Effect of GEM and/or GW, ACPA, or SR1 on growth of pancreatic adenocarcinoma cell lines and normal fibroblasts. (a) Cells were seeded in 96-well plates and incubated overnight. The compounds were added at the concentrations of 200 nM GEM, 16 μM SR1 and GW, and 90 μM ACPA; cells were incubated for additional 48 h. Values are reported as percentage of treated versus untreated cells, and are the means of triplicate samples from three independent experiments (±S.D.). Statistical analysis: P<0.001, GEM versus each combination; P<0.01, each cannabinoid versus its combination with GEM in Panc1 cells, no significance between the various treatments in fibroblasts. (b) Panc1 cells were seeded in 96-well plates and incubated overnight. The compounds were added at the following concentrations for 24 h: 25 nM GEM, 2 μM SR1 and GW, and 11.25 μM ACPA. The growth ratio on the y axis was obtained by dividing the absorbance of untreated or treated cell lines by the mean absorbance of each cell line measured at time 0. Values are the means of triplicate samples from three independent experiments (±S.D.). The statistical analysis was performed for each combined treatment versus control. (c) Antiproliferative synergism by GEM/cannabinoids. The percentage values were obtained by analyzing CI/effect curves, as described in Materials and Methods. Statistical analysis: P<0.001, % CI<1 in all cancer cell lines versus normal fibroblasts

Effect of GEM and/or cannabinoids on intracellular ROS production. Panc1 cells were treated with increasing concentrations of the compounds for 4 h at constant dose ratios, as reported in Materials and Methods. The DCF fluorescence intensity, corresponding to the level of ROS production, was measured by a multimode plate reader. Values are the means of triplicate samples from three independent experiments. The statistical analysis was performed for each combined treatment versus single treatments

Role of NF-κB in CB1 and CB2 activation by GEM and in the antiproliferative synergism by GEM/cannabinoids. (a) Panc1 cells were seeded in 60-mm plates and incubated overnight. Cells were pretreated with 5 μg/ml ActD for 1 h, then 2 μM GEM was added, and the treatments prolonged up to 16 h. Total RNA extraction and real-time PCR were performed, as described in Materials and Methods. Values are the means of triplicate samples from four independent experiments (±S.D.). (b) qPCR analysis of CB1 and CB2 mRNAs from cells treated with 2 μM GEM or 10 μg/ml IL-1. In all, 10 mM NAC, 10 μM BAY, 100 μM PDTC, or 100 μM MG132, where indicated, were added 1 h before treatments. CB1 and CB2 mRNAs were analyzed at 16 h. Values are the means of triplicate samples from three independent experiments (±S.D.). Statistical analysis: P<0.001, control versus GEM or GEM+NAC and P<0.001, GEM versus GEM+MG132, GEM+BAY, or GEM+PDTC. P<0.001, IL-1 versus IL-1+MG132 (for both CB1 and CB2). (c) Analysis of the antiproliferative synergism by 2 μM GEM and 40 μM GW, 225 μM ACPA, or 40 μM SR1 in the absence or presence of 100 nM MG132 or 1 μM BAY. Values are the means of three independent experiments (±S.D.). Statistical analysis for total synergism (0.3<CI<1): P<0.001, GEM+GW versus GEM+GW+BAY; P<0.001, GEM+ACPA versus GEM+ACPA+BAY; P<0.001, GEM+SR1 versus GEM+SR1+MG or GEM+SR1+BAY; P<0.05, GEM+GW versus GEM+GW+MG; and P<0.05, GEM+ACPA versus GEM+ACPA+MG. Statistical analysis for high synergism (CI<0.3): P<0.001, GEM+GW versus GEM+GW+MG or GEM+GW+BAY; P<0.001, GEM+SR1 versus GEM+SR1+BAY; P<0.01, GEM+ACPA versus GEM+ACPA+BAY; P<0.01, GEM+SR1 versus GEM+SR1+BAY; and P<0.05, GEM+ACPA versus GEM+ACPA+MG

Effect of GEM and/or cannabinoids on ER stress-related gene expression. Panc1 cells were treated with 500 nM GEM and/or 40 μM GW, 225 μM ACPA, or 40 μM SR1 for 8 h. RT-PCR for XBP-1(S) and qPCR for Grp78 and CHOP were performed, as described in Materials and Methods. Densitometric analysis of XBP-1(S) was normalized to β-actin and performed, as described in Materials and Methods. Values are the means of triplicate samples from three independent experiments (±S.D.). Statistical analysis: P<0.001, GEM versus each cannabinoid and each cannabinoid versus its combination with GEM (for all the three genes)

