Near-infrared fluorescent probe for fast track of cyclooxygenase-2 in Golgi apparatus in cancer cells

doi:10.1007/s11705-019-1796-1

Front. Chem. Sci. Eng.

2020, Vol. 14

Issue (1) : 41-52 https://doi.org/10.1007/s11705-019-1796-1

RESEARCH ARTICLE

Near-infrared fluorescent probe for fast track of cyclooxygenase-2 in Golgi apparatus in cancer cells

Bhaskar Gurram¹, Miao Li¹, Jiangli Fan¹, Jingyun Wang², Xiaojun Peng¹(

)

¹. State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China
². School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China

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Abstract

Cyclooxygenase-2 (COX-2) has been used as an excellent traceable biomarker, and exists maximally in Golgi apparatus (Cancer cells). Celecoxib (CCB) is a selective inhibitor for COX-2, and has been used as one of non-steroidal anti-inflammatory drug. Herein we report the conjugation of nile blue (NB) with CCB via a six-carbon linkage to form a fluorescence probe NB-C6-CCB for the detection of COX-2. NB-C6-CCB displays strong fluorescence with the emission peak centered at near-infrared wavelength (700 nm) in tumor cells or tumor tissues with high expression of COX-2. Importantly, NB-C6-CCB can discriminate cancer cells (MCF-7) fluorescence intensity from normal ones (COS-7) in the co-culture medium under confocal microscope. Subcellular localization of the NB-C6-CCB preferentially points to the Golgi apparatus and increases the fluorescent intensity. The competitive analysis (with CCB) and Native-PAGE analysis confirmed that NB-C6-CCB shows selective binding affinity towards COX-2 enzyme. Competitive analysis with CCB (flow cytometry assay) revealed the fluorescence intensity fluctuation due to pretreatment of CCB with different concentrations, indicating that the NB-C6-CCB is a precise or sensitive probe for the COX-2. Tumor tissue (depth: 500 µm), organs and mice imaging tests show excellent near-infrared visualization, specific localization and identification of tumors.

Keywords cyclooxygenase-2 nile blue CCB Golgi apparatus NIR imaging

Corresponding Author(s): Xiaojun Peng

Online First Date: 28 April 2019 Issue Date: 09 March 2020

Cite this article:

Bhaskar Gurram,Miao Li,Jiangli Fan, et al. Near-infrared fluorescent probe for fast track of cyclooxygenase-2 in Golgi apparatus in cancer cells[J]. Front. Chem. Sci. Eng., 2020, 14(1): 41-52.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-019-1796-1
https://academic.hep.com.cn/fcse/EN/Y2020/V14/I1/41

Fig.1 Chemical structures of NB-C6-CCB. (a) NB, (b) CCB derivative (CCB) and (c) NB-C6-CCB

Fig.2 Scheme 1 Synthesis of NB-C6-CCB. Reagents and conditions: (a) methoxyethanol, CuI, CsCO₃, 125°C, 24 h, (b) ethanol, HCl, 90°C, 4.5 h, (c) THF, CCB derivative, TEA, 4 h, r.t.

Fig.3 Native-PAGE analysis of NB-C6-CCB labeling. (a) Coomassie brilliant blue staining, (b) fluorescence image with excitation 630 nm and emission wavelengths 640–700 nm (lane 1: purified cyclooxygenases-2; lanes 2–4: protein extracts of MCF-7 cells incubated with various concentrations of NB-C6-CCB for 30 min)

Fig.4 Fluorescent images of live cells stained with NB-C6-CCB (2.5 µmol/L). (a) HL-7702, (b) COS-7, (c) MCF-7, (d) HeLa and (e) fluorescence intensity of live cells (l_ex = 630 nm, l_em = 640–700 nm)

Fig.5 Normal and cancer cells co-cultured and stained with NB-C6-CCB (2.5 µmol/L). (a) and (c) Fluorescence of co-cultured cancer (MCF-7) and normal cells (COS-7), (b) and (d) overly of co-cultured cancer (MCF-7) and normal cells (COS-7). Images were acquired by confocal microscopy (l_ex = 630 nm, scan range l_em = 640–700 nm)

Fig.6 Selectivity conformation of NB-C6-CCB. (A) MCF-7 and (B) HeLa were pre-incubated with CCB 0, 5, 10 µmol/L for 3 h and then 1.0 µmol/L of NB-C6-CCB was added. l_ex = 630 nm, scan range l_em = 640–700 nm; quantitative image analysis of the average total fluorescence of (C) MCF-7 cells and (D) HeLa cells

Fig.7 Flow cytometry analysis of competition of CCB with NB-C6-CCB for COX-2 in MDA-MB-231 cells. (a) Control group, (b) stained with only NB-C6-CCB (1.0 µmol/L), (c) pre-incubated with CCB 10 µmol/L for 2.0 h and then added 1.0 µmol/L of NB-C6-CCB, (d) pre-incubated with CCB 15 µmol/L for 2.0 h and then added 1.0 µmol/L of NB-C6-CCB (l_ex = 630 nm, scan range l_em = 640–700 nm)

Fig.8 Fluorescence images of NB-C6-CCB (2.5 µmol/L) co-stained with NBD-C6-Ceramide (2.5 µmol/L) in MDA-MB-231 cells. (a) Green emission of Golgi tracker (l_ex = 488 nm, l_em = 500–540 nm), (b) red emission of NB-C6-CCB (λ_ex = 630 nm, l_em = 640–700 nm), (c) overlay of the green and red channels, (d) overlay zoom image from picture c, (e) intensity profile of cross co-stain image, (f) Pearson’s co-efficient graph of overlay

Fig.9 Fluorescence images of NB-C6-CCB (2.5 µmol/L) co-stained with NBD-C6-ceramide (2.5 µmol/L) and Hoechst 33342 (2.5 µmol/L) in MDA-MB-231 cells. (a) Red emission of NB-C6-CCB (l_ex = 630 nm, l_em = 640–700 nm), (b) blue emission of Hoechst 33342 (l_ex = 405 nm, l_em = 440–480 nm), (c) green emission of Golgi tracker (l_ex = 488 nm, l_em = 500–540 nm), (d) overlay of the blue and red channels, (e) overlay of NB-C6-CCB, hoechst 33342 and NBD-C6-ceramide

Fig.10 Fluorescence imaging of cancer and normal tissues with stained NB-C6-CCB (2.0 µmol/L). A(a): bright field, A(b) merged, A(c): fluorescence image; B(a): bright field, B(b): 2D image, B(c): 3D images of tumor tissue (length: 500 mm); C(a): bright field, C(b): merged, C(c): fluorescence images of normal mouse liver tissue

Fig.11 Fluorescence imaging of tumor mouse and organs by using NB-C6-CCB. A(a): white light, A(b): merged light; B(a): tumor lump, B(b): liver, B(c): kidney, B(d): spleen, B(e): heart

Fig.12 MTT assay of NB-C6-CCB in live cells (MCF-7)

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