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Visualization of Boronic Acid-Containing Pharmaceuticals in Live Tumor Cells Using a Fluorescent Boronic Acid Sensor Yoshihide Hattori, Miki Ishimura, Yoichiro Ohta, Hiroshi Takenaka, and Mitsunori Kirihata ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00522 • Publication Date (Web): 25 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016
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Visualization of Boronic Acid-Containing Pharmaceuticals in Live Tumor Cells Using a Fluorescent Boronic Acid Sensor Yoshihide Hattori,* † Miki Ishimura,† Yoichiro Ohta,† Hiroshi Takenaka,† and Mitsunori Kirihata† †Research
Center of Boron Neutron Capture Therapy, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Japan. Supporting Information Available: The following files are available free of charge. SI.doc Broef description of contents. ABSTRACT: Boronic acid-containing compounds are
regarded as a new class of pharmaceuticals, and the distribution in live cells is crucial for the development of novel drugs. However, generic detection methods for boron compounds are not suitable for live-cell imaging, fluorescencelabeling technique is challenging to apply for small molecules. Fluorescent boron-sensor DAHMI which based on boron chelating-ligand was developed for a novel visualization method of boron-based pharmaceuticals in live cell. In this study, we analyzed the distribution of boronic acid derivatives in live cells using a fluorescent boronic acid sensor.
As increasing number of studies have been performed to assess the interaction between boronic acid-containing compounds and biomolecules in recent years. 1-6 In the pharmaceutical science field, boronic acid-containing compounds have been developed as a boron carrier for boron neutron capture therapy (BNCT)7, protease inhibitor, sugar sensor, and cell-penetrating peptide. Recently, new boroncontaining drug, such as p-boronophenylalanine (L-BPA, for BNCT)8-10, o-fluoro-p-boronophenylalanine (L-FBPA, for PET imaging)11,12, and tavaborole (antifungal drug for onychomycosis)13 have been touted as a new class of pharmaceuticals (Figure 1). 14,15 Assessment of drug distribution, especially the distribu-
a particular compound in cells and tissues, an immunostaining or fluorescence-labeling technique is typically employed. However, these methods would require the development of novel antibodies or fluorescence-labeled pharmaceuticals, which are challenging to apply for live-cell imaging. The distribution of boron-based pharmaceuticals in cells and biological tissues can be evaluated by the detecting the boron atom, because biological tissues contain little or no boron. However, most boron-detection methods, such as those using immunostaining17,18 and -autoradiography19, are costly and/or require extensive sample pretreatment. Furthermore, these detection methods are generally not suitable for live-cell imaging. Thus the distribution of boron-based pharmaceuticals in live cells has not been fully characterized. Recently, we disigned and synthesized a novel fluorescent sensor for boronic acid, DAHMI (Figure 2)20. DAHMI reacted rapidly with boron-based pharmaceuticals in aqueous media, resulting in the emission of blue fluorescence by the DAHMI-boron complex (Figure S1, S2). Furthermore, DAHMI can passs throgh the cellmembranes; thus it, can be used to visualize L-BPA distribution in immobilized cells whithout requireing perior cell permeabilization. Therefore, we aimed to develop a novel detection method for boronic acid derivatives in live cells using DAHMI as the fluorescent boron-sensors. In the present study, we elucidated the distribution of boronic acid-containing compounds in live cells by using DAHMI as a fluorescent boronic acid sensor.
Figure 1. Example of boron-containing pharmaceuticals. Figure 2. The boronic acid sensor DAHMI. tion in live cells, is crucial for the development of novel drugs. For example, boron accumulation in the cell nucleus was reported to efficiently kill cells during BNCT16, and konwledge on the precise intracellular localization, distribution, and tumor/normal tissue ratio of boron-based pahrmaceuticals is critical. To elucidate the distribution of
In order to investigate the potential of DAHMI for livecell imaging of boron pharmaceuticals, DAHMI cytotoxicity in the C6 rat glioma cells, B16 mouse melanoma cells, MCF7 human breast cancer cells, and Hst-578T human breast cancer cells which incubated with/without L-BPA was de-
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ACS Sensors termined using the WST-8 cell counting kit. As shown in Table 1, the cytotoxicity of DAHMI and DAHMI-BPA complex was considerably low at 1 h post treatment. However, DAHMI cytotoxicity increased after 2 h of treatment, which may be due to the effect of N-methylamine and 4diethylaminosalicylaldehyde generated by the hydrolysis of DAHMI. In our previous study, DAHMI reacted rapidly with boron-based pharmaceuticals in less than 30 min, and the DAHMI-boron complex generated was stable in aqueous media20. These results suggested that DAHMI can be applied for live-cell imaging in 0.5-1mM within 1hour after treatment of DAHMI.
Fluorescence Micrograph
Time
Differential Interference Contrast Microscope
0 min
1 min
Table 1. DAHMI Cytotoxicity in various cell lines. IC50 (after at 1h)
[a]
IC50 (after at 2h)
[b]
5 min
Cell Line DAHMI
DAHMI [c]
+BPA
DAHMI
DAHMI [c]
+BPA
C6
> 1.0 mM
> 1.0 mM
0.46 mM
0.70 mM
B16
> 1.0 mM
> 1.0 mM
0.55 mM
> 1.0 mM
MCF-7
> 1.0 mM
> 1.0 mM
0.42 mM
> 1.0 mM
Hst-578T
> 1.0 mM
> 1.0 mM
0.37 mM
> 1.0 mM
10 min
[a] Cytotoxicity at 1 h-post DAHMI. [b] Cytotoxicity at 2 h-post DAHMI. [c] Cytotoxicity against L-BPA uptook cells. IC50, 50% inhibitory concentration.
