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

Abstract

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that delta(9)-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.

Figures

Figure 1. Inhibition of autophagy prevents THC-induced cancer cell death.

(A–C) Effect of THC on U87MG cell morphology. Representative electron microscopy photomicrographs are shown (6 h). Scale bars: 500 nm. Note the presence of early (A, open arrows, and B) and late (A, filled arrows, and C) autophagosomes in THC-treated but not vehicle-treated (veh-treated) cells. (D) Top: Effect of SR141716 (SR1; 1 μM) and THC on LC3 immunostaining (green) in U87MG cells (18 h; n = 3). The percentage of cells with LC3 dots relative to the total cell number is shown in the corner of each panel (mean ± SD). Scale bar: 20 μm. Bottom: Effect of SR1 and THC on LC3 lipidation in U87MG cells (18 h; n = 3). (E) Effect of E64d (10 μM) and pepstatin A (PA; 10 μg/ml) on THC-induced LC3 lipidation in U87MG cells (18 h; n = 3). (F and G) Effect of THC treatment and transfection with control siRNAs (siC) or ATG1-selective siRNAs (siATG1) on cell viability (F; mean ± SD; n = 3), LC3 immunostaining (G, left panels; 18 h; percentage of cells with LC3 dots relative to the total number of cells cotransfected with a red fluorescent control siRNA, mean ± SD; n = 3; scale bar: 20 μm), and LC3 lipidation (G, right panel; 18 h; n = 3) in U87MG cells. (H and I) Effect of THC on cell viability (H; mean ± SD; n = 3), LC3 immunostaining (I, left panels; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean ± SD; n = 3; scale bar: 20 μm), and LC3 lipidation (I, right panel; 18 h; n = 3) in Atg5+/+ and Atg5–/– RasV12/T-large antigen MEFs. *P < 0.05 and **P < 0.01 compared with THC-treated U87MG (D) and Atg5+/+ (H and I) cells and compared with siC-transfected, THC-treated U87MG cells (F and G). THC concentration was 6 μM.

Figure 2. ER stress precedes autophagy in cannabinoid action.

(A) Effect of THC on U87MG cell morphology. Note the presence of the dilated ER in THC- but not vehicle-treated cells (6 h). Arrows point to the ER. Scale bars: 500 nm. (B) Effect of SR1 (1 μM) and THC on PDI immunostaining (red) in U87MG cells (8 h; n = 3). The percentage of cells with PDI dots relative to the total cell number is shown in the corner of each panel (mean ± SD). Scale bar: 20 μm. (C) Effect of SR1 (1 μM) on THC-induced eIF2α phosphorylation of U87MG cells (3 h; OD relative to vehicle-treated cells, mean ± SD; n = 3). (D) Effect of THC on PDI (red) and LC3 (green) immunostaining in U87MG cells (n = 3). The percentage of cells with PDI or LC3 dots relative to total cell number at each time point (mean ± SD) is shown. Scale bar: 20 μm. (E) Effect of THC on eIF2α phosphorylation and LC3 lipidation in U87MG cells (n = 3). **P < 0.01 compared with THC-treated (B) or vehicle-treated (C and D) cells.

Figure 3. THC induces autophagy via ER stress–evoked p8 and TRB3 upregulation.

(A and B) Effect of ISP-1 (1 μM) on THC-induced eIF2α phosphorylation (A; 3 h; n = 3) and LC3 immunostaining (B, left panels; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean ± SD; n = 3; scale bar: 20 μm) in U87MG cells. sip8, p8-selective siRNA; siTRB3, TRB3-selective siRNA. (C) Effect of THC on p8, ATF4, CHOP, and TRB3 mRNA levels of eIF2α WT and eIF2α S51A MEFs as determined by real-time quantitative PCR (8 h; n = 3). Numbers indicate the mean fold increase ± SD relative to vehicle-treated eIF2α WT MEFs. (D) Top: Analysis of p8 and TRB3 mRNA levels. Results from a representative RT-PCR experiment are shown. The numbers indicate gene expression levels as determined by real-time quantitative PCR (mean fold change ± SD relative to siC-transfected cells; n = 5). Bottom: Effect of THC on LC3 immunostaining (green) of U87MG cells transfected with siC, sip8, or siTRB3 (18 h; n = 4). The percentage of cells with LC3 dots relative to cells cotransfected with a red fluorescent control siRNA is shown in each panel (mean ± SD). Scale bar: 20 μm. (E) Effect of THC on LC3 lipidation in U87MG cells transfected with siC, sip8, or siTRB3 (18 h; n = 6). (F) Effect of THC on LC3 lipidation (top; 18 h; n = 5) and LC3 immunostaining (bottom; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean ± SD; n = 4; scale bar: 40 μm) in p8+/+ or p8–/– MEFs. *P < 0.05 and **P < 0.01 compared with THC-treated U87MG (B), eIF2α WT (C), or p8+/+ (F) cells and compared with siC-transfected, THC-treated U87MG cells (D).

