Cerivastatin, pravastatin and fluvastatin were purchased from LKT Laboratories, Inc (USA), lovastatin and simvastatin from Santa Cruz Biotechnology (Dallas, TX, USA). Bovine serum albumin (BSA), hemin, reduced nicotinamide adenine dinucleotide (NADPH), sulfosalicylic acid, Dulbecco’s Modified Essential Media (DMEM), and RPMI-1640 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and L-glutamine (L-Glu) were purchased from Biosera (Boussens, France), 15-deoxy-Δ-12,14-prostaglandin J2 (PGJ2) was purchased from Merck (Darmstadt, Germany).
For cell culture studies, the following pancreatic cancer cell lines were used: PA-TU-8902 (DSMZ, Braunschweig, Germany), MiaPaCa-2 and BxPC-3 (ATCC, Manassas, VA, USA). All cell lines were maintained and grown in a humidified atmosphere containing 5 % CO2 at 37 °C. PA-TU-8902 and MiaPaCa-2 were cultured in DMEM supplemented with 10 % FBS, antibiotics and 1 % L-Glu, BxPC-3 in RPMI-1640 supplemented with 10 % FBS, antibiotics and 2 % L-Glu. For all experiments, medium with reduced content of FBS to the final concentration of 0.5 % was used. All statins in the study were used at 12 μM (corresponding to IC50 of simvastatin for MiaPaCa-2 cells after 24 h incubation ) diluted in methanol (vehicle) and hemin (methemalbumin) was prepared as previously described and used in the final concentration of 30 μM (pH = 7.4) .
Ethical approval for work on cell lines was not required by our Institution.
HMOX RNA interference (RNAi)
Pancreatic cancer cells were transfected with 10 pmol of HMOX1 esiRNA and 10 pmol of HMOX2 esiRNA (Sigma-Aldrich) per 5 x 103 seeded cells using the Lipofectamine RNAiMAX reagent (Life Technologies, Carlsbad, CA, USA) for 24 h in ATB-free DMEM medium. The esiRNA Universal control was used as negative control in all experiments. Data were expressed as % of esiRNA Universal control (Sigma-Aldrich).
Cell proliferation assay
For the cell proliferation assay, cells were seeded into 96 well (5–12.5 x 104 cells per ml according to the cell line) and kept at 37 °C and 5 % CO2. After 24 h, cells were treated with statins or/and hemin, followed by the MTT test (Sigma-Aldrich) as a general cell proliferation assay. As we experienced difficulties with hemin-treated samples using MTT test due to interfering effects of hemin, we further used the more sensitive CellTiter-Glo Luminescent Cell Viability Assay (Promega, Fichburg, WI, USA). Both tests were used according to the manufacturer's instructions. Results were expressed as % of controls.
HMOX activity measurement
Cells in plates were treated with statins and hemin. After 12 h, cells were washed twice with ice-cold phosphate buffer and finally collected into freshly added phosphate buffer and centrifuged. The pellet was resuspended in 150 μl of 0.1 M potassium phosphate buffer (pH = 7.4) and sonicated with an ultrasonic cell disruptor (Model XL2000, Misonics, Farmingdale, NY, USA). The protein concentration was assessed using the DC™ Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instruction. A total of 0.15 mg of protein was incubated for 15 min at 37 °C in CO-free septum-sealed vials containing 20 μl of 4.5 mM NADPH as previously described . The amount of CO generated by HMOX activity was quantified by gas chromatography with a reduction gas analyzer (Peak Laboratories LLC, Mountain View, CA, USA) and calculated as pmol CO/h/mg protein. Five μM PGJ2 was used as a positive control of heme regulation. Results were expressed as % of control.
Western blot analyses
For protein expression analyses, cells were transfected with esiRNA universal control or esiRNA HMOX1/2 as mentioned previously. After 24 h, cells were treated with 30 μm hemin for 20 h. Hemin treatment was used to upregulate HMOX1 protein expression to cumulate detectable levels of HMOX1 protein. Thirty μg of total protein were separated on 12 % polyacrylamide gel and then transferred to nitrocellulose membrane (Bio-Rad Laboratories). After blocking in Tween-PBS with 5 % milk (Sigma-Aldrich) for at least 1 h, membranes were incubated with HMOX1 antibody (1:1000; Thermo Fisher, Rockford, IL, USA), or β-actin (1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing, membranes were incubated with anti-mouse IgG-HRP (Abcam, Cambridge, UK) for 1 h. Immunocomplexes on the membranes were visualized with ECL Western Blotting Detection Reagents (Cell Signaling Technology).
