Abstract
Nitric oxide (NO), an uncharged free radical is implicated in various physiological and pathological processes. The present study is an investigation on the effect of NO on proliferation, apoptosis and migration of colon cancer cells. Colon adenocarcinoma cells, WiDr, were used for the in vitro experiments. Tissues from colon adenocarcinoma, adjacent normal and inflammatory tissue and lymph node with metastasis were evaluated for iNOS, MMP-2/9 and Fra-1/Fra-2. NO increases the proliferation of cancer cells and simultaneously prevents apoptosis. Expression of MMP-2/9, RhoB and Rac-1 was enhanced by NO in a time dependent manner. Further, NO increased phosphorylation of ERK1/2 and induced nuclear translocation of Fra-1 and Fra-2. Electrophoretic mobility shift analysis and use of deletion mutant promoter constructs identified role of AP-1 in NO-mediated regulation of MMP-2/9. iNOS, MMP-2/9, Fra-1 and Fra-2 in normal and colon adenocarcinoma tissues were analyzed and it was found that increased expression of these proteins in cancer when compared to normal provides support to our in vitro findings. The study showed that the NO-cGMP-PKG promotes MMP-2/9 expression by activating ERK-1/2 and AP-1. This study reveals the insidious role of NO in imparting tumor aggressiveness.
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Acknowledgments
Suboj Babykutty acknowledges Indian Council for Medical Research (ICMR), Government of India, for SRF fellowship and Srinivas Gopala acknowledges Department of Biotechnology (DBT), Government of India, for funds to carry out this work (BT/PR4201/Med/14/513/2004). The authors acknowledge Prof. M. Radhakrishna Pillai, Director, RGCB for extending Flow Cytometry facility; Dr. Kannan S., Professor, RCC for providing support for real-time PCR; Mr. Deepak Roshan V. G. for helping to do the real-time PCR experiments; Mr. Radhakrishnan N. S., Scientific Assistant, Ms Abitha Thomas, for helping in IHC protocol, Mrs. Shirley Stewart, Associate Professor, Mar Ivanios College and Mrs. Nandini R. J., for editing and technical support.
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Supplementary Figure 1. Nitrite production estimated using Griess assay. (A) The nitrite level was determined by adding the Griess reagent to the supernatant and incubated as described in materials and methods. After incubation the absorbance was measured at 548 nm. Optical densities were converted into nitrite concentration using recent calibration curve of the NaNO2. Each value is presented as the mean ± SD of determinations from three independent experiments. (B) The protein expression of cyclin D1 levels were expressed as ratio to the expression of β-actin. The above experiment was repeated at least three times. Each value is presented as the mean ± SD of determinations from three independent experiments ***P < 0.001. Real Time RT-PCR analysis of HIF 1-α transcription by NO. (C) The real-time PCR was performed in 7900 HT Fast Real-Time PCR system using MESA green qPCR Mastermix for SYBR Assay. Fold increase of the HIF 1-α was normalized with that of β-actin and was plotted as a graph. Each value is presented as the mean ± SD of determinations from two independent experiments. The mean fold change was significantly higher from the corresponding untreated treated group as analysed by one way ANOVA and Student’s t-test. ***P < 0.001. Flow cytomteric histograms of cell cycle progression (D) and (E). WiDr cells were grown to confluence and treated with or without SNAP (15.6 μM) and then analyzed by FACS as described in materials and methods. The distributions of cells in different phases of the cell cycle are represented in each histogram and figure shown here is the representative histogram from three independent experiments. (JPEG 353 kb)
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Supplementary Figure 2. Graphical representation of MMPs and RhoGTPase fold change (A and B) The cells were grown in 60 mm dish and cells were exposed to SNAP (15.6 μM) for 4 h and the medium was removed and fresh medium was added and incubated for different time points. After treatment, total RNA was isolated using Pure Link RNA Mini Kit following manufacture’s protocol. RNA was reverse transcribed into cDNA and then amplified by using specific primers by protocols described in the Materials and Methods. Fold change of the target mRNAs (MMPs and Rho GTPases) were normalized with that of β-actin and is plotted as a graph. Each value is presented as the mean ± SD of determinations from two independent experiments. (JPEG 281 kb)
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Supplementary Figure 3. Graphical representation of TIMP-1/2 fold change (A). After treatment, total RNA was isolated and RNA was reverse transcribed into cDNA and then amplified by using specific primers by protocols described in the Materials and Methods. Fold change of the target mRNAs (TIMPs) were normalized with that of β-actin and is plotted as a graph. Each value is presented as the mean ± SD of determinations from two independent experiments. The mean fold decrease was significantly higher from the corresponding untreated treated group as analysed by Student’s t-test. *P < 0.05; **P < 0.01. Effect of inhibitors on cell viability (B) WiDr cells grown in 96-well plates were treated with or without SNAP(15.6 μM),ODQ(30 μM),KT5823 (180 nm) and PD98059 (10 μM) for 4 h, then the medium was removed and fresh medium was added and incubated for another 48 h. At the end of treatment, cell viability was assessed by MTT assay as described in Materials and Methods. All results were expressed as the mean percentage of control ± S.D. of quadruplicate determinations from three independent experiments. The differences among the mean values were analyzed using 1-way ANOVA followed by Tukey’s post hoc t-test analysis. ***P < 0.001. (JPEG 288 kb)
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Supplementary Figure 4. Effect of DetaNONOate on viability of WiDr (A) /HUVECs (B). The cells grown in 96-well plates were treated with or without the indicated concentrations of DetaNONOate and at the end of experiment cell viability was assessed by MTT assay as described in Materials and Methods. All results were expressed as the mean percentage over control ± S.D. of quadruplicate determinations from three independent experiments. ***P < 0.001. (JPEG 221 kb)
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Supplementary Figure 5. (A) Detection of endogenous NO production by DAF2-DA. NO levels in colon carcinoma cell line; HCT 116 was detected using DAF-2DA. Cells were incubated with DAF-2-DA and visualized using an inverted fluorescent and light microscope. Images were captured using ProgRes CapturePro v2.8.0. Emission of green fluorescence is an indicative of endogenous NO production. Pictures shown were representative of those that were independently repeated at least two times. (B) Effect of SNAP on viability of colon cancer cells. HCT 116 cells grown in 96-well plates were treated with or without the indicated concentrations of SNAP and at the end of experiment cell viability was assessed by MTT assay as described in Materials and Methods. All results were expressed as the mean percentage of control ± S.D. of quadruplicate determinations from three independent experiments. There was no significant difference between control and SNAP treated HCT cells. (C) NO induced migration of HCT 116 cells. Cells were seeded in 24-well plates and then pre-incubated for 24 h in serum-free DMEM before creating a wound across the cell monolayer with a sterile plastic tip. Cells were allowed to migrate with or without SNAP (15.6 μM). Cell migration into the wound surface was then monitored by microscopy after 24 h and reported as the width of remaining wounded area relative to the initial wounded area. (D) Scratch wound assay was independently repeated two times and the values are plotted as graph. ***P < 0.001. (TIFF 2901 kb)
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Babykutty, S., Suboj, P., Srinivas, P. et al. Insidious role of nitric oxide in migration/invasion of colon cancer cells by upregulating MMP-2/9 via activation of cGMP-PKG-ERK signaling pathways. Clin Exp Metastasis 29, 471–492 (2012). https://doi.org/10.1007/s10585-012-9464-6
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DOI: https://doi.org/10.1007/s10585-012-9464-6