Abstract
Pancreatic cancer is an aggressive malignancy with poor prognosis and the efficacy of chemotherapy is limited. KML001 (sodium meta-arsenite) has been demonstrated to have anticancer activity against some solid cancer cells. The aim of the present study was to determine the effect of KML001 on cell proliferation, migration, and invasion of pancreatic cancer cells. The Dojindo Cell Counting Kit-8 assay was used to determine the inhibition of pancreatic cancer cell proliferation by drugs. Cell migration and invasion were examined using 24-well inserts and Matrigel™-coated invasion chambers. The activity of nuclear factor-kappa B (NF-κB) p65, vascular endothelial growth factor-C (VEGF-C), and matrix metalloproteinase-9 (MMP-9) were measured by enzyme-linked immunosorbent assay (ELISA). KML001 inhibited the proliferation of pancreatic cancer cells in a dose- and time-dependent manner. KML001 also inhibited the migration and invasion of pancreatic cancer cells in a dose-dependent manner. KML001 significantly decreased NF-κB p65 and VEGF-C activities in the pancreatic cancer cells. KML001 inhibited cell proliferation, migration, and invasion in pancreatic cancer cells. Suppression of NF-κB and VEGF-C activation may partly be associated with the anticancer activity of KML001. These results suggest that KML001 could be a novel potential therapeutic agent for treatment of pancreatic cancer.
Abbreviations: NF-κB, nuclear factor kappa-light-chain-enhancer of activated B-cells; ELISA, enzyme-linked immunosorbent assay; VEGF-C, Vascular endothelial growth factor-C; MMP-9, matrix metalloproteinase-9; hTERT, human telomerase reverse transcriptase; TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand; CCK-8, Cell Counting Kit-8; EGF, endothelial growth factor; BPE, bovine pituitary extract; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MEM, minimum essential medium; PBS, phosphate buffered saline; SEM, standard error of the mean; IκB, inhibitor of κB; ROS, reactive oxygen species.
Pancreatic cancer is one of the most aggressive malignancies with poor prognosis (1). In the United States, it is the fourth-leading cause of cancer-related death and the 5-year survival rate is only 6% (2). The poor prognosis of pancreatic cancer is caused by its tendency for late presentation, aggressive local invasion, early metastases, and poor response to chemotherapy (3). Since only 10-15% of patients are candidates for surgery and the recurrence rate is high even with radical surgery, postoperative chemotherapy is necessary (4). Gemcitabine has been widely used as a standard systemic chemotherapeutic agent for advanced pancreatic cancer (5). However, it induces only a 5.4% partial response rate and the progression-free survival ranges from 0.9 to 4.2 months (5, 6). Thus, novel agents are required to treat pancreatic cancer.
Arsenic has been used as a therapeutic agent for more than 2,400 years (7). Arsenic trioxide (As2O3), a traditional Chinese medicine, is the most widely used and studied of the arsenic-based cancer drugs (8). Arsenic trioxide has a therapeutic effect on refractory or relapsed acute promyelocytic leukemia (9). Arsenic trioxide also induces apoptosis in a variety of human solid tumor cell lines, including neuroblastoma, esophagus, stomach, prostate, and ovary carcinomas (10-13).
KML001 (sodium meta-arsenite, NaAsO2) is an orally-bioavailable, water-soluble, trivalent arsenical that has entered phase I/II clinical trials in prostate cancer (14). Because arsenic trioxide is poorly water-soluble and must be dissolved with sodium hydroxide, arsenic trioxide and KML001 are identical in solution (14). KML001 inhibits cell growth and induces apoptosis in leukemia cell lines. The induction of apoptosis was accompanied by down-regulation of human telomerase reverse transcriptase (hTERT) mRNA. There were no major differences encountered with respect to the effects of two arsenic compounds, arsenic trioxide and KML001, on gene expression of apoptosis and cellular proliferation related genes (15). KML001 cytotoxic activity is associated with its binding to telomeric sequences and telomere erosion in prostate cancer cells (14). Sequential treatment by ionizing radiation and sodium arsenite accelerated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-medicated apoptosis of human melanoma cells (16). However, few studies have been conducted to demonstrate the anticancer activity of KML001 in pancreatic cancer cells.
