Gemcitabine (GEM, 2′,2′-difluorodeoxycytidine) is currently used in advanced pancreatic adenocarcinoma, with a response rate of < 20%. The purpose of our work was to improve GEM activity by addition of cannabinoids. Here, we show that GEM induces both cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) receptors by an NF-κB-dependent mechanism and that its association with cannabinoids synergistically inhibits pancreatic adenocarcinoma cell growth and increases reactive oxygen species (ROS) induced by single treatments. The antiproliferative synergism is prevented by the radical scavenger N-acetyl–cysteine and by the specific NF-κB inhibitor BAY 11-7085, demonstrating that the induction of ROS by GEM/cannabinoids and of NF-κB by GEM is required for this effect. In addition, we report that neither apoptotic nor cytostatic mechanisms are responsible for the synergistic cell growth inhibition, which is strictly associated with the enhancement of endoplasmic reticulum stress and autophagic cell death. Noteworthy, the antiproliferative synergism is stronger in GEM-resistant pancreatic cancer cell lines compared with GEM-sensitive pancreatic cancer cell lines. The combined treatment strongly inhibits growth of human pancreatic tumor cells xenografted in nude mice without apparent toxic effects. These findings support a key role of the ROS-dependent activation of an autophagic program in the synergistic growth inhibition induced by GEM/cannabinoid combination in human pancreatic cancer cells.

GEM/cannabinoid combined treatments synergistically inhibited pancreatic adenocarcinoma cell proliferation

The antiproliferative effect of the synthetic cannabinoids GW, ACPA, and SR1 in combination with GEM was examined on six pancreatic adenocarcinoma cell lines characterized by different sensitivities to GEM. As reported in our previous papers,⁵ CFPAC1, T3M4, and MiaPaCa2 cells are more sensitive to GEM treatment than PaCa3, PaCa44, and Panc1 cells. Figure 1a shows that the combined treatments significantly enhanced Panc1 cell growth inhibition compared with single treatments. Similar results were obtained with all the pancreatic adenocarcinoma cell lines assessed in this study (data not shown). On the contrary, normal fibroblasts were significantly less sensitive to cannabinoids and their growth was not further inhibited by GEM addition. To analyze the trend of the inhibitory effect over the time, we performed a time-dependent analysis of the antiproliferative activity following a 24-h single-step treatment with low concentrations of GEM and/or cannabinoids. Figure 1b shows that, starting from the fourth day, only the combined treatments were able to significantly inhibit Panc1 cell proliferation, with a growth ratio inhibition at the sixth day in treated versus untreated cells of 48, 36, and 57% for GEM/GW, GEM/ACPA, and GEM/SR1, respectively.

Figure 1

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 200nM GEM, 16 …

To evaluate whether cell growth inhibition by GEM/cannabinoids was synergistic, we analyzed cell growth inhibition curves by using the dedicated software CalcuSyn (Biosoft, Ferguson, MO, USA; see ‘Materials and Methods’). Figure 1c reports the percentages of the combination index (CI) values encompassed between 1 and 0.3 (synergism) or lower than 0.3 (strong synergism) for all combinations. Although GEM-resistant cell lines showed percentages of the overall synergism (CI<1) similar to those of GEM-sensitive cell lines, they had a level of strong synergism (CI<0.3) significantly higher than that of the latter cell lines (Supplementary Figure 1). This result suggests that cannabinoids sensitize cancer cells to the antiproliferative effects caused by GEM. Supplementary Table 1 shows that in the resistant Panc1 cells, cannabinoids potentiated the effects of GEM from 5- to 10-fold (PF). Similar results were obtained with the other GEM-resistant cells (data not shown). On the other hand, in agreement with data shown in Figure 1a, GEM/cannabinoid combinations did not determine any synergism in normal fibroblasts.

