Drugs and chemicals:
PNU-74654 was obtained from Cayman (Michigan, USA) while gemcitabine was received as a gift from Eli-Lilly (Indianapolis, IN) which were dissolved in dimethyl sulfoxide (DMSO) or aseptic water, DMEM (Dulbecco’s Modified Eagle Medium), FBS (fetal calf serum), PCN (c = 50 IU/ml) and STREPTO-Fatol (c = 50 µg/ml) were procured from Sigma or Gibco Chemical company.
Cell culture:
The MiaPaCa-2 cell line was originally acquired from ATCC (American Type Culture Collection). The cells were cultured in DMEM containing heat-inactivated FBS (10%) and streptomycin/penicillin (1%), after that, incubated (at 37°C in 5% CO2 atmosphere) and finally, harvested with trypsin-EDTA [25].
Growth inhibition studies:
The MTT assay was employed to determine the effects of PNU-74654, gemcitabine, and their combination on cell growth and proliferation of PC cells, which was performed before and after 24 hr of treatment. Thereafter, PNU-74654 (1-1000 nM), Gemcitabine (0.001-500 nM), and thier combination were used to treat MiaPaCa-2 cells for 24 hours at an established ratio according to IC50 of the drugs. After that, the plates were then organized furthermore for MTT assay as reported previously [26].
Assessment of synergistic and/or antagonistic interactions with gemcitabine:
The analysis of median drug effect was determined based on bio-activity, including synergistic/antagonistic interaction of PNU-74654 and gemcitabine against PC cells as described previously [27]. Briefly, cell growth inhibition is measured by the combination index (CI) which was used to calculate drug effects in both solo and combinatorial forms. CalcuSyn software was applied to analyze the results (Biosoft, Oxford, UK).
Multicellular spheroids:
To form the spheroids, 105 cells/ml were seeded in DMEM/F12 + GlutaMAX-I (with ratio of 1:1) in 96-well plates. After 10 days, important factors including the cell attachment, cell growth, and cytotoxicity of the formulated drugs were measured under DMI300B inverted microscope (Leica, Wetzlar, Germany). Spheroid volume (V) was calculated by the mean perpendicular diameters using D= (Dmax + Dmin)/2 as shown in the following formula: V= (4/3) ×π (D/2)3 [15].
In vitro cell migration and invasion assays:
Cell invasion assays were used to determine the interactions of PC cells and extracellular matrix (ECM). Trans-well chambers with polycarbonate membranes and 8-µm pores were implemented for this goal [27, 28]. To further proceed, the trans-well filters should be coated by collagen I solution (applying 100 µl of 0.1 mg/mL solution). In short, 105 cells were plated and in serum free media, they were incubated by PNU-74654 at 5xIC50. Paraformaldehyde solution as a fixative agent was applied to preserve the Migratory cells in polycarbonate membranes. In order to find the sum of Migrartory cells, they were counted after imaging with Giemsa stain.
Quantitative reverse transcription-polymerase chain reaction (RT-qPCR):
RT-qPCR was implemented due to the manufacturer’s protocol initially and after the treatment procedure by PNU-74654, hence, the overal RNAs obtained from the PC cells could be determined at 5XIC50 of PNU-74654 utilizing the RNXPLOS (CinaColon, Tehran, Iran). cDNA preparation was done by applying cDNA Synthesis Kit (CinaColon, Tehran, Iran). RT-qPCR for CyclinD1, and E-cadherin mRNA (Macrogene Ink., South Korea) was executed by ABI-instrument. The normalization of the gene expression data were fulfilled by implementing GAPDH as described previously [29].
Western blot (WB):
WB analyses were undertaken as it stated preveously [30]. Briefly, a 10% SDS-polyacrylamide gel was used to isolate 40 µg of proteins, which was later resettled onto Immobilon-FL PVDF Membrane. After the incubation of the membrane with anti-CyclinD1 (obtain from rabbit, Scbt) and anti-β-actin (1:10000; Sigma–Aldrich)., it was diluted with 1:1000 ratio in the blocking solution. The goat anti-rabbit secondary antibody was originally obtained from Westburg (1:10000, Leusden, Netherlands). In the next step, the membrane was visualized and detected, utilizing chemiluminescent methods [31, 32].
Statistical analysis:
All data were obtain by averaging triplicate measurements and replicated at least twice to be ensured of the accuracy of the results. The data analyses was performed by Student’s t-test in parallel with Tukey's multiple comparison test and expressed as mean values ± SD. IBM SPSS statistics was employed to analyze data statistically. All data analysis rely on statistical significance of P < 0.05 [33].