Analysis of apoptosis, cell cycle, and autophagy by GEM and/or cannabinoids. (a) Panc1 cells were treated with 200 nM GEM and/or 16 μM GW, 90 μM ACPA, or 16 μM SR1 for 48 h and analyzed by flow cytometry to determine the percentages of apoptotic cells. Values are the means of three independent experiments (±S.D.). Statistical analysis: P<0.001, control versus GEM; P<0.05, control versus each combination; P<0.01, GEM versus each combination, no significance between control and each cannabinoid. (b) Cells were treated with 200 nM GEM and/or 16 μM GW, 90 μM ACPA, or 16 μM SR1 for 48 h. Cell cycle distribution was analyzed by a flow cytometer after DNA staining with PI. Values are the means of three independent experiments (±S.D.). Statistical analysis: no significance of GEM versus each combination. (c) Western blot analysis of LC3 was performed using total protein extracts from Panc1 cells treated with 500nM GEM and/or 40 μM GW, 225 μM ACPA, or 40 μM SR1 for 24 h in the presence of acid lysosomal protease inhibitors E64d (10 μM) and pepstatin A (10 μg/ml). Densitometric quantification of LC3-II bands was normalized to α-tubulin and performed, as described in Materials and Methods. Values are the means of triplicate samples from three independent experiments (±S.D.). Statistical analysis: P<0.01, control versus GW or ACPA; P<0.05, control versus SR1; and P<0.01, each cannabinoid versus its combination

Involvement of ROS in GEM/cannabinoid-induced autophagy. (a) Fluorescence microscopy analysis of autophagosome formation in Panc1 cells after acridine orange staining treated with 500 nM GEM and/or 40 μM GW, 225 μM ACPA, or 40 μM SR1 in the absence or presence of 20 mM NAC or 10 μM CQ or 1 mM 3-MA for 24 h. (b) The MFIs were calculated, as described in ‘Materials and Methods', at FACS after trypsinization of the acridine orange-labeled cells. Values are the means of triplicate samples from three independent experiments (±S.D.). Statistical analysis: P<0.05, control versus GEM, GW, ACPA, or SR1; P<0.001, each cannabinoid or GEM versus their combination; and P<0.001, GEM/cannabinoids versus GEM/cannabinoids+NAC, GEM/cannabinoids+CQ, or GEM/cannabinoid+3-MA. (c) Flow cytometric analyses of autophagosomes formation (MDC incorporation) in Panc1 cells treated with 500 nM GEM and/or 40 μM GW, 225 μM ACPA, or 40 μM SR1 in the absence or presence of 20 mM NAC or 10 μM CQ or 1 mM 3-MA for 24 h. The MFIs were calculated, as described in ‘Materials and Methods'. Values are the means of three independent experiments (±S.D.). Statistical analysis: P<0.01, control versus GEM, SR1, or ACPA; P<0.001, control versus GW; P<0.001, each cannabinoid or GEM versus their combination; and P<0.001, GEM/cannabinoids versus GEM/cannabinoids+NAC, GEM/cannabinoids+CQ, or GEM/cannabinoid+3-MA

Involvement of ROS and autophagy in the antiproliferative synergism by GEM/cannabinoids and kinetic analysis of ROS, Grp78, and LC3-II induction by GEM/cannabinoids. (a) Analysis of the antiproliferative synergism by 500 nM GEM and 40 μM GW, 225 μM ACPA, or 40 μM SR1 in the absence or presence of 20 mM NAC or 10 μM CQ or 2.5 mM 3-MA. Values are the means of three independent experiments (±S.D.). Statistical analysis: P<0.001, GEM/cannabinoids versus GEM/cannabinoids+NAC, GEM/cannabinoids+CQ, or GEM/cannabinoid+3-MA. (b) Panc1 cells were treated with 500 nM GEM and 40 μM GW, 225 μM ACPA, or 40 μM SR1 for the indicated time points. ROS, Grp78, and LC3-II were analyzed, as described in Materials and Methods. Values are the means of three independent experiments. (c) Schematic representation of the kinetic analysis of oxidative stress (ROS), ER stress (Grp78), and autophagy (LC3-II) marker induction by GEM/cannabinoids

Effect of GEM+SR1 treatment on xenografts of PaCa44 cells in nude mice. Cells were subcutaneously injected into female nude mice. After 1 week, i.p. injections with PBS (solution vehicle), GEM, or/and SR1 were administered twice a week for 4 weeks, as described in Materials and Methods. (a) Values are the means of mice tumor volume measured at 3 days after each injection. (b) Values are the means of mice body mass measured at 3 days after each injection. (c) Values are the means of mice tumor mass (±S.D.) measured after 8 injections