GEM/cannabinoid combined treatments enhanced intracellular ROS production

As it was previously reported that the antiproliferative effect of GEM or cannabinoids is mediated by oxidative stress,^5, 21 we measured ROS levels in Panc1 cells treated with increasing concentrations of the single compounds or their combinations. Figure 2 shows that GEM/cannabinoids were able to significantly enhance ROS production, induced by single treatments, at 4h. Similar enhancement was obtained at 16h (data not shown).

Figure 2

Effect of GEM and/or cannabinoids on intracellular ROS production. Panc1 cells were treated with increasing concentrations of the compounds for 4h at constant dose ratios, as reported in Materials and Methods. The DCF fluorescence intensity, …

GEM induced cannabinoid receptor expression by NF-κB-mediated mechanism

To investigate whether GEM was able to regulate cannabinoid receptor expression, we performed a time-dependent analysis of the mRNA for CB1 and CB2 receptors on Panc1 cells. As shown in Figure 3a and Supplementary Figure 2, GEM determined an approximately fourfold and sevenfold induction of CB1 and CB2 mRNAs, respectively. CB mRNAs induction by GEM was transcriptionally regulated, as their increase was completely antagonized by the addition of actinomycin D (ActD). Similarly, both CB1 and CB2 mRNA GEM-mediated increase was completely blocked by three NF-κB inhibitors (BAY, pyrrolidine dithiocarbamate (PDTC), and MG132), but not by the free radical scavenger N-acetyl–cysteine (NAC; Figure 3b). IL-1, a known NF-κB inducer, stimulated both CB1 and CB2 expression, which was antagonized by MG132 addition. Moreover, the antiproliferative synergism was significantly reduced by MG132 and completely abrogated by BAY (Figure 3c). Altogether, these data indicate that NF-κB is involved in both ROS-independent cannabinoid receptor induction by GEM and in the antiproliferative synergism by GEM/cannabinoid combinations, suggesting a role for CB1 and CB2 activation by GEM in the latter effect.

Figure 3

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 5μg/ml ActD for 1h, …

GEM enhanced cannabinoid-induced ER stress

It was previously reported that ER stress is a molecular mechanism involved in cannabinoid antiproliferative effect.¹³ To investigate whether GEM was able to enhance the cannabinoid-induced ER stress, we analyzed mRNA expression of the ER stress sensors XBP-1, Grp78, and CHOP following single or combined treatments. Figure 4 shows that all the three mRNAs were induced by GW, ACPA, or SR1, and their levels were significantly enhanced by the addition of GEM, even if GEM alone was ineffective.

Figure 4

Effect of GEM and/or cannabinoids on ER stress-related gene expression. Panc1 cells were treated with 500nM GEM and/or 40μM GW, 225μM ACPA, or 40μM SR1 for 8h. RT-PCR for XBP-1(S) and …

GEM enhanced cannabinoid-induced autophagy by a ROS-mediated mechanism

To determine whether apoptosis and/or cell cycle arrest were involved in the antiproliferative synergism between GEM and cannabinoids, we performed both annexin V-FITC/propidium iodide (PI) assay and cell cycle analysis by flow cytometry after single or combined treatments. Figure 5a shows that GEM, but not cannabinoids, significantly induced apoptosis at 48h. This effect was partially reduced by the addition of cannabinoids. Similar results were obtained at 24h, and with PaCa44 and T3M4 cell lines (data not shown). Figure 5b shows that GEM increased the percentages of cells in G1 and S phases and cannabinoids in G1 phase, and the combined treatments did not induce a potentiation of the accumulation of cells in a particular phase of cell cycle when compared with the changes induced by GEM or cannabinoids alone. Altogether, these data indicate that GEM/cannabinoid synergism is likely mediated by neither apoptotic nor cell cycle modulation.

Figure 5

Analysis of apoptosis, cell cycle, and autophagy by GEM and/or cannabinoids. (a) Panc1 cells were treated with 200nM GEM and/or 16μM GW, 90μM ACPA, or 16μM SR1 for 48h and analyzed by …

Recently, Salazar et al.¹³ reported that tetrahydrocannabinol (THC) is able to induce autophagy-mediated cell death in human glioma cells. Therefore, we investigated whether GW, ACPA, or SR1 also were able to induce autophagy and whether GEM could additionally enhance this effect.