Real-time PCR analysis of mRNA
Cells grown in plates were treated with statins, hemin or PGJ2. After 4 h, they were washed twice with ice-cold PBS and collected in the lysis buffer. Total cell RNA was isolated using Perfect Pure RNA Cultured Cell Kit (5Prime, Gaithersburg, MD, USA) and cDNA was generated using High Capacity RNA-to-cDNA Master Mix (Life Technologies) according to the manufacturer’s instructions. Real-time PCR for HMOX1 (OMIM *141250) and HMOX2 (OMIM *141251) was performed using the SYBR master mix (Life Technologies) according to the manufacturer’s instructions with optimized primers (Generi Biotech, Hradec Králové, Czech Republic). Results were calculated using the comparative Ct method with HPRT as a house-keeping gene and were expressed as % of control.
Markers of invasiveness
Cells were treated for 12, 24 and 48 h with individual statins. Total RNA was collected and cDNA generated as mentioned above. For real-time qPCR, cDNA corresponding to 10 ng of starting total RNA was diluted with water in 3.6 μl; 0.2 μl of the combined 10 μM forward and reverse primers were added and, finally, 3.8 μl of 2x iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) was added. The reaction was carried out using the Eco real-time PCR system (Illumina, San Diego, CA, USA) using three-step PCR. The relative mRNA expression levels of osteopontin (secreted phosphoprotein 1, SPP1, OMIM*166490) and sex-determining region Y-related HMG box 2 (SOX2, OMIM*184429) were calculated using the comparative Ct (ΔΔCt) method, with ribosomal phosphoprotein (P0, OMIM*180510) as a reference gene.
Sequences of primers used for real-time PCR: SPP1 forward, AGA CCT GAC ATC CAG TAC CCT, reverse - CAA CGG GGA TGG CCT TGT AT; SOX2 forward - AGG ACC AGC TGG GCT ACC CG, reverse - GCC AAG AGC CAT GCC AGG GG.
Apoptosis was quantified using the annexin V-FITC method, which detects phosphatidyl serine externalized in the early phases of apoptosis, in combination with propidium iodide (PI) staining. After exposure to cerivastatin and/or hemin, floating and attached cells were collected, washed with PBS, re-suspended in 100 μl binding buffer and incubated for 20 min at room temperature with 0.3 μl annexin V-FITC (Apronex, Vestec, Czech Republic). PI (10 μg/ml) was added directly before flow cytometry analysis (BD FASC Calibur, BD, Franklin Lakes, NJ, USA). Annexin V positive (An+) and PI negative (PI-) are cells in early apoptosis, An + and/or PI positive (PI+) are cells in late apoptosis or post-apoptotic necrosis.
Reactive oxygen species (ROS) generation
For assessment of ROS generation, dichlordihydrofluorescein diacetate (H2DCFDA) (Life Technologies) was used. After treatment, cells were washed and exposed to 10 μM H2DCFDA in 37 °C for 20 min. Cells were then washed, lysed and the fluorescent signal at 492/520 (Ex/Em) was evaluated in 100 μl aliquots. Total fluorescence was related to protein concentration. Results were expressed as % of control.
Ras protein translocation assay
PA-TU-8902 cells were seeded in dishes with glass bottom 6 h before transfection by pEGFP-KrasWT (GFP – green fluorescent protein, WT-wild type) plasmid prepared as described previously . Transfection was carried out using FuGene HD according to the manufacturer’s instructions. Cerivastatin (12 μM), pravastatin (12 μM) and hemin (30 μM) were added 12 h post transfection and the cells incubated with the agents for 24 h. Intracellular localization of the GFP-K-Ras protein was visualized by confocal microscopy, using a spinning disk confocal microscope (Olympus, Tokyo, Japan; Andor, Belfast, UK) equipped with solid state laser (488 nm for continual excitation). Emission was collected through a single-band filter (BrightLine® FF01-525 nm, Semrock Inc., NY, USA). The images were obtained and analyzed with the iQ2 software (Andor).
All data were expressed as mean ± SD. For normally distributed datasets, one-way ANOVA with post-hoc Holm-Sidak test for multiple comparisons was used for analysis. For non-normally distributed data and small datasets (n ≤ 6), Mann–Whitney rank sum test and Kruskal-Wallis ANOVA with Dunn’s test for multiple comparisons were used. P-values less than 0.05 were considered statistically significant.
All datasets of the results discussed in the manuscript are available on request.