo, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.008
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target of rapamycin (mTOR) inhibitor, and its primary active metabolite is rapamycin. It also shows preclinical potential in hematological malignancies and pancreatic cancer [14,15]. But its effect on human salivary ACC is still unknown. In our study, we investigated the role of temsirolimus in ACC as autophagy revulsant, and detected that temsirolimus not only significantly inhibited cells growth in vitro, up-regulated the expression of gene regulating autophagy in ACC cells, but also obviously restrained the xenografts development in vivo, relating with the inhibition of mTOR pathway. Materials and methods
and expressed in mg per ml. Samples containing equal amounts of protein were resolved on SDS-polyacrylamide gel in a 10–15% gel, transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were then incubated with primary antibodies against human LC3B (Sigma–Aldrich, St. Louis, MO; at a dilution of 1:1000), human Beclin1 (Abcam, MA, UK; at a dilution of 1:1000) antibodies overnight. Thereafter membranes were incubated with peroxidaseconjugated secondary antibodies (at 1:10,000 dilution) for 60 min. Protein bands were visualized by the Alpha Imager 2200 system (Alpha Innotech, San Leandro, CA, USA). To ensure equal protein loading, each membrane was stripped and reprobed with anti-actin antibody (at 1:1000 dilution).
Cell culture
Nude mice xenografts
The high metastasis cell lines of human ACC (ACC-M) was provided by Professor Wantao Chen (Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University, Shanghai, China), and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 U/ml penicillin and 100 g/ml streptomycin (Invitrogen, Carlsbad, CA) at 37 ◦ C in a humidified atmosphere of 95% air with 5% CO2 .
Female BALB/c nude mice (18–20 g; 6 weeks of age) were purchased from the Weitonglihua Experimental Animal Corporation (Beijing, China), in pressurized ventilated cage according to institutional regulations. All studies were approved and supervised by Animal Care and Use Committee of Shandong Provincial Hospital Affiliated to Shandong University. Exponentially growing ACC-M cells (6 × 106 in 0.1 ml medium) were inoculated subcutaneously into the back of the mouse. Ten days later, the tumor-bearing mouse was euthanatized, the tumors were captured and cut into several isometrical tissue blocks, 2 mm in diameter. Then, each tissue block was put into the back of each mouse under aseptic conditions. Xenografted tumors were allowed to grow until reaching about 200 mm3 , at which time mice were randomly divided into treatment groups consisting of control and experimental groups. Two groups were treated with temsirolimus (2 mg/kg intraperitoneal administration, every two days; n = 8) or PBS (100 l, intraperitoneal administration, every two days; n = 8), and last for 32 days before experimental termination. Tumor growth was determined by measuring the size of the tumors with vernier caliper every two days, before the mice were intraperitoneally injected. Tumor volumes (V) were calculated as V = (width2 × length)/2. The mice were euthanatized at day 32, and the tumors were acquired, photographed and embedded in paraformaldehyde or frozen at −80 ◦ C for the following immunohistochemical analysis or for western blot analysis.
Cell growth inhibition assay Temsirolimus was provided by Wyeth-Ayerst (Pearl River, NY, USA). The cytotoxic activity of temsirolimus was determined using a standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazodium bromide (MTT; Sigma–Aldrich, St. Louis, MO), based colorimetric assay. In brief, ACC-M cells (1 × 104 cells/well) were seeded in 96well plates in a final volume of 100 l. After overnight incubation, cells were treated with the 0.1–50 mol/l indicated concentrations of temsirolimus for 24 h, 48 h or 72 h. After completion of the treatment, 10 l of MTT (5 mg/ml) solution was added to each well and incubated for 4 h at 37 ◦ C to allow metabolization. Blotting nutrient solution, DMSO (150 l) was placed in each well and then set table on low speed oscillation 10 min, measure the absorption value of each well at 570 nm by the enzyme linked immune detector. The concentration of drug that produced cytotoxicity against 50% of the cultured cells (half maximal inhibitory concentration, IC50) was calculated using the linear regression method. All the experiments were carried out in triplicate. Transmission electron microscopy The transmission electron microscopy (TEM) was performed according to our acknowledged procedures. In brief, ACC-M cells were incubated with DMSO (Control), 10 M or 20 M temsirolimus for 48 h. And then, the cells were fixed in ice-cold 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 detected with the JEM-1200EX Transmission Electron Microscope (JEOL, Japan). Western blot analysis Cells were treated with 0, 10 M or 20 M temsirolimus, and then collected after 48 h, centrifuged and washed with ice cold phosphate-buffered saline (PBS) for three times. The cell aggregate was then resuspended in lysis buffer, and protein extraction was executed in the same manner as the previously published protocol [16]. The BCA protein assay kit (Shenneng Bocai Biotechnology Co., Ltd.) was used for quantification of the total protein content
Immunohistochemical staining The tumors loaded on the back of the mice were disposed as following, triformol-fixed, dehydrated in a graded alcohol series, embedded in paraffin, cut into slices. Sections (3 m thick) were deparaffinized. Antigen retrieval procedure was performed by microwave in 10 mmol/citrate buffer (pH 6.0). The slices were subjected to blocking with hydrogen peroxide, sealed with serum, and incubated for 30 min at 37 ◦ C with primary monoclonal antibodies for LC3B (1:200) and Beclin1 (1:50), using a biotin-free polymeric horseradish peroxidase linker antibody conjugate system, and nuclei were counterstained with hematoxylin in a Bench-Mark ULTRA Advanced Staining System (Ventana Medical Systems, AZ, USA). The positive controls were supplied by Abcam, and negative controls were obtained by omitting primary antibody replaced by PBS. All immunohistochemical markers were assessed by light microscopy. Beclin1 and LC3B immunohistochemical staining was evaluated on the basis of the proportion of stained cells and the immunostaining intensity. Statistical analysis Data were expressed as the mean ± SEM of at least three independent experiments. Statistical comparisons between groups
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Fig. 1. Effect of temsirolimus on the proliferation in ACC-M cells. (A) Temsirolimus induced cell death in ACC-M cells. Cells were cultured with various concentrations (0.1–50.0 M) of temsirolimus for 48 h or the indicated times and cell viability was analyzed by MTT assay. (B) Dose–response curve that shows the in vitro cytostatic and cytotoxic effects of temsirolimus versus ACC-M cells for 48 h. All data are presented as mean ± SEM from three independent experiments with duplicate.
