Increase of gap junction activities in SW480 human colorectal cancer cells
© Bigelow and Nguyen; licensee BioMed Central Ltd. 2014
Received: 18 March 2014
Accepted: 30 June 2014
Published: 9 July 2014
Colorectal cancer is one of the most common cancers in the United States with an early detection rate of only 39%. Colorectal cancer cells along with other cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, and cell signaling. Since the regulation of gap junctions is lost in colorectal cancer cells, the goal of this study is to determine the effect of GJIC restoration in colorectal cancer cells.
Gap Junction Activity Assay and protein analysis were performed to evaluate the effects of overexpression of connexin 43 (Cx43) and treatment of PQ1, a small molecule, on GJIC.
Overexpression of Cx43 in SW480 colorectal cancer cells causes a 6-fold increase of gap junction activity compared to control. This suggests that overexpressing Cx43 can restore GJIC. Furthermore, small molecule like PQ1 directly targeting gap junction channel was used to increase GJIC. Gap junction enhancers, PQ1, at 200 nM showed a 4-fold increase of gap junction activity in SW480 cells. A shift from the P0 to the P2 isoform of Cx43 was seen after 1 hour treatment with 200 nM PQ1.
Overexpression of Cx43 and treatment of PQ1 can directly increase gap junction activity. The findings provide an important implication in which restoration of gap junction activity can be targeted for drug development.
KeywordsGap junction intercellular communication PQ1 Kinase activity
Colorectal cancer is the third most common cancer and the third leading cause of cancer related death in the United States [1, 2]. In 2013, approximately 136,830 people were diagnosed with colorectal cancer. Approximately 50,310 deaths in the past year were due to colorectal cancer . Thus, understanding the etiology of colorectal cancer is critical for the treatment of the disease.
GJIC has been shown to be decreased in cancerous cells and at tumor borders [4, 5]. Gap junctions are intercellular channels made of the protein known as connexin. There are 21 isoforms of connexin . Six connexins make up a connexon; two connexons, each on an adjacent cell, interact and form a gap junction. Gap junctions mediate the direct passage of small molecules (<1000 Da) from one cell to the next . They are involved in the regulation of the cell cycle, cell differentiation, and cell signaling . The life cycle of gap junctions is regulated by phosphorylation events [9–11].
Regulation of Cx43 in GJ formation has been shown to be due to phosphorylation by different kinases at multiple phosphorylation sites on the carboxy(C)-terminus domain [9–14]. Mitogen-activated protein kinase (MAPK) and active protein kinase B (pAKT) are known regulators of Cx43 [12–14]. Early studies showed MAPK phosphorylation of Cx43 which leads to a decrease in GJs. However, recent literature suggests that it can lead to an upregulation in Cx43 causing an increase in functional GJs . Another kinase, Akt, has been shown to stabilize gap junctions via phosphorylation, the exact site of Akt phosphorylation Cx43 is unknown. Dunn et al., found that upon inhibition of Akt by Akt VIII inhibitor or with a dominate negative version of Akt gap junctions were smaller and less phosphorylated Cx43 was present .
Phosphorylation events correlate with three known isoforms of Cx43, the isoforms are known as P0, P1 and P2. The P0 form localizes on internal membranes like the Golgi apparatus and the endolysosomal system [16, 17]. The P1 and P2 forms are associated with certain phosphorylation cites. The P1 form has phosphorylation site at S364/S365. When phosphorylated at S365 of Cx43, the assembly conformation of the gap junction was observed . The P2 form has two different sets of phosphorylation sites. One of these sets is phosphorylated at S325/S328/S330; this form has been found at the stage of gap junctional plaques. When phosphorylated at S262 and/or S368 of Cx43, a decrease in GJIC is found [17, 19, 20].
Gap junction enhancers, 6-Methoxy-8-[(3-amionpropyl) amino]-4-methyl-5-(3-trifluoromethyl-phenloxy) quinolone (PQ1), has been demonstrated to increase gap junction activity in breast cancer cells . PQ1 caused an 8.5-fold increase in gap junction activity in T47D breast cancer cells and subsequently a decrease of 70% growth in a xenograft tumor . Furthermore, PQ1 has also been shown to induce apoptosis via caspase 8 and 9 . Oral bioavailability studies indicate that administration of PQ1 via oral gavage has a low toxicity to normal tissue with no observable adverse effects , while significantly attenuating tumor growth .
