Torres et al. Supplementary Figure 1
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1 Torres et al. Supplementary Figure 1 A 9 tetrahydrocannabinol (THC) B Cannabidiol (CBD) C Temozolomide (TMZ)
2 Supplementary Figure 1. Chemical structures of THC, CBD and TMZ.
3 A 3 TMZ (μm) Torres et al. Supplementary Figure THC (μm) B LN 5 THC (μm) TMZ (μm) HG14 # THC (μm) TMZ (μm) C THC(μM) TMZ (μm) # THC(μM) TMZ (μm)
4 Supplementary Figure 2. THC enhances TMZinduced glioma cell death (A) Effect of TMZ (left panel) and THC (right panel) on the viability (72 h) of cells (n = 3; p <.1 from vehicletreated cells). (B) Effect of THC, TMZ and THCTMZ on the viability (72 h) of LN5 (human glioma cell line) and HG14 (primary culture of human glioma cells) as determined by the MTT test (mean ± s.d.; n = 6 for LN5 and n = 4 for HG14 cells; p <.1 from vehicletreated cells; # p <.5 or p <.1 from THCtreated cells; p <.5 or p <.1 from TMZtreated cells). Red lines correspond to the reduction of cell viability obtained from the addition of the individual cell deathpromoting actions of THC and TMZ at each concentration of these agents. (C) Effect of THC and TMZ on the viability (72 h) of cells (n = 3; p <.1 from vehicletreated cells; # p <.5 or p <.1 from THCtreated cells; p <.5 or p <.1 from TMZtreated cells).
5 Torres et al. Supplementary Figure 3 A LC3 THC 1.9 M THC 2.1 M THC 2.3 M 2.1 ± 1.2% 16.1 ± 2.7% 46.2 ± 2.7% 76. ± 8.3% TMZ 25 M TMZ 5 M TMZ 75 M 2.1 ± 1.2% 4.4 ± 1.2% 5.6 ± 1.7% 4.7 ±.8% B LC3 I LC3 II tub LC3 I LC3 II tub THC 2.3 M TMZ ( M) C THC ( M).9 TMZ ( M) MA QVDOPH
6 Supplementary Figure 3. Autophagy and apoptosis are involved in the mechanism of THCTMZinduced cell death (A) Effect of THC and TMZ (24 h) on LC3 immunostaining of cells. Values in the lower right corner of each photomicrograph correspond to the percentage of cells with LC3 dots relative to the total number of cells (mean ± s.d;; n = 3; representative photomicrographs of each condition are shown; p <.1 or p <.5 from vehicletreated cells. (B) Effect of THC and TMZ on LC3 lipidation. A representative experiment of 3 is shown. (C) Effect of 3MA (5 mm) and QVDPOH (15 M) on the viability (72 h) of cells treated with THC, TMZ or THCTMZ (n = 6; mean ± s.d, p <.1 from vehicletreated cells; p <.1 from THCtreated cells; p <.1 from TMZtreated cells and p <.5 or p <.1 fom THCTMZtreated cells). Additional controls are omitted for clarity.
7 A THC (μm) CBD (μm) SW Torres et al. Supplementary Figure 4 THC (μm) CBD (μm) LN A172 1 U373 THC (μm) CBD (μm) THC (μm) CBD (μm) B 1 THC (μm) CBD (μm) HG THC (μm) CBD (μm) HG
8 Supplementary Figure 4. Effect of THC and CBD on the viability of human glioma cell lines and primary cultures of human glioma cells Effect of THC and CBD on the viability (72 h) of several human glioma cell lines (A) and primary cultures of human glioma cells (B) as determined by the MTT test (mean ± s.d.; n = 6 for SW1783, LN5, A172 and U373 and n = 4 for HG2 and HG21 cells; p <.1 from vehicletreated cells; p <.1 from THCtreated cells; p <.1 or p <.5 from CBDtreated cells). Red lines correspond to the reduction of cell viability obtained from the addition of the individual cell deathpromoting actions of THC and CBD at each concentration of these agents.