Figure 4. THC inhibits the Akt/mTORC1 pathway via TRB3.

(A) Effect of THC on p70S6K and S6 phosphorylation of U87MG cells (n = 6). (B) Effect of THC on cell viability (left panel; 24 h; mean ± SD; n = 6) and LC3 lipidation (right panel; 18 h; n = 4) in Tsc2+/+ and Tsc2–/– MEFs. (C) Effect of THC on Akt, TSC2, PRAS40, p70S6K, and S6 phosphorylation of U87MG cells (18 h; OD relative to vehicle-treated cells, mean ± SD; n = 7). (D) Effect of THC on cell viability (left panel; 24 h; mean ± SD; n = 4) and LC3 lipidation (right panel; 18 h; n = 4) of pBABE and myristoylated Akt (myr-Akt) MEFs. (E) Effect of THC on Akt co-immunoprecipitation with TRB3 in U87MG cell extracts (8 h; OD relative to vehicle-treated cells, mean ± SD; n = 9; input: TRB3). (F and G) Effect of THC on Akt, TSC2, PRAS40, p70S6K, and S6 phosphorylation and LC3 lipidation (G only) of siC- and siTRB3-transfected (F; 18 h; OD relative to vehicle-treated siC-transfected U87MG cells, mean ± SD; n = 7; upper panel shows an analysis of TRB3 mRNA levels) and EGFP (Ad-EGFP) or rat TRB3 (Ad-TRB3) adenoviral vector–infected (G; 18 h; OD relative to vehicle-treated Ad-EGFP–infected U87MG cells, mean ± SD; n = 4; upper panel shows an analysis of rTRB3 mRNA levels) U87MG cells. (H) Effect of THC on Akt, p70S6K, and S6 phosphorylation of p8+/+ and p8–/– MEFs (n = 7). *P < 0.05 and **P < 0.01 compared with THC-treated Tsc2+/+ (B) and pBABE (D) MEFs and compared with vehicle-treated (C and E), vehicle-treated siC-transfected (F), or Ad-EGFP–infected (G) U87MG cells.

Figure 5. Autophagy is upstream of apoptosis in cannabinoid-induced cancer cell death.

(A) Effect of THC and the pan-caspase inhibitor ZVAD (10 μM) on the viability of Atg5+/+ and Atg5–/– MEFs (36 h; percentage of viable cells relative to the corresponding Atg5+/+ vehicle-treated cells, mean ± SD; n = 3). (B) Effect of THC on the apoptosis of Bax/Bak WT and Bax/Bak DKO MEFs as determined by cytofluorometric analysis of Annexin V/propidium iodide (PI) (24 h; mean ± SD; n = 3). The mean ± SD percentage of Annexin V–positive/PI-positive and Annexin V–positive, PI-negative cells is shown in the upper and lower corners, respectively. (C) Effect of THC on eIF2α phosphorylation (3 h; n = 3) and LC3 lipidation (18 h; n = 4) of Bax/Bak WT and DKO MEFs. (D) Left: Effect of THC on autophagy and apoptosis of U87MG cells transfected with siC or siATG1. Green bars, cells with LC3 dots; red bars, active caspase-3–positive cells; white bars, cells with both LC3 dots and active caspase-3 staining. Data correspond to the percentage of cells with LC3 dots (green bars), active caspase-3–positive cells (red bars), and cells with LC3 dots and active caspse-3 staining (white bars) relative to the total number of transfected cells at each time point (mean ± SD; n = 3). Right: Representative photomicrographs (36 h; scale bar: 20 μm). (E and F) Effect of THC on apoptosis (E; 24 h; n = 3) and loss of mitochondrial membrane potential as determined by DiOC6(3) staining (F; 24 h; n = 4) of Atg5+/+ and Atg5–/– MEFs. In E, the mean ± SD percentage of Annexin V–positive/PI-positive and Annexin V–positive, PI-negative cells is shown in the upper and lower corners, respectively. **P < 0.01 compared with THC-treated Atg5+/+ (A, E, and F) and Bax/Bak WT (B) MEFs and from THC-treated, siC-transfected cells (D).