were performed using two-tailed Student’s t-test (SPSS 15.0). Statistical significance was set at *p < 0.05; **p < 0.01. Results Cell growth inhibition effect MTT assay was used to quantify the cytotoxicity of temsirolimus in ACC-M cells. As shown in our data, temsirolimus caused a decrease in the cell viability of ACC-M cells (Fig. 1), the inhibition effect assumed an obvious dose–response relationship. According to Fig. 1A, it could be revealed that there were no significant differences between various treatments for different time (24 h, 48 h or 72 h) on inhibition effects. As shown in Fig. 1B, we demonstrated the growth-inhibitory curve of ACC-M cells treated with temsirolimus, the 50% inhibitory concentration (IC50 ) of temsirolimus in this culture system approximated 20 mol/l. Temsirolimus induced the formation of autophagosome in ACC cells In order to explore whether autophagy was induced in ACCM cell, we conducted a transmission electron microscopy (TEM) study. As shown in Fig. 2A and B, compared with the control group, numerous autophagosomes or lysosomes containing the segregation and degradation of organelles were recognized ultrastructurally in temsirolimus treated groups. Furthermore, compared to low dose group, we confirmed that high concentration of temsirolimus treatment revealed more acute response. Many
vesicles including autophagosomes and autophagolysosomes, containing entrapped cytoplasm organelles were found (Fig. 2A). Taken together, our findings suggested that temsirolimus induced cell death in ACC-M cells was related to autophagy. Temsirolimus induces high expression of autophagy related proteins in ACC-M cells After 48 h of treatment with temsirolimus, we examined whether autophagy was induced in ACC-M cells by western blot. Since the protein is a good indicator of autophagosome formation, we examined levels of LC3B and Beclin1 induced by temsirolimus treatment in ACC-M cells. As shown in Fig. 3, in the control cells, only few LC3 and Beclin1could be detected. However, after treatment with temsirolimus for 48 h, the levels of autophagy related proteins LC3 and Beclin1 were dramatically increased with high dose of temsirolimus. Furthermore, the ratio of LC3-II/LC3-I was increased obviously compared with the control group. Temsirolimus inhibits ACC xenograft growth by inducing autophagy Animal experiment was designed to detect whether temsirolimus could induce autophagy and inhibit xenograft growth in vivo, in order to further prove the effect of temsirolimus as described above. As the results showed, the growth of the ACC xenograft was significantly inhibited by temsirolimus, (Fig. 4A and D), and the body weight of nude mice showed no significant difference between two groups, either pretreatment or
Fig. 2. Detection and quantification for temsirolimus-induced autophagosomes by transmission electron microscopy (TEM). (A) Numerous autophagosomes or lysosomes (short black arrows) containing the segregation and degradation of organelles were recognized ultrastructurally in temsirolimus treated groups. Magnification, 10,000×. (B) The quantitative analysis of temsirolimus-induced autophagosomes among control and treatment groups. All data are presented as mean ± SEM from three independent experiments with duplicate. **p < 0.01 versus the control group.
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mTOR activation as well as up-regulate Beclin1 expression, sequentially induce autophagic in ACC-M cell in vivo, which might be the most important mechanism of action about the anti-tumor effect of temsirolimus. Discussion
Fig. 3. Temsirolimus induced autophagy in ACC-M cells. (A) The differences in LC3 (the ratio of LC3-II/LC3-I) and Beclin1 expression levels between control and temsirolimus-treated samples were compared by western blotting. (B and C) Gray values was compared between groups. All data are presented as mean ± SEM from three independent experiments with duplicate. **p < 0.01 versus the control group, and *p < 0.05 versus the control group.
posttreatment (Fig. 4B). In addition, as shown in Fig. 4C, the tumor from temsirolimus treatment mice exhibited significantly increased expression of LC3 (C1, C2) and Beclin1 (B1, B2). All of the above findings demonstrated that temsirolimus could suppress
Beyond all doubt, apoptosis, the type I programmed cell death, is the main cellular control mechanism for cell death [16], but it has been debated with respect to whether autophagy is beneficial or harmful for survival about the role of autophagy in neoplasm development and treatment. According to previous reports, autophagy may play dual and diametrically opposed role in cancer development [17,18]. Due to the activation of autophagy, cancer cells may survive after anticancer treatment, whereas in other neoplasms the cancer cells may undergo autophagic cell death [19]. In our research, the results indicated that temsirolimus induced autophagy indeed inhibited the development of ACC in vivo and in vitro. In vitro study, ACC-M cells were treated with assay concentration of temsirolimus, the date had distinctly suggested that temsirolimus induced cell death primely resulted from autophagy in ACC-M cells. Meanwhile, temsirolimus also displayed a distinct inhibitional effect in vivo. Our present study provides the first experimental evidence of autophagy induction in ACC cells disposed with temsirolimus. Temsirolimus-mediated autophagy in ACC-M cells was reflected by the induction of LC3B followed by the recruitment of processed LC3B to autophagosomes and the formation of autophagosome by technique of electron microscopy (Fig. 2), as well as the tumor volume growth curve and immunohistochemical staining in vivo (Fig. 4).