As colorectal cancer forms, there is a decrease in gap junction activity and Cx43 expression as well as a shift in localization of Cx43 [4, 5, 24]. Thus, this study addresses whether overexpression of Cx43 or increase gap junction activity can be achieved in human colorectal cancer cells, SW480. Using overexpression of Cx43 and treatment of PQ1 approaches, the gap junction activity of SW480 cells was restored. Overall, this study provides evidence for the first time that regain of GJIC can be achieved by a small molecule gap junction enhancer, PQ1, on SW480 colorectal cancer cells.
All experiments in this manuscript have been approved by the Kansas State University Institutional Biosafety Committee (IBC).
The SW480 human colorectal cancer cell line was purchased from American Type Cell Culture (ATCC, Manassas, VA). Cells were grown with 0% CO2 in Leibovitz’s L-15 Medium with 10% Gibco Fetal Bovine Serum (FBS) purchased from Life Technologies (Grand Island, NY, USA).
Cells were seeded to 50% density in a T-25 cm2 flask for 24 hours and allow the density to reach 90% prior to treatment. cells were harvested with lysis buffer (20 mM Tris–HCl pH 7.6, 0.5 mM EDTA, 0.5 mM EGTA, and 0.5% Triton-X 100) (Cell Signaling Technology Inc., Danver, Massachusetts, USA). The mixture was centrifuged at 13,000 rpm (15,700 g using an Eppendorf centrifuge 5415R with rotor F-45-24-11, Eppendorf North America, Hauppauge, New York, USA) for 30 minutes at 4˚C, and the supernatant was collected. Total protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Life Science Research, Hercules, California, USA). 25 μg of whole cell extract was separated by 5-10% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose membrane. The nitrocellulose membrane was immunoblotted against the protein of interest. The primary antibodies were purchased from two different companies; mouse anti-Cx43 antibody and the mouse anti-GAPDH antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Primary antibodies purchased from Cell Signaling Techonolgy (Danvers, Massachusetts, USA) were; rabbit anti-phospho-Akt and rabbit anti-phospho-p44/42 MAPK. Secondary antibodies were anti-mouse and anti-rabbit IGg HRP linked, they were purchased from Cell Signaling Technology (Danver, Massachusetts, USA). Proteins were detected using the FluorChem E System purchased from Protein Simple (Santa Clara, California, USA).
Eight hundred thousand SW480 cells were seeded into six-well plates for 24 hours. Cells were transfected with 3.5 μg of Gja1, NM 012567.2, subcloned into pEGFP-N3 vector  and Optifect reagent in 0% FBS tissue culture media.
Gap junction activity
Scrape Load/Dye Transfer (SL/DT) assay was used to measure gap junction activity. Eight hundred thousand cells were grown on a cover slip in a six-well plate. Cells were grown for 24 hours; cells designated for overexpression of Cx43 were transfected, 24 hours later treatments of 200 nM 12-O-Tetradecanoylphorbol-13-Acetate (TPA) and/or with 50 nM, 200 nM and 500 nM PQ1 for 1 hour. Cells were then washed with Phosphate Buffered Saline (PBS) 3 times. A mixture of 1% Lucifer yellow and 0.75% Rhodamine dextran was added in the center of the cover slip. Two cuts crossing one another in the center of the coverslip were made. After 3 minutes, cells were washed with PBS 3 times and incubated at 37˚C in tissue culture media for 20 minutes. The cells were then washed with PBS and fixed with 2.5% paraformaldehyde for 30 minutes. Cells were mounted on a slide and then sealed and visualized under a fluorescent microscope (Nikon Eclipse 80i, Nikon Instruments, Melville, NY, USA) (X-Cite 120 PC fluorescence illumination system, EXFO Photonic Solutions Inc., Mississauga, Ontario, Canada) at 10x objective (Nikon Instruments, Melville, NY, USA). Images were captured using Nikon Digital Sight Fi1 (Nikon Instrument, Melville, NY, USA). The distance between the designated cut and the dye transfer was measured. The distance of dye uptake indicates that cells are active and have allowed the dye to pass from one cell to the next cell.