To assess the use of DAHMI for live-cell imaging, we monitored the L-BPA distribution in live B16 cells. Following DAHMI treatment, live B16 cells that took up L-BPA displayed immediate blue fluorescence (Figure 3). In image analysis of the fluorescence micrograph using ImageJ21, the fluorescent intensity quickly increased in 5 min, and the maximum intensity was reached at 20 min post-treatment. The blue fluorescence was distributed throughout the cyto plasm and nucleus of the cells. Fluorescent images obtained in this study were comparable to those that showed L-BPA distribution in immobilized cell22,23. To further elucidate the distribution of L-BPA in several types of live tumor cells, DAHMI was used to monitor C6, MCF-7 and Hst-578T that took up L-BPA (Table 2). As shown in Table 2, DAHMI was successfully used to visualize the distribution of L-BPA in these live tumor cell lines. LBPA was distributed throughout the tumor cells, including in the cell nucleus. Lastly, to confirm the utility of DAHMI for live-cell imaging of boron-based pharmaceuticals, we stained live B16 cells that were incubated with L-BPA, L-FBPA, LBPA(2CF3),24 phenylboric acid, and tavaborole with DAHMI (Table 3). The results showed that the distribution pattern of fluorinated BPA derivatives was similar to that of L-BPA. In contrast, phenylboric acid and tavaborole were localized in the cytosol and excluded from the nucleus. These results suggested that DAHMI-based imaging method can be used to visualize the distribution of various boron-based pharmaceuticals in live cells.
15 min
20 min
Time couse of mean fluorescent intensity of fluorescence micrographs 1.6 1.4 Mean Intensity
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1.2 1 0.8 0.6 0.4 0.2 0 0
5
10
15
20
Time after treatment of DAHMI (min)
Figure 3. Fluorescence (ex: 405nm) and differential interference contrast microscope showing the distribution of L-BPA over a 20 min period in melanoma cells using DAHMI as the boron sensor, and time corse of mean fluorescent intensity in fluorescence micrographs.
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Table 2. Micro-distribution of L-BPA in tumor cells.
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[a]
Cell Line
DAHMI stain
[b]
Nuclear Stain
[c]
Merged Image
C6
In conclusion, the properties of DAHMI allowed for its application in live-cell imaging of boron-based pharmaceuticals in several tumor cell lines. Thus, we were able to elucidated the distribution of boron-based pharmaceuticals in live cells using DAHMI. Our study showed that BPA and fluorinated BPAs were distributed throughout the cell, while tavaborole and phenylboric acid were distributed in the cytosol but excluded from the cell nucleus. These results demonstrated the utility of DAHMI as a tool to assess the distribution of boronic acid derivatives in live cells. Further studies on DAHMI application are currently underway.
MCF-7
AUTHOR INFORMATION Corresponding author *E-mail:
[email protected] HST-578T
Supporting Information [a]
[b]
Cells were incubated in DMEM containing L-BPA (2.0 mM). Cells were subsequently treated with DAHMI (1.0 mM) for 20 min at 37°C (ex: [c] 405nm). Cell nuclei were stained with CellLight™ Nucleus-GFP (Thermo Fisher Scientific, Waltham, MA).
The Supporting Information is available free of charge SI.doc. Detailed cell culture condition; WST-8 assay; protocol of live-cell imaging; excitation spectra; fluorescent spectra. Author Contributions
Table 3. Micro-distribution of boron-based compounds in B16 melanoma cells. Compound
DAHMI stain
[a]
Nuclear Stain
[b]
Merged Image
Y.H. and M.K. designed the experiments. Y.H., Y.O., and H.T. performed the synthesis of the compounds. Y.H. and, M.I. performed the biological assays. Y.H. wrote the manuscript. All authors discussed the results and implications of the study and approved the final manuscript.
Notes
The authors declare no competing financial interests. L-BPA (1 mM)
Keywords
L-FBPA (1 mM)
ACKNOWLEDGMENT
fluorescent boronic acid sensor, live-cell imaging, molecular probe, boron-based pharmaceuticals, tavaborole, pboronophenylalanine
We would like to thank Prof. Yoshihiro Yamaguchi of the Kindai University for the measurement of fluorescence properties. This work was supported by the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering grant program for biomedical engineering research, and by the Project for Cancer Research And Therapeutic Evolution (PCREATE) from the Japan Agency for Medical Research and Development, AMED.
BPA(2CF3) (1 mM)
Phenylboric acid (1 mM)
REFERENCES
Tavaborole (0.5 mM) [a]
Cells were stained with DAHMI (1.0 mM) for 20 min at 37°C (ex: [b] 405nm). Cell nuclei were stained with CellLight™ Nucleus-GFP (Thermo Fisher Scientific, Waltham, MA).
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