Lysosomal Membrane Permeabilization is an Early Event in Sigma-2 Receptor Ligand Mediated Cell Death in Pancreatic Cancer

Multiple structurally distinct compounds (Figure 1) with high affinity for sigma-2 receptors were tested for cytotoxicity against multiple pancreatic cancer cell lines in vitro (Table1) and screened for efficacy in a mouse model of pancreatic cancer with Panc02 cells (Additional file 1: figure S1). Compounds were further tested in athymic nude mice bearing human Bxpc3 subcutaneous tumorsand treated daily with equimolar doses of these sigma-2 receptor ligands. These mice with established tumors were treated for eleven days and compared to vehicle, SV119, SW43, PB28, and PB282 each significantly decreased tumor volume (Figure 2).

Recently characterized fluorescent sigma-2 receptor ligands SW120 (derivative of SW43) [16] and PB385 (derivative of PB282) [17], colocalize with LysoTracker Red in Bxpc3 pancreatic cancer cells by confocal microscopy (Figure 3), and also appreciated by fluorescent microscopy in Bxpc3 and Aspc1 (Additional file 2 figure S2A). Bxpc3 cells preloaded with acridine orange (AO, 2 μg/mL), had decreased retention of orange dye in the lysosome (Figure 3, bottom panel) following treatment with SW43 and PB282, and displayed increased green fluorescence as dye escaped and bound with nucleic acids. Similar results were observed in Aspc1 cells (Additional file 2 figure S2A). The pH gradient of the lysosome is actively driven by a V-Type ATPase H⁺ pump [18], and its inhibition with concanamycin A (CMA) prevented dye retention in the lysosome. As well, hydroxychloroquine (HCQ), originally used for treatment of malaria [19] and extensively studied as a lysosomotropic detergent [20] showed decreased dye retention (positive control) (Additional file 3 figure S3A). SV119 and PB28, with high affinity to sigma-2 receptors, displayed leakeage of lysosomal dyes by acridine orange,and leakage by all compounds was confirmed with LysoTracker Green (Additional file 3 figure S3B).

LAMP1 and LAMP2 are large, closely homologous, glycoprotein constituents of the lysosomal membrane that contribute to protection of the membrane against the acidic enviroment within this organelle [21]. We hypothesized that decreasing the content of LAMP1 in the lysosome would subject the membrane to increased stress and susceptibility to permeabilization. pLKO.1-LAMP1 and pLKO.1-Neg shRNA lentiviral constructs were used to transform and select Bxpc3 (Figure 4A) cells with decreased expression of LAMP1 and LAMP2 (Figure 4A). LAMP1 shRNA-expressing cells significantly retained less fluorescence of LysoTracker Green (Figure 4B), mean fluorescence 61.6 ± 0.1 percent of vehicle, with moderate decreases following treatment with SW43 or PB282. LAMP1 knockdown significantly increased susceptibility of Bxpc3 cells to cell death following treatment with SW43 and PB28, with less protection observed in the lower range of toxicity with HCQ (Figure 4C).

Bxpc3 cells were treated with CMA (10 nM) for 60 minutes in order to effectively inhibit the pH gradient across the lysosomal membrane. Subsequent accumulation of the fluorescently labeled sigma-2 receptor ligands SW120 and PB385 showed marked decrease of fluorescence intensity by flow cytometry (Figure 5A). Bxpc3 cells pretreated with CMA were more viable following treatment with sigma-2 ligands, but the response was greater for SW43 and HCQ compared to PB282 (Figure 5B). To determine whether LMP lead to release of proteases into the cytoplasm, the cytosolic fraction was isolated from the lysosomal fraction by selective permeabilization of the plasma membrane with digitonin, and cleavage ofcathepsin B substrate Z-RR-AMC was assessed. All compounds increased Z-RR-AMC cleavage within one hour of treatment, and CMA decreased this Z-RR-AMC cleavage to baseline (Figure 5C). CA-074-Me and pepstatin A decreased cleavage for all compounds as well, with the exception that pepstatin A was not observed to inhibit cleavage following SW43 treatment. Functional rescue of viability in the presence of CA-074-Me and pepstatin A was assessed at 24 hours following treament, and pepstatin A was observed to rescue viability across a titrated dose range for all compounds, while CA-074-Me had a lesser effect, though the observed differences did not reach statistical significance (Figure 5D).

Sufficient LMP leads to apoptosis through release of specific mediators of crosstalk with the mitochondria such as cathepsins, and release or stimulation of reactive oxygen species (ROS) [22]. Though cancer cells typically have a higher than normal content of ROS due to relative anoxia, additional oxidative stress is lethal due to oxidation and disruption of membrane lipids, proteins, and DNA [23]. To assess the involvement of ROS in apoptosis following sigma-2 receptor ligand treatement, we examined the influence of antioxidants on cell death. ROS production in Bxpc3 cells following 24 hour treatment with SW43 (60 μM), PB282 (90 μM), and H₂O₂(100 μM) was detected with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) as described in the Materials and Methods. Substantial amounts of ROS were detected with SW43 and H₂O₂, but no ROS was detectable after treatment with PB282. ROS was decreased following SW43 treatment in the presence of antioxidants α-tocopherol (α-toco) and n-acetylcysteine (NAC), while ROS from H₂O₂ was only decreased by NAC (Figure 6A). The impact of antioxidants on cell viability was assessed following 24 hour treatment with SW43 and PB282. Antioxidants protected against sigma-2 receptor ligand induced cell death, with NAC protecting against SW43 to a greater extent than α-toco. Interestingly, while PB282 treatment did not result in detectable ROS release, both antioxidants increased tumor cell viability after PB282 exposure (Figure 6B).

