J Cancer Res Clin Oncol (2014) 140:769–781 DOI 10.1007/s00432-014-1628-0

Original Article – Cancer Research

Temsirolimus and chloroquine cooperatively exhibit a potent antitumor effect against colorectal cancer cells Manabu Kaneko · Hiroaki Nozawa · Masaya Hiyoshi · Noriko Tada · Koji Murono · Takako Nirei · Shigenobu Emoto · Junko Kishikawa · Yuuki Iida · Eiji Sunami · Nelson H. Tsuno · Joji Kitayama · Koki Takahashi · Toshiaki Watanabe 

Received: 15 December 2013 / Accepted: 22 February 2014 / Published online: 12 March 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Purpose Temsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in preclinical models, but its efficacy against colorectal cancer (CRC) has not been fully explored. Methods  We evaluated the antitumor effect of TEM in CRC cell lines (CaR-1, HT-29, Colon26) in vitro and in vivo. In vitro, cell growth inhibition was assessed using a MTS assay. Apoptosis induction and cell cycle effects were measured using flow cytometry. Modulation of mTOR signaling was measured using immunoblotting. Antitumor activity as a single agent was evaluated in a mouse subcutaneous tumor model of CRC. The effects of adding chloroquine, an autophagy inhibitor, to TEM were evaluated in vitro and in vivo. Results  In vitro, TEM was effective in inhibiting the growth of two CRC cell lines with highly activated AKT, possibly through the induction of G1 cell cycle arrest via a reduction in cyclin D1 expression, whereas TEM reduced HIF-1α and VEGF in all three cell lines. In a mouse subcutaneous tumor model, TEM inhibited the growth of tumors in all cell lines, not only through direct growth inhibition but also via an anti-angiogenic effect. We also explored the M. Kaneko (*) · H. Nozawa · M. Hiyoshi · N. Tada · K. Murono · T. Nirei · S. Emoto · J. Kishikawa · Y. Iida · E. Sunami · J. Kitayama · T. Watanabe  Department of Surgical Oncology, Faculty of Medicine, The University of Tokyo, 7‑3‑1 Hongo, Bunkyo‑ku, Tokyo 113‑8655, Japan e-mail: [email protected] N. H. Tsuno · K. Takahashi  Department of Transfusion Medicine, Faculty of Medicine, The University of Tokyo, 7‑3‑1 Hongo, Bunkyo‑ku, Tokyo 113‑8655, Japan

effects of adding chloroquine, an autophagy inhibitor, to TEM. Chloroquine significantly potentiated the antitumor activity of TEM in vitro and in vivo. Moreover, the combination therapy triggered enhanced apoptosis, which corresponded to an increased Bax/Bcl-2 ratio. Conclusions  Based on these data, we propose TEM with or without chloroquine as a new treatment option for CRC. Keywords Temsirolimus · Chloroquine · Colorectal cancer · mTOR · Autophagy · Angiogenesis

Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females worldwide. It was estimated that over 1.2 million new CRC cases and 600,000 deaths due to CRC occurred in 2008 (Jemal et al. 2011). A treatment option for unresectable patients is systemic chemotherapy such as FOLFOX, CapeOX or FOLFIRI regimens with or without biologics as first-line treatment (Van Cutsem et al. 2010). Although these regimens have achieved tolerable response rates (14–48 %) and longer survivals (16.5–24.5 months) than ever (Chau and Cunningham 2009), there are still many CRC patients not responding to these treatments, and there is a pressing need for new drugs and a broader range of therapeutic options to treat CRC. Temsirolimus (TEM) is a derivative of rapamycin, targeting a serine/threonine kinase, mammalian target of rapamycin (mTOR), in the PI3 K/AKT/mTOR signaling pathway (Nguyen et al. 2012). Inhibition of mTOR decreases the phosphorylation of two downstream targets, 4E-BP1 and p70S6 K, and blocks protein synthesis concerning cell growth, survival, proliferation, motility, apoptosis