Figure 5c shows that at 24h LC3-II protein, the phospho-ethanolaminated form of the autophagosome protein LC3-I, was increased by single treatments with GEM or cannabinoids and additionally enhanced by GEM/cannabinoid combinations. Similar results were obtained at 48h (data not shown). A late step in the autophagic cell death process is the fusion of lysosomes with autophagosomes into autolysosomes, which can be detected by measuring their acidification with acridine orange staining. Interestingly, we found acridine orange staining (characterized by a punctuation, suggesting vacuole formation) slightly increased in GEM or cannabinoid-treated cells and significantly potentiated in cells treated with GEM/cannabinoid combinations (Figure 6a). These effects were almost completely antagonized by the addition of either the scavenger NAC or the autophagy inhibitors CQ or 3-methyladenine (3-MA). This observation was confirmed by FACS analysis, which shows a significant difference in the acidification of the acidic vesicular organelles (AVOs) in pancreatic cancer cells treated with GEM or cannabinoids compared with control, and with GEM/cannabinoid combination compared with single treatments (Figure 6b). Blocking autophagy with 3-MA or CQ resulted in a decrease in autolysosomal acidification, demonstrating that acidification induced by the different combinations was linked to the autophagic pathway.

Figure 6

Involvement of ROS in GEM/cannabinoid-induced autophagy. (a) Fluorescence microscopy analysis of autophagosome formation in Panc1 cells after acridine orange staining treated with 500nM GEM and/or 40μM GW, 225μ …

To ascertain whether the presence of numerous cytoplasmic vacuoles in GEM/combination-treated cells was really due to the induction of autophagy, the autofluorescent drug monodansylcadaverine (MDC), a selective marker for AVOs, such as autophagic vacuoles and especially autolysosomes, was used.²² Figure 6c shows the quantitative evaluation of MDC staining performed by FACS. We again found that GEM, SR-1, and ACPA induced a similar increase in AVO formation, which was more prominent in GW-treated cells. This effect was strongly potentiated by the GEM/cannabinoid combinations and antagonized by the autophagy inhibitors CQ or 3-MA or the scavenger NAC.

Figure 7a shows that either NAC, CQ, or 3-MA strongly reduced the percentages of the antiproliferative synergism by GEM/cannabinoids. Similar results were obtained with PaCa44 and PaCa3 cell lines (data not shown). It is worth to note that the NAC concentration used in these experiments (20mM) was non-toxic by itself and was able to completely abolish ROS induction by GEM/cannabinoids (data not shown). Altogether, these data demonstrate that GEM enhances cannabinoid-induced autophagy by a ROS-mediated mechanism and that this event is required for GEM/cannabinoid synergism. Figures 7b and c show a kinetic analysis of the events involved in GEM/cannabinoid antiproliferative synergism, that is, oxidative stress, ER stress, and autophagy. Although ROS were induced within 4h, the peak of Grp78 and LC3-II protein expression appeared at 8 and 12/16h, respectively, suggesting that ER stress could be a mechanism connecting ROS induction with autophagic cell death.

Figure 7

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 500nM GEM and 40 …

GEM and cannabinoids strongly inhibited growth of human pancreatic adenocarcinoma cells in vivo

The effect of GEM and/or SR1 on growth inhibition of PaCa44 cells subcutaneously xenografted in nude mice was also investigated. Figure 8a shows that the volume of tumor in mice treated with the combination GEM+SR1 remained essentially unchanged during the observation time, whereas it increased considerably in the control group and, at a lower extent, in either GEM- or SR1-treated groups. Figure 8b shows that mice body masses did not change during the experiment, suggesting that the treatments did not produce any apparent toxicity. At the end of the treatment time, the percentages of mean tumor mass reduction relative to control were 65, 34, or 92% in mice treated with GEM, SR1, or GEM+SR1, respectively (Figure 8c).