Fig. 4. Temsirolimus inhibits ACC xenograft growth according to induced autophagy. (A and B) Tumor volume and body weight of animal in vehicle-treated control mice and temsirolimus-treated mice. (C) Immunohistochemical analysis of the indicated biomarkers in both control and temsirolimus-treated ACC-M tumor tissues. The negative Beclin1 expression in control group (B1) and positive expression in treatment group (B2); the negative LC3 expression in control group (C1) and positive expression in treatment group (C2); HE staining of tumor tissue (A1) and negative controls by PBS (A2). Magnification, 200×. (D) Real-time morphological changes of xenografts. **p < 0.01 versus the control group.
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Rapamycin is a classical mTOR inhibitor. The poor solubility that compromised rapamycin as an intravenous agent led to the development of a more soluble ester analog of rapamycin, CCI-779 (temsirolimus) [20]. Frost et al. [21], confirmed that CCI-779 is effective in vivo against myeloma cells by inhibiting proliferation, angiogenesis, and induction of tumor cell apoptosis. This phenomenon is reminiscent of the experience with mTOR inhibitor temsirolimus used in the observed tumor cell apoptosis and antitumor response in vivo on hematological malignancies [22]. In support of this hypothesis existed in ACC, ACC-M cells were treated with temsirolimus in series of concentration, and it revealed significative effect on human adenoid cystic carcinoma (Fig. 1A). Dose–response curve showed that half maximal inhibitory concentration of temsirolimus in vitro cytostatic and cytotoxic effects on ACC-M cells was about 20 mol/l. We next investigated whether autophagy was induced by temsirolimus, which had been shown to trigger cell growth inhibition in ACC-M cells. The differential between control and treatment groups we studied was reflected by their discrepant expression of autophagy related proteins LC3 and Beclin1 in vitro treatment. LC3 is composed of a soluble LC3-I and a lipidated LC3-II, and is expressed as three isoforms in mammalian cells: LC3A, LC3B, and LC3C. LC3A and LC3B are related to autophagy [10,23]. Once autophagy is initiated by ATGs, LC3 is converted to the active LC3-II form, which is inserted into the double membrane of autophagophores and autophagosomes [24,25]. LC3-II, the marker protein of autophagy, is aggregated in membranes of autophagosomes and the ratio of LC3-II/LC3-I is increased. Beclin1 is responsible for both the signaling pathway activating autophagy and in the initial step of autophagosome formation in mammals, which functions as a tumor suppressor [26]. Our data confirmed that Temsirolimus, as the mTOR inhibitor, induced autophagy (Fig. 3). In several reports, blocking mTOR activity decreased proliferation, and promoted the formation of autophagosome [27]. Here transmission electron microscopy was used to observe the autophagosomes in our study. Consistent with the above observations, numerous autophagosomes or lysosomes could be found in temsirolimus treated groups (Fig. 2). This result proved that temsirolimus could activate autophagy through inhibiting mTOR pathway, which was associated with increased LC3 and Beclin1 levels. The animal experiment was designed to detect whether temsirolimus could induce autophagy and inhibit xenograft growth in vivo. Just as expected, the date and photos provided first-hand evidence that temsirolimus could inhibit the tumor growth though promoting mTOR-mediated autophagy. The inhibition effect was more significant along with the growth of nude mice. The body weight showed no significant difference between groups, from which we realized that temsirolimus did not impact the mice growth (Fig. 4). According to our previous study, we demonstrated that the progression of head and neck adenoid cystic carcinoma was associated with the down-regulated expression of LC3 and Beclin1, and the positive LC3 was directly correlated with absent lymph node involvement and TNM stage. Beclin1 expression showed a statistically significant correlation with the histological growth pattern and the histological grade, and the log-rank test showed that the survival in the positive Beclin1 group tended to be better than in the negative Beclin1 group [13]. This phenomenon also emerged when LC3 expression decreased in brain and ovary cancer and decreased expression of Beclin1 correlated with tumor progression in breast, ovarian, and brain cancer [28–31]. All of the above also could be reappeared in our in vivo study. As the control group shown, weak positive results were detected while the model was dealt with PBS. On the contrary, the expression of LC3 and Beclin1 appeared strong positive response in treatment group
