Cannabidiol’s cytotoxicity in pancreatic cancer is induced via an upregulation of ceramide synthase 1 and ER stress | Journal of Cannabis Research

The findings presented in this work, indicate dose-dependent and time-dependent cytotoxic effects of CBD in both human and murine pancreatic cancer cells. Gemcitabine and CBD in combination upregulate CerS1 greater in gemcitabine sensitive cells and in resistant cells CBD alone can upregulate CerS1. The downstream effects of CerS1 upregulation induce ER stress which activate the GRP78/ATF4/CHOP arm of the UPR response in gemcitabine resistant cells. Our pilot in vivo study showed a significant reduction in tumour volume in both double chemotherapy and combination of all three; CBD, gemcitabine and abraxane. No significant difference in survival was observed for CBD alone and as an adjunct therapy. The in vitro platform points to a potential novel mechanism of CBD’s mechanism of action in pancreatic cancer cells and further animal studies are needed to validate ceramide’s involvement in CBD therapy for PDAC.

Experimental procedures

Cell lines, culture media and conditions

Panc03.27, Panc1, HPAF-II, CFPAC-I, IMR-90 were cultured according to ATCC’s guidelines. and murine-3275 were cultured in RPMI supplemented with 10% FBS. Panc03.27 cells were seeded in respective 96 well plates at 1 × 10⁴ and Panc1 at 8 × 10³ for the use of CellTiter-Glo® (Promega, Cat No G9241) and Sulforhodamine B (SRB) (Abcam Cat No ab235935) assays to analyse cytotoxicity of drug treatments.

Transfections

Cell lines Panc03.27 and Panc1 were seeded in a 24-well plate at 7.5 × 10 and 5 × 10 respectively and treated with following siRNA targets, Silencer™ Select Negative Control No. 1 siRNA (cat no.: 4390843), Silencer® Select Pre-Designed & Validated siRNA s6980 (cat no.: 4392420), Silencer® Select Pre-Designed & Validated siRNA s230924 (cat no.: 4390816) and DsiRNA DDIT3, Integrated DNA Technologies (cat no.: hs. Ri. DDIT3.13.1). Transfection was performed after 4–6 h of seeding (until cells adhered) with the indicated oligonucleotide concentration using 1µL per well of Lipofectamine RNAiMAX according to manufacturer’s instructions (ThermoFisher, Invitrogen, Protocol Pub. No. MAN0007825 Rev.1.0). Transfections were conducted with Lipofectamine RNAiMAX (ThermoFisher, Cat No 13,778,150). Replacement of medium took place 24 h later containing drugs for treatment. At the 24- and 48-hours’ time point post treatment, cells were harvested for analysis. Silencer™ Select Negative/Scrambled Control No.1 siRNA, catalgoue number; 4,390,843, Silencer® Select Pre-designed & validated siRNA s6980 (BiP/GRP78), catalogue number; 4,392,420, Silencer® Select Pre-designed & validated siRNA s230924 (CERS1), catalogue number; 4,390,816.

RNA extraction and real-time PCR

Total RNA was isolated from cultured cells using the RNeasy Mini Kit according to manufacturer’s instructions (QIAGEN). The RNA was quantitated using a QIAxpert microfluidic spectrophotometer (QIAGEN) and reverse transcribed using the Quantitect Reverse Transcription Kit according to manufacturer’s instructions (QIAGEN). Relative mRNA expression levels were determined by real-time PCR using either Applied BiosystemsTM PowerUpTM SYBRTM Green Master Mix (Applied Biosystems) with validated QuantiTect SYBR Probes from QIAGEN or TaqMan Fast Advanced Master Mix (Life Technologies) with validated FAM Probes from Applied Biosystems. Following Quantitect Primer Assays (QIAGEN) were used for data analysis to ensure accurate interpretation of the data and in line with the MIQE guidelines: Hs_B2M_1_SG (QT00088935), Ms_B2m_1_SG (QT01149547), Mm_Cers1_1_SG (QT00151081), Mm_Cers2_1_SG (QT00144025), Mm_Cers3_1_SG (QT00525952), Mm_Cers4_1_SG (QT00122101), Mm_Cers5_1_SG (QT00101605), Mm_Cers6_1_SG (QT00137291). The following FAM Probes purchased from Applied Biosystems that were used are listed: B2M (Hs00187842_m1), AHSA1 (Hs00201602_m1), ATF4 (Hs00909569), GRP78 (Hs00607129), CERS1 (Hs04195319_s1), CERS2 (Hs00371958_g1), CERS3 (Hs00698859_m1), CERS4 (Hs00226114_m1), CERS5 (Hs00332291_m1), CERS6 (Hs00826756_m1). Relative gene expression values were calculated using the Livak method 2^-(ΔΔCt), where target genes were normalised to a housekeeping gene (Livak and Schmittgen 2001).