Figure 6. THC activates the autophagic cell death pathway in vivo.

(A) Effect of peritumoral THC administration on TRB3 and p-S6 immunostaining in U87MG tumors. TRB3- or p-S6–stained area normalized to the total number of nuclei in each section; numbers indicate the mean fold change ± SD; 18 sections were counted for each of 3 dissected tumors for each condition. Scale bar: 50 μm. (B) Left: Effect of peritumoral THC administration on LC3 and active caspase-3 immunostaining in U87MG tumors. Arrows point to cells with LC3 dots. The numbers indicate the percentage of active caspase-3–positive cells relative to the total number of nuclei in each section ± SD. Ten sections were counted for each of 3 dissected tumors for each condition. Scale bars: 20 μm. Right: Effect of peritumoral THC administration on LC3 lipidation in U87MG tumors. Representative samples from 1 vehicle-treated and 1 THC-treated tumor are shown. Numbers indicate the LC3-I and LC3-II OD values relative to vehicle-treated tumors (mean ± SD). n = 3. (C) Left: Effect of THC administration on LC3 immunostaining (green) and TUNEL (red) in RasV12/E1A p8+/+ and p8–/– tumor xenografts. Arrows point to cells with LC3 dots and TUNEL-positive nuclei. Right: Bar graph shows the percentage of TUNEL-positive nuclei or cells with TUNEL-positive nuclei and LC3 dots relative to the total number of nuclei in each section (mean ± SD). Eighteen sections were counted from 3 dissected tumors for each condition. Scale bars: 50 μm. Inset shows the magnification of 1 selected cell (arrows point to LC3 dots; scale bar: 10 μm). *P < 0.05 and **P < 0.01 compared with vehicle-treated tumors.

Figure 7. Autophagy is essential for cannabinoid antitumoral action.

(A) Effect of peritumoral THC administration on the growth of Atg5+/+ (upper panel) and Atg5–/– (lower panel) RasV12/T-large antigen MEF tumor xenografts generated in nude mice (mean ± SD; n = 7 for each condition). Photographs show representative images of vehicle- and THC-treated tumors. (B) Left: Effect of THC administration on LC3 immunostaining (green) and apoptosis as determined by TUNEL (red) in Atg5+/+ and Atg5–/– MEF tumor xenografts. Representative images from 1 vehicle-treated and 1 THC-treated Atg5+/+ and Atg5–/– tumors are shown. Right: Bar graphs show the percentage of TUNEL-positive nuclei and cells with TUNEL-positive nuclei and LC3 dots relative to the total number of nuclei in each section (mean ± SD). Eighteen sections were counted from 3 dissected tumors for each condition (vehicle-treated and THC-treated). Scale bar: 50 μm. (C) Schematic of the proposed mechanism of THC-induced cell death (see text for details). **P < 0.01 compared with vehicle-treated tumors.

Figure 8. THC administration promotes autophagy in glioblastomas of 2 patients.

Analysis of different parameters in 2 patients with glioblastoma multiforme before and after intracranial THC treatment (it was estimated that doses of 6–10 μM were reached at the site of administration). (A) TRB3 and p-S6 immunostaining. Representative photomicrographs are shown. Numbers indicate the TRB3- or p-S6–stained area normalized to the total number of nuclei in each section (mean fold change ± SD) relative to the corresponding pre-treatment sample. Fifteen sections were counted for each tumor and each condition (before and after treatment). Scale bar: 50 μm. (B) Representative photomicrographs of LC3 diaminobenzidine immunostaining. The mean percentage of cells with LC3 dots ± SD relative to the total number of nuclei in each section is noted in the corner of each panel. Ten sections were counted from each biopsy for each condition. Arrows point to cells with LC3 dots. Scale bar: 20 μm. (C) Representative photomicrographs of active caspase-3 diaminobenzidine immunostaining. Numbers indicate the percentage of cells with active caspase-3 staining ± SD relative to the total number of nuclei in each section. Ten sections were counted from each biopsy for each condition. Arrows point to cells with active caspase-3 staining. Scale bar: 20 μm. *P < 0.05 and **P < 0.01 compared with before treatment.