13

770

and autophagy (Nguyen et al. 2012; Kim and Eng 2012). Unlike rapamycin or everolimus, TEM is water soluble so that it can be easily administered to animals by intraperitoneal injection in vivo experiments. In addition, TEM was shown to have anti-angiogenic effects, including inhibition of human umbilical vein endothelial cell growth in vitro, and reductions in the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in human breast cancer and rhabdomyosarcoma cells (Del Bufalo et al. 2006; Wan et al. 2006). Similar observations were reported for rapamycin in murine colon cancer, rat hepatoma and glioma cells (Guba et al. 2005; Semela et al. 2007; Phung et al. 2006), and for everolimus in murine melanoma and colon cancer, and human pancreatic, ovarian, colon and cervical cancer cells (Mabuchi et al. 2007a, b; Manegold et al. 2008; Shinohara et al. 2005; Lane et al. 2009). Furthermore, as well as everolimus, TEM was already approved for clinical use in patients with advanced renal cell carcinoma (Kapoor and Figlin 2009). These results motivated us to investigate the potential antitumor effects of TEM on CRC cells. TEM is also known to be a potent inducer of autophagy, which is thought to be a possible mechanism for resistance to an mTOR inhibitor (Carew et al. 2011a, b). The induction of autophagy has been reported following treatment with many other anticancer agents, including arsenic trioxide, histone deacetylase inhibitors, etoposide, tamoxifen, temozolomide, bortezomib and imatinib (Bursch et al. 1996; Kanzawa et al. 2003, 2004; Paglin et al. 2001; Shao et al. 2004; Takeuchi et al. 2005; Zhu et al. 2010). In addition, previous reports showed that the inhibition of autophagy enhanced the efficacy of many of these therapeutic agents (Amaravadi et al. 2007; Bellodi et al. 2009; Carew et al. 2007, 2010, 2011a, b). Based on the above-mentioned findings, we hypothesized that autophagy inhibition could increase the efficacy of TEM. In the present study, we investigated whether TEM inhibited tumor angiogenesis in human and murine colorectal cancer cells in mouse subcutaneous tumor models using CRC cell lines with or without activation of the PI3 K/AKT signaling axis. We also determined whether the inhibition of autophagy could increase the activities of TEM on CRC cells, in in vitro and in vivo experiments.

Materials and methods

J Cancer Res Clin Oncol (2014) 140:769–781

calf serum and 1 % antibiotics/antimycotics, and then incubated in a 5 % CO2 and 21 % O2 incubator at 37 °C. In the hypoxic condition, cells were cultured in a modular incubator chamber filled with 5 % CO2 and 1 % O2 at 37 °C. The TEM (Sigma-Aldrich) used for the in vitro experiments was dissolved in dimethyl sulfoxide at a concentration of 1 mM. For the in vivo experiments, TEM (provided by Pfizer Inc.) was dissolved in 100 % ethanol at a concentration of 50 mg/mL. Chloroquine (CQ) (Sigma-Aldrich) was dissolved in calcium- and magnesium-free phosphate-buffered saline [PBS(−)] at a concentration of 10 mM. Stock solutions were diluted to the desired final concentrations with growth medium just before use. In vitro drug treatments To evaluate the independent effects of TEM on proliferation, cell cycle/apoptosis and the protein expressions in CRC cells, the cells were treated with TEM (0, 0.1, 1, 10 or 100 nM) for 96 h, TEM (0, 1, 10 or 100 nM) for 48 h or TEM (0, 1, 10 or 100 nM) for 24 and 48 h, respectively. For Western blotting analyses, the cells of control groups were incubated for 24 h in the presence of the vehicle alone. To assess the effects of the co-treatment with TEM and CQ on cellular functions, cells were treated with TEM (100 nM) and/or CQ (20 μM) for 24 and/or 48 h, respectively. In vitro cell proliferation Cell proliferation was evaluated by MTS assay using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI), according to the manufacturer’s recommendation. The absorbance of formazan at 490 nm was measured directly using a microtiter plate reader (Becton–Dickinson, Mountain View, CA). Detection of apoptosis by flow cytometry Treated cells were stained with the combination of Annexin V/fluorescein isothiocyanate (FITC) and propidium iodide (PI) using the Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, San Jose, CA), according to the manufacturer’s instructions. Cells were analyzed using a fluorescence-activated cell sorting (FACS) flow cytometer (Becton–Dickinson), and the data were analyzed with CellQuest software (Becton–Dickinson). The population of Annexin V (+) cell populations was considered to be apoptotic cells.