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(Fig. 4C). These findings suggested that temsirolimus could suppress the development of tumor growth by inducing autophagy. These results suggested that temsirolimus could act as an mTOR inhibitor to induce autophagy in adenoid cystic carcinoma both in vitro and in vivo. However, we only focused our attention on the basic research of temsirolimus at present. It was far from clear whether it could play a similar role in human body. In some other neoplasm, investigators had make progress on the preclinical or clinical research. Trafalis et al. [32], confirmed that temsirolimus could be significantly effective for head and neck squamous cell carcinoma (HNSCC), all in vitro, in vivo, and clinical, especially combined with bevacizumab (antivascular endothelial growth factor antibody). Temsirolimus also showed efficacy in heavily pretreated and elderly patients in mantle cell lymphoma, however followed with some partial responses [33]. In this study, we successfully demonstrated that autophagy could inhibit the development of ACC, both in vitro and in vivo. Next, we may proceed the preclinical or clinical study, in order to clarify its effect on ACC in human. In conclusion, our investigation revealed for the first time that mTOR inhibitor temsirolimus induced autophagy suppressed the development of ACC by promoting the formation of autophagosome, and we demonstrated that mTOR pathway played a particularly important role in autophagy regulation. So, the findings suggested that autophagy was activated as a protective mechanism against ACC. It showed us a bright prospect in improving anticancer therapeutics. Nevertheless, the dual and diametrically opposed role of autophagy in various neoplasms, and more precise molecular mechanism, for instance the regulation between autophagy and apoptosis in ACC cells, are still far from clear. Moreover, further studies will be needed to clarify the anti-cancer effect of temsirolimus. Acknowledgments This study is supported by Shandong Provincial Science and Technology Development Plan (No. 2006GG2202034), the Science and Technology Foundation of Shandong Province (2006GG20002046) and Shandong Provincial Natural Science Foundation (ZR2012HQ022). References [1] M. Persson, Y. Andrén, J. Mark, et al., Recurrent fusion of MYB and NFIB transcription factor genes in carcinomas of the breast and head and neck, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 18740–18744. [2] D. Bell, D. Roberts, M. Kies, A.K. Weber, El-Naggar, et al., Cell type-dependent biomarker expression in adenoid cystic carcinoma: biologic and therapeutic implications, Cancer 116 (2010) 5749–5756. [3] O. Tetsu, J. Phuchareon, A. Chou, R.C. Eisele, Jordan, et al., Mutations in the c-Kit gene disrupt mitogen-activated protein kinase signaling during tumor development in adenoid cystic carcinoma of the salivary glands, Neoplasia 12 (2010) 708–717. [4] J. Fordice, C. Kershaw, A. El-Naggar, et al., Adenoid cystic carcinoma of the head and neck: predictors of morbidity and mortality, Arch. Otolaryngol. Head Neck Surg. 125 (1999) 149–152. [5] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (2008) 27–42. [6] N. Chen, J. Debnath, Autophagy and tumorigenesis, FEBS Lett. 584 (2010) 1427–1435. [7] A. Notte, L. Leclere, C. Michiels, Autophagy as a mediator of chemotherapyinduced cell death in cancer, Biochem. Pharmacol. 82 (2011) 427–434. [8] Y. Kondo, T. Kanzawa, R. Sawaya, et al., The role of autophagy in cancer development and response to therapy, Nat. Rev. Cancer 5 (2005) 726–734. [9] S. Chen, S.K. Rehman, W. Zhang, et al., Autophagy is a therapeutic target in anticancer drug resistance, Biochim. Biophys. Acta 1806 (2010) 220–229. [10] N. Mizushima, D.J. Klionsky, Protein turnover via autophagy: implications formetabolism, Annu. Rev. Nutr. 27 (2007) 19–40. [11] G. Viola, R. Bortolozzi, E. Hamel, et al., MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells, Biochem. Pharmacol. 83 (2012) 16–26. [12] J. Hao, Y. Pei, G. Ji, et al., Autophagy is induced by 3-O-succinyl-lupeol (LD9-4) in A549 cells via up-regulation of Beclin1 and down-regulation mTOR pathway, Eur. J. Pharmacol. 670 (2011) 29–38.
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Animal and In Vitro Models
Temsirolimus, the mTOR inhibitor, induces autophagy in adenoid cystic carcinoma: In vitro and in vivo Wenlei Liu 1 , Shengyun Huang 1 , Zhanwei Chen, Huachun Wang, Haiwei Wu, Dongsheng Zhang ∗ Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China
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Article history: Received 13 November 2013 Received in revised form 2 February 2014 Accepted 10 March 2014 Keywords: Autophagy mTOR Adenoid cystic carcinoma Temsirolimus
a b s t r a c t Temsirolimus acts as a mammalian target of rapamycin (mTOR)-dependent autophagic inhibitor. In order to clarify its effects and mechanisms on human salivary adenoid cystic carcinoma (ACC), we examined whether temsirolimus induced autophagy as the mTOR inhibitor in ACC, both in vitro and in vivo. In this study, MTT assay showed that the inhibition effect of temsirolimus assumed an obvious dose–response relationship on ACC-M cells, and the 50% inhibitory concentration (IC50 ) approached 20 mol/l; numerous autophagosomes were observed by the transmission electron microscopy (TEM) in temsirolimus treatment groups; notably, expression of LC3 and Beclin1 was significantly up-regulated by temsirolimus. More importantly, the xenograft model provided further evidence of temsirolimus-induced autophagy in vivo by inhibiting mTOR activation as well as up-regulation the expression of Beclin1. These results suggest that temsirolimus could act as an mTOR inhibitor to induce autophagy in adenoid cystic carcinoma both in vitro and in vivo. © 2014 Published by Elsevier GmbH.
