Autophagy mediates THC-induced cancer cell death. As a first approach to gain insight into the morphological changes induced in cancer cells by cannabinoid administration, we performed electron microscopy analysis of U87MG human astrocytoma cells. Interestingly, double membrane vacuolar structures with the morphological features of autophagosomes were observed in THC-treated cells (Figure 1, A–C). The conversion of the soluble form of LC3 (LC3-I) to the lipidated and autophagosome-associated form (LC3-II) is considered one of the hallmarks of autophagy (1), and thus we observed the occurrence of LC3-positive dots as well as the appearance of LC3-II (Figure 1D) in cannabinoid-challenged cells. In addition, co-incubation with the lysosomal protease inhibitors E64d and pepstatin A, which blocks the last steps of autophagic degradation (14), enhanced THC-induced accumulation of LC3-II (Figure 1E), confirming that cannabinoids induce dynamic autophagy in U87MG cells. Furthermore, incubation with the cannabinoid receptor 1 (CB1) antagonist SR141716 prevented THC-induced LC3 lipidation and formation of LC3 dots (Figure 1D), indicating that induction of autophagy by cannabinoids relies on CB1 receptor activation.

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.

Since autophagy has been implicated in promotion and inhibition of cell survival, we next investigated its participation in the cancer cell death–inducing action of THC. Pharmacological inhibition of autophagy at different levels (Supplemental Figure 1, A–C; supplemental material available online with this article; doi:10.1172/JCI37948DS1) or selective knockdown of ATG1 (an essential protein in the initiation of autophagy; ref. 1) (Figure 1, F and G), ATG5 (an essential protein in the formation of the autophagosome; ref. 1) (Supplemental Figure 1, D–F), or AMBRA1 (a recently identified beclin-1–interacting protein that regulates autophagy; ref. 15) (Supplemental Figure 1, D–F) strongly reduced cannabinoid-induced autophagy and cell death. Moreover, transformed Atg5-deficient mouse embryonic fibroblasts (MEFs), which are defective in autophagy (16), were more resistant than their wild-type counterparts to THC-induced cell death (Figure 1H) and did not undergo autophagy upon cannabinoid treatment (Figure 1I). Taken together, these findings demonstrate that autophagy plays a prominent role in THC-induced cancer cell death.

THC induces autophagy via ER stress–dependent upregulation of p8 and TRB3. In addition to the presence of autophagosomes, electron microscopy analysis of cannabinoid-treated cells revealed the presence of numerous cells with dilated ER (Figure 2A). In line with this observation, immunostaining of the ER luminal marker protein disulphide isomerase (PDI) showed a striking dilation in the ER of THC-treated U87MG cells (Figure 2B), an event that was associated with an increased phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), a hallmark of the ER stress response (17) (Figure 2C). In addition, THC-induced ER dilation and eIF2α phosphorylation were prevented by pharmacological blockade of the CB1 receptor (Figure 2, B and C).

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.

Time-course analysis of PDI and LC3 immunostaining, eIF2α phosphorylation, and LC3 lipidation of cannabinoid-treated cells revealed that ER stress occurred earlier than autophagy (Figure 2, D and E). Of interest, cannabinoid administration produced similar activation of ER stress and autophagy, as well as cell death, in other human astrocytoma cell lines (Supplemental Figure 2, A–F), a primary culture of human glioma cells (Supplemental Figure 2, G–I), and several human cancer cell lines of different origin, including pancreatic cancer (Supplemental Figure 2, J–L), breast cancer, and hepatoma (data not shown). However, neither ER dilation nor eIF2α phosphorylation or autophagy was evident in normal, nontransformed primary astrocytes (Supplemental Figure 3), which are resistant to cannabinoid-induced cell death (13).

