Research ArticleExperimental Studies

470 nm LED Irradiation Inhibits the Invasiveness of CD133-positive Human Colorectal Cancer Stem Cells by Suppressing the Cyclooxygenase-2/prostaglandin E2 Pathway

SANGJOON MO, HYUN JEONG KU, SUN-HYANG CHOI, HYE JUNG JEONG, DONG-GUK PARK, MAN HWAN OH and JIN CHUL AHN
Anticancer Research March 2021, 41 (3) 1407-1420; DOI: https://doi.org/10.21873/anticanres.14898

Abstract

Background/Aim: Recurrence and metastasis of cancer caused by cancer stem cells (CSCs) is a challenge to overcome. Low level laser therapy is a new treatment strategy to suppress their invasiveness. We have assessed the inhibitory effects of 470 nm blue LED on the invasiveness of them to determine the molecular mechanisms of anti-invasiveness. Materials and Methods: The effects of blue LEDs on their viability, proliferation and invasion were analyzed using MTT and transwell methods. In addition, the anti-invasiveness effect of blue LED on them was evaluated by zymography, semi-quantitative RT-PCR and western blot analysis. Results: Irradiation with blue LED at 3 J/cm2 resulted in inhibition of their viability, proliferation and invasiveness. Their matrix metalloproteinase 2 (MMP-2) and MMP-9 activities were reduced by blue LED irradiation. Semi-quantitative RT-PCR also showed similar results. In addition, western blotting analyses showed that cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthesis were significantly inhibited by LED irradiation in CD133+ colorectal CSCs. Conclusion: Down-regulation of the COX-2/PGE2 signaling pathway by blue LED irradiation led to reduce expression of MMP-2 and MMP-9, inhibiting the invasiveness of CD133+ colorectal CSC.

Key Words:

Colorectal cancer (CRC) is the tumor with the third highest incidence worldwide and the fourth mortality rate, with approximately 1,400,000 new cases and 700,000 deaths worldwide annually (1). Cancer cells of the colon can diffuse to other organs of the body through the bloodstream or lymphatic system, including the lungs, bones, brain or spinal cord, and most often the liver. Tumor metastasis is a multistage process that includes breaking cell–matrix or cell– cell adhesions, which promotes primary tumor cell mobility and migration/invasion into neighboring tissue as well as to remote tissues through the circulatory system (2). Dysregulation of the expression of cytokeratin and integrin proteins contribute to the metastatic process (3, 4). Bai and colleagues have reported that β1-integrin activity in lung cancer cell lines was increased by cyclooxygenase-2 (COX-2) over-expression or prostaglandin E2 (PGE2) addition (5). To date, the most general approach to cancer treatment has been to target tumor development founded on clonal progression models. However, therapeutic strategies that target large numbers of tumor cells have been relatively restricted because of cancer relapse (6).

The cancer stem cell (CSC) hypothesis is a more definite model of tumor incidence, progression, and relapse after cancer treatment, as the different heterogeneity of malignant tumors is consistent with the differentiation of stem cells. Despite many targeted therapies, the drug resistance of cancer cells can be explained by the basic characteristics of stem cells, and the association between these tumors and stem cells may be a new field of targeted therapy (7). To better understand tumor progression, metastasis, and drug resistance of CSCs, biomarkers including cluster of differentiation 133 (CD133), CD44, and aldehyde dehydrogenase 1 (ALDH1) were identified on the surface of CSCs to distinguish them from normal tumor cells. In particular, CD133 is a cell surface antigen used as a major biomarker for the study of oncogenesis and metastasis of CSCs in various solid tumors. Its expression is associated with cancer stem cell metabolism (7, 8).

Human CD133 with a molecular weight of 120 kDa, consisting 865 amino acids, is a pentaspan transmembrane protein, localized in membrane protuberances (9). In 2007, two studies reported that a small population of CD133+ CRC cells could produce tumors when implanted subcutaneously into the kidney capsules of immunodeficient mice, whereas CD133– CRC cells could not produce tumors (10, 11). These results confirmed that CD133+ cells are characterized by the ability to sustain themselves, differentiate and restore tumor heterogeneity in xenograft models (10). Following this initial study, a few additional studies have estimated the relationship between expression of CD133 and parameters of clinical pathology in CRC (12-14). CD133+ CRC shows a high degree of chemo- or radio-resistance, although the clinical and prognostic implication of CD133 expression in CRC remain uncertain (15, 16). Moreover, a meta-analysis has demonstrated that CD133 expression is correlated with low survival rates of 5 years or less in CRC patients and thus may play a significant role in improving the survival rate of these patients (17).

Low-level light therapy (LLLT), which uses various wavelength regions (blue, green, red, near infrared) of the laser or light-emitting diode (LED) electromagnetic spectrums, is a promising alternative option for treating wounds and inflammation as well as removing bacteria (18-20). Blue LEDs with a wavelength of 400-500 nm specifically inhibit the growth of various tumor cells by light toxic and anti-growth effects via the formation of intracellular reactive oxygen species (ROS) in vitro and in vivo (21-24). Recent studies have shown that blue LED irradiation promotes the survival rate of leukemia mice and induces cell death in B-cell lymphoma through a mitochondria-apoptosis pathway. In addition, blue LED emanation increases intracellular ROS levels and DNA impairment, which in turn leads to mitochondrial disorder as well as autophagosome generation in circulating tumor cells (22). However, the mechanisms underlying the ability of LED irradiation to prevent metastasis and migration of solid tumor cells remain unclear.

In this study, the effect of 470 nm LED irradiation on the invasiveness of CD133-positive human colorectal CSCs isolated from CRC patients was analyzed and the underlying mechanism of the inhibitory effect on the invasiveness in colorectal CSCs is proposed.

Materials and Methods

Cell culture reagents and chemicals. Dulbecco’s modified Eagle Medium (DMEM)/F-12, fetal bovine serum (FBS), penicillin/ streptomycin, and Dulbecco’s phosphate-buffered saline were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Protease and phosphatase inhibitor cocktails and all other chemicals were from Sigma-Aldrich (St. Louis, MO, USA). All antibodies used in this study were purchased from Cell Signaling (Beverley, MA, USA) and Bioss (Woburn, MA, USA).

Isolation and culture of primary CRC cells. This study was accomplished in accordance to the International Conference on Harmonization Good Clinical Practices guidelines and the Declaration of Helsinki. Patients with study-related colorectal cancer gave written consent prior to sample collection. Among the 30 CRC ascites samples of metastatic CRC patients undergoing surgery or without radiotherapy and chemotherapy, ascites of patients with highly metastatic CRC were provided by the Dankook University School Hospital Human Resources BioBank (BB No. BB17-24, Cheonan, Republic of Korea). Ascites transferred to a 15 ml conical tube were pelleted by centrifugation at 3,000 × g for 10 min. To remove contaminating red blood cells, cells were rinsed twice in cold phosphate-buffered saline (PBS). Then cells were resuspended in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin and seeded in 0.2% gelatin coated T-25 flasks (Sigma-Aldrich). Cells were incubated in 5% CO2-humidified atmosphere at 37°C. Suitability for cell propagation and morphology were determined with an inverted phase-contrast microscope (CKX53; Olympus, Tokyo, Japan). Primary CRC cells were cryopreserved in liquid nitrogen for future study. This study was reviewed and approved by the Institutional Review Board (IRB) of Dankook University Hospital (IRB No. 2020-03-30).