In the present study, we investigated the anticancer activity of KML001 in pancreatic cancer cells. Our results demonstrated that KML001 inhibited cell proliferation, migration, and invasion of pancreatic cancer cells. KML001 also suppressed NF-κB p65 and VEGF-C activation in pancreatic cancer cells.
Materials and Methods
Cell lines and reagents. The human pancreatic duct cell HPDE-4 was obtained from the Applied Biological Meterials Inc. (Richmond, BC, Canada). Cells were cultured in keratinocyte serum-free medium supplemented with endothelial growth factor (EGF), bovine pituitary extract (BPE, Invitrogen, Grand Island, NY, USA), and 1% antibiotic/antimycotic mixture (Gibco, Rockville, MD) at 37°C in a humidified incubator containing an atmosphere of 5% carbon dioxide/95% air. The human pancreatic carcinoma cell line MIA PaCa-2 was obtained from the American Type Culture Collection (Manassas, VA, USA). MIA PaCa-2 cells were sourced from Caucasian male 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 stocks prepared in phosphate buffered saline (PBS), and aliquots stored at −20°C. Stock solutions were stable for more than 1 year. Working concentrations were freshly prepared daily by diluting the stock with DMEM.
Determination of cell proliferation. Cell proliferation was determined by the Dojindo Cell Counting Kit-8 (CCK-8; Dojindo, Gaithersburg, MD, USA). This assay is based on the cleavage of the tetrazolium salt WST-8 by mitochondrial dehydrogenase in viable cells (17). Cells were seeded in 96-well plates at 5×103 cells in 100 μl of culture medium and allowed to adhere overnight. After cells were treated with 0.5, 10, 20 or 40 μM KML001 for 24, 48 and 72 h, 10 μl of tetrazolium substrate were incubated at 37°C for 1 h. The absorbance at 450 nm was measured with a thermomax microplate reader (TECAN, Grodig, Austria). The cell proliferation index was calculated by the following formula: Cell proliferation index (%)=(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 (Becton Dickinson Labware, Franklin Lakes, NJ, USA) with 8-μm pores according to the manufacturer's protocol. 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 Scan Scope 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. Invasion studies were performed similar to migration assay with the exception that membranes were Matrigel™-coated invasion chambers (BD Biosciences Bedford, MA, USA) prehydrated in serum-free medium.
Enzyme-linked immunosorbent assay. Enzyme-linked immuno-sorbent assay (ELISA) kits for measurements of VEGF-C and matrix metalloproteinase-9 (MMP-9) were from R&D Systems (Minneapolis, MN, USA). MiaPaCa-2 cells were treated with 5, 10, 15, or 20 μM KML001 for 24 h. After the treatment, nuclear protein was extracted by using a Nuclear Extract Kit (Active Motif, Carlsbad, Ca, USA) according to the instructions from the manufacturer (18). DNA binding activity of NF-κB p65 was evaluated using an ELISA kit (TransAM NF-κB Chemi; Active Motif) on nuclear protein extract. TransAM NF-κB kits contain a 96-well plate to which an oligonucleotide containing the NF-κB consensus site (5’-GGGACTTTCC-3’) has been immobilized. The active form of NF-κB contained in nuclear extract specifically binds to this oligonucleotide. The primary antibodies used to detect NF-κB recognize an epitope on p65 that is accessible only when NF-κB is activated and bound to its target DNA. A horseradish peroxidase-conjugated secondary antibody was added according to the instructions from the manufacturer (21). The culture medium was collected and used to determine the secretion levels of VEGF-C and matrix metalloproteinase -9 (MMP-9) using ELISA.
Densitometric and statistical analysis. All statistical analyses were performed using PASW Statistics version 18.0 software (SPSS, Cary, NC, USA). Results are expressed as the mean±standard error of the mean (SEM). Statistical analysis of data collected for cell proliferation assay was performed using the repeated measures ANOVA test. Group differences were statistically analyzed using the Kruskall-Wallis test. The post hoc analysis for multiples was compared with Tukey test using ranks. A p-value <0.05 was considered statistically significant. Each assay was repeated in triplicate to check the validity of the results.