Western blot

Cells were seeded at different seeding densities per well in a 24-well plate depending on each cell line and transfected as described above. A pool of 4 wells per condition was used for total protein extraction. 48, 72 and 96 h post drug/siRNA treatment and prior to cell lysis, all wells were rinsed twice with either ice cold Phosphate Buffered Saline (PBS) or Tris Buffered Saline (TBS) buffer in the case of phosphoproteins only. RIPA lysis buffer was then added to cells (100µL/well) containing 50mM of Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40 and 5mM EDTA supplemented with protease inhibitor cocktail (Sigma, Cat No P8340) and phosphatase inhibitor cocktail (Sigma, Cat No 524,629). Thereafter, the cells were transferred into pre-chilled tubes using a cell scraper and centrifuged gently for 15 min at 5 °C. Cell debris was subsequently removed, and the protein supernatant was transferred into a pre-chilled tube. The amount of protein per condition was thereafter quantified using the RC-DC Bradford Assay Kit following the manufacturer’s protocol (Bio-Rad, Cat No 5,000,120), and 20 µg of total protein was then loaded onto SDS-PAGE (ThermoFisher, Cat No XP04122BOX). A recombinant protein of human CERS1 (ORIGENE, Cat No TP311201) was also purchased and loaded as a positive control alongside with the CERS1 samples for the validation of CERS1 protein levels. The acrylamide gels were then transferred onto polyvinylidene fluoride (PVDF) (Sigma, Cat No GE10600023) membranes for western blotting using the antibodies; Anti-ATF4 (Cell signalling, 11,815), Anti-Ceramide Synthase 1 (Abcam, ab85696), Anti-alpha tubulin (Abcam, ab7291), Anti-GRP78 (Cell Signalling, 3177), WesternSure® HRP Goat anti-Mouse IgG (H + L) (LI-COR, 926-80010), WesternSure® HRP Goat anti-Rabbit IgG (H + L) (LI-COR, 926-80011), Donkey Anti-Goat IgG H&L (HRP) (Abcam, ab205723).

ELISA

Following cell lysis (see 4.5 western blot), 100µL of sample, standards, blanks were added to the pre-coated microtiter plate wells with a biotin-conjugated antibody preparation specific for target antigen and then avidin conjugated to Horseradish Peroxidase (HRP) was added to each microplate well and incubated followed by the addition of a TMB substrate solution added to each well. Only those wells that contain target antigen, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in colour. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the colour change measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of target antigen in the samples was then determined by comparing the O.D. of the samples to the standard curve, as referred to in MyBioSource, Human CERS1 ELISA kit (Cat no. MBS2890964).

Mice, tumour induction and cell isolation

3275-Luc⁺ cell line was derived from a KPC model on a C57/BL6 background harbouring mutation in KRAS, INK4A and p53 (derived from the Swiss Institute for Experimental Cancer Research (ISREC)). Tumours were excised, meshed, and processed to form the cell line. 3275-Luc⁺ cells were cultured in RPMI 1640 (Lonza, Cologne, Germany) supplemented with 10% FCS for injection preparations.