LLLT. A 470 nm LED light was used for the irradiation experiments (WON TECH, Daejeon, Republic of Korea). During the irradiation, temperature was measured with a thermometer (Fluke, Everett, WA, USA), and a fan was operated to avoid heat generation in all experiments. A photo-radiometer (PD300-TP; Ophir Optronics, Jerusalem, Israel) was used to measure the light intensity. The LED array system was designed to fit over a standard cell culture plate (12.5×8.5 cm). The distance from the light source to the cell was fixed at 2 cm, irradiation was performed in continuous mode, and the light energy was equally conveyed to experimental cells at 10 mW/cm2, except for energy-dependent experiments. Detailed information on the laser parameters, such as the full width at half maximum and bandwidth of the 470 nm LED, is illustrated in Table I.

View this table:
Table 1.

Laser information and parameters.

CD133+ cell sorting with immunomagnetic beads. CD133 positive cells were isolated from cultured primary CRC cells by magnetic-activated cell sorting (MACS) with CD133 MicroBeads (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s instructions. A single cell suspension of primary CRC cells (~1×108) was reacted with MicroBeads conjugated to monoclonal anti-human CD133 antibodies (Miltenyi Biotec) for 30 min in the dark at 4°C. For the magnetic separation, a MACS column (Miltenyi Biotec) was used to hold the positive cells attached to the beads. Cells were rinsed and sorted to separate CD133-positive (CD133+) and CD133-negative (CD133–) cell populations. After isolation, cells were cultured for additional analysis, and some of these cells were used to assess the efficiency of magnetic separation by flow cytometry (BD Accuri™ C6 Plus; BD Biosciences, San Jose, CA, USA), semi-quantitative RT-PCR, and western blotting analyses.

Cell surface marker analysis by flow cytometry. CD133+ cells were detached using pre-warmed 0.25% trypsin-EDTA solution (Sigma-Aldrich) and rinsed two times with PBS containing 1% FBS. According to the manufacturer’s protocol, dissociated 1×105 living cells were suspended in 100 μl PBS buffer with 0.5% bovine serum albumin and 2 mM EDTA and incubated with 10 μl phycoerythrin-conjugated anti-CD133/1 (AC133) antibody (1:11, Miltenyi Biotec) for 10 min at 4°C. As a negative control, unlabeled cells passing through the column were collected. Cells were analyzed on the BD Accuri™ C6 Plus flow cytometer (BD Biosciences).

Cell viability and proliferation assay. Cell viability was measured by the MTT assay according to the manufacturer’s protocol. Primary CRC cells and CD133+ cells were seeded in 96-well plates (1×105 cells/well) and incubated at 37°C for 24 h in a 5% CO2 atmosphere. After incubation, cells were treated with different irradiation energy doses (3, 9, 15 J/cm2) with 470 nm LED in the dark, and then incubated for another 24 h. Then cells were incubated with MTT (0.25 mg/ml) for 2-4 h at 37°C. After completely removing the MTT solution and drying the culture plate, the formazan crystals in the cells were dissolved with 200 μl dimethyl sulfoxide (Sigma-Aldrich), and absorbance was measured at 570 nm with an ELISA plate reader (SPECTRA; Tecan Group, Mannedorf, Switzerland).

To determine cell proliferation, primary CRC cells and CD133+ cells were plated at a density of ~1×105 cells per well in 96-well plates, incubated them for 24 h, and then were refed with culture medium. The cells were divided into four groups: non-irradiated primary CRC cells, non-irradiated CD133+ cells, irradiated primary CRC cells, and irradiated CD133+ cells. The cells were irradiated for 5 min with 470 nm LED at a power density of 10 mW/cm2 and then further cultured at 37°C in a 5% CO2 atmosphere for 24, 48, and 72 h. Then cells were incubated with MTT and analyzed as mentioned above.

Invasion assay. The cell invasion assay was performed using transwell chambers (Corning, Corning, NY, USA) in 24-well culture plates. The transwell chambers were coated with Matrigel (1:3 in DMEM/F-12 without FBS, BD Biosciences). Culture medium (500 μl) containing 10% FBS was poured to the bottom well and a polyethylene membrane (pore size 8 μm) was put between the bottom and top wells. Non-irradiated and irradiated primary CRC cells and CD133+ cells were added to the top well of the transwell chamber at a concentration of ~1×105 cells per well in culture medium without FBS. After 48 h incubation under the same conditions as above, cells that did not migrated were removed from the upper compartment of the polyethylene membrane using a cotton swab, and those that migrated to the lower compartment were fixed with 10% formaldehyde and stained with 0.1% (w/v) crystal violet. For light microscopic analysis, the recovered polyethylene membrane was mounted on a glass slide. Images were taken with a high-resolution digital camera at 200x magnification. Invasiveness was determined by counting the total number of cells using ImageJ (NIH, Bethesda, MD, USA). The assay was repeated three times for each group (non-irradiated group: primary CRC and CD133+ cells, irradiated group with 470 nm LED: primary CRC and CD133+ cells). The median value of cells that had migrated the membranes was determined as the number of invading cells per group.

Gelatin zymography. To analyze the enzyme activity of matrix metalloproteinase 2 (MMP-2) and MMP-9, primary CRC and CD133+ cells were seeded at a density of 1×106 cells per well in culture dishes (Falcon #1007, Φ60 mm). Cells were grown until approximately 70-80% confluence in culture dish. After incubation, the culture medium was drained, and cells were refed with serum-free DMEM/F-12 and irradiated with 470 nm LED (10 mW/cm2) at an energy density of 3 J/cm2 for 48 h. Control cells were primary CRC cells that did not receive irradiation. After 48 h, the supernatants were centrifuged at 3,000 × g for 10 min at room temperature, and the remaining cell pellet was stored at –80°C for use in semi-quantitative RT-PCR and western blotting analyses. The centrifuged supernatants were concentrated with Amicon Ultra-0.5 Centrifugal Filter Units (10 kDa, EMD Millipore, Billerica, MA, USA), and total protein was quantified using the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Total protein (20 μg) was loaded onto a gelatin-containing gel (10% acrylamide gel containing 1 mg/ml gelatin) and separated by electrophoresis. The gels were rinsed twice in wash buffer (2.5% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl2, 1 μM ZnCl2, pH 7.5) with gentle rocking for 30 min at room temperature, placed in incubation buffer (1% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl2, 1 μM ZnCl2, pH 7.5) for another 30 min, and then incubated overnight at 37°C. After staining with Coomassie Brilliant Blue R-250, gelatinolytic enzymes were identified as a clear zone against a blue background. Densitometric analyses were performed with ImageJ.