9 Torres et al. Supplementary Figure 5 A Tumor volume (fold from day 1) THC (3.7 mg/kg) THC (7.5 mg/kg) THC (15 mg/kg) Tumor volume at day 15 (fold from day 1) THC 3.7 THC 7.5 THC 15 B Time ( days) Tumor volume (fold from day 1) CBD (3.7 mg/kg) CBD(7.5 mg/kg) CBD (15 mg/kg) Tumor volume at day 15 (fold from day 1) CBD 3.7 CBD 7.5 CBD C Time ( days) Tumor volume (fold from day 1) 12 THC (3.7 mg/kg) CBD (3.7 mg/kg) 1 THC (7.5 mg/kg) CBD (7.5 mg/kg) Time ( days) Tumor volumeatn day 15 (fold from day 1) THC 3.7 CBD 3.7 THC 7.5 CBD 7.5
10 Supplementary Figure 5. Antitumoral action of different doses of THC and CBD Effect of THC (A), CBD (B) or THCCBD (C) on the growth of cellderived tumor xenografts. Once the tumors reached 25 mm 3, THC, CDB or THCCBD were daily administered for 14 days with a single peritumoral injection (n = 68 for each condition; mean ± S.E.M; In panels A, B and C, symbols of significance are omitted for clarity; see below for a description of the statistical differences for each treatment). Right panels, data correspond to the mean foldincrease in tumor growth ± S.E.M at the last day of the treatment. (n = 8; p <.1 or p <.5 from vehicletreated tumors.). Photographs of representative tumors at the last day of the treatment are shown. Legend to Supplementary Figure 5 (Statistical analyses) Panel A: THC (15 mg/kg)treated tumors were significantly different from Vehtreated tumors at day 5 (p <.5), and from day 6 until the end of the treatment (p <.1). THC (7.5 mg/kg)treated tumors were significantly different from Vehtreatedtumors at days 14 and 15 (p <.5). THC (3.7 mg/kg)treated tumors were significantly different from Vehtreated tumors at days 6, 7 and 8 (p <.5) and from day 9 until day 11 (p <.1). Panel B: CBD (15 mg/kg)treated tumors were significantly different from Vehtreated tumors at days 5 to 13 (p <.5) and at days 14 and 15 (p <.1). Panel C: THC (3.7 mg/kg) CBD (3.7 mg/kg)treated tumors were significantly different from Vehtreated tumors at days 6 and 12 (p <.5), and from day 7 until day 1 (p <.1). THC (7.5 mg/kg) CBD (7.5 mg/kg)treated tumors were significantly different from Vehtreated tumors at days 6 and 7 (p <.5), and from day 8 until the end of the treatment (p <.1).
11 A Torres et al. Supplementary Figure 6 Cell viability (% from vehicle) THC THC BDS Concentration (μg/ml) Cell viability (% from vehicle) T98G THC THC BDS Concentration (μg/ml) Cell viability (% from vehicle) CBD CBD BDS Concentration (μg/ml) Cell viability (% from vehicle) T98G CBD CBD BDS Concentration (μg/ml) IC5 g/ml (Pure cannabinoid) IC5 g/ml (BDS) IC5 g/ml (Pure cannabinoid in BDS) IC5 g/ml (Pure cannabinoid) IC5 g/ml (BDS) IC5 g/ml (Pure cannabinoid in BDS) THC CBD THC CBD B T98G Cell viability (% from vehicle) THC CBD THCCBD (1:1) SATL (THCBDSCBDBDS) (1:1) SATL (Amount of pure THCCBD present in SATL Concentration (μg/ml) Cell viability (% from vehicle) THC CBD THCCBD (1:1) SATL (THCBDSCBDBDS) (1:1) SATL (Amount of pure THCCBD present in SATL) Concentration (μg/ml)
12 Supplementary Figure 6. Effect of pure cannabinoids and their corresponding botanical drug substances on the viability of human glioma cells (A) Effect of THC and THCBDS (upper panels), CBD and CBDBDS (lower panels) on the viability (72 h) of and T98G cells (mean ± s.d.; n = 6; the IC 5 values for each pure cannabinoid, their respective BDSs or the amount of pure cannabinoid present in each BDS are included in the table. (B) Effect of THCCBD and Sativexlike (SAT L; THCBDSCBDBDS) on the viability (72 h) of and T98G cells (mean ± s.d.; n = 6).