We next investigated whether activation of ER stress is involved in the induction of autophagy in response to cannabinoid treatment of cancer cells. We have previously shown that THC-induced accumulation of de novo–synthesized ceramide, an event that occurs in the ER (18), leads to upregulation of the stress-regulated protein p8 and its ER stress–related downstream targets, ATF4, CHOP, and TRB3, to induce cancer cell death (13). Of importance, incubation with ISP-1 (a selective inhibitor of serine palmitoyltransferase, the enzyme that catalyzes the first step of sphingolipid biosynthesis; ref. 18) prevented ceramide accumulation (Supplemental Figure 4A); THC-induced ER dilation (Supplemental Figure 4B); eIF2α phosphorylation (Figure 3A); p8, ATF4, CHOP, and TRB3 upregulation (Supplemental Figure 4C); and autophagy (Figure 3B), supporting that ceramide accumulation is involved in cannabinoid-triggered ER stress and autophagy. We also verified by means of RNA interference that CaCMKKβ — which had been previously implicated in activating autophagy in response to ER stress–associated calcium release (19) — was not involved in THC-induced autophagy and cell death (data not shown). As phosphorylation of eIF2α on Ser51 attenuates general protein synthesis while enhancing the expression of several ER stress response genes (17), we used cells derived from eIF2α S51A knockin mice to test whether eIF2α phosphorylation regulates the expression of p8 and its downstream targets. In agreement with this hypothesis, THC treatment (which promoted ceramide accumulation in both wild-type and eIF2α S51A immortalized MEFs; Supplemental Figure 5A) triggered p8, ATF4, CHOP, and TRB3 upregulation (Figure 3C) as well as autophagy (Supplemental Figure 5B) in wild-type cells but not in their eIF2α S51A counterparts.

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).

We subsequently asked whether p8 and its downstream targets regulate autophagy. Knockdown of p8 or TRB3 prevented THC-induced autophagy (Figure 3, D and E) but not ER dilation (Supplemental Figure 4D) in U87MG cells. Furthermore, THC induced autophagy in p8+/+ but not p8-deficient transformed MEFs (Figure 3F and Supplemental Figure 5C). Altogether, these findings reveal that THC induces autophagy of cancer cells via activation of an ER stress–triggered signaling route that involves stimulation of ceramide synthesis de novo, eIF2α phosphorylation, and p8 and TRB3 upregulation.

THC inhibits Akt and mTORC1 via TRB3. Inhibition of mTORC1 is considered a key step in the early triggering of autophagy (6). We therefore tested whether cannabinoid-induced upregulation of the p8 pathway leads to autophagy via inhibition of this complex. THC treatment of U87MG cells reduced the phosphorylation of p70S6 kinase (a well-established mTORC1 substrate) and the ribosomal protein S6 (a well-established p70S6 kinase substrate) (Figure 4, A and C), indicating that mTORC1 is inhibited in cannabinoid-challenged cells. In addition, the cannabinoid-induced decrease in p70S6 kinase and S6 phosphorylation, autophagy, and cell death were not evident in Tsc2–/– cells, in which mTORC1 is constitutively active (20) (Figure 4B and Supplemental Figure 6, A and B), further supporting a major role for mTORC1 inhibition in the induction of autophagic cell death by cannabinoids.

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.

The protein kinase Akt positively regulates the activity of the mTORC1 complex by phosphorylating and inhibiting TSC2 and PRAS40 (a well-established Akt substrate within the mTORC1 complex). Thus, Akt inhibition decreases mTORC1 activity and promotes autophagy (20). In line with this idea, THC decreased the phosphorylation of Akt, TSC2, and PRAS40 as well as p70S6 kinase and S6 (Figure 4C). This inhibition of the Akt/mTORC1 pathway was abrogated by incubation with a CB1 receptor antagonist (Supplemental Figure 6C) or a ceramide synthesis inhibitor (Supplemental Figure 6D). Likewise, cells overexpressing a myristoylated (constitutively active) form of Akt were resistant to THC-induced mTORC1 inhibition, autophagy, and cell death (Figure 4D and Supplemental Figure 6, E and F), further supporting that THC induces autophagy via Akt inhibition.

Since TRB3 has been shown to directly interact with and inhibit Akt (21, 22), we investigated whether upregulation of TRB3 was responsible for THC-induced Akt/mTORC1 inhibition. Several observations support that this is indeed the case: (a) THC increased the amount of Akt coimmunoprecipitated with TRB3 from U87MG extracts (Figure 4E), (b) knockdown of TRB3 prevented the effect of THC on Akt, TSC2, PRAS-40, p70S6 kinase, and S6 phosphorylation (Figure 4F), and (c) TRB3 overexpression decreased Akt, TSC2, PRAS40, p70S6 kinase, and S6 phosphorylation, enhanced the inhibitory effect of THC on the phosphorylation of these proteins, and promoted autophagy (Figure 4G). In line with these observations, THC failed to inhibit Akt, p70S6 kinase, and S6 phosphorylation of eIF2α S51A knockin or p8-deficient MEFs, in which TRB3 did not become upregulated upon cannabinoid treatment (Figure 4H and Supplemental Figure 6, G and H). Altogether, these data demonstrate that upregulation of p8 and TRB3 induce autophagy of tumor cells via inhibition of the Akt/mTORC1 pathway.