Semi-quantitative RT-PCR. For semi-quantitative RT-PCR analysis, RNA isolation and RT-PCR amplification were conducted according to our previous our study (25) with modifications. Standard PCR was performed with the EmeraldAmp PCR Master Mix (TaKaRa Bioscience, Kyoto, Japan) on a T100 Thermal Cycler PCR system (Bio-Rad) as follows: PCR was run at 95°C for 3 min, followed by 35 cycles of 95°C for 1 min, 60°C (65°C for MMP-9) for 1 min, 72°C for 2 min, and then 72°C for 7 min. The optimum number of amplification cycles of each gene were chosen to obtain sufficient visibility of the RT-PCR band within the linear range of the amplification. After electrophoresis and staining, the PCR product was quantified by measuring the band density using Quantity One (Bio-Rad). The experiments were repeated three times. Gene expression was normalized to that of GAPDH. The following primers were used: CD133, 5’-CACTCTATACCAAAGCGTCAA-3’ (forward), 5’-CACG ATGCCACTTTCTCAC-3’ (reverse); MMP-2: 5’-CTCAGATCCG TGGTGAGATCT-3’ (forward), 5’-CTTTGGTTCTCCAGCTTCAGG-3’ (reverse); MMP-9: 5’-ATCCAGTTTGGTGTCGCGGAGC-3’ (forward), 5’-GAAGGGGAAGACGCACAGCT-3’ (reverse); and GAPDH: 5’-AGAAGGCTGGGGCTCATTTG-3’ (forward), 5’-AGGG GCCATCCACAGTCTTC-3’ (reverse).

Western blotting analysis. The cells pellets isolated above were lysed for 30 min in cold RIPA buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 2 mM (w/v) EDTA (pH 8.0), Biosesang, Songnam, Republic of Korea] containing 1% (w/v) sodium deoxycholate (Roche Diagnostics, Mannheim, Germany) for 30 min. The lysates were centrifuged at 13,000 × g for 15 min at 4°C. The amount of protein in the supernatant was measured as described above. Finally, 20 μg of total protein were loaded onto 12% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (GE Healthcare Life Sciences, Piscataway, NJ, USA). The PVDF membranes were immersed in Tris-buffered saline containing 0.1% Tween 20 (TBST) and wiped by gently rocking, then blocked with 5% bovine serum albumin in TBST for 1 h at room temperature and incubated with the following antibodies overnight at 4°C: rabbit polyclonal anti-CD133 (1:1000; #ac19898; Abcam, Cambridge, MA, USA), rabbit monoclonal anti-COX-2 (1:1,000; #12282S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PGE2 polyclonal antibody (1:1,000; #bs-2639R; Bioss), or rabbit anti-GAPDH (1:5,000; ab181602; Abcam). The membrane was rinsed twice with PBS with 0.1% Tween 20 and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, goat anti-rabbit IgG-HRP; #7074; Cell Signaling Technology) for 1 h at 37°C. The proteins were detected with ECL detection system according to the manufacturer’s instructions (Bio-Rad). Protein quantification was performed with Quantity One (Bio-Rad) and GAPDH used as the loading control.

Statistical analysis. All statistical analyses were performed using GraphPad Prism 7 software for Windows (San Diego, CA, USA). Each experiment was carried out three times and data are presented as the mean±standard deviation (SD). Comparisons between groups were analyzed using one-way ANOVA or two-way ANOVA. A p-value of <0.05 was considered to show a statistically significant difference.

Results

Establishment of primary CRC cells. Ascites of a CRC patient with severe metastasis (BB17-24) who had not received chemotherapy or radiotherapy were selected and stored for the establishment of primary CRC cells. Fresh CRC cells isolated from ascites were suspended in DMEM/F-12 and then seeded in T-25 flasks pre-coated with 0.2% gelatin and allowed to attach to the tissue culture flasks for 48-72 h. At this time, most of the cells except CRC cells were observed to degenerate and/or die. Initial primary cell cultures could be maintained for at least 2 weeks before their first passage. For all additional experiments presented in this study, primary CRC cells were used in the early passages. Cultured primary CRC cells were established through morphological evaluation and the characteristic patterns of cancer cell growth.

Purification of CD133+ cells from primary CRC cells. Primary CRC cells were divided into CD133+ and CD133– groups by immunomagnetic cell sorting using the CD133 marker and cultured in DMEM/F12 supplemented with 10% FBS (Figure 1A). After 5 days, numerous individual cells in the CD133+ cell culture were examined for survival and proliferation in suspension. The CD133+ cells gradually formed colonies of different sizes and irregular shapes. Most CD133– cells also formed colonies of different sizes and irregular shapes but had thin and long processes at both ends (Figure 1B). Flow cytometry showed that the percentage of the CD133+ subset in the CD133+ group was remarkably increased compared to that in the CD133– group (60.3±5% and 4.6±2%, respectively; p<0.001; Figure 1C). Semi-quantitative RT-PCR revealed that the relative intensity of CD133 PCR band was significantly increased in the CD133+ group compared to the CD133– group (1.2774±0.0619 and 0.0516±0.0374, respectively; p<0.001; Figure 1D). In addition, western blotting analysis determined that the relative intensity of the CD133 protein band was significantly increased in the CD133+ group compared to the CD133– group (0.3689±0.0375 and 0.0545±0.0040, respectively; p<0.001; Figure 1E).

Figure 1.
Figure 1.
Figure 1.

Summary of the methodological approach used to generate CD133+ CSCs from CRC patients using the immunomagnetic beads approach. Human primary CRC cells were obtained from the ascites of CRC patients with metastatic cancer. (A) Schematic diagram depicting the experimental design. Isolated human primary CRC cells were cultured to single-cell suspensions containing colorectal CSCs. Incubation with magnetic beads conjugated to antibodies that recognize the CSC surface protein CD133 was followed by the passage of the sample through separation columns attached to a magnet (gray bars). CD133+ cells were ultimately separated from the cell culture. (B) Phase-contrast morphology of CD133+ and CD133– cells derived from human primary CRC cells at the same passage number (magnification 200×). Scale bar, 100 μm. (C) Proportion of the CD133+ subset in the CD133+ and CD133– groups in human CRC cells, determined with flow cytometry. (D) Detection of CD133 mRNA expression with the polymerase chain reaction. GAPDH was used as the control. (E) Detection of protein expression of CD133 with western blotting analyses. GAPDH was used as the loading control.

Inhibition of viability and proliferation of CD133+ cells by LED irradiation. We examined the effects of LED irradiation on the viability and proliferation of CD133+ cells by MTT assay. After LED irradiation, viability tended to decrease in both primary CRC cells and CD133+ cells compared to the control. In particular, the viability of CD133+ cells was further reduced by 18.1% and 14.3% (p<0.01) compared to primary CRC cells (reduced by 17.3% and 34.5%) at 3 and 9 J/cm2, respectively (Figure 2A). After irradiating the primary CRC cells and CD133+ cells with LED for 5 min at an optical power density of 10 mW/cm2, we investigated cell proliferation for 3 days to reconfirm the growth inhibitory activity. As shown in Figure 2B, proliferation of all non-irradiated CRC cells tended to increase in a time-dependent manner, but the trend was not significant. However, the proliferation of CD133+ cells was reduced by approximately 0.48-fold (p<0.001) after 24 h LED irradiation compared to immediately after irradiation, whereas proliferation of primary CRC cells was decreased by only approximately 0.12-fold (p<0.05; Figure 2B). This result shows that the proliferation of CD133+ cells was effectively inhibited 24 h after irradiation with 470 nm LED.