13 Torres et al. Supplementary Figure 7 A THCBDS (15 mg/kg) Tumor volume (fold from day 1) CBDBDS (15 mg/kg) THC (15 mg/kg) SATL (15 mg/kg) Time ( days) Tumor volume at day 15 (fold from day 1) CBDBDS THCBDS THC 15 SATL B CBDBDS THCBDS THC SATL 1. ±,1 1.1 ± ± ±.1 2. ±.2 TUNEL 3.7 ± 1,1% 5.7 ± 2,5% 12.7 ± 6.7% 19.3 ± 7.% 21.6 ± 5.8% nuclei LC3 nuclei
14 Supplementary Figure 7. Combined administration of THCBDS and CBDBDS strongly reduces the growth of cellderived tumor xenografts (A) Effect of THC (15 mg/kg), THCBDS (15 mg/kg containing 1 mg/kg THC), CBDBDS (15 mg/kg containing 1 mg/kg CBD) or Sativexlike [SATL; THCBDS (7.5 mg/kg) CBDBDS (7.5 mg/kg)] on the growth of cellderived tumor xenografts (n = 68 for each condition; mean ± S.E.M; in left panel A symbols of significance are omitted for clarity) [THC (15 mg/kg)treated tumors were significantly different from Vehtreated tumors on days 7 to 1 (p <.5), and from day 11 until the end of the treatment (p <.1). THCBDStreated tumors were significantly different from Vehtreated tumors on day 13 (p <.5) and on days 14 and 15 (p <.1). SAT Ltreated tumors were significantly different from Vehtreated tumors on day 7 (p <.5) and from day 8 until the end of the treatment (p <.1)]. Right panels, data correspond to the mean foldincrease in tumor growth ± S.E.M at the last day of the treatment. [n = 8; p <.1 from vehicletreated tumors; p <.1 from THCBDS (15 mg/kg)treated tumors; and p <.1 from CBDBDS (15 mg/kg)treated tumors]. (B) Analysis of LC3 immunostaining and TUNEL. Values correspond to the LC3stained area normalized to the total number of nuclei in each section (mean fold change ± s.d.; arrows point cells with LC3 dots) or to the percentage of TUNELpositive cells relative to the total number of nuclei in each section ± s.d. [1 sections of 3 different tumors from each condition were analyzed; p <.1 from vehicletreated tumors; p<.1 or # p <.5 from THCBDS treated tumors; p<.1 or p <.5 from CBDBDS treated tumors].
15 A Torres et al. Supplementary Figure 8 SR1 SR2 SR1 SR2 THC CBD B # ISP 3MA QVDOPH THC CBD C THC CBD SI C SI ATG1 D TOCOPHEROL NAC THC CBD
16 Supplementary Figure 8. Effect of SR1, SR2, ISP1, 3MA and QVDPOH on THC or CBDinduced cell death (A and B) Effect of SR (SR1; 1 M), SR (SR2; 1 M) or SR1SR2 (1 M 1 M) (panel A) or ISP1 (ISP; 1 M), 3MA (5 mm) and QVDPOH (15 M) (panel B) on the viability of cells treated with THC (2 M,) or CBD (2 M) (mean ± s.d; n = 6; p <.1 from vehicletreated cells; p <.1 from THCtreated cells; p <.1 from CBDtreated cells). (C) Effect of THC or CBD on the viability (72 h) of cells transfected with control (sic) or Atg1selective (siatg1) sirna. (n = 6; mean ± s.d, p <.1 from sictransfected, vehicletreated cells; p <.1 from sictransfected, THCtreated cells; p <.1 from sictransfected, CBDtreated cells). Atg1 mrna levels (as determined by realtime quantitative PCR) were reduced in siatg1transfected cells relative to their corresponding sictransfected cells by 72% (n = 5). (D) Effect of tocopherol (1 M) or Nacetylcysteine (3 mm) on the viability of cells treated with THC (2 M,) or CBD (2 M) (mean ± s.d; n = 6; p <.1 from vehicletreated cells; p <.1 from THCtreated cells; p <.1 from CBDtreated cells).
