Cannabinoid Action Induces Autophagy-Mediated Cell

[EDDIEKIRK]

Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells



Macro-autophagy, hereafter referred to as autophagy, is a highly conserved cellular process in which cytoplasmic materials including organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation or recycling (1). In many cellular settings, triggering of autophagy relies on the inhibition of mammalian target of rapamycin complex 1 (mTORC1), an event that promotes the activation (de-inhibition) of several autophagy proteins (Atgs) involved in the initial phase of membrane isolation (1). Enlargement of this complex to form the autophagosome requires the participation of 2 ubiquitin-like conjugation systems. One involves the conjugation of ATG12 to ATG5 and the other of phosphatidylethanolamine to LC3/ATG8 (1). The final outcome of the activation of the autophagy program is highly dependent on the cellular context and the strength and duration of the stress-inducing signals (25). Thus, besides its role in cellular homeostasis, autophagy can be a form of programmed cell death, designated type II programmed cell death, or play a cytoprotective role, for example in situations of nutrient starvation (6). Accordingly, autophagy has been proposed to play an important role in both tumor progression and promotion of cancer cell death (24), although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated.

⁹-Tetrahydrocannabinol (THC), the main active component of marijuana (7), exerts a wide variety of biological effects by mimicking endogenous substances the endocannabinoids that bind to and activate specific cannabinoid receptors (8). One of the most exciting areas of research in the cannabinoid field is the study of the potential application of cannabinoids as antitumoral agents (9). Cannabinoid administration has been found to curb the growth of several types of tumor xenografts in rats and mice (9, 10). Based on this preclinical evidence, a pilot clinical trial has been recently run to investigate the antitumoral action of THC on recurrent gliomas (11). Recent findings have also shown that the pro-apoptotic and tumor growthinhibiting activity of cannabinoids relies on the upregulation of the transcriptional co-activator p8 (12) and its target the pseudo-kinase tribbles homolog 3 (TRB3) (13). However, the mechanisms that promote the activation of this signaling route as well as the targets downstream of TRB3 that mediate its tumor cellkilling action remain elusive. In this study we found that ER stressevoked upregulation of the p8/TRB3 pathway induced autophagy via inhibition of the Akt/mTORC1 axis and that activation of autophagy promoted the apoptotic death of tumor cells. The uncovering of this pathway, which we believe is novel, for promoting tumor cell death may have therapeutic implications in the treatment of cancer.







.....................................









Cell culture and viability. Cortical astrocytes were prepared from 24-hour-old mice as previously described (13). Primary cultures of brain tumor cells were prepared and cultured as described in the Supplemental Methods. U87MG, T98G, U373MG, and MiaPaCa2 cells, p8^+/+ and p8^/ Ras^V12/E1A MEFs, Atg5^+/+ and Atg5^/ T-large antigen MEFs (provided by Noboru Mizushima, Tokyo Medical and Dental University, Tokyo, Japan), Bax/Bak wild-type and Bax/Bak DKO T-large antigen MEFs (provided by Luca Scorrano, Dulbecco Telethon Institute, Milan, Italy, and Patrizia Agostinis, Catholic University of Leuven, Leuven, Belgium), eIF2a S51S WT and eIF2a S51A T-large antigen MEFs (provided by Richard Kaufman, University of Michigan, Ann Arbor, Michigan, USA, and Cesar de Haro and Juan J. Berlanga, Centro de Biologa Molecular Severo Ochoa, Autonoma University, Madrid, Spain), Tsc2^+/+ and Tsc2^/ p53^/ MEFs, empty vector (pBABE) and pBABE-myr-Akt MEFs, and Atg5^+/+ and Atg5^/ Ras^V12/T-large antigen MEFs were cultured in DMEM containing 10% FBS and transferred to medium containing 0.5% FBS (except Ras^V12/E1A-transformed MEFs, which were transferred to medium containing 2% FBS) 18 h before performing the different treatments. p8^+/+ and p8^/ Ras^V12/E1A MEFs as well as Atg5^+/+ and Atg5^/ Ras^V12/T-large antigen MEFs correspond to a polyclonal mix of at least 20 different selected clones. Unless otherwise indicated, THC was used at a final concentration of 5 M. Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test (Sigma-Aldrich).