Figure 2.
Figure 2.

Viability and proliferation of human primary CRC and CD133+ cells after 470 nm LED irradiation. (A) Viability of human primary CRC and CD133+ cells after 3, 9, and 15 J/cm2 irradiation with 470 nm LED. Non-irradiated cells were used as a control. Cell viability was determined by MTT assay after incubation for 24 h after LED irradiation. The control values were set to 100%. Data are means±SDs (n=3). **p<0.01, significant difference from primary CRC cells. (B) Proliferation of human primary CRC and CD133+ cells after 3 J/cm2 irradiation with 470 nm LED. Non-irradiated cells were used as a control. Effects of 470 nm LED on cell proliferation were analyzed (at 3 J/cm2) by MTT assay. Cell proliferation was quantified at 24, 48, and 72 h after irradiation. Data are means±SDs (n=3). ***p<0.001 and *p<0.01, significant difference from non-irradiated cells.

Effects of 470 nm LED irradiation on CD133+ cell invasion. Cell migration and invasion are involved in the cancer development (26). A transwell invasion assay was performed to analyze the ability of CD133+ cells to directionally respond to chemoattractants and 470 nm LED. To assess the invasiveness of primary CRC and CD133+ cells after 10 mW/cm2 irradiation for 5 min, we measured and compared the cell count values obtained in the invasion assay after seeding the same number of cells. As shown in Figure 3, the invasiveness of all CRC cells was inhibited by 470 nm LED irradiation. The invasiveness of primary CRC cells was approximately 2.4-fold (p<0.01) lower than that of non-irradiated primary CRC cells 24 h after 470 nm LED irradiation. However, the invasiveness of irradiated CD133+ cells was approximately 12.5-fold (p<0.001) lower than that of non-irradiated cells (Figure 3B). Comparing the invasiveness of primary CRC and CD133+ cells irradiated with 470 nm LED showed that CD133+ cells were about 5.3-fold (p<0.05) less invasive than primary CRC cells. Taken together, these results suggest that irradiation with 470 nm LED had significantly greater inhibitory effects on the invasiveness of CD133+ cells than primary CRC cells. In addition, these results provide further evidence that CD133+ cell populations are rich in CSCs sensitive to the 470 nm wavelength compared to primary CRC cells.

Figure 3.
Figure 3.

Transwell invasion assay. Irradiation with 470 nm LED decreased invasive activity in primary CRC and CS133+ cells. (A) The invasion assay was conducted in both cells after irradiation with 470 nm LED. Primary CRC and CS133+ cells that penetrated the membrane were fixed and stained with 0.1% crystal violet after 48 h as described in Materials and Methods. (B) Histogram of the results of the invasion assay for the two groups of cells. The results show a significant decrease in the number of invasive primary CRC and CD133+ cells after irradiation with 470 nm LED (**p<0.01 and ***p<0.001). Results for CD133+ cells show a significant decrease in relative cell invasion after 470 nm LED irradiation compared to primary CRC cells (#p<0.05). Non-irradiated cells were used as a control. Data are means±SDs (n=3).

Effects of 470 nm LED irradiation on the activity of MMP-2 and MMP-9 in CD133+ cells. Invasion of tumor cells has been linked to the production of proteolytic enzymes capable of digesting the extracellular matrix (ECM) barrier, including MMPs (27). In this study, the proteolytic activity of MMP-2 and MMP-9 were investigated in primary CRC and CD133+ cells after 10 mW/cm2 irradiation for 5 min. MMP-2 and MMP-9 enzyme activity determined by gelatin zymography was significantly increased in CD133+ cell supernatants compared to primary CRC cells (Figure 4A and B). As shown in Figure 4B, MMP-2 dimer and pro-MMP-2 activity were both significantly increased by approximately 1.6-fold (p<0.001 and p<0.01, respectively) in CD133+ cells compared to primary CRC cells. Pro-MMP-9 activity was particularly increased, by approximately 3-fold (p<0.001) and active-MMP-9 activity was increased by approximately 1.6-fold (p<0.01) in CD133+ cells compared to primary CRC cells. When cells were irradiated with 470 nm LED, MMP-2 and MMP-9 activity was significantly decreased in both cells. Regarding primary CRC cells, only MMP-2 dimer activity was significantly decreased by approximately 2.4-fold (p<0.001) in 470 nm LED irradiated cells as compared to non-irradiated cells. However, MMP-2 dimer, pro-MMP-2, pro-MMP-9, and active-MMP-9 activities of CD133+ cells irradiated with 470 nm LED were decreased by approximately 1.4-, 1.5-, 3.5- and 1.6-fold, as compared with non-irradiated CD133+ cells, respectively (p<0.01, p<0.01, p<0.001, and p<0.001, respectively). These results show that down-regulation of MMP-2 and MMP-9 activities by 470 nm LED irradiation reduced the invasiveness of CD133+ cells.

Figure 4.
Figure 4.

Gelatin zymogram of primary CRC and CD133+ cells. (A) MMP zymography identified the amount of active MMP-2 and MMP-9 in primary CRC and CD133+ cells with and without irradiation with 470 nm LED. The clear bands of MMP-9 (92 kDa) and MMP-2 (72 kDa) signify their gelatinolytic activity. Human pro-MMP-9 (92 kDa) and pro-MMP-2 (72 kDa) standards were used as positive controls (Sigma-Aldrich). (B) Densitometric analyses showed MMP-2 and MMP-9 activity in non-irradiated cells versus cells irradiated with 470 nm LED in response to serum free for 48 h. Graphical presentation of the relative intensity of MMPs (fold induction over non-irradiated primary CRC cells) following 470 nm LED irradiation as indicated. Data are means±SDs of three independent experiments. ###p<0.001 and ##p<0.01, significant difference between primary CRC cells and CD133+ cells. ***p<0.001 and **p<0.01, significant difference between non-irradiated and irradiated cells.

Effects of 470 nm LED irradiation on MMP-2 and MMP-9 mRNA expression in CD133+ cells. In order to analyze transcript levels of MMP-2 and MMP-9 after irradiation with 470 nm LED in primary CRC and CD133+ cells, the transcript expression levels of these genes were determined using semi-quantitative RT-PCR and normalized relative to the expression of GAPDH (Figure 5A and B). The expression of MMP-2 and MMP-9 increased by approximately 1.3- and 1.5-fold (p<0.05 and p<0.001) in CD133+ cells compared to primary CRC cells under the non-irradiated conditions, consistent with the results of zymography. Whereas, the MMP-2 and MMP-9 mRNA expressions were reduced by irradiation with 470 nm LED in primary CRC cells, but the effects were not significant. However, their expression was reduced slightly but significantly by about 1.5- and 1.6-fold in CD133+ cells irradiated with 470 nm LED compared to non-irradiated CD133+ cells (p<0.01 and p<0.001). This demonstrates that the proteolytic activity of MMP-2 and MMP-9 decreased only in cells irradiated with 470 nm LED. Taken together, these results show that irradiation with 470 nm LED reduced MMP-2 and MMP-9 expression and functional activity.