17 Torres et al. Supplementary Figure 9 A B C D THC.9 M CBD.9 M THC.9 M CBD.9 M THC.9 M CBD.9 M THC.9 M CBD.9 M THC.9 M CBD.9 M THC.9 M CBD.9 M THC.9 M CBD.9 M THC.9 M CBD.9 M THC 1.2 M CBD 1.2 M THC 1.2 M CBD 1.2 M THC 1.2 M CBD 1.2 M THC 1.2 M CBD 1.2 M THC 1.2 M CBD 1.2 M THC 1.2 M CBD 1.2 M THC 1.2 M CBD 1.2 M THC 1.2 M CBD 1.2 M SR1 SR2 SR1 SR2 ISP 3MA QVDOPH SI C SI ATG1 TOCOPHEROL NAC
18 Supplementary Figure 9. Effect of SR1, SR2, ISP1, 3MA and QVDPOH on THCCBDinduced cell death (A and B) Effect of SR (SR1; 1 M), SR (SR2; 1 M) or SR1SR2 (1 M 1 M) (panel A) or ISP1 (ISP; 1 M), 3MA (5 mm) and QVDPOH (15 M) (panel B) on the viability of cells treated with THCCBD (mean ± s.d; n = 6; p <.1 from vehicletreated cells; p <.1 from THCtreated cells; p <.1 from CBDtreated cells and p <.1 or p <.5 from THCCBDtreated cells). (C) Effect of THC, CBD and THCCBD on the viability (72 h) of cells transfected with control (sic) or Atg1selective (siatg1) sirna (n = 6; mean ± s.d, p <.1 from sictransfected, vehicletreated cells; p <.1 from sictransfected, THCtreated cells; p <.1 from sictransfected, CBDtreated cells and p <.1 from sictransfected THCCBDtreated cells). Atg1 mrna levels (as determined by realtime quantitative PCR) were reduced in siatg1transfected cells relative to their corresponding sictransfected cells by 72% (n = 5). (D) Effect of tocopherol (2 M) or Nacetylcysteine (3 mm) on the viability of cells treated with THC, CBD or THC CBD (mean ± s.d; n = 6; p <.1 from vehicletreated cells; p <.1 from THCtreated cells; p <.1 from CBDtreated cells and p <.1 from THCCBDtreated cells). (AD). Additional controls are omitted for clarity
19 A Torres et al. Supplementary Figure 1 Cell Viability (% from vehicle) T98G TMZ (75 M) THC (,9 M) CBD (,9 M) TMZ ( M) THC (1.1 M) CBD (1.1 M) B LC3 THCCBD C TMZ 2.5 ± 1.1% 49.1 ± 5.1% THCCBDTMZ 3.7 ± 1.6 % 94. ± 6.% % Cleaved Caspase 3 positive cells TMZ ( M).9.9 THC ( M) CBD ( M) D TMZ ( M) THC ( M) CBD ( M) MA QVDOPH
20 Supplementary Figure 1. Combined administration of THC, CBD and TMZ strongly reduces the viability of human glioma cell lines. (A) Effect of THC, CBD, TMZ, THCCBD, THCTMZ and THCCBDTMZ on the viability (72 h) of (left panel) and T98G (right panel) cells as determined by the MTT test (n = 6; mean ± s.d; p <.1 from vehicletreated cells; p <.1 from THCtreated cells; p <.1 from TMZtreated cells, p <.1 from CBDtreated cells, p <.1 from THCCBDtreated cells and p <.1 from THCTMZtreated cells). (B) Effect of THCCBD (.9 M.9 M), TMZ (75 M) and THCCBDTMZ (.9 M.9 M 75 M) (24 h) on LC3 immunostaining of cells. Values in the lower right corner of each photomicrograph correspond to the percentage of cells with LC3 dots relative to the total number of cells (mean ± s.d; n = 3; representative photomicrographs of each condition are shown; p <.1 from vehicletreated cells; p <.1 from TMZtreated cells and p <.1 from THCCBDtreated cells). (C) Effect of THCCBD, TMZ and THCCBDTMZ (24 h) on apoptosis (as determined by activecaspase 3 immunostaining) of cells. Data correspond to the percentage of activecaspase 3positive cells relative to the total number of cells (mean ± s.d; n = 3; p <.1 from vehicletreated cells; p <.1 from TMZtreated cells and p <.1 from THCCBDtreated cells). (D) Effect of 3MA (5 mm) and QVDPOH (15 M) on the viability of cells treated with THCCBD, TMZ and THCCBDTMZ (mean ± s.d; n = 6; p <.1 or p <.5 from vehicletreated cells; p <.1 from TMZtreated cells, p <.1 from THCCBDtreated cells and p <.1 or p <.5 from THCCBDTMZtreated cells). Additional controls are omitted for clarity.
21 A Torres et al. Supplementary Figure 11 B 3 TMZ (μm) 6 In vitro T98G MGMT expression n.d n.d THC THC C 6 U87 T98 In vivo MGMT expression n.d n.d THC n.d n.d n.d TMZ THC TMZ SATL THC TMZ THC TMZ SATL SATL TMZ T98G
22 Supplementary Figure 11. Resistance to TMZ treatment correlates with higher MGMT expression (A) Effect of TMZ on the viability (72 h) of and T98G cells (n = 3; p <.1 or p <.5 from vehicletreated cells). (B) MGMT mrna levels as determined by real time quantitative PCR of and T98G cells treated with THC. Data correspond to the MGMT mrna levels (n = 5; mean fold change ± s.d. relative to MGMT mrna levels of vehicletreated cells in one of the experiments). (C) Effect of the different treatments on MGMT mrna levels (as determined by realtime quantitative PCR) of and T98G tumor xenografts. Data correspond to MGMT mrna levels relative to one of the vehicletreated tumors (mean ± s.d; n = 5 for each condition). n.d: non detected.
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