Introduction Adenoid cystic carcinoma (ACC), a highly aggressive neoplasm mostly occurring in the salivary gland, accounts for approximately 15–25% of all the carcinomas at this location [1,2]. This neoplasm often presents a prolonged clinical course [3]. After curative surgery, radiotherapy, and chemotherapy, the disease-specific survival at 10 years for patients with ACC remains to be 29–40% [4]. Most deaths from salivary ACC are caused by local recurrence and distant metastasis following treatment with those conventional therapies. Therefore, more effective agents with minimal side-effect are still needed for treatment of ACC. Autophagy is a process whereby cellular components such as proteins and organelles are engulfed by autophagosomes, and are delivered to the lysosomes for degradation [5]. Autophagy facilitates cellular survival by maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation; it also functions in many physiopathologic processes (i.e.,
∗ Corresponding author at: Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwu Road, Jinan 250021, China. Tel.: +86 531 68776950; fax: +86 531 87035697. E-mail addresses: [email protected], [email protected] (D. Zhang). 1 These two authors contributed equally to this study.
differentiation and development, antiaging, innate and adaptive immunity) and cancer [6]. According to the recent report, mild and/or slow autophagy may enhance cell survival while more severe and/or rapid autophagy would take part in inducing cell death [7]. Several anticancer agents have been reported to induce autophagy and either protect or further sensitize cells to drug treatment [8]. Furthermore, autophagy is now considered to be a therapeutic target in tumor cells that are resistant to anti-tumor drugs [9]. The autophagy pathway has been conserved among all eukaryotes, and in the last decades, many autophagyspecific genes regulating autophagy (Atg) have been identified, including 16 homologues in human [10]. In these homologues, it has been proved that LC3 (microtubule-associated protein 1 light chain 3) and Beclin1 genes play a pivotal role in the autophagy of mammalian, which are identified as the mammalian orthologue of yeast Atg8 and Atg6, respectively. The regulation and role of autophagy in human carcinoma has been researched, and the expression of Beclin1 and LC3 has been explored in detail in various cancers [11,12]. According to our previous study, we demonstrated that the progression of head and neck ACC was associated with the down-regulated expression of LC3 and Beclin1 [13]. However, the mechanism of autophagy is still far from clear in ACC. Temsirolimus (CCI-779), approved for the treatment of advanced renal cell carcinoma (RCC), is a prodrug as a mammalian
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Please cite this article in press as: W. Liu, et al., Temsirolimus, the mTOR inhibitor, induces autophagy in adenoid cystic carcinoma: In vitro and in vivo, Pathol. – Res. Pract (2014), http://dx.doi.org/10.1016/j.prp.2014.03.008
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target of rapamycin (mTOR) inhibitor, and its primary active metabolite is rapamycin. It also shows preclinical potential in hematological malignancies and pancreatic cancer [14,15]. But its effect on human salivary ACC is still unknown. In our study, we investigated the role of temsirolimus in ACC as autophagy revulsant, and detected that temsirolimus not only significantly inhibited cells growth in vitro, up-regulated the expression of gene regulating autophagy in ACC cells, but also obviously restrained the xenografts development in vivo, relating with the inhibition of mTOR pathway. Materials and methods
and expressed in mg per ml. Samples containing equal amounts of protein were resolved on SDS-polyacrylamide gel in a 10–15% gel, transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were then incubated with primary antibodies against human LC3B (Sigma–Aldrich, St. Louis, MO; at a dilution of 1:1000), human Beclin1 (Abcam, MA, UK; at a dilution of 1:1000) antibodies overnight. Thereafter membranes were incubated with peroxidaseconjugated secondary antibodies (at 1:10,000 dilution) for 60 min. Protein bands were visualized by the Alpha Imager 2200 system (Alpha Innotech, San Leandro, CA, USA). To ensure equal protein loading, each membrane was stripped and reprobed with anti-actin antibody (at 1:1000 dilution).
Cell culture
Nude mice xenografts
The high metastasis cell lines of human ACC (ACC-M) was provided by Professor Wantao Chen (Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University, Shanghai, China), and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 U/ml penicillin and 100 g/ml streptomycin (Invitrogen, Carlsbad, CA) at 37 ◦ C in a humidified atmosphere of 95% air with 5% CO2 .
Female BALB/c nude mice (18–20 g; 6 weeks of age) were purchased from the Weitonglihua Experimental Animal Corporation (Beijing, China), in pressurized ventilated cage according to institutional regulations. All studies were approved and supervised by Animal Care and Use Committee of Shandong Provincial Hospital Affiliated to Shandong University. Exponentially growing ACC-M cells (6 × 106 in 0.1 ml medium) were inoculated subcutaneously into the back of the mouse. Ten days later, the tumor-bearing mouse was euthanatized, the tumors were captured and cut into several isometrical tissue blocks, 2 mm in diameter. Then, each tissue block was put into the back of each mouse under aseptic conditions. Xenografted tumors were allowed to grow until reaching about 200 mm3 , at which time mice were randomly divided into treatment groups consisting of control and experimental groups. Two groups were treated with temsirolimus (2 mg/kg intraperitoneal administration, every two days; n = 8) or PBS (100 l, intraperitoneal administration, every two days; n = 8), and last for 32 days before experimental termination. Tumor growth was determined by measuring the size of the tumors with vernier caliper every two days, before the mice were intraperitoneally injected. Tumor volumes (V) were calculated as V = (width2 × length)/2. The mice were euthanatized at day 32, and the tumors were acquired, photographed and embedded in paraformaldehyde or frozen at −80 ◦ C for the following immunohistochemical analysis or for western blot analysis.