Figure 5.
Figure 5.

Effects of 470 nm LED irradiation on mRNA expression of MMPs in primary CRC and CD133+ cells. Primary CRC and CD133+ cells were incubated for 24 h after irradiation or without irradiation with 470 nm LED. The control was non-irradiated primary CRC cells. After incubation, total RNA was purified, and mRNA expression of MMP-2 and MMP-9 was evaluated by RT-PCR. PCR products were resolved by 1% agarose gel electrophoresis and stained with ethidium bromide. (A) mRNA levels of MMP-2 and MMP-9. Data are from one of three independent experiments. (B) Quantification of mRNA levels of MMP-2 and MMP-9. GAPDH expression was evaluated as an internal standard. The relative mRNA expression of MMP-2 and MMP-9 corrected for the expression of GAPDH is expressed as a ratio to non-irradiated primary CRC cells. Data are means±SDs of three independent experiments. ###p<0.001 and #p<0.05, significant difference between primary CRC cells and CD133+ cells. ***p<0.001 and **p<0.01, significant difference between non-irradiated and irradiated cells. SD: Standard deviation.

Effects of 470 nm LED irradiation on the expression of COX-2 and PGE2 in CD133+ cells. Abnormal over-expression of COX-2 and PGE2 inhibits apoptosis in cancer cells, accelerates their growth, and increases invasiveness (28, 29). To examine the inhibition of COX-2 and PGE2 expression by 470 nm LED irradiation, western blotting analysis was used to quantify the expression of COX-2 and PGE2 after 470 nm LED irradiation in primary CRC and CD133+ cells (Figure 6A and B). The expression levels of COX-2 and PGE2 decreased significantly, by approximately 1.3- and 1.6-fold (p<0.01 and p<0.05), in CD133+ cells irradiated with 470 nm LED compared to non-irradiated CD133+ cells. The expression levels of both proteins were slightly, but not significantly, decreased in irradiated primary CRC cells compared to non-irradiated primary CRC cells. However, the expression of COX-2 and PGE2 was significantly increased under the non-irradiated conditions, by approximately 1.6- and 2.0-fold, in CD133+ cells compared to primary CRC cells (p<0.001 and p<0.05). This indicates that the relative over-expression of COX-2 and PGE2 in CD133+ cells was reduced by 470nm LED irradiation. The results of western blotting analyses corroborated the zymography data, which demonstrated the cancer-suppressive effects through the reduction in CD133+ cells migration and invasion by 470 nm LED irradiation.

Figure 6.
Figure 6.

Evaluation of the effect of 470 nm LED irradiation on COX-2 expression and PGE2 synthesis by in primary CRC and CD133+ cells. The control was non-irradiated primary CRC cells. (A) COX-2 and PGE2 levels were determined by western blotting analyses. (B) COX-2 expression and PGE2 synthesis were normalized to GAPDH expression. The relative protein expression of COX-2 and PGE2 corrected for that of GAPDH is expressed as a ratio to non-irradiated primary CRC cells. ###p<0.001 and #p<0.05, significant difference between primary CRC cells and CD133+ cells. **p<0.001 and *p<0.01, significant difference between non-irradiated and irradiated cells.

Discussion

Despite the progress in new treatment strategies for cancer patients, 30-50% of CRC patients die from relapse of cancer within the first two years after surgery (30, 31). These limitations can be explained by the CSC model. This study was conducted to determine whether 470 nm LED irradiation can inhibit the metastasis of colorectal CD133+ CSC cells. We found that two factors might affect viability and proliferation of human primary CRC cells and CD133+ cells: the light intensity of the 470 nm LED and the incubation time after light exposure. When CD133+ cells were irradiated with 470 nm LED with 9 J/cm2 energy, their viability was reduced further compared to 3 J/cm2, but the viability of primary CRC cells was also significantly reduced. Therefore, 3 J/cm2 was chosen as the optimal irradiation energy that can only suppress the viability of CD133+ cells without affecting the viability of CRC cells. Cell proliferation was most inhibited 24 h after 470 nm LED irradiation and tended to recover gradually thereafter. However, the proliferation of primary CRC cells was not markedly inhibited by irradiation with 470 nm LED. These results suggest that 470 nm LED irradiation may inhibit growth of colorectal CSCs. Blue LED generates intracellular ROS, which have photoirritation and anti-proliferation effects (32, 33). Oh et al. have reported that 450 nm blue LED irradiation leads to apoptosis by mitochondria-associated signaling pathways, reducing the initial growth of tumors in melanoma cells (21). These results may also explain the inhibition of viability and proliferation of CD133+ cells by 470 nm LED irradiation in our study. In addition, the invasiveness of primary CRC cells and CD133+ cells were measured after irradiation with 470 nm LED using invasion assay. Although there was a difference in invasiveness between the two irradiated cells, both cells were inhibited by blue LED irradiation, which is consistent with results of Oh et al., which demonstrated that blue LED irradiation suppresses the migration and invasion of murine colorectal carcinoma (CT-26) and human fibrosarcoma (HT-1080) cell lines (34). However, the anti-invasive effect of blue LED irradiation in human solid tumors and its effects on the regulation of human CRC cell invasion via signaling pathways remain unclear.

In malignant tumors, the initial steps of metastasis and invasion tumor cells include disruption of the ECM and invasion of the basement membrane. MMPs play significant role in this mechanism by degrading ECM components. Among MMPs, MMP-2 and MMP-9 are related primarily to malignant tumor invasion and metastasis in human tumors. Zhang et al. have reported that increased MMP-9 expression stimulates metastasis of non-small cell lung cancer (35). It has also been shown that MMP-2 and MMP-9 may also lead to metastasis in gastric adenocarcinoma. Moreover, Simbulan-Rosenthal et al. (36) have reported that doxycycline-induced CD133 expression in BAK-P cells upregulated the secretion of active MMP-2 and 9. Their results confirm the interaction of CD133 with MMP in tumor growth and explain the importance of their correlation. The gelatinolytic activity of MMP-2 and MMP-9 was significantly higher in CD133+ cells than primary CRC cells, consistent with previous studies (36). In addition, the proteolytic activity of MMP-2 and MMP-9 (except the MMP-2 dimer) in primary CRC cells was not significantly reduced by irradiation with 470 nm LED compared to CD133+ cells but the gelatinase (MMP-2 and MMP-9) activity in CD133+ cells was reduced by irradiation with LED. Although Oh and colleagues reported decreased MMP-2 and MMP-9 expression in CT-26 and HT-1080 cells irradiated with blue LEDs (34), this study is the first to show that their expression decreased in CD133+ cells by irradiation with 470 nm blue LED. Semi-quantitative RT-PCR showed that 470 nm LED irradiation inhibited MMP-2 and MMP-9 mRNA expression levels in primary CRC and CD133+ cells. In particular, mRNA expression was markedly inhibited in CD133+ cells compared to primary CRC cells, consistent with the results of gelatin zymography.