All in vivo experiments were performed in accordance with the local ethical review panel, the UK Home Office Animals (Scientific Procedures) Act 1986, the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research, and the ARRIVE guidelines. 70 FVB/NJ female mice aged between 6 and 8 weeks old were purchased from Charles Rivers, Germany at a mean weight of 23 g and used in this study. 3275-Luc⁺ cells were screened for mycoplasma. Animals were housed in specific pathogen-free rooms in autoclaved, aseptic micro isolator cages with a maximum of five animals per cage. For tumour induction, 0.5 × 10⁶ 3275-Luc⁺ cells in 100uL volume of HBSS were injected subcutaneously in the right flank of FVB/N mice post-acclimatization. Animals were checked twice weekly for good health, ulceration grade and tumour growth. However, any mice displaying the following pre-assigned effects were culled. The pre-assigned end points included mice displaying one of the following: development of abdominal ascites, severe cachexia, significant weight loss (approaching 20% of initial weight), extreme weakness, inactivity, discomfort, or pain. No major side/adverse effects and no weight loss were observed in mice treated with CBD. Tumour measurements were performed using a caliper with width and height noted and volume calculated using the formula: 4.19*(d/4 + d/4)³. Once tumour reached 5 mm x 5 mm in size, mice were randomised to treatment/control groups based on mean tumour volumes using a record table on excel software. Mice were then treated according to their appropriate arm of treatment. Endpoint for each animal was determined by tumour burden or G4 ulceration. Mice were sacrificed and tumours were removed for cell isolation in MACS® Tissue Storage Solution (Miltenyi Biotec). Tumours were spun at 200xg for 5 min, then meshed and passed through a 100 μm cell strainer (Miltenyi SmartStrainer) to obtain a single cell suspension using collagenase and DNase (Sigma-Aldrich, Munich, Germany) and incubated at 37 °C for 40 min. Tumour cells were filtered through a 100 μm mesh, washed, and stained for flow cytometer analysis.

Drug and vehicle injections

Drug treatments were administered via intraperitoneal means and for the triple combination arm, injection sites were altered from right to left flank. Gemcitabine (purchased from MedChemExpress, United Kingdom) and vehicle (DMSO) were injected at 10 mg/kg twice weekly, Abraxane (nab-paclitaxel purchased from Celgene, Netherlands) and vehicle Albumin human (purchased from Sigma Life Science) were injected at 10 mg/kg twice weekly and Cannabidiol and vehicle (DMSO) were injected at 100 mg/kg thrice weekly. Cannabidiol, 100% purity, was obtained from EMMAC life sciences (Batch no. MCE/CBD/19 − 001) and dissolved in Tween-80 (Sigma-Aldrich), sunflower oil (Sigma Life Science) and PBS (Gibco) at a 1:1:8 ratio in 0.01% DMSO.

Immunohistochemistry on formalin fixed and paraffin embedded tissue

3 μm sections of formalin-fixed, paraffin-embedded (FFPE) tissues were cut using the Leica RM2255 microtome (Leica Biosystems Ltd., Newcastle). Prior to immunostaining, sections were deparaffinised in xylene, re-hydrated through a series of decreasing concentrations of ethanol and transferred to water. In detail, slides were immersed in xylene solution for x2 10 min following by re-hydration progressively in ethanol 100% for 5 min, ethanol 96% for 5 min, ethanol 80% for 5 min and H₂O for 5 min. Sodium citrate buffer [10mM; 0.05%Tween-20 (pH = 6.0)] was used for antigen retrieval process and slides were placed in glass jars and boiled in water bath for 30 min at 110 °C. Sections were left to cool down gradually at room temperature. Slides were washed by Tris-buffered saline (TBS) − 0.025% Tween 86 twice for 5 min each, incubated in a dark humidified chamber at room temperature with 5% BSA (400µL of BSA (12.5%) and 600µL of PBS-Triton 0.25%). Immediately, without washing, primary antibody in PBS + TritonX100 + 1% BSA added and incubated overnight at 4˚C temperature. Slides were washed three times for 5 min each in TBS-0.025% Tween 20 and secondary antibodies added and left at room temperature for 60 min. Following by washing with TBS-0.025% three times for 5 min each in order to remove excess of secondary polymer antibody. Signal detection was done using diaminobenzidine tetrahydrochloride (DAB) as the reaction of chromogenic for 5 min. Reaction stopped by immersing the slides with ddH2O and then counterstained with Haematoxylin (50% Harris-50%Mayer) (VWR International Ltd., Leicestershire, UK) for 5 s and briefly washed in tap water. Dehydration applied with 3 min incubation with 96% ethanol, 10 min with 100% ethanol and 5 min in xylene. Slides were sealed with a drop of mounting reagent (VWR International Ltd., Leicestershire, UK) and coverslips (VWR International Ltd., Leicestershire, UK).

Statistical analysis

Students two-tailed t test was used to compare vehicle and drug/siRNA treated groups. A p-value of less than 0.05 was considered significant. As shown on the graphs, * denotes p < 0.05, ** denotes p < 0.01 and *** denotes p < 0.001 in comparison to vehicle. All graphs were generated with GraphPad Prism 8 (GraphPad Software, USA).

Kaplan-Meier estimator used to estimate the proportion of mice alive at any one time-point in the study groups. It is a nonparametric test and appropriate to use when the data are right skewed and censored.