Cell growth inhibition assay Temsirolimus was provided by Wyeth-Ayerst (Pearl River, NY, USA). The cytotoxic activity of temsirolimus was determined using a standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazodium bromide (MTT; Sigma–Aldrich, St. Louis, MO), based colorimetric assay. In brief, ACC-M cells (1 × 104 cells/well) were seeded in 96well plates in a final volume of 100 l. After overnight incubation, cells were treated with the 0.1–50 mol/l indicated concentrations of temsirolimus for 24 h, 48 h or 72 h. After completion of the treatment, 10 l of MTT (5 mg/ml) solution was added to each well and incubated for 4 h at 37 ◦ C to allow metabolization. Blotting nutrient solution, DMSO (150 l) was placed in each well and then set table on low speed oscillation 10 min, measure the absorption value of each well at 570 nm by the enzyme linked immune detector. The concentration of drug that produced cytotoxicity against 50% of the cultured cells (half maximal inhibitory concentration, IC50) was calculated using the linear regression method. All the experiments were carried out in triplicate. Transmission electron microscopy The transmission electron microscopy (TEM) was performed according to our acknowledged procedures. In brief, ACC-M cells were incubated with DMSO (Control), 10 M or 20 M temsirolimus for 48 h. And then, the cells were fixed in ice-cold 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 detected with the JEM-1200EX Transmission Electron Microscope (JEOL, Japan). Western blot analysis Cells were treated with 0, 10 M or 20 M temsirolimus, and then collected after 48 h, centrifuged and washed with ice cold phosphate-buffered saline (PBS) for three times. The cell aggregate was then resuspended in lysis buffer, and protein extraction was executed in the same manner as the previously published protocol [16]. The BCA protein assay kit (Shenneng Bocai Biotechnology Co., Ltd.) was used for quantification of the total protein content
Immunohistochemical staining The tumors loaded on the back of the mice were disposed as following, triformol-fixed, dehydrated in a graded alcohol series, embedded in paraffin, cut into slices. Sections (3 m thick) were deparaffinized. Antigen retrieval procedure was performed by microwave in 10 mmol/citrate buffer (pH 6.0). The slices were subjected to blocking with hydrogen peroxide, sealed with serum, and incubated for 30 min at 37 ◦ C with primary monoclonal antibodies for LC3B (1:200) and Beclin1 (1:50), using a biotin-free polymeric horseradish peroxidase linker antibody conjugate system, and nuclei were counterstained with hematoxylin in a Bench-Mark ULTRA Advanced Staining System (Ventana Medical Systems, AZ, USA). The positive controls were supplied by Abcam, and negative controls were obtained by omitting primary antibody replaced by PBS. All immunohistochemical markers were assessed by light microscopy. Beclin1 and LC3B immunohistochemical staining was evaluated on the basis of the proportion of stained cells and the immunostaining intensity. Statistical analysis Data were expressed as the mean ± SEM of at least three independent experiments. Statistical comparisons between groups
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Fig. 1. Effect of temsirolimus on the proliferation in ACC-M cells. (A) Temsirolimus induced cell death in ACC-M cells. Cells were cultured with various concentrations (0.1–50.0 M) of temsirolimus for 48 h or the indicated times and cell viability was analyzed by MTT assay. (B) Dose–response curve that shows the in vitro cytostatic and cytotoxic effects of temsirolimus versus ACC-M cells for 48 h. All data are presented as mean ± SEM from three independent experiments with duplicate.
were performed using two-tailed Student’s t-test (SPSS 15.0). Statistical significance was set at *p < 0.05; **p < 0.01. Results Cell growth inhibition effect MTT assay was used to quantify the cytotoxicity of temsirolimus in ACC-M cells. As shown in our data, temsirolimus caused a decrease in the cell viability of ACC-M cells (Fig. 1), the inhibition effect assumed an obvious dose–response relationship. According to Fig. 1A, it could be revealed that there were no significant differences between various treatments for different time (24 h, 48 h or 72 h) on inhibition effects. As shown in Fig. 1B, we demonstrated the growth-inhibitory curve of ACC-M cells treated with temsirolimus, the 50% inhibitory concentration (IC50 ) of temsirolimus in this culture system approximated 20 mol/l. Temsirolimus induced the formation of autophagosome in ACC cells In order to explore whether autophagy was induced in ACCM cell, we conducted a transmission electron microscopy (TEM) study. As shown in Fig. 2A and B, compared with the control group, numerous autophagosomes or lysosomes containing the segregation and degradation of organelles were recognized ultrastructurally in temsirolimus treated groups. Furthermore, compared to low dose group, we confirmed that high concentration of temsirolimus treatment revealed more acute response. Many
vesicles including autophagosomes and autophagolysosomes, containing entrapped cytoplasm organelles were found (Fig. 2A). Taken together, our findings suggested that temsirolimus induced cell death in ACC-M cells was related to autophagy. Temsirolimus induces high expression of autophagy related proteins in ACC-M cells After 48 h of treatment with temsirolimus, we examined whether autophagy was induced in ACC-M cells by western blot. Since the protein is a good indicator of autophagosome formation, we examined levels of LC3B and Beclin1 induced by temsirolimus treatment in ACC-M cells. As shown in Fig. 3, in the control cells, only few LC3 and Beclin1could be detected. However, after treatment with temsirolimus for 48 h, the levels of autophagy related proteins LC3 and Beclin1 were dramatically increased with high dose of temsirolimus. Furthermore, the ratio of LC3-II/LC3-I was increased obviously compared with the control group. Temsirolimus inhibits ACC xenograft growth by inducing autophagy Animal experiment was designed to detect whether temsirolimus could induce autophagy and inhibit xenograft growth in vivo, in order to further prove the effect of temsirolimus as described above. As the results showed, the growth of the ACC xenograft was significantly inhibited by temsirolimus, (Fig. 4A and D), and the body weight of nude mice showed no significant difference between two groups, either pretreatment or
Fig. 2. Detection and quantification for temsirolimus-induced autophagosomes by transmission electron microscopy (TEM). (A) Numerous autophagosomes or lysosomes (short black arrows) containing the segregation and degradation of organelles were recognized ultrastructurally in temsirolimus treated groups. Magnification, 10,000×. (B) The quantitative analysis of temsirolimus-induced autophagosomes among control and treatment groups. All data are presented as mean ± SEM from three independent experiments with duplicate. **p < 0.01 versus the control group.