COX-2 is a core enzyme that promotes production of PGE2 and arachidonic acid. Changes in the expression of COX-2 as well as its synthetic product PGE2 affect the progression of cancer since their levels are markedly raised in cancerous tissue (37). Luo et al. have demonstrated that the expression of COX-2 and PGE2 was significantly increased in 786-O/S cells, which over-express CD133, compared to control cells (38). They suggested that COX-2 may influence the activation of CSCs. Our results show a significant increase in COX-2 expression and PGE2 synthesis in CD133+ cells compared to primary CRC cells, which is consistent with their suggestion. Moreover, it was confirmed that 470 nm LED irradiation markedly inhibited the expression of COX-2 and the synthesis of PGE2 in CD133+ cells and could inhibit the proteolytic activity of MMP-2 and MMP-9. We hypothesize that suppression of COX-2/PGE2 expression by irradiation with 470 nm LEDs leads to suppression of ERK1/2 and NF-κB activity, which prevents the migration and invasion of colorectal CSCs. However, the precise mechanisms regulating this process requires further study.

Our results showed that 470 nm LED irradiation effectively reduced the proliferation and invasion of primary CRC and CD133+ cells, suppressed the growth of tumor cells by counteracting the expression of COX-2 and PGE2 in CD133+ cells. As explained above, inhibition of COX-2 expression is associated with the inhibition of PGE2 synthetic signaling pathway, whereas activation of this mechanism promotes higher levels of metastasis and invasion of cancer stem cells. This is because activation of the COX-2 expression/PGE2 synthesis axis is associated with progression of malignancies, and immunosuppression through an autocrine/paracrine mode action that accelerates tumor growth and survival. Therefore, inhibition of COX-2 by the 470nm LED interferes with the synthesis of prostaglandins, a cytokine-like factor that stimulates the growth and metastasis of CSCs, blocking the tumor’s growth-stimulating effects that are generated from COX-2 activation.

Overall, the results of this study suggest that the anti-invasion properties of 470 nm LED irradiation in primary CRC and CD133+ cells may result from the low expression of MMP-2 and MMP-9 via the down-regulation of the upstream COX-2/PGE2 signaling pathway in CRC cells. The exact mechanism of the effects of 470nm LEDs in tumors is unclear, but there is a possibility that irradiation with 470 nm LEDs could suppress the proliferation and invasion of CSCs in vivo. Therefore, controlling the expression or proteolytic activity of ECM-degrading enzymes with 470 nm LED may be a powerful treatment strategy for inhibiting the invasion–metastasis cascade of CSCs in solid tumors.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1A6A1A03043283), supported by the Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (MIST) (grant NRF: 2018K1A4A3A02060572). National Research Facilities & Equipment Center (NFEC) grant funded by the Korea government (Ministry of Education) (No. 2019R1A6C1010033). This research was a part of the project titled ‘Development of marine material based near infrared fluorophore complex and diagnostic imaging instruments’, funded by the Ministry of Oceans and Fisheries, Korea (Grant Number 20170263), and also supported by the Ministry of Trade, Industry & Energy, Republic of Korea (Grant Numbers 20002831).

Footnotes

  • Authors’ Contributions

    SM, HJK, SHC, and HJJ performed the experiment. SM, MHO, and JCA analyzed the results. SM and JCA designed all the experiments. DGP provided the samples of colorectal cancer patients. SM wrote the manuscript. SM and JCA edited the manuscript. All Authors contributed to the revision of the manuscript and approved the final version for publication. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    All Authors of this manuscript declare that there are no conflicts of interest regarding this study.

  • Received December 20, 2020.
  • Revision received January 19, 2021.
  • Accepted January 21, 2021.

