Cell culture
PANC-1 (CRL-1469™) and MIA PaCa-2 (CRL-1420™) human pancreatic cancer cell lines obtained from ATCC. Cells were grown in high glucose DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1% non-essential amino acids and 1% sodium pyruvate at 37°C, 5% CO2 and 95% humidity.
Cytotoxicity assay
The cytotoxic effect of tomentosin on pancreatic cancer cells was evaluated by XTT analysis. PANC-1 and MIA PaCa-2 cells were seeded at 2x103 cell in 96-well plates. After 24 hours, varying concentrations of tomentosin (0-300 µM) were treated to cells for 24, 48 and 72 hours. At the end of the specified hours, XTT solution (Biological Industries, 20-300-1000) was added to wells and after approximately 4 hours, absorbance value of each well was read in a microplate reader (BioTek Epoch) at 450 nm wavelength and 630 nm reference range. % cell viability was calculated and IC50 doses of tomentosin in PANC-1 and MIA PaCa-2 cells were determined with GraphPad Prism 8.0.2 software.
Apoptosis assay
After tomentosin treatment, % apoptosis rate was determined by a flow cytometry using FITC Annexin V apoptosis kit (BioLegend, 640.922). Cell suspensions (1x106 cells/ml) were prepared from control and dose group cells with Dulbecco’s PBS. And, 5 µl of FITC Annexin V and 5 µl of 7-ADD dye were added to 100 µl of cell suspensions. After incubation at room temperature for 15 minutes in the dark, 400 µl of Annexin V binding buffer was added to cell suspensions and % apoptotic cells were determined in a flow cytometer (BD FACSAriaTM III).
Wound healing assay
The effect of tomentosin on cell migration in pancreatic cancer cells was determined by wound healing assay. For this, an artificial wound was created with the help of a yellow pipette tip in control and dose group cells, which were seeded in 24-well plates (1x105 cells/well). Cell migration was evaluated by imaging under the microscope at 24, 48 and 72 hours. Wound areas were measured using ImageJ software and % rate of wound closure was determined.
Invasion assay
The effect of tomentosin on invasion of pancreatic cancer cells was determined using the 24-well Corning BioCoatMatrigel Invasion Chamber (Corning, 354480) Cell suspensions (2.5x105cells/ml) were prepared with serum-free medium from control and dose group cells. 500 µl of cell suspension was added to upper chamber and medium containing serum was added to lower chamber. After 22 hours at 37°C, non-invasive cells were removed from upper surface of membrane with a swab, while invasive cells that migrated to lower part of membrane were fixed with 100% methanol and stained with 0.1% crystal violet. After washing, three random fields were counted under a light microscope.
Colony assay
For the colony assay, control and dose group cells, which were seeded in 6-well plates (2x103), were cultured for 10 days and medium changed every 2 days. At the end of 10 days, washing was done with PBS and 100% methanol was added to wells for fixation. After 10 minutes, methanol was removed and 5% crystal violet was added to wells. Colonies were counted after washing.
Measurement of mitochondrial membrane potential (MMP)
The effect of tomentosin on MMP in pancreatic cancer cells was performed according to manufacturer's instructions (Abcam, ab113850). Briefly, control and dose group cells (2x104 cells/well) were washed with 1X dilution buffer in 96-well plates. Then, cells were stained with dilution buffer containing JC-1 dye. After washing with 1X dilution buffer, 100 μl of medium was added to wells. The fluorescence of JC-1 was measured at Ex/Em = 475/530 nm for green fluorescence and Ex/Em = 535/590 nm for red fluorescence in a fluorescent microplate reader (BioTek Epoch).
Intracellular reactive oxygen species (ROS) detection
After tomentosin treatment, intracellular ROS level was determined using ROS red dye, which reacts with cellular ROS to produce a red fluorescent signal (Abcam, ab186027). For this, 100 µl of ROS Red Working Solution was added to control and dose group cells (2x104 cells/well) in 96-well plates and plates were incubated for 1 hour at 37°C. After incubation, 20 µL of Assay buffer was added to wells. After 15 minutes, the red fluorescence measured in a fluorescent microplate reader (Ex:520 nm/Em:605 nm) (BioTek Epoch).
RNA extraction and qRT-PCR analysis
qRT-PCR analysis was performed to determine the effect of tomentosin on expression levels of apoptosis-related genes in pancreatic cancer cells. Firstly, total RNA isolation was performed using RiboEx (GeneAll, 301-001) from control and dose group cells seeded in 6-well plates (3x105 cells/well). Then, cDNA synthesis was performed from total RNAs in accordance with the manufacturer's instructions (Bio-Rad, 170-8891). Primers for target genes and ACTB used in qPCR analysis were designed with IDT PrimerQuest (https://eu. idtdna. com/ Prime rquest/ Home/ Index). 5 μl of BrightGreen 2X qPCR MasterMix, 5 pMol forward primer, 5 pMol reverse primer and 2 μl cDNA were used in qRT-PCR. The PCR protocol, consisting of 10 minutes at 95˚C, 15 seconds at 95˚C and 60 seconds at 60˚C, was carried out in Bio-Rad CFX ConnectTM Real-Time System as 40 cycles. The analysis was performed with 2–ΔΔCT method using "RT2 Profiler ™ PCR Array Data Analysis" software.
Wetern blot analysis
The effect of tomentosin on Caspase-3 and Caspase-9 protein levels in pancreatic cancer cells was evaluated by western blot analysis. Protein isolation was performed using RIPA solution (Cell signaling, 9806) from control and dose group cells, which were seeded in 6-well plates (5x105 cells/well). 50 µg of protein was separated in 8-15% SDS-PAGE and transferred to PVDF membrane. After blocking with 5% non-fat milk, membrane was incubated with anti-Caspase-3 (StJohn’s Laboratory, STJ92021, 1:500), anti-Caspase-9 (StJohn’s Laboratory, STJ92027, 1:500) and anti-β-Actin (Bioss Antibodies, BS-0061R, 1:1000) primary antibodies at +4°C overnight. At the end of the incubation, washing was done with TBST and membrane was treated with secondary antibody (Jackson Immuno Research, 211-035-109) for 2 hours. After washing, chemiluminescence solution (Biovision, K820-500) was added to membrane for imaging and chemiluminescence was detected on Azure Biosystems™ c280. ImageJ software was used for analysis.
Statistical analysis
All experiments were performed in triplicate and data were presented as mean ± standard deviation. Comparison between control and dose groups was performed with GraphPad Prism version 8.0.2 using Student's t test. In all analyzes, p < 0.05 was considered statistically significant.