Figure 8

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, …

Chemicals

GEM (Gemzar) was provided by Eli Lilly (Florence, Italy). SR141716 (N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-pyrazole-3-carboxamide); SR1 (rimonabant; Acomplia, Sanofi-Aventis, Milan, Italy) was kindly provided by Dr. Maurizio Bifulco (University of Salerno, Italy); ACPA was obtained from Cayman Chemicals (Inalco, Milan, Italy); and GW405833hydrochloride (1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-(1-morpholine)ethyl)-5-methoxyindole; GW) from Sigma (Milan, Italy). ActD, PDTC, NAC, E64d, chloroquine diphosphate (CQ; N⁴-(7-chloro-4-quinolinyl)-N¹,N¹-dimethyl-1,4-pentanediamine), and 3-MA were obtained from Sigma. MG132 and BAY 11-7085 (2E)-3-94-(1,1-dimethylethyl)-phenyl-sulfonyl-2-propenenitrile) (BAY) were obtained from Enzo Life Sciences (VinciBiochem, Florence, Italy), pepstatin A from AppliChem (Darmstadt, Germany), and the recombinant human IL-l from PeproTech (Inalco, Milan, Italy).

Cell culture

PaCa44, PaCa3, Panc1, CFPAC1, T3M4, and MiaPaCa2 cell lines³⁸ were grown in RPMI 1640 supplemented with 2mM glutamine, 10% FBS, and 50μg/ml gentamicin sulfate (BioWhittaker, Lonza, Bergamo, Italy), and incubated at 37°C with 5% CO₂. Normal primary fibroblasts (PromoCell, PBI, Milan, Italy) were grown in DMEM supplemented with 2mM glutamine, 10% FBS, and 50μg/ml gentamicin sulfate, and were incubated at 37°C with 5% CO₂.

Cell proliferation assay

Cells were plated in 96-well cell culture plates (5 × 10³ cells per well) and treated with various compounds, as indicated in the legends to figures. At the end of the treatments, cell proliferation was evaluated by Crystal Violet (Sigma) staining. The dye was solubilized in 1% SDS in PBS and measured photometrically (A_595nm) to determine cell viability. Three or four independent experiments were performed for each assay condition.

Drug combination studies

The compounds were added for 48h at the following concentration ranges: 1nM–1μM for GEM, 80nM–80μM for SR1 and GW, and 450nM–450μM for ACPA in GEM-resistant cell lines; 0.2nM–200nM for GEM, 100nM–100μM for SR1 and GW, and 560nM–560μM for ACPA in GEM-sensitive cell lines. The CI was calculated by the Chou–Talalay equation, which takes into account both the potency (IC₅₀) and the shape of the dose–effect curve,³⁹ by using the CalcuSyn software. The general equation for the classic isobologram is given by CI=(D)1/(Dx)1+(D)2/(Dx)2+((D)1 (D)2)/((Dx)1^˙(Dx)2), where (Dx)1 and (Dx)2 in the denominators are the doses (or concentrations) for D1 (drug 1) and D2 (drug 2) alone that give x% growth inhibition, whereas (D)1 and (D)2 in the numerators are the doses of drug 1 and drug 2 in combination that also inhibited x% cell viability (i.e., isoeffective). CI<1 and CI<0.3 indicated synergism and strong synergism, whereas CI=1 and CI>1 indicated additive effect and antagonism, respectively. CI/effect curves represent the CI versus the fraction (0–1) of cells killed by drug combination. The synergism percentage was obtained analyzing CI/effect curve and measuring the CI values at each 0.05 fraction, that is, 5% growth inhibition, of the antiproliferative effect. Throughout all the experiments, we obtained a linear correlation coefficient (r)>0.90. Drug combination studies were performed using the following concentration ratios: [GEM]:[SR1] and [GEM]:[GW]=1:500 and [GEM]:[ACPA]=1:2800 in GEM-sensitive cell lines, whereas [GEM]:[SR1] and [GEM]:[GW]=1:80 and [GEM]:[ACPA]=1:450 in GEM-resistant cell lines. These molar ratios were calculated on the basis of GEM and cannabinoid IC₅₀ mean values at 48h. For GEM, they correspond to 36nM for the GEM-sensitive cell lines and 220nM for the GEM-resistant cell lines, as reported in our previous works.⁵ The three cannabinoids were all effective in the six cell lines, with IC₅₀ mean values corresponding to 18μM for SR1 and GW and 100μM for ACPA. By inverting the molar ratios of the compounds between the two groups of cell lines, no significant alteration of the results was observed (data not shown), indicating that the data obtained were not influenced by the specific experimental condition tested.