Caspase-3 has been extensively studied as a mechanism of sigma-2 receptor ligand mediated apoptosis, and we wished to examine the impact of ROS stimulation by structurally different ligands. Basal caspase-3 activity by SW43, PB282, and HCQ treatment following 24 hours was detected by cleavage of Z-DEVD-AMC as previously described [10] (Figure 7A). This activation was inhibited by α-toco following PB282 treatment, but not following SW43 or HCQ treatment. NAC, however, decreased caspase-3 activation by all compounds. DEVD-FMK caspase-3 inhibitor was used as a positive control for inhibition in all experiments. One hour pretreatment with DEVD-FMK, followed by 24 hour treatment with SW43, PB282, or HCQ showed little protection for SW43 and HCQ, while PB282 mediated cytotoxicity was protected to a much greater extent following caspase-3 inhibition (Figure 7B). Interestingly, caspase-3 activity was not observed in Aspc1 cells (Additional file 3 figure S3C), a cell line with less sensitivity to PB282 (Additional file 3 figure S3D).

Cell lines were maintained in RPMI media (GIBCO) supplemented with L-glutamine (2 mM), (HEPES) (1 mM), pyruvate (1 mM), sodium bicarbonate (0.075 % w/v), penicillin and streptomycin (100 IU/mL), amphotericin (0.25 μg/mL), and 10 % fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA). Cells were seeded at a density of 2 x 10⁵/mL unless otherwise stated and maintained in a humidified atmosphere of 5 % CO₂ at 37°C.

Sigma-2 receptor ligands were synthesized as previously described [10, 16, 17, 40‐42]. The imaging dyes acridine orange and LysoTracker Red were obtained from Invitrogen (Carlsbad, CA), FITC mouse anti-human CD107a (LAMP1) and CD107b (LAMP2) antibodies from BD Biosciences (Franklin Lakes, NJ), peptidase inhibitors CA-074-Me and pepstatin A, and fluorogenic peptidase substrate Z-RR-AMC from Enzo Life Sciences (Plymouth Meeting, PA), caspase-3 inhibitor Z-DEVD-FMK from R&D Systems (Minneapolis, MN); caspase-3 substrate Ac-DEVD-AMC from Bachem Biosciences, Inc (King of Prussia, PA); All other reagents were obtained from Sigma-Alrich (St. Louis, MO) unless otherwise stated. Compounds were dissolved in DMSO with final concentrations less than 0.3 %.

Athymic nude mice from Harlan Bioproducts, Inc. were inoculated subcutaneously with 1x10⁶ Bxpc3 cells in the right flank. Tumor sizes were monitored with calipers and when tumors reached an average of 5 mm in diameter, mice were randomized and treated daily with equimolar doses of sigma-2 receptor ligands SV119 (1.0 mg), SW43 (1.1 mg), PB28 (0.9 mg), or PB282 (0.9 mg) resuspended in vehicle consisting of 5 % DMSO, 5 % EtOH, and 10 % Cremophor in 1X PBS and injected intraperitoneally. Data represents mean ± SEM, n = 7–10 per group.

Cells grown on glass cover slips were incubated with SW120 or PB385 (100 nM) in the presence of LysoTracker Red (25 nM) for 30 minutes at 37°C. Cells were washed with PBS and fixed in 2 % paraformaldehyde for 30 minutes at 37°C prior to additional washing and mounting with ProLong Gold antifade reagent. Confocal imaging was performed on a Carl Zeiss Axiovert 100 inverted microscope, fitted with LSM 510 laser scanning microscope camera and software. Images were collected with filter bandwidths corresponding to 505–530 nm for green, 560–615 nm for red, and > 650 nm for far red, with 4 scans over 11.8 seconds.

Cells grown on glass cover slips were loaded with acridine orange (2 μg/mL) for 15 minutes at 37°C prior to treatment for one hour with compounds. Cover slips were inverted onto slides and images taken immediately at 40X magnification on anOlympus BX51 microscope fitted with a U-LH100HE reflective fluorescence system and equipped with a Diagnositic Instruments, Inc. SPOT camera and software. Chroma Technology Corp filter sets were used for green (exciter: D480/30x, emitter: 535/40 m, beamsplitter: 505dclp), red (exciter: D540/25x, emitter: 606/55 m, beamsplitter: 556dclp), and blue (exciter: D360/40x, emitter: 460/50 m, beamsplitter: 400dclp). Scale bar equals 20 μm.