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mTOR activation as well as up-regulate Beclin1 expression, sequentially induce autophagic in ACC-M cell in vivo, which might be the most important mechanism of action about the anti-tumor effect of temsirolimus. Discussion
Fig. 3. Temsirolimus induced autophagy in ACC-M cells. (A) The differences in LC3 (the ratio of LC3-II/LC3-I) and Beclin1 expression levels between control and temsirolimus-treated samples were compared by western blotting. (B and C) Gray values was compared between groups. All data are presented as mean ± SEM from three independent experiments with duplicate. **p < 0.01 versus the control group, and *p < 0.05 versus the control group.
posttreatment (Fig. 4B). In addition, as shown in Fig. 4C, the tumor from temsirolimus treatment mice exhibited significantly increased expression of LC3 (C1, C2) and Beclin1 (B1, B2). All of the above findings demonstrated that temsirolimus could suppress
Beyond all doubt, apoptosis, the type I programmed cell death, is the main cellular control mechanism for cell death [16], but it has been debated with respect to whether autophagy is beneficial or harmful for survival about the role of autophagy in neoplasm development and treatment. According to previous reports, autophagy may play dual and diametrically opposed role in cancer development [17,18]. Due to the activation of autophagy, cancer cells may survive after anticancer treatment, whereas in other neoplasms the cancer cells may undergo autophagic cell death [19]. In our research, the results indicated that temsirolimus induced autophagy indeed inhibited the development of ACC in vivo and in vitro. In vitro study, ACC-M cells were treated with assay concentration of temsirolimus, the date had distinctly suggested that temsirolimus induced cell death primely resulted from autophagy in ACC-M cells. Meanwhile, temsirolimus also displayed a distinct inhibitional effect in vivo. Our present study provides the first experimental evidence of autophagy induction in ACC cells disposed with temsirolimus. Temsirolimus-mediated autophagy in ACC-M cells was reflected by the induction of LC3B followed by the recruitment of processed LC3B to autophagosomes and the formation of autophagosome by technique of electron microscopy (Fig. 2), as well as the tumor volume growth curve and immunohistochemical staining in vivo (Fig. 4).
Fig. 4. Temsirolimus inhibits ACC xenograft growth according to induced autophagy. (A and B) Tumor volume and body weight of animal in vehicle-treated control mice and temsirolimus-treated mice. (C) Immunohistochemical analysis of the indicated biomarkers in both control and temsirolimus-treated ACC-M tumor tissues. The negative Beclin1 expression in control group (B1) and positive expression in treatment group (B2); the negative LC3 expression in control group (C1) and positive expression in treatment group (C2); HE staining of tumor tissue (A1) and negative controls by PBS (A2). Magnification, 200×. (D) Real-time morphological changes of xenografts. **p < 0.01 versus the control group.
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Rapamycin is a classical mTOR inhibitor. The poor solubility that compromised rapamycin as an intravenous agent led to the development of a more soluble ester analog of rapamycin, CCI-779 (temsirolimus) [20]. Frost et al. [21], confirmed that CCI-779 is effective in vivo against myeloma cells by inhibiting proliferation, angiogenesis, and induction of tumor cell apoptosis. This phenomenon is reminiscent of the experience with mTOR inhibitor temsirolimus used in the observed tumor cell apoptosis and antitumor response in vivo on hematological malignancies [22]. In support of this hypothesis existed in ACC, ACC-M cells were treated with temsirolimus in series of concentration, and it revealed significative effect on human adenoid cystic carcinoma (Fig. 1A). Dose–response curve showed that half maximal inhibitory concentration of temsirolimus in vitro cytostatic and cytotoxic effects on ACC-M cells was about 20 mol/l. We next investigated whether autophagy was induced by temsirolimus, which had been shown to trigger cell growth inhibition in ACC-M cells. The differential between control and treatment groups we studied was reflected by their discrepant expression of autophagy related proteins LC3 and Beclin1 in vitro treatment. LC3 is composed of a soluble LC3-I and a lipidated LC3-II, and is expressed as three isoforms in mammalian cells: LC3A, LC3B, and LC3C. LC3A and LC3B are related to autophagy [10,23]. Once autophagy is initiated by ATGs, LC3 is converted to the active LC3-II form, which is inserted into the double membrane of autophagophores and autophagosomes [24,25]. LC3-II, the marker protein of autophagy, is aggregated in membranes of autophagosomes and the ratio of LC3-II/LC3-I is increased. Beclin1 is responsible for both the signaling pathway activating autophagy and in the initial step of autophagosome formation in mammals, which functions as a tumor suppressor [26]. Our data confirmed that Temsirolimus, as the mTOR inhibitor, induced autophagy (Fig. 3). In several reports, blocking mTOR activity decreased proliferation, and promoted the formation of autophagosome [27]. Here transmission electron microscopy was used to observe the autophagosomes in our study. Consistent with the above observations, numerous autophagosomes or lysosomes could be found in temsirolimus treated groups (Fig. 2). This result proved that temsirolimus could activate autophagy through inhibiting mTOR pathway, which was associated with increased LC3 and Beclin1 levels. The animal experiment was designed to detect whether temsirolimus could