Analysis of ROS

The non-fluorescent diacetylated 2′,7′-dichlorofluorescein (DCF-DA) probe (Sigma), which becomes highly fluorescent upon oxidation, was used to evaluate intracellular ROS production. Briefly, cells were plated in 96-well plates (5 × 10³cells per well) and, 24h later, treated with various compounds, as indicated in the legends to figures. Then, cells were incubated with 10μM DCF-DA for 15min at 37°C, and the DCF fluorescence was measured by using a multimode plate reader (Ex 485nm and Em 535nm). Three independent experiments were performed for each assay condition.

RNA extraction qPCR, RT-PCR, and image analysis

Total RNA was extracted from 9 × 10⁵ cells using TRIzol Reagent (Invitrogen, Milan, Italy), and 1μg of RNA was reverse transcribed using first-strand cDNA synthesis. Real-time quantification was performed in triplicate samples by SYBR Green detection chemistry with SYBR Green PCR Master Mix (Applied Biosystems, Invitrogen) on a 7000 Sequence Detection System. The primers used were: CB1For 5′-GAGAGGTGCCAAGGGAGCTT-3′ and CB1Rev 5′-GGTGCGGAAGGTGGTATCTG-3′ CB2For 5′-CACAGCCTCTGTGGGTAGCC-3′ and CB2Rev 5′-ACGGGTGAGCAGAGCTTTGT-3′ Grp78For 5′-GACGGGCAAAGATGTCAGGAA-3′ and Grp78Rev 5′-TCATAGTAGACCGGAACAGATCCA-3′ CHOPFor 5′-GCAGCCCATGAAGGAGAAAG-3′ and CHOPRev 5′-CGGTCGATTTCCTGCTTGAG-3′ and GAPDHFor 5′-TGTGTCCGTCGTGGATCTGA-3′ and GAPDHRev 5′-R-GATGCCTGCTTCACCACCTT-3′. The following cycling conditions were used: 95°C for 10min, 40 cycles at 95°C for 15s, 60°C for 1min, and 72°C for 30s. The average of cycle threshold of each triplicate was analyzed according to the 2^(−ΔΔCt) method. For RT-PCR, one-tenth of the cDNA was used as a template for PCR amplification using the following primers and cycling conditions: β-actinFor 5′-ACCAACTGGGACGACATGGAGAA-3′ and β-actinRev 5′-GTGGTGGTGAAGCTGTAGCC-3′, 25 cycles of 94°C for 60s, 55°C for 60s, and 72°C for 60s; XBP-1For 5′-CCTTGTAGTTGAGAACCAGG-3′ and XBP-1Rev 5′-GGGGCTTGGTATATATGTGG-3′, 40 cycles of 93°C for 60s, 55°C for 60s, and 72°C for 60s. PCR products were separated by electrophoresis through ethidium bromide-stained 3.5% agarose gel and visualized by ultraviolet light. To quantify XBP-1 splicing, XBP-1(S) bands were scanned as digital peaks and the areas of the peaks were calculated using the public domain NIH Image software (http://imagej.nih.gov), normalized with β-actin mRNA expression, and reported as fold induction relative to controls.