References

  1. ↵
    1. Arnold M,
    2. Sierra MS,
    3. Laversanne M,
    4. Soerjomataram I,
    5. Jemal A and
    6. Bray F
    : Global patterns and trends in colorectal cancer incidence and mortality. Gut 66(4): 683-691, 2017. PMID: 26818619. DOI: 10.1136/gutjnl-2015-310912
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Hashimoto T,
    2. Kusakabe T,
    3. Watanabe K,
    4. Sugino T,
    5. Fukuda T,
    6. Nashimoto A,
    7. Honma K,
    8. Sato Y,
    9. Kimura H,
    10. Fujii H and
    11. Suzuki T
    : Liver-type fatty acid-binding protein is highly expressed in intestinal metaplasia and in a subset of carcinomas of the stomach without association with the fatty acid synthase status in the carcinoma. Pathobiology 71(3): 115-122, 2004. PMID: 15051923. DOI: 10.1159/000076465
    OpenUrlCrossRefPubMed
  3. ↵
    1. Visca P,
    2. Sebastiani V,
    3. Pizer ES,
    4. Botti C,
    5. De Carli P,
    6. Filippi S,
    7. Monaco S and
    8. Alo PL
    : Immunohistochemical expression and prognostic significance of FAS and GLUT1 in bladder carcinoma. Anticancer Res 23(1A): 335-339, 2003. PMID: 12680233.
    OpenUrlPubMed
  4. ↵
    1. de Leval X,
    2. Benoit V,
    3. Delarge J,
    4. Julemont F,
    5. Masereel B,
    6. Pirotte B,
    7. Merville MP,
    8. David JL and
    9. Dogné JM
    : Pharmacological evaluation of the novel thromboxane modulator BM-567 (II/II). Effects of BM-567 on osteogenic sarcoma-cell-induced platelet aggregation. Prostaglandins Leukot Essent Fatty Acids 68(1): 55-59, 2003. PMID: 12538091. DOI: 10.1016/s0952-3278(02)00235-1
    OpenUrlCrossRefPubMed
  5. ↵
    1. Pan J,
    2. Yang Q,
    3. Shao J,
    4. Zhang L,
    5. Ma J,
    6. Wang Y,
    7. Jiang BH,
    8. Leng J and
    9. Bai X
    : Cyclooxygenase-2 induced β1-integrin expression in NSCLC and promoted cell invasion via the EP1/MAPK/E2F-1/FoxC2 signal pathway. Sci Rep 6: 33823, 2016. PMID: 27654511. DOI: 10.1038/srep33823
    OpenUrlCrossRefPubMed
  6. ↵
    1. Rahman M,
    2. Deleyrolle L,
    3. Vedam-Mai V,
    4. Azari H,
    5. Abd-El-Barr M and
    6. Reynolds BA
    : The cancer stem cell hypothesis: failures and pitfalls. Neurosurgery 68(2): 531-545, 2011. PMID: 21135745. DOI: 10.1227/NEU.0b013e3181ff9eb5
    OpenUrlCrossRefPubMed
  7. ↵
    1. Glumac PM and
    2. LeBeau AM
    : The role of CD133 in cancer: a concise review. Clin Transl Med 7(1): 18, 2018. PMID: 29984391. DOI: 10.1186/s40169-018-0198-1
    OpenUrlCrossRefPubMed
  8. ↵
    1. Li Z
    : CD133: a stem cell biomarker and beyond. Exp Hematol Oncol 2(1): 17, 2013. PMID: 23815814. DOI: 10.1186/2162-3619-2-17
    OpenUrlCrossRefPubMed
  9. ↵
    1. Shmelkov SV,
    2. St Clair R,
    3. Lyden D and
    4. Rafii S
    : AC133/CD133/Prominin-1. Int J Biochem Cell Biol 37(4): 715-719, 2005. PMID: 15694831. DOI: 10.1016/j.biocel.2004.08.010
    OpenUrlCrossRefPubMed
  10. ↵
    1. O’Brien CA,
    2. Pollett A,
    3. Gallinger S and
    4. Dick JE
    : A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445(7123): 106-110, 2007. PMID: 17122772. DOI: 10.1038/nature05372
    OpenUrlCrossRefPubMed
  11. ↵
    1. Ricci-Vitiani L,
    2. Lombardi DG,
    3. Pilozzi E,
    4. Bifoni M,
    5. Todaro M,
    6. Peschle C and
    7. De Maria R
    : Identifcation and expansion of human colon-cancer-initiat-ing cells. Nature 445(7123): 111-115, 2007. PMID: 17122771. DOI: 10.1038/nature05384
    OpenUrlCrossRefPubMed
  12. ↵
    1. Takahashi S,
    2. Kamiyama T,
    3. Tomaru U,
    4. Ishizu A,
    5. Shida T,
    6. Osaka M,
    7. Sato Y,
    8. Saji Y,
    9. Ozaki M and
    10. Todo S
    : Frequency and pattern of expression of the stem cell marker CD133 have strong prognostic effect on the surgical outcome of colorectal cancer patients. Oncol Rep 24(5): 1201-1212, 2010. PMID: 20878111. DOI: 10.3892/or_00000973
    OpenUrlCrossRefPubMed
    1. Xi HQ and
    2. Zhao P
    : Clinicopathological significance and prognostic value of EphA3 and CD133 expression in colorectal carcinoma. J Clin Pathol 64(6): 498-503, 2011. PMID: 21415057. DOI: 10.1136/jcp.2010.087213
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Zhou F,
    2. Mu YD,
    3. Liang J,
    4. Liu ZX,
    5. Chen HS and
    6. Zhang JF
    : Expression and prognostic value of tumor stem cell markers ALDH1 and CD133 in colorectal carcinoma. Oncol Lett 7(2): 507-512, 2014. PMID: 24396478. DOI: 10.3892/ol.2013.1723
    OpenUrlCrossRefPubMed
  15. ↵
    1. Hongo K,
    2. Kazama S,
    3. Sunami E,
    4. Tsuno NH,
    5. Takahashi K,
    6. Nagawa H and
    7. Kitayama J
    : Immunohistochemical detection of CD133 is associated with tumor regression grade after chemoradiotherapy in rectal cancer. Med Oncol 29(4): 2849-2857, 2012. PMID: 22246526. DOI: 10.1007/s12032-012-0161-8
    OpenUrlCrossRefPubMed
  16. ↵
    1. Nagata T,
    2. Sakakura C,
    3. Komiyama S,
    4. Miyashita A,
    5. Nishio M,
    6. Murayama Y,
    7. Komatsu S,
    8. Shiozaki A,
    9. Kuriu Y,
    10. Ikoma H,
    11. Nakanishi M,
    12. Ichikawa D,
    13. Fujiwara H,
    14. Okamoto K,
    15. Ochiai T,
    16. Kokuba Y,
    17. Sonoyama T and
    18. Otsuji E
    : Expression of cancer stem cell markers CD133 and CD44 in locoregional recurrence of rectal cancer. Anticancer Res 31(2): 495-500, 2011. PMID: 21378329.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Chen S,
    2. Song X,
    3. Chen Z,
    4. Li X,
    5. Li M,
    6. Liu H and
    7. Li J
    : CD133 expression and the prognosis of colorectal cancer: a systematic review and meta-analysis. PloS One 8(2): e56380, 2013. PMID: 23409180. DOI: 10.1371/journal.pone.0056380
    OpenUrlCrossRefPubMed
  18. ↵
    1. Desmet KD,
    2. Paz DA,
    3. Corry JJ,
    4. Eells JT,
    5. Wong-Riley MT,
    6. Henry MM,
    7. Buchmann EV,
    8. Connelly MP,
    9. Dovi JV,
    10. Liang HL,
    11. Henshel DS,
    12. Yeager RL,
    13. Millsap DS,
    14. Lim J,
    15. Gould LJ,
    16. Das R,
    17. Jett M,
    18. Hodgson BD,
    19. Margolis D and
    20. Whelan HT
    : Clinical and experimental applications of NIR-LED photobiomodulation. Photomed Laser Surg 24(2): 121-128, 2006. PMID: 16706690. DOI: 10.1089/pho.2006.24.121
    OpenUrlCrossRefPubMed
    1. Lim W,
    2. Lee S,
    3. Kim I,
    4. Chung M,
    5. Kim M,
    6. Lim H,
    7. Park J,
    8. Kim O and
    9. Choi H
    : The anti-inflammatory mechanism of 635 nm light-emitting-diode irradiation compared with existing COX inhibitors. Lasers Surg Med 39(7): 614-621, 2007. PMID: 17868110. DOI: 10.1002/lsm.20533
    OpenUrlCrossRefPubMed
  20. ↵
    1. Nandakumar K,
    2. Keeler W,
    3. Schraft H and
    4. Leung KT