Flow cytometry. Briefly, cells (approximately 5 10⁵ cells per assay) were trypsinized, divided in 2 tubes, washed, and collected by centrifugation at 1,500 g for 5 min. One aliquot was incubated for 10 min at 37C with Annexin VFITC (BD Biosciences). Propidium iodide (1 g/ml) was added just before cytofluorometric analysis. The other aliquot was simultaneously labeled with 3,3-dihexyloxacarbocyanine iodide (DiOC₆[3], 40 nM; Invitrogen) and hydroethidium (5 M; Invitrogen) for 10 minutes at 37C, followed by cytofluorometric analysis. Cells (10,000) were recorded in each analysis. Fluorescence intensity was analyzed in an EPICS XL flow cytometer (Beckman Coulter).

Western blot. Western blot analysis was performed following standard procedures. A list of the antibodies used can be found in Supplemental Methods. Densitometric analysis was performed with Quantity One software (Bio-Rad).

Transfections. U87MG cells (75% confluent) were transfected with siRNA duplexes using the DharmaFECT 1 Transfection reagent (Dharmacon). Cells were trypsinized and seeded 24 h after transfection, at a density of 5,000 cells/cm². Transfection efficiency was greater than 70% as monitored with a control fluorescent (red) siRNA (siGLO RISC-Free siRNA; Dharmacon). In immunofluorescence experiments, control and selective siRNAs were used in a 1:5 ratio, and cells with red spots were scored as transfected.

Infections with adenoviral vectors. U87MG cells (75% confluent) were transduced for 1 h with supernatants obtained from HEK293 cells infected with adenoviral vectors carrying EGFP (provided by Javier G. Castro, Hospital Infantil Universitario Nio Jess, Madrid, Spain), rat HA-tagged TRB3 (donated by Patrick Iynedjian, University of Geneva, Geneva, Switzerland) (32), or human EGFP-LC3 (provided by Aviva Tolkovsky and Christoph Goemans, University of Cambridge, Cambridge, United Kingdom). Infection efficiency was greater than 80% as determined by EGFP fluorescence.

RNA interference. Double-stranded RNA duplexes were purchased from Dharmacon. A list of sequences can be found in the Supplemental Methods.

RT-PCR analysis. RNA was isolated using Trizol Reagent (Invitrogen). cDNA was obtained with Transcriptor Reverse transcriptase (Roche Applied Science). Primers and amplification conditions can be found in the Supplemental Methods.

Real-time quantitative PCR. cDNA was obtained using Transcriptor (Roche Applied Science). Real-time quantitative PCR assays were performed using the FastStart Universal Probe Master mix with Rox (Roche Applied Science), and probes were obtained from the Universal ProbeLibrary Set (Roche Applied Science). Primer sequences can be found in the Supplemental Methods. Amplifications were run in a 7900 HT-Fast Real-Time PCR System (Applied Biosystems). Each value was adjusted by using 18S RNA levels as a reference.

Immunoprecipitation. U87MG cells were lysed in HEPES lysis buffer (see Supplemental Methods for buffer composition). Lysate (14 mg) was precleared by incubating with 520 l of protein GSepharose conjugated to pre-immune IgG. The lysate extracts were then incubated with 520 l of protein GSepharose conjugated to 520 g of the anti-TRB3 antibody or pre-immune IgG. TRB3 antibody (aminoterminal end, ab50516; Abcam) was covalently conjugated to protein GSepharose using dimethyl pimelimidate. Immunoprecipitations were carried out for 1 h at 4C on a rotatory wheel. The immunoprecipitates were washed 4 times with HEPES lysis buffer, followed by 2 washes with HEPES kinase buffer. The immunoprecipitates were resuspended in 30 l of sample buffer (not containing 2-mercaptoethanol) and filtered through a 0.22-m Spin-X filter, and 2-mercaptoethanol was added to a concentration of 1% (vol/vol). Samples were subjected to electrophoresis and immunoblot analysis.