THC-induced autophagy promotes the apoptotic death of cancer cells. While analyzing the mechanism of cannabinoid cell-killing action, we observed that incubation with the pan-caspase inhibitor ZVAD-fmk prevented cell death to the same extent as genetic (Figure 5A) or pharmacological (Supplemental Figure 7) inhibition of autophagy. Furthermore, Bax/Bak double knockout (DKO) immortalized MEFs, which are protected against mitochondrial apoptosis (23), were resistant to THC-induced cell death and apoptosis (Figure 5B) but underwent eIF2α phosphorylation and autophagy (Figure 5C) upon THC treatment. We therefore investigated whether cannabinoid-induced autophagy promoted the apoptotic death of cancer cells. Time-course analysis of LC3 and active caspase-3 immunostaining in U87MG cells revealed that autophagy preceded the appearance of apoptotic features in THC-treated cells (Figure 5D). In addition, selective knockdown of ATG1 (Figure 5D) as well as of AMBRA1 or ATG5 (Supplemental Figure 8) prevented THC-induced caspase-3 activation. Moreover, unlike their wild-type counterparts, Atg5-deficient immortalized MEFs did not undergo phosphatidylserine translocation to the outer leaflet of the plasma membrane (Figure 5E), loss of mitochondrial membrane potential (Figure 5F), or increased production of reactive oxygen species (Supplemental Figure 9) in response to cannabinoid treatment. These findings indicate that activation of the autophagy-mediated cell death pathway occurs upstream of apoptosis in cannabinoid antitumoral action.

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 DiOC 6 (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).

Activation of autophagy is necessary for cannabinoid antitumoral action in vivo. To determine the in vivo relevance of our findings, we first investigated whether THC promotes the activation of the above-described autophagy-mediated cell death pathway in U87MG cell–derived tumor xenografts, in which we have recently shown that cannabinoid treatment reduces tumor growth (specifically, THC administration for 14 days decreased tumor growth by 50%; ref. 13). Analysis of these tumors revealed that cannabinoid administration increases TRB3 expression and decreases S6 phosphorylation (Figure 6A). Likewise, formation of LC3 dots as well as increase in LC3-II and active caspase-3 immunostaining were observed in THC-treated, but not vehicle-treated, tumors (Figure 6B).

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.

To further investigate whether activation of the p8 pathway mediates cannabinoid antitumoral action, we also analyzed tumors derived from p8+/+ and p8–/– RasV12/E1A-transformed MEFs (in this case, THC administration for 8 days decreased by 45% the growth of p8+/+ tumors but had no significant effect on p8–/– tumors; ref. 13). THC treatment increased TRB3 expression, decreased S6 phosphorylation, and increased autophagy as well as TUNEL and active caspase-3 immunostaining in p8+/+ but not p8–/– tumors (Figure 6C and Supplemental Figure 10). Moreover, THC treatment enhanced the number of cells with LC3 dots and TUNEL-positive nuclei in p8+/+ but not in p8–/– tumors (Figure 6C).

In order to verify the importance of autophagy for cannabinoid antitumoral action, we next generated tumors with Atg5+/+ and Atg5–/– RasV12/T-large antigen transformed MEFs. THC administration reduced by more than 80% the growth of tumors derived from wild-type cells but had no significant effect on those tumors generated by autophagy-deficient cells (Figure 7A). Furthermore, cannabinoid administration increased autophagy, TUNEL (Figure 7B), and active caspase-3 immunostaining (Supplemental Figure 11) in Atg5+/+ but not Atg5–/– tumors. Likewise, cannabinoid administration increased the number of cells with LC3 dots and TUNEL-positive nuclei in Atg5+/+ but not Atg5–/– tumors (Figure 7B). Taken together, these findings demonstrate that activation of the autophagy-mediated cell death pathway is indispensable for cannabinoid antitumoral action.

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.

Finally, we analyzed the tumors of 2 patients enrolled in a clinical trial aimed at investigating the effect of THC on recurrent glioblastoma multiforme. The patients were subjected to intracranial THC administration, and biopsies were taken before and after the treatment (11). In the 2 patients, cannabinoid inoculation increased TRB3 immunostaining and decreased S6 phosphorylation (Figure 8A). Interestingly, the number of cells with autophagic phenotype (Figure 8B) as well as with active caspase-3 immunostaining (Figure 8C) was increased in the tumor samples obtained after THC treatment. Although these studies were only conducted in specimens from 2 patients, they are in line with the preclinical evidence shown above and suggest that cannabinoid administration might also trigger autophagy-mediated cell death in human tumors.