    : Visible laser and UV-A radiation impact on a PNP degrading Moraxella strain and its rpoS mutant. Biotechnol Bioeng 94(4): 793-802, 2006. PMID: 16489628. DOI: 10.1002/bit.20898
    OpenUrlCrossRefPubMed
  21. ↵
    1. Oh PS,
    2. Na KS,
    3. Hwang H,
    4. Jeong HS,
    5. Lim S,
    6. Sohn MH and
    7. Jeong HJ
    : Effect of blue light emitting diodes on melanoma cells: involvement of apoptotic signaling. J Photochem Photobiol B 142: 197-203, 2015. PMID: 25550119. DOI: 10.1016/j.jphotobiol.2014.12.006
    OpenUrlCrossRef
  22. ↵
    1. Oh PS,
    2. Hwang H,
    3. Jeong HS,
    4. Kwon J,
    5. Kim HS,
    6. Kim M,
    7. Lim S,
    8. Sohn MH and
    9. Jeong HJ
    : Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy. Int J Biochem Cell Biol 70: 13-22, 2016. PMID: 26555754. DOI: 10.1016/j.biocel.2015.11.004
    OpenUrlCrossRefPubMed
    1. del Olmo-Aguado S,
    2. Núñez-Álvarez C and
    3. Osborne NN
    : Blue light action on mitochondria leads to cell death by necroptosis. Neurochem Res 41(9): 2324-2335, 2016. PMID: 27216620. DOI: 10.1007/s11064-016-1946-5
    OpenUrlCrossRefPubMed
  24. ↵
    1. Sparsa A,
    2. Faucher K,
    3. Sol V,
    4. Durox H,
    5. Boulinguez S,
    6. Doffoel-Hantz V,
    7. Calliste CA,
    8. Cook-Moreau J,
    9. Krausz P,
    10. Sturtz FG,
    11. Bedane C,
    12. Jauberteau-Marchan MO,
    13. Ratinaud MH and
    14. Bonnetblanc JM
    : Blue light is phototoxic for B16F10 murine melanoma and bovine endothelial cell lines by direct cytocidal effect. Anticancer Res 30(1): 143-147, 2010. PMID: 20150629.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Mo S,
    2. Chung P-S and
    3. Ahn JC
    : 630 nm-OLED accelerates wound healing in mice via regulation of cytokine release and genes expression of growth factors. Curr Opt Photon 3(6): 485-495, 2019. DOI: 10.3807/COPP.2019.3.6.485
    OpenUrlCrossRef
  26. ↵
    1. Bozzuto G,
    2. Ruggieri P and
    3. Molinari A
    : Molecular aspects of tumor cell migration and invasion. Ann Ist Super Sanita 46(1): 66-80, 2010. PMID: 20348621. DOI: 10.4415/ANN_10_01_09
    OpenUrlCrossRefPubMed
  27. ↵
    1. Cheng JC,
    2. Chou CH,
    3. Kuo ML and
    4. Hsieh CY
    : Radiation-enhanced hepatocellular carcinoma cell invasion with MMP-9 expression through PI3K/Akt/NF-kappaB signal transduction pathway. Oncogene 25(53): 7009-7018, 2006. PMID: 16732316. DOI: 10.1038/sj.onc.1209706
    OpenUrlCrossRefPubMed
  28. ↵
    1. Solomon SD,
    2. McMurray JJ,
    3. Pfeffer MA,
    4. Wittes J,
    5. Fowler R,
    6. Finn P,
    7. Anderson WF,
    8. Zauber A,
    9. Hawk E and
    10. Bertagnolli M
    : Adenoma Prevention with Celecoxib (APC) Study Investigators: Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med 352(11): 1071-1080, 2005. PMID: 15713944. DOI: 10.1056/NEJMoa050405
    OpenUrlCrossRefPubMed
  29. ↵
    1. Grösch S,
    2. Maier TJ,
    3. Schiffmann S and
    4. Geisslinger G
    : Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst 98(11): 736-747, 2006. PMID: 16757698. DOI: 10.1093/jnci/djj206
    OpenUrlCrossRefPubMed
  30. ↵
    1. O’Connell MJ,
    2. Campbell ME,
    3. Goldberg RM,
    4. Grothey A,
    5. Seitz J-F,
    6. Benedetti JK,
    7. André T,
    8. Haller DG and
    9. Sargent DJ
    : Survival following recurrence in stage II and III colon cancer: findings from the ACCENT data set. J Clin Oncol 26(14): 2336-2341, 2008. PMID: 18467725. DOI: 10.1200/JCO.2007.15.8261
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Ryuk JP,
    2. Choi GS,
    3. Park JS,
    4. Kim HJ,
    5. Park SY,
    6. Yoon GS,
    7. Jun SH and
    8. Kwon YC
    : Predictive factors and the prognosis of recurrence of colorectal cancer within 2 years after curative resection. Ann Surg Treat Res 86(3): 143-151, 2014. PMID: 24761423. DOI: 10.4174/astr.2014.86.3.143
    OpenUrlCrossRefPubMed
  32. ↵
    1. Kim S,
    2. Kim J,
    3. Lim W,
    4. Jeon S,
    5. Kim O,
    6. Koh JT,
    7. Kim CS,
    8. Choi H and
    9. Kim O
    : In vitro bactericidal effects of 625, 525, and 425 nm wavelength (red, green, and blue) light-emitting diode irradiation. Photomed Laser Surg 31(11): 554-562, 2013. PMID: 24138193. DOI: 10.1089/pho.2012.3343
    OpenUrlCrossRefPubMed
  33. ↵
    1. Yoshida A,
    2. Yoshino F,
    3. Makita T,
    4. Maehata Y,
    5. Higashi K,
    6. Miyamoto C,
    7. Wada-Takahashi S,
    8. Takahashi SS,
    9. Takahashi O and
    10. Lee MC
    : Reactive oxygen species production in mitochondria of human gingival fibroblast induced by blue light irradiation. J Photochem Photobiolo B 129: 1-5, 2013. PMID: 24141287. DOI: 10.1016/j.jphotobiol.2013.09.003
    OpenUrlCrossRef
  34. ↵
    1. Oh PS,
    2. Kim HS,
    3. Kim EM,
    4. Hwang H,
    5. Ryu HH,
    6. Lim S,
    7. Sohn MH and
    8. Jeong HJ
    : Inhibitory effect of blue light emitting diode on migration and invasion of cancer cells. J Cell Physiol 232(12): 3444-3453, 2017. PMID: 28098340. DOI: 10.1002/jcp.25805
    OpenUrlCrossRefPubMed
  35. ↵
    1. Zhang W,
    2. Zhang T,
    3. Lou Y,
    4. Yan B,
    5. Cui S,
    6. Jiang L and
    7. Han B
    : Placental growth factor promotes metastases of non-small cell lung cancer through MMP9. Cell Physiol Biochem 37(3): 1210-1218, 2015. PMID: 26418253. DOI: 10.1159/000430244
    OpenUrlCrossRefPubMed
  36. ↵
    1. Simbulan-Rosenthal CM,
    2. Dougherty R,
    3. Vakili S,
    4. Ferraro AM,
    5. Kuo LW,
    6. Alobaidi R,
    7. Aljehane L,
    8. Gaur A,
    9. Sykora P,
    10. Glasgow E,
    11. Agarwal S and
    12. Rosenthal DS
    : CRISPR-Cas9 knockdown and induced expression of CD133 reveal essential roles in melanoma invasion and metastasis. Cancers (Basel) 11(10): 1490, 2019. PMID: 31623313. DOI: 10.3390/cancers11101490
    OpenUrlCrossRefPubMed
  37. ↵
    1. Liu B,
    2. Qu L and
    3. Yan S
    : Cyclooxygenase-2 promotes tumor growth and suppresses tumor immunity. Cancer Cell Int 15: 106, 2015. PMID: 26549987. DOI: 10.1186/s12935-015-0260-7
    OpenUrlCrossRefPubMed
  38. ↵
    1. Luo L,
    2. Liang Y,
    3. Ding X,
    4. Ma X,
    5. Zhang G and
    6. Sun L
    : Significance of cyclooxygenase-2, prostaglandin E2 and CD133 levels in sunitinib-resistant renal cell carcinoma. Oncol Lett 18(2): 1442-1450, 2019. PMID: 31423209. DOI: 10.3892/ol.2019.10442
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
470 nm LED Irradiation Inhibits the Invasiveness of CD133-positive Human Colorectal Cancer Stem Cells by Suppressing the Cyclooxygenase-2/prostaglandin E2 Pathway
SANGJOON MO, HYUN JEONG KU, SUN-HYANG CHOI, HYE JUNG JEONG, DONG-GUK PARK, MAN HWAN OH, JIN CHUL AHN
Anticancer Research Mar 2021, 41 (3) 1407-1420; DOI: 10.21873/anticanres.14898
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords