KML001 Enhances Anticancer Activity of Gemcitabine Against Pancreatic Cancer Cells

Materials and Methods

Cell lines and reagents. The human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were purchased from the American Type Culture Collection (Manassas, VA, USA). Both MIA PaCa-2 and PANC-1 cells were obtained from Caucasian males diagnosed with pancreatic carcinoma. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA), 1 mM L-glutamine, 1 mM minimum essential medium (MEM), 100 μg/ml streptomycin, and 100 U/ml penicillin. KML001 (Kominox®) was from Komipharm International (Shiheung, Korea), with 20 mmol/l stock prepared in phosphate-buffered saline (PBS), and aliquots stored at −20°C. Stock solutions were stable for over one year. Working concentrations were prepared daily by diluting the stock with DMEM.

Determination of cell proliferation. Dojindo Cell Counting Kit-8 (CCK-8; Dojindo, Gaithersburg, MD, USA) was used to assess cell proliferation. This assay is based on the cleavage of the water-soluble tetrazolium salt WST-8 by mitochondrial dehydrogenase in viable cells (9). Cells were seeded in 96-well plates at 5×10³ cells in 100 μl of culture medium and allowed to adhere overnight. Cells were then treated with 1 μM gemcitabine either alone or in combination with 20 μM KML001 (herein referred to as the combination treatment) for 24, 48, or 72 h, after which tetrazolium substrate (10 μl) was added and cells were incubated at 37°C for 1 h. Considering the potential for cytotoxicity, a KML001 concentration of 20 μM was used for combination treatment because our prior studies have shown that exposure to this concentration exerts a suboptimal inhibition of cell proliferation (8). The absorbance at 450 nm was assessed with a thermomax microplate reader (Tecan, Grödig, Austria). The cell proliferation index (%) was calculated using the following formula: (mean absorbance in all wells of the treatment group) ÷ (mean absorbance in all wells of the control group) ×100.

Cell migration and invasion assay. Cell migration was assessed using 24-well inserts (BD, Franklin Lakes, NJ, USA) with 8-μm pores according to the manufacturer's protocol. MIA PaCa-2 and PANC-1 cells were treated with gemcitabine (1 μM) alone or in combination with KML001 (20 μM) for 48 h. Cells were placed in serum-free culture medium and plated into the upper compartment of a migration chamber. After 24 h of incubation, cells in the upper chamber were removed, fixed in ice-cold methanol, and stained with Wright-Giemsa solution (Polysciences, Warrington, PA, USA). Digital images were obtained from the membranes, and cell areas were selected using the ScanScope CS system (Aperio Technologies, Vista, CA, USA). Migrating cells on each membrane were quantified in five randomly selected fields at ×40 magnification in each membrane, and the average value was defined as a migration or invasion index on three independent membranes. Methods for invasion studies were similar to those of the migration assay with the exception that membranes were Matrigel™-coated invasion chambers (BD Biosciences, Bedford, MA, USA) pre-hydrated in serum-free medium. The relative-fold change calculated for migration and invasion of cells treated with gemcitabine alone or in combination with KML001 was normalized to that of PBS-treated cells and expressed as a percentage of the control, which was assumed to be 100%.

Enzyme-linked immunosorbent assay (ELISA). ELISA kits for measurement of EGFR, MMP2, and VEGFC were purchased from R&D Systems (Minneapolis, MN, USA). MIA PaCa-2 and PANC-1 cells were treated with 1 μM gemcitabine alone or in combination with 20 μM KML001 (combination treatment) for 24 h. After treatment, nuclear protein was extracted using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the instructions from the manufacturer (10). DNA binding activity of NFκB p65 was evaluated using an ELISA kit (TransAM NF-κB Chemi; Active Motif) on nuclear protein extract. The TransAM NFκB kit contains a 96-well plate in which an oligonucleotide containing the NFκB consensus site (5’-GGGACTTTCC-3’) has been immobilized. The active form of NFκB contained in nuclear extract specifically binds to this oligonucleotide. The primary antibodies used to detect NF-κB recognize an epitope on subunit p65 that is accessible only when NFκB is activated and bound to its target DNA. A horseradish peroxidase-conjugated secondary antibody was added according to the manufacturer's instructions (10). The culture medium was collected and used to determine secretion levels of MMP2, EGFR, and VEGFC using ELISA. The relative-fold change of NF-κB p65, EGFR, MMP2, and VEGFC activity in cells treated with gemcitabine alone or in combination with KML001 was normalized against the level of control cells. Values in control cells were arbitrarily set to one.

Densitometric and statistical analyses. To assure the validity of the results, each assay was performed in triplicate. Results from three independent experiments were expressed as the mean±standard error of the mean (SEM). Cell proliferation indices for each group were compared using the repeated-measures ANOVA test. Group differences were analyzed using the Kruskall–Wallis test. The post hoc analysis for multiples was compared with Tukey test using ranks. A p-value of less than 0.05 was considered statistically significant. Statistical analyses were performed using PASW Statistics v. 18.0 (SPSS, Cary, NC, USA).

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Figure 1.

KML001 enhances the antiproliferative effects of gemcitabine in MIA PaCa-2 cells. MIA PaCa-2 and PANC-1 cells were treated with gemcitabine (1 μM) alone and in combination with KML001 (20 μM) for the indicated time periods prior to estimation of the cell number using the Dojindo Cell Counting Kit-8 (CCK-8) assay. There was a significant reduction in cell proliferation in MIA PaCa-2 cells treated with a combination of gemcitabine and KML001 compared to cells treated with gemcitabine alone (p<0.001). The experiment was performed in triplicate. PBS, Control treated with phosphate-buffered saline; Gem, gemcitabine; KML001, sodium meta-arsenite. Data are the mean±standard error of the mean (SEM) of three independent experiments.

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Figure 2.

KML001 enhances the anti-migrative and anti-invasive effects of gemcitabine on pancreatic cancer cells. MIA PaCa-2 and PANC-1 cells were treated with gemcitabine (1 μM) alone and in combination with KML001 (20 μM) for 48 h. Cells were placed in serum-free culture medium and plated into the upper compartment of either a migration or invasion chamber. After 24 h, cells in the upper chamber were removed and cells that had migrated (A) or invaded (B) into the lower surface of the membrane were fixed and stained with Wright-Giemsa. The relative-fold change calculated for migration and invasion of cells treated with gemcitabine alone or in combination with KML001 was normalized to that of the phosphate-buffered saline (PBS)-treated cells and expressed as a percentage of the control, which was assumed to be 100%. Cell migration was significantly reduced in both cell lines. Cell invasion was significantly reduced in MIA PaCa-2 cells. The experiment was performed in triplicate. PBS, Control treated with phosphate-buffered saline; Gem, gemcitabine; KML001, sodium meta-arsenite. Data are the mean±SEM of three independent experiments. *p<0.005.