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. 2020 May 6;10(1):7668.
doi: 10.1038/s41598-020-64790-y.

Design of Fluorescent Probes for Bioorthogonal Labeling of Carbonylation in Live Cells

Hazel Erkan  1   2 , Dilek Telci  3 , Ozlem Dilek  4
Affiliations

Design of Fluorescent Probes for Bioorthogonal Labeling of Carbonylation in Live Cells

Hazel Erkan et al. Sci Rep. .

Abstract

With the rapid development of chemical biology, many diagnostic fluorophore-based tools were introduced to specific biomolecules by covalent binding. Bioorthogonal reactions have been widely utilized to manage challenges faced in clinical practice for early diagnosis and treatment of several tumor samples. Herein, we designed a small molecule fluorescent-based biosensor, 2Hydrazine-5nitrophenol (2Hzin5NP), which reacts with the carbonyl moiety of biomolecules through bioorthogonal reaction, therefore can be utilized for the detection of biomolecule carbonylation in various cancer cell lines. Our almost non-fluorescent chemical probe has a fast covalent binding with carbonyl moieties at neutral pH to form a stable fluorescent hydrazone product leading to a spectroscopic alteration in live cells. Microscopic and fluorometric analyses were used to distinguish the exogenous and endogenous ROS induced carbonylation profile in human dermal fibroblasts along with A498 primary site and ACHN metastatic site renal cell carcinoma (RRC) cell lines. Our results showed that carbonylation level that differs in response to exogenous and endogenous stress in healthy and cancer cells can be detected by the newly synthesized bioorthogonal fluorescent probe. Our results provide new insights into the development of novel bioorthogonal probes that can be utilized in site-specific carbonylation labeling to enhance new diagnostic approaches in cancer.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Scheme 1
Scheme 1
Structure of 2-Hydrazine 5nitrophenol, and its aliphatic hydrazone and aromatic hydrazone.
Figure 1
Figure 1
(A) Absorption spectra of 2Hzin5NP and its hydrazone products in methanol. From left to right, 2Hzin5NP (blue), aliphatic hydrazone; 2 (red), aromatic hydrazone; 3 (green). (B) Emission spectra of compounds in methanol. 2Hzin5NP (blue, λex = 354 nm, λem = 469 nm), 2 (red, λex = 397 nm, λem = 502 nm), 3 (green, λex = 418 nm, λem = 517 nm). (C) Solutions of 2Hzin5NP, its aromatic hydrazone in methanol under room light (left) and long wavelength fluorescent light (right). (D) Solutions of 2Hzin5NP, its aliphatic hydrazone in methanol under room light (left) and long wavelength fluorescent light (right).
Figure 2
Figure 2
(A) Cytotoxic effect of H2O2 treatment on cell viability of HDF, A498 and ACHN cell lines. Each data point represents the mean percentage of viable cells treated H2O2 (0.5–2.5 mM) at different time points from three separate experiments. (B) Cytotoxic effect of 2Hzin5NP on HDF, A498 and ACHN cell lines. Cells were treated with (5–50 µM) 2Hzin5NP for 30 minutes and then incubated with standard DMEM for 24 hours. Each data point represents the mean percentage of viable cells at different time points from three independent experiments. The percentage of cell viability was calculated by assigning the absorbance value obtained from non-treated cells as 100% for each time point.
Figure 3
Figure 3
Detection of H2O2 induced carbonylation levels of HDF, A498 and ACHN cells in the presence and absence of pyruvate. The cells were incubated DMEM with (1 and 2 mM) or without sodium pyruvate prior to 2 mM H2O2 treatment. Carbonylation was detected by labeling HDF and ACHN cells with 15 µM 2Hzin5NP and A498 with 20 µM 2Hzin5NP for 30 minutes. A 488 nm diode laser was used for excitation and LP 518 filter was used for emission. Images were captured using Zeiss LSM 800 confocal microscope. Figure displays representative images from three independent experiments. Scale bar is equal to 10 µm.
Figure 4
Figure 4
Effect of pyruvate on carbonylation levels in (A) HDF, (B) A498 and (C) ACHN cells. Cells were incubated DMEM with and without 2 mM sodium pyruvate prior to 2 mM H2O2 treatment. Cells were then lysed by six freeze-thaw cycles and fluorescence intensities were measured by Varioskan Multimode Plate Reader at 396 nm excitation and 506 nm emission. Autofluorescence intensity of control groups was respectively subtracted from all experimental groups. Each data point represents the mean of fluorescence intensity (RFU) at least from three separate experiments. The percentage of RFU obtained for each cell line for H2O2 + 2Hzin5NP treatment in the absence of pyruvate was set to 100%. (D) Detection of carbonylation levels in HDF, A498 and ACHN cells in response to H2O2 treatment in pyruvate-free conditions. Each data point corresponds to average RFU from three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 5
Figure 5
Detection of serum starvation-induced carbonylation in HDF, A498 and ACHN cells. Cells were incubated with and without 10 percent FBS in DMEM for 16 hours. While A498 cells were labeled with 20 µM, ACHN and HDF cells were labeled with 15 µM 2Hzin5NP for 30 minutes. (A) Representative images from there independent experiments were captured using Zeiss LSM 800 confocal microscope at 40x objective. 405 nm and 488 nm diode lasers were used for excitation and LP 435 and 518 filters were used for the emission. Scale bar is equal to 10 µm. (B) Quantitative analysis of carbonylation levels in HDF, A498 and ACHN cells using Varioskan Multimode Plate Reader. Cell lysates obtained by six freeze-thaw cycles in lysis buffer were analyzed by measuring fluorescence intensity (RFU) at 396 nm excitation and 506 nm emission. The autofluorescence intensity of control groups was respectively subtracted from all experimental groups. Each data point represents average of at least three independent experiments. The percentage of RFU obtained for each 2Hzin5NP labeled cell line in the serum free conditions was set to 100%. (C) Detection of carbonylation levels in HDF, A498 and ACHN cells in response to serum starvation. Each data point corresponds to average RFU from three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
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