Results
KML001 inhibited cell proliferation of pancreatic cancer cells. To determine the effect of KML001 on cell proliferation of normal pancreatic cells and pancreatic cancer cells, the growth of HPDE-4 and MIA PaCa-2 cell lines was tested using the CCK-8 assay after treatment with KML001. KML001 significantly inhibited cell proliferation of HPDE-4 and MIA PaCa-2 cells in a dose-dependent (p<0.001 and p<0.001, respectively) and time-dependent manner (p<0.001 and p<0.001, respectively) (Figure 1A and 1B). Since higher than 40 μM concentrations caused cell necrosis, a non-cytotoxic dosage of KML001 was used for subsequent experiments.
KML001 inhibited cell migration and invasion of pancreatic cancer cells. To determine whether KML001 inhibits cell migration or invasion of cancer cells, we examined MIA PaCa-2 cells with a cell migration and invasion assay after treatment with KML001 for 24 h. The results showed that treatment with KML001 reduced migrative and invasive ability of MIA PaCa-2 cells in a dose-dependent manner (p=0.037 and p=0.036, respectively) (Figure 2A and 2B).
KML001 suppressed NF-κB p65 and VEGF-C activities in pancreatic cancer cells. NF-κB is an important component in cell proliferation, migration, invasion, angiogenesis and metastatic process in pancreatic cancer (19). The primary NF-κB member responsible for transcriptional activation of target genes is the p64 (RelA) subunit (20). To determine whether the anticancer activity of KML001 correlated with NF-κB activity, we examined NF-κB p65 activity in nuclear extract of MIA PaCa-2 cells after treatment with KML001. Treatment with KML001 significantly down-regulated NF-κB p65 activation in a dose-dependent manner (p=0.018) (Figure 3). VEGF-C has been associated with lymphangiogenesis, lymph node metastasis and angiogenesis (21, 22). We next assessed the level of VEGF-C activity and found that KML001 significantly suppressed VEGF-C activity in a dose dependent manner (p=0.029). MMP-9 has been associated with the progression and invasion of tumors (23). To investigate whether the anti-migratory and anti-invasive activities of KML001 are associated with MMP-9 activity, we examined the levels of MMP-9 activity. Our results showed that MMP-9 activity was decreased as concentration of KML001 increased. This difference however, was not statistically significant (p=0.291).
Discussion
KML001 has been demonstrated to have a anticancer activity in leukemia and certain solid cancer cells including prostate cancer cells (14-16,24). However, the antitumor effect of KML001 on pancreatic cancer cells has not been investigated. Our results showed, for the first time, that KML001 inhibited cell proliferation, migration and invasion in pancreatic cancer cells. The mechanism by which KML001 exerts its anticancer activity in pancreatic cancer cells may partly involve suppression of NF-κB p65 and VEGF-C activities.
KML001 inhibits cell proliferation in pancreatic cancer cells. HPDE-4 and MIA PaCa-2 cells were treated with 0.5, 10, 20 and 40 μM KML001 or PBS for the indicated time periods prior to estimation of cell number using the CCK-8 assay. The experiment was performed in triplicate. KML001 significantly inhibited cell proliferation of HPDE-4 (A) and MIA PaCa-2 (B) cells in a dose- dependent manner (p<0.001 and p<0.001, respectively). PBS, Control; KML001, sodium meta-arsenite. Bars, standard error of the mean (SEM).
KML001 inhibits cell migration and invasion in pancreatic cancer cells. MIA PaCa-2 cells were treated with 5, 10, 15 and 20 μM KML001 for 48 h. Cells were placed in serum-free culture media 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 value of KML001-treated cells was normalized to the PBS-treated cells and expressed as the percentages of the control, which was assumed to be 100%. The experiment was performed in triplicate. KML001 significantly inhibited cell migration and invasion of MIA PaCa-2 cells in a dose-dependent manner (p=0.037 and p=0.036, respectively). PBS, Control; KML001, sodium meta-arsenite. Columns, mean of three independent experiments; bars, SEM.
A prior study reported that sodium meta-arsenite effectively induced cellular toxicity by inhibiting telomerase and reverse transcriptase activity, and down-regulating transcript levels in cancer cells with shorter telomere lengths (24). In prostate cancer cells with short telomeres, KML001 causes telomere-associated DNA damage and rapid telomere erosion (14). KML001 exhibited cytotoxic activity in tumor cells with short telomeres, but telomerase is not directly inhibited by KML001 (14). We found that KML001 inhibited proliferation of pancreatic cancer cells in a dose- and time-dependent manner. The telomeres of the human pancreatic cancer cell line MIA PaCa-2 are short compared to normal human fibroblasts (25). Thus, KML001 might have the anticancer activity in MIA PaCa-2 cells similar to those in prostate cancer cells. Our results also showed that KML001 inhibited cell proliferation of the normal cell line HPDE-4 and suggested that the cytotoxic effect of KML001 was not specific against pancreatic cancer cells.