For ActD experiments, 2.5 × 10⁵ Panc1 cells were seeded in 60-mm plates and incubated overnight. Cells were pretreated with 5μg/ml ActD for 1h, then 2μM GEM was added, and the treatments prolonged up to 16h. Total RNA extraction and real-time PCR were performed, as above described.

Apoptosis

The percentage of apoptotic cells was evaluated by staining 3 × 10⁵ cells with annexin V-FITC (Bender Med System, Prodotti Gianni, Milan, Italy) and 5μg/ml PI in binding buffer (10mM HEPES/NaOH (pH 7.4), 140mM NaOH, and 2.5mM CaCl₂) for 10min at room temperature in the dark. The samples were analyzed by flow cytometry (FACScalibur, Becton Dickinson, San Jose, CA, USA) to determine the percentage of cells displaying annexin V+/PI− (early apoptosis) or annexin V+/PI+ staining (late apoptosis). For each sample, we report the percentage values corresponding to the addition of early and late apoptosis. Three independent experiments were performed for each assay condition.

Cell cycle analysis

Cell cycle distribution was analyzed by staining 3 × 10⁵ cells with PI. After the indicated treatments, cells were washed with PBS, incubated with 0.1% sodium citrate dihydrate, 0.1% Triton X-100, 200μg/ml RNase A, 50μg/ml PI (Roche Diagnostics, Milan, Italy), and analyzed using a flow cytometer (FACScalibur, Becton Dickinson). The percentage of cells in the various stages of the cell cycle was determined using the ModFitLT software (Verity Software House, Topsham, ME, USA). Three independent experiments were performed for each assay condition.

Immunoblot analysis

In all, 1.2 × 10⁶ cells were treated with the various compounds, as indicated in the legends to figures, collected, washed in PBS, and resuspended in RIPA buffer, pH 8 (150mM NaCl, pH 8; 50mM Tris-HCl; 1% NP-40; 0.5% Na-Doc; and 0.1% SDS), 1mM PMSF, 1mM Na₃VO₄, 1mM NaF, 2.5mM EDTA, and 1 × protease inhibitor cocktail (Roche Diagnostics) for 30min on ice. The lysate was centrifuged at 14000 × g for 10min at 4°C, and the supernatant was used for western blot. Protein concentration was measured with the Bradford protein assay reagent (Pierce, Celbio, Milan, Italy) using bovine serum albumin as standard. Fifty (for LC3-II) or thirty (for Grp78) micrograms of protein extracts were electrophoresed through a 15% SDS-polyacrylamide gel and electroblotted onto PVDF membranes (Millipore, Milan, Italy). Membranes were then incubated for 2h at room temperature with blocking solution (5% low-fat milk in TBST (100mM Tris, pH 7.5, 0.9% NaCl, and 0.1% Tween 20)) and probed overnight at 4°C with the appropriate primary antibody (1:1000 in blocking solution of LC3B (Cell Signaling, Milan, Italy), Grp78 (Santa Cruz Biotechnology, DBA, Milan, Italy), or α-tubulin (oncogene) antibodies). Horseradish peroxidase-conjugated IgG (1:2000 in blocking solution, Upstate Biotechnology, Milan, Italy) was used to detect specific proteins. Immunodetection was carried out using chemiluminescent substrates (Amersham Pharmacia Biotech, Milan, Italy) and recorded using a Hyperfilm ECL (Amersham Pharmacia Biotech). To quantify LC3-II and Grp78 expression, bands were scanned as digital peaks and the areas of the peaks were calculated in arbitrary units using the public domain NIH Image software (http://rsb.info.nih.gov/nihimage/), normalized with α-tubulin expression, and reported as fold induction relative to controls.