Cells were incubated with acridine orange (2 μg/mL) or LysoTracker Green (25 nM) for 30 minutes at 37°C prior to treatment with compounds for one hour. Cells were washed and mean fluorescence quantified with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Mean fluorescence was normalized to DMSO to determine the degree of lysosomal permeabilization. In addition, cells were pretreated with concanamycin A (10 μM) for one hour at 37°C prior to staining with either SW120 or PB385 (100 nM) for 30 minutes at 37°C and the difference in uptake represented by histogram.

shRNAlentiviral constructs in pLKO.1 against human LAMP1 was purchased from Sigma Aldrich, and following verification of knockdown, clone ID NM_005561.2-1183s1c1 used to compromise lysosomal integrity. Packaging vectors were obtained through Addgene, Inc. (Cambridge, MA). Lentivirus particles were prepared by transfection of 293 T cells in T75 flasks with 3 μg construct, 2.8 μgpRSV-Rev, 2.4 μgpMDLg/pRRE, and 0.6 μg pMD2.G utilizing FuGENE® 6 Transfection Reagent from F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Forty-eight and 72 hours following transfection, supernatant was transferred to Bxpc3 cells in the presence of polybrene (8 μg/mL). Transformed cells were selected with puromycin (1 μg/mL) and assayed accordingly.

Cells were washed once with PBS prior to fixation with IC Fixation Buffer (eBiosciences) for 15 minutes at 37°C. Fixed cells were washed with PBS, resuspended in Permeabilization Buffer (eBiosciences), and incubated for 30 minutes at room temperature. Intracellular antigen staining was performed with FITC-antibody dilution of 1:100 in Permeabilization Buffer for 60 minutes at room temperature. Mean fluorescence in FL1 was quantified with a FACSCalibur flow cytometer.

Cell lines maintained at optimal culture conditions were seeded into 96-well white, clear-bottom plates and following treatment, viability determined with CellTiter-Glo Luminescent Viability Assay from Promega (Madison, WI). Luminescence was quantified with a SpectraMax Gemini microplate spectrofluorometer from Molecular Devices (Silicon Valley, CA). Viability relative to vehicle was fit by non-linear regression and plotted against concentration.

Cells were treated in the presence of inhibitors and cytosolic extracts prepared using the digitonin extraction method as previously described [43]. Washed cells were resuspended at 1x10⁶ cells/mL in extraction buffer consisting of sucrose (250 mM), HEPES (20 mM), KCl (10 mM), MgCl₂ (1.5 mM), EDTA (1 mM), and digitonin (30 μM). Cells were placed on ice on an orbital shaker for 10 minutes prior to centrifugation for 1 min at 14,000 rpm at 4°C. Supernatants were collected and 20 μL used to detect cleavage of Z-RR-AMC in and equal volume of reaction buffer consisting of sodium acetate (100 mM), NaCl (200 mM), EDTA (4 mM), DTT (10 mM), and Z-RR-AMC (10 μM). Plates were read following incubation at 37 ° for 60 minutes with SpectraMax Gemini microplate spectrofluorometer, Molecular Devices (Silicon Valley, CA) (ex 355 nm, em 450 nm).

Cells were seeded into 12-well plates one day prior to treatment, stained with 25 μM 5-(and-6)-carboxy-2’,7’-dichlorodihydro-fluorescein diacetate (carboxy-H₂DCFDA) (Image-iT Live Green Reactive Oxygen Species Detection Kit, Molecular Probes, Eugene, OR) for 30 minutes at 37°C and treated overnight. Fluorescence intensity (max 529 nM) was quantified in the FL1 channel with a FACSCalibur flow cytometer.

Cells were maintained at optimal conditions and seeded in 96-well black-bottom plates in a volume of 100 μL. Following treatment, 5X assay buffer containing EDTA (10 mM), CHAPS (5 %), HEPES (100 mM), DTT (25 mM), and Ac-DEVD-AMC (250 μM) was added directly to the cell media and incubated for two hours at 37°C on a microplate shaker, and liberated AMC quantified with a SpectraMax Gemini microplate spectrofluorometer, Molecular Devices (ex 355 nm, em 450 nm). Caspase-3 activity is normalized to the absence of inhibitor.

Statistical analysis and data plotting was conducted using GraphPad Prism (GraphPad Software, San Diego, CA). Data represents the mean ± SEM. Viability IC₅₀ values at 18 hours were calculated by line fitting normalized viability versus concentration with non-linear regression and statistical significance determined using one-way ANOVA. Differences in viability, caspase-3 activity, apoptosis, and oxidation status were analyzed using two-way ANOVA to identify differences and confirmed with paired two-tailed t-tests. Blood cytology and biochemistry results were analyzed using one-way ANOVA with Tukey’s multiple comparison test. Statistical analysis for the difference in tumor volume between treatments groups was determined with the repeated measures ANOVA. Kaplan-Meier survival curves were plotted and differences compared with a log-rank test. A p-value of less than 0.05 was considered significant for all tests.