KML001 suppressed NF-κB p65 and VEGF-C activities in pancreatic cancer cells. (A) Nuclear extracts were prepared from control or KML001 treated MIA PaCa-2 cells and subjected to analysis for NF-κB p65 activity as measured by the Active Motif enzyme-linked immunosorbent assay (ELISA). The culture media of control or KML001-treated MIA PaCa-2 cells were harvested for the detection of VEGF-C (B) and MMP-9 (C) using ELISA. The relative-fold change of NF-κB p65, VEGF-C, or MMP-9 activity in KML001 treated cells was normalized against control cells. Values in control cells were arbitrarily set to 1. The experiment was performed in triplicate. Treatment with KML001 significantly down-regulated NF-κB p65 activation in a dose-dependent manner (p=0.018, p=0.029, and p=0.291). PBS, Control; KML001, sodium meta-arsenite. Columns, mean of three independent experiments; bars, SEM.
The migration and invasion of tumor cells through the extracellular matrix is a crucial step in tumor dissemination and metastasis (8). Information of the effects of KML001 on migration and invasion of cancer cells is limited. Our results showed that KML001 significantly inhibited migration and invasion of pancreatic cancer cells in a dose-dependent manner.
The underlying mechanisms of anticancer activity of KML001 have yet to be elucidated. The NF-κB signaling pathway is involved in cancer cell proliferation, migration, invasion, and the metastatic process (22). Increased NF-κB activity can promote growth and tumorigenesis, inhibit apoptosis, promote angiogenesis, promote invasion and metastasis, and promote chemoresistance in pancreatic cancer (26). Thus, different NF-κB-inhibiting drugs can improve the treatment of pancreatic cancer (26). Arsenite inhibit NF-κB-mediated gene transcription by specifically blocking inhibitor of κB (IκB) kinase activity and IκBα phosphorylation and degradation (27). In human fibrosarcoma cells, arsenic trioxide inhibits cell invasion through generation of reactive oxygen species (ROS) and suppression of the NF-κB activity (28). We found that NF-κB p65 activation was significantly decreased in a dose-dependent manner in pancreatic cancer cells treated with KML001. These results have suggested that KML001 inhibit proliferation and invasion of pancreatic cancer cells partly through down-regulation of NF-κB activation.
VEGF-C has been associated with tumor growth, lymphangiogenesis and angiogenesis in many cancers including gallbladder cancer and esophageal cancer (29, 30). VEGF-C produced by tumor cells in the marginal portion of the tumors significantly associated with lymphatic metastasis in pancreatic cancer (31). Our results showed that KML001 significantly suppressed VEGF-C activity in KML001-treated pancreatic cancer cells. Thus, it suggested that the anti-migratory and anti-invasive activities of KML001 may be mediated by the suppression of VEGF-C secretion.
MMP-9 is overexpressed in various human cancer tissues and is associated with tumor initiation, invasion, angiogenesis and metastasis (32). MMP-9 has been significantly associated with nodal metastasis and nervous metastasis in pancreatic cancer tissue (31). In this study, KML001 slightly decreased the secretion of MMP-9 in KML001-treated pancreatic cancer cells but the difference was not statistically significant.
In conclusion, our results showed that KML001 inhibited cell proliferation, migration, and invasion of pancreatic cancer cells. Suppression of NF-κB and VEGF-C activation may partly be associated with the anticancer activity of KML001. Our findings suggest that KML001 appears as a potential complementary agent for treatment of pancreatic cancer. Further investigation is required to confirm the mechanism and anticancer activity of KML001 in animal models and in human pancreatic cancer.
Acknowledgements
This work was supported by the Samsung Biomedical Research Institute grant, # SBRI C-B1-118-2.
Footnotes
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* MH Yang and HT Kim equally contributed to the study and are both first authors.
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Conflicts of Interest
None.
- Received April 2, 2014.
- Revision received May 28, 2014.
- Accepted May 29, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