Ceramide levels. Ceramide levels were determined as previously described (37).

Confocal laser scanning microscopy. Standard protocols for immunofluorescence microscopy were used (see Supplemental Methods for the antibodies used). To quantify the percentage of cells with LC3 or PDI dots, at least 200 cells per condition were counted in randomly selected fields. In all cases, only those cells with 4 or more prominent dots of either LC3 or PDI were scored positively.

In vivo treatments. Tumors derived from U87MG cells and p8^+/+ and p8^/ MEFs were induced and treated as previously described (13). Tumors derived from Atg5^+/+ or Atg5^/ Ras^V12/T-large antigen MEFs (see Supplemental Methods for the procedure used to generate these cells) were induced in nude mice by subcutaneous injection of 10⁷ cells in PBS supplemented with 0.1% glucose. Tumors were allowed to grow until an average volume of 200250 mm³, and animals were assigned randomly to the different groups. At this point, vehicle or THC (15 mg/kg/d) in 100 l of PBS supplemented with 5 mg/ml BSA was administered daily in a single peritumoral injection. Tumors were measured with an external caliper, and volume was calculated as (4/3) (width/2)² (length/2). All procedures involving animals were performed with the approval of the Complutense University Animal Experimentation Committee according to Spanish official regulations.

Human tumor samples. Tumor biopsies were obtained from 2 recurrent glioblastoma multiforme patients who had been treated with THC. The characteristics of the patients and the clinical study have been described in detail elsewhere (11). Briefly, THC dissolved in 30 ml of physiological saline solution plus 0.5% (wt/vol) human serum albumin was administered intratumorally to the patients. Patient 1 received a total of 1.46 mg of THC for 30 days, while patient 2 received a total of 1.29 mg of THC for 26 days (it was estimated that doses of 610 M THC were reached at the site of administration; ref. 11). Samples were fixed in formalin, embedded in paraffin, and used for immunomicroscopy.

Immunomicroscopy of tumor samples. Samples from tumor xenografts were dissected, Tissue-Tek (Sakura) embedded, frozen, and, before the staining procedures were performed, fixed in acetone for 10 min at room temperature. Samples from human tumors were subjected to deparaffinization, rehydration, and antigen retrieval before the staining procedures were performed. Standard protocols for immunofluorescence or immunohistochemistry microscopy were used (see Supplemental Methods). Nuclei were counterstained with TOTO-3 iodide (U87MG and human tumor samples; Invitrogen) or Hoechst 33342 (MEF tumors; Invitrogen). Fluorescence images were acquired using Metamorph-Offline 6.2 software (Universal Imaging) and Zeiss Axioplan 2 Microscope.

TUNEL. Tumor samples were fixed, blocked, and permeabilized, and TUNEL was performed as previously described (13).

Electron microscopy. Ultrastructural analysis of vehicle- and THC-treated cells was assessed by conventional embedding in the epoxy-resin EML-812 (Taab Laboratories). Ultrathin (20- to 30-nm-thick) sections of the samples were obtained using a Leica-Reichert-Jung ultramicrotome and then stained with saturated uranyl acetatelead citrate by standard procedures. Ultrathin sections were analyzed in a JEOL 1200-EX II transmission electron microscope operating at 100 kV.

Statistics. Statistical analysis was performed by ANOVA with a post-hoc analysis using the Student-Neuman-Keuls test. Differences were considered significant when the P value was less than 0.05.



http://www.jci.org/articles/view/37948