induce autophagy and inhibit xenograft growth in vivo. Just as expected, the date and photos provided first-hand evidence that temsirolimus could inhibit the tumor growth though promoting mTOR-mediated autophagy. The inhibition effect was more significant along with the growth of nude mice. The body weight showed no significant difference between groups, from which we realized that temsirolimus did not impact the mice growth (Fig. 4). According to our previous study, we demonstrated that the progression of head and neck adenoid cystic carcinoma was associated with the down-regulated expression of LC3 and Beclin1, and the positive LC3 was directly correlated with absent lymph node involvement and TNM stage. Beclin1 expression showed a statistically significant correlation with the histological growth pattern and the histological grade, and the log-rank test showed that the survival in the positive Beclin1 group tended to be better than in the negative Beclin1 group [13]. This phenomenon also emerged when LC3 expression decreased in brain and ovary cancer and decreased expression of Beclin1 correlated with tumor progression in breast, ovarian, and brain cancer [28–31]. All of the above also could be reappeared in our in vivo study. As the control group shown, weak positive results were detected while the model was dealt with PBS. On the contrary, the expression of LC3 and Beclin1 appeared strong positive response in treatment group
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(Fig. 4C). These findings suggested that temsirolimus could suppress the development of tumor growth by inducing autophagy. These results suggested that temsirolimus could act as an mTOR inhibitor to induce autophagy in adenoid cystic carcinoma both in vitro and in vivo. However, we only focused our attention on the basic research of temsirolimus at present. It was far from clear whether it could play a similar role in human body. In some other neoplasm, investigators had make progress on the preclinical or clinical research. Trafalis et al. [32], confirmed that temsirolimus could be significantly effective for head and neck squamous cell carcinoma (HNSCC), all in vitro, in vivo, and clinical, especially combined with bevacizumab (antivascular endothelial growth factor antibody). Temsirolimus also showed efficacy in heavily pretreated and elderly patients in mantle cell lymphoma, however followed with some partial responses [33]. In this study, we successfully demonstrated that autophagy could inhibit the development of ACC, both in vitro and in vivo. Next, we may proceed the preclinical or clinical study, in order to clarify its effect on ACC in human. In conclusion, our investigation revealed for the first time that mTOR inhibitor temsirolimus induced autophagy suppressed the development of ACC by promoting the formation of autophagosome, and we demonstrated that mTOR pathway played a particularly important role in autophagy regulation. So, the findings suggested that autophagy was activated as a protective mechanism against ACC. It showed us a bright prospect in improving anticancer therapeutics. Nevertheless, the dual and diametrically opposed role of autophagy in various neoplasms, and more precise molecular mechanism, for instance the regulation between autophagy and apoptosis in ACC cells, are still far from clear. Moreover, further studies will be needed to clarify the anti-cancer effect of temsirolimus. Acknowledgments This study is supported by Shandong Provincial Science and Technology Development Plan (No. 2006GG2202034), the Science and Technology Foundation of Shandong Province (2006GG20002046) and Shandong Provincial Natural Science Foundation (ZR2012HQ022). References [1] M. Persson, Y. Andrén, J. Mark, et al., Recurrent fusion of MYB and NFIB transcription factor genes in carcinomas of the breast and head and neck, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 18740–18744. [2] D. Bell, D. Roberts, M. Kies, A.K. Weber, El-Naggar, et al., Cell type-dependent biomarker expression in adenoid cystic carcinoma: biologic and therapeutic implications, Cancer 116 (2010) 5749–5756. [3] O. Tetsu, J. Phuchareon, A. Chou, R.C. Eisele, Jordan, et al., Mutations in the c-Kit gene disrupt mitogen-activated protein kinase signaling during tumor development in adenoid cystic carcinoma of the salivary glands, Neoplasia 12 (2010) 708–717. [4] J. Fordice, C. Kershaw, A. El-Naggar, et al., Adenoid cystic carcinoma of the head and neck: predictors of morbidity and mortality, Arch. Otolaryngol. Head Neck Surg. 125 (1999) 149–152. [5] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (2008) 27–42. [6] N. Chen, J. Debnath, Autophagy and tumorigenesis, FEBS Lett. 584 (2010) 1427–1435. [7] A. Notte, L. Leclere, C. Michiels, Autophagy as a mediator of chemotherapyinduced cell death in cancer, Biochem. Pharmacol. 82 (2011) 427–434. [8] Y. Kondo, T. Kanzawa, R. Sawaya, et al., The role of autophagy in cancer development and response to therapy, Nat. Rev. Cancer 5 (2005) 726–734. [9] S. Chen, S.K. Rehman, W. Zhang, et al., Autophagy is a therapeutic target in anticancer drug resistance, Biochim. Biophys. Acta 1806 (2010) 220–229. [10] N. Mizushima, D.J. Klionsky, Protein turnover via autophagy: implications formetabolism, Annu. Rev. Nutr. 27 (2007) 19–40. [11] G. Viola, R. Bortolozzi, E. Hamel, et al., MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells, Biochem. Pharmacol. 83 (2012) 16–26. [12] J. Hao, Y. Pei, G. Ji, et al., Autophagy is induced by 3-O-succinyl-lupeol (LD9-4) in A549 cells via up-regulation of Beclin1 and down-regulation mTOR pathway, Eur. J. Pharmacol. 670 (2011) 29–38.
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