Cells and reagents Analysis of the cell cycle by flow cytometry The human CRC cell lines CaR-1 and HT-29 and the murine CRC cell line Colon26 were obtained from the Japanese Cancer Research Resource Bank. Cells were cultured in RPMI-1640 medium supplemented with 10 % fetal

13

The cell cycle of the treated cells was analyzed by flow cytometry using the Cycle TEST PLUS DNA Reagent Kit (BD Pharmingen), according to the manufacturer’s

J Cancer Res Clin Oncol (2014) 140:769–781

instructions. Samples were analyzed with the FACS and the Cell Quest software. Western blotting of cultured cell extracts Cultured cells were lysed with 0.5 mL Tris-saline (TS; 50 mM Tris–HCl, pH 7.6, 150 mM sodium chloride) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich), and 1 % Triton-X for 1 h at 4 °C. The total protein concentration was determined by the BCA protein assay reagent (Pierce Biomedical Co., Rockford, IL). Cell lysates were separated on a 7–15 % Ready Gel J SDS-PAGE system (Bio-Rad, Hercules, CA) for Hybond ECL nitrocellulose membrane blotting (Amersham Pharmacia Biotech, Buckinghamshire, UK). The secondary antibodies used were biotinylated antirabbit IgG or biotinylated antimouse IgG (Vector Laboratories Inc., Burlingame, CA). The immunoreactive bands were visualized by enhanced chemiluminescence using the ECL detection system (Amersham Pharmacia Biotech). The density of each band was measured using Image J software (open source Image J software available at http://rsb.info.nih. gov/ij/), standardized by the density of β-actin. The antibodies used for Western blotting were against phosphoAKT (p-AKT, Ser473) (Cell Signaling Technology, Beverly, MA), phospho-mTOR (p-mTOR, Ser2448) (Cell Signaling Technology), phospho-4E-BP1 (p-4E-BP1, Thr37/46) (Cell Signaling Technology), phospho-p70 S6 kinase (p-p70S6 K, Thr389) (Cell Signaling Technology), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), HIF-1α (Santa Cruz Biotechnology), VEGF (Santa Cruz Biotechnology), Bax (Santa Cruz Biotechnology), Bcl-2 (Santa Cruz Biotechnology), LC3 (Medical Biological Laboratories Co., Nagoya, Japan), p62 (Medical Biological Laboratories Co.) and β-actin (Sigma-Aldrich). Animal experiments Balb/c nu/nu mice and Balb/c mice (4–6 weeks old) were purchased from Oriental Yeast Co. (Tokyo, Japan) and housed under specific pathogen-free conditions. The protocol of the animal experiments was approved by the Animal Care Facility of the University of Tokyo. CaR-1 (5 × 106 cells) or HT-29 (5 × 106 cells) were implanted subcutaneously in the right flank of Balb/c nu/nu mice, and Colon26 (5  × 105 cells) were implanted subcutaneously in that of Balb/c mice. When their tumors reached a mean volume of 100–200 mm3, the mice were divided into four treatment groups (n = 6–8 each), namely the untreated controls and TEM at a dose of 0.1, 1 and 10 mg/kg. TEM dissolved in PBS with 5 % Tween 80 and 5 % polyethyleneglycol 400 (total volume: 0.2 mL) was injected in the mice intraperitoneally three times a week for 3 weeks (long-term