Proliferation and viability
Eight hundred thousand cells seeded into 6-well plates for 24 hours. Cells were treated with PQ1 at various concentrations or overexpressed with Cx43. After another 24, 48 or 72 hours tissue culture media of respective treatments was saved and 0.5 mL of trypsin was added to the cells for 5 minutes. Three mL of PBS was used to harvest cells. Cells were spun down for 5 minutes at 13,000 rpm; afterwards solution of tissue culture media, trypsin and PBS was removed. Nine hundred μL of PBS and 100 μL of trypan blue were added to the pellet and left to stand for 5 minutes. Cellometer Auto 2000 from Nexcelom Bioscience was used to measure number of cells for proliferation and viability.
Pixel intensities of protein bands were normalized to pixel intensities of loading control (GAPDH). All protein expression data were expressed as mean ± standard deviation of three independent experiments. Significant differences were analyzed by comparing the data of treated samples and control (untreated) samples and indicated as P value > 0.05 using Student’s t-test.
Transfection of Cx43 leads to increased GJIC in SW480 colorectal cancer cells
PQ1, gap junction enhancer, increases GJIC in SW480 colorectal cancer cells
PQ1’s effects on Kinase activity
In colorectal cancer, a decrease in GJIC has been found. In this study, overexpression of Cx43 increased GJIC through the increase of gap junction protein, Cx43 (Figure 1). It was shown that overexpression of Cx43 lead to a change in isoform expression from P0 to P1 leading to an increase in GJIC. This suggests that the SW480 cells have the key kinases needed to regulate GJIC. Other approach of increasing GJIC was also evaluated since transfection of Cx43 is not a viable therapeutic target. A small molecule, PQ1, was tested as a potential GJ enhancer in SW480 colorectal cancer cells. The results show that PQ1 can increase gap junction activities.
Gap junctions allow for intercellular communication between adjacent connecting cells. GJs play a major role in the life cycle of cells; they are involved in tissue homeostasis and proliferation as well other aspects of the cell cycle [28–30]. In cancer cells, there is a significant change of Cx43 localization at the plasma membrane and the cytoplasmic membrane such as the golgi apparatus and the endolysosomal system [18, 24]. PQ1, does not cause an increase in Cx43 expression, it causes a shift in the isoform expression causing Cx43 to once again be localized to the plasma membrane and to form functional gap junctions (Figures 4B and C and 5).
The regulation of gap junctions is controlled by phosphorylation of specific sites (mostly serine sites) on the carboxy-terminal tail region of the connexins . Previously, Akt and MAPK have demonstrated to modulate Cx43 phosphorylation and subsequently increase gap junction activity. Active Akt has been found to stabilize gap junctions and active p44/42 MAPK has been shown to increase GJIC [12, 13]. This study was also determined whether the increase of gap junction activity by PQ1 was due to the activation of Akt and MAPK. Interestingly, PQ1 was shown to cause an increase in activated Akt and p44/42 MAPK (Figures 6 and 7). This suggests the possibility that PQ1’s ability to increase GJIC is through activation of Akt and MAPK. PQ1’s ability to increase GJIC by kinase activity was tested using kinase inhibitors (Figure 8A and B). Results show that the increase in GJIC by PQ1 is at least in part due to kinase activity (Figure 8A and B).
We have shown that GJIC can be restored via overexpression of Cx43 and by small molecule PQ1. PQ1 was shown to cause an increase in GJIC by changing the isoform expression of Cx43. Since PQ1 was designed using the structure of the c-terminus of Cx43 a potential mechanism may by direct binding to the c-terminus of Cx43. However this study has provided evidence of PQ1’s ability to cause an activation of Akt and p44/42 MAPK and subsequently increase GJIC in SW480 cells. Further studies are needed to elucidate the direct impact of PQ1 on specific site of phosphorylation of Cx43. Overall, the initial data provide insight into PQ1 being a mediator of kinase activity and via this activity cause an increase in GJIC of colorectal cancer cells. These data also conclude that PQ1 is a GJ enhancer in the SW480 colorectal cancer cells. This leads to the possibility of PQ1 being able to enhance the effects of current chemotherapeutic drugs by way of gap junctions.
Gap junction intercellular communication
Scrape load/dye transfer
Protein Kinase B
Mitogen activated protein kinase.
We gratefully acknowledge the financial support from the Developing Scholars Program, the Terry Johnson Cancer Center at Kansas State University, and the National Center for Research Resources (5P20RR016475) and the National Institute of General Medical Sciences (8P20GM103418) from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources and the National Institute of General Medical Sciences of the National Institutes of Health.
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