Labeling of autophagic vacuoles with MDC

To quantify the induction of the autophagic process in Panc-1 cells treated with the various compounds as indicated in the legend, MDC staining was performed, as previously described.⁴⁰ Following the treatments, cells were incubated with 50μM MDC in PBS at 37°C for 15min. After incubation, cells were washed with PBS, trypsinized, and immediately analyzed by flow cytometry. All fluorescences were analyzed with a FACScalibur flow cytometer (Becton Dickinson). The fluorescent emissions were collected through a 530nm band pass filter (FL1 channel). At least 10000 events were acquired in log mode. For the quantitative evaluation of MDC, CellQuest software (Becton Dickinson) was used to calculate mean fluorescence intensities (MFIs). The MFIs were calculated by the formula (MFItreated/MFIcontrol), where MFItreated is the fluorescence intensity of cells treated with the various compounds and MFIcontrol is the fluorescence intensity of untreated and unstained cells. Values reported in the figures are the means±S.D.s from three independent experiments.

Quantification of AVOs with acridine orange

In acridine orange-stained cells, the cytoplasm and nucleus are bright green and dim red, whereas acidic compartments are bright red. The intensity of the red fluorescence is proportional to the degree of acidity. Following the treatments, as specified in the legend, cells were incubated with acridine orange solution (1μg/ml) at 37°C; after 15min in drug-free medium at 37°C, they were washed with PBS, immediately stained with 1μg/ml of acridine orange for 15min, and observed with a Nikon Eclipse TE300 Inverted microscope (Nikon Instruments, Inc., Melville, NY, USA). Then, cells were trypsinized and analyzed by flow cytometry using FACScan cytometer and CellQuest software, as previously described. Statistical analyses were performed, as described above.

In vivo studies

On the basis of our previous experience, we could not make use of Panc1 cells forin vivo studies because of their low growth rate in nude mice. Thus, we chose PaCa44 cells because of their similar behavior to Panc1 cells in in vitro growth inhibition studies. PaCa44 cells (2 × 10⁶ cells/mice) were s.c. injected into female nude mice (4 weeks of age, Harlan laboratories, Udine, Italy). One week after cell inoculation, five randomized animals for each experimental group received solution vehicle (PBS), 25mg/kg GEM, or/and 0.28mg/kg SR1 by intraperitoneal injection biweekly for 4 weeks. Drug doses were chosen on the basis of their respective clinical use following US Food and Drug Administration directives. Tumor volume and body mass were biweekly recorded for each animal. Animals were killed at the end of the 4-week study period, and the tumors were resected and weighted. Animal studies were approved by the Verona University Review Board.

Statistical analysis

ANOVA (post hoc Bonferroni) analysis was performed by GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P-values <0.05, 0.01, or 0.001 were indicated as ^*, ^**, or ^***, respectively.

This work was supported by Associazione Italiana Ricerca Cancro, Milan, Italy; Fondazione CariPaRo, Padova, Italy; Joint Project of University of Verona, Verona, Italy; and MIUR, PRIN 2008 (Alberto Abbruzzese). SR141716 (Rimonabant) was provided by Sanofi-Aventis (grant to M Bifulco, no. 051309). We thank Dr. Federica Cioffi (University of Verona) for FACS analysis support, and Dr. Giorgio Malpeli (University of Verona) for qPCR technical support.

IRE1

inositol-requiring ER to nucleus signal kinase 1

XBP-1

X-box-binding protein-1

PERK

RNA-dependent protein kinase-like ER kinase

ATF-6

activating transcription factor-6

CHOP

CCAAT/enhancer-binding protein (C/EBP) homologous protein

GRP78

glucose-regulated protein 78kDa

LC3

microtubule-associated protein 1 light-chain 3α

ATG5

autophagy-related 5 homolog

BIM

Bcl-2-like protein 11

Bcl-2

B-cell lymphoma gene-2

Supplementary Information accompanies the paper on Cell Death and Disease website (http://www.nature.com/cddis)

Edited by G Melino

Supplementary Figure 1

Supplementary Figure 2

Supplementary Figure Legends

Supplementary Table 1

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