771

treatment) or 1 week (short-term treatment). The control group received injections of 0.2 mL of vehicle instead. In the long-term treatment, the diameters of the subcutaneous tumors were measured three times per week with a caliper, and the tumor volume was calculated using the formula: (shortest diameter)2  × (longest diameter) × 0.5. In the short-term treatment, subcutaneous tumors were resected and either stored at −80 °C for Western blotting or embedded in paraffin for immunohistochemistry on day 7 after the start of treatment. For the combinatorial treatment of TEM and CQ, CQ was dissolved in 0.1 mL PBS at 50 mg/kg/day. Four treatment groups were designed: vehicle control, CQ alone (50 mg/kg), TEM alone (10 mg/kg) and TEM + CQ (the same doses). Experimental procedures similar to those for the single TEM treatment were carried out. Histology and immunohistochemistry of in vivo tumors Five-mm-thick sections were prepared, and immunohistochemistry was performed by standard methods. In brief, de-waxed sections were processed using 0.05 % citraconic anhydride buffer (pH 7.4) at 98 °C for 45 min to expose the antigen epitopes. The primary antibody was incubated with tissues overnight at 4 °C. Peroxidase activity from the secondary antibody was detected by adding the substrate 3, 3′-diaminobenzidine (DAB) (Wako Pure Chemical Industries), and the sections were counterstained with hematoxylin. The primary antibodies used were against HIF1α (Santa Cruz Biotechnology) and VEGF (Santa Cruz Biotechnology). Apoptosis assay of in vivo tumors Apoptosis was analyzed by a terminal transferase uridyl end labeling (TUNEL) assay. Five-μm sections were immersed in 0.5 % H2O2 in methanol for 20 min to block the endogenous peroxidase. The sections were then incubated in 100 mM potassium cacodylate, 0.15 M NaCl, 0.05 % bovine serum albumin (BSA), 2 mM cobalt(II) chloride, 0.015 mM biotin-dUTP and 200 U/mL terminal deoxynucleotidyl transferase (Invitrogen, Carlsbad, CA) at 37 °C for 30 min. After being rinsed in PBS, the sections were exposed to peroxidase-conjugated antidigoxigenin at 37 °C for 30 min. Cells undergoing apoptosis were visualized with DAB. The numbers of stained tumor cells were counted in six fields from two different tumors for each group at 400× magnification. Proliferation activity of in vivo tumors Mice were injected intraperitoneally with bromodeoxyuridine (BrdU) at 50 mg/g of body weight 1 h before killing.

13

772

Tissues were processed, and immunohistochemistry was performed on paraffin sections using M.O.M. Immunodetection Kit (Vector Laboratories, Burlingame, CA). The primary antibody used was mouse anti-BrdU antibody (Roche Diagnostics, Indianapolis, IN) and colored with DAB. The quantification of BrdU-positive cells was performed on digital images captured under 400× magnification of each section. The numbers of stained tumor cells were counted in six fields with 400× magnification from two tumors for each group. In vivo angiogenesis Animals received an intravenous injection of 0.2 mg FITClabeled Lycopersicon esculentum (tomato) lectin (Vector Laboratories). After 2 min, the subcutaneous tumors were resected, placed in OCT compound (Sakura Finetek Japan Co., Tokyo) and snap-frozen on dry ice. Sections were hydrated by immersion in xylene solutions. All sections were examined using a fluorescence microscope (Keyence, Osaka, Japan). Cell nuclei were also stained with mounting medium with DAPI (Vector Laboratories). Microvessel density (MVD) was evaluated as follows: The entire tumor section was first carefully scanned at 40× magnification to find the area that presented the most intense neovascularization. The six most highly vascularized areas from two different tumors for each group were selected in 200× magnification fields, and the vascular endothelium visualized by green fluorescence was counted. Western blotting for proteins expressed in subcutaneous tumors Homogenates of subcutaneous tumors were used, and the same procedures were performed as that for cultured cell extracts mentioned above. The primary antibodies used were the ones mentioned above. Statistical analysis All experiments were repeated at least three times. The statistical significance of the differences was evaluated by the unpaired, two-tailed Student’s t test, and an association was considered significant when the exact significant level of the test was P 

Temsirolimus and chloroquine cooperatively exhibit a potent antitumor effect against colorectal cancer cells.

Temsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in...
5MB Sizes 1 Downloads 3 Views