6,6′-Bis(O-4-pyren-1-yl-ethynylbenzoyl)-α,α-trehalose(aryl-substituted trehalose) activates the NF-κB signaling pathway
and is an effective fluorescent probe forcell imaging in HeLa CD4+ cells
Rumiko Saito1), Takanobu Nakayama2), Kahoko Hashimoto 1), 2), and Naoko Miyano-Kurosaki 1) ,2) *Abstract
Cell imaging is expected to contribute not only to basic life science research but also to clinical diagnosis,
treatment, and drug discovery as a method for exploring and interpreting biological phenomena. We haveshown that 6,6′-bis(O-4-pyren-1-yl-ethynylbenzoyl)-α,α-trehalose (aryl-substituted trehalose) is taken up
by HeLa CD4+ cells, a human cervical cancer cell line, and shows bright green luminescence. In this study,
we evaluated the effect of aryl-substituted trehalose on HeLa CD4+ cells and its utilization as a cell
imaging material. An analysis of the change in intracellular fluorescence of aryl-substituted trehalose overtime by fluorescence microscopy revealed uptake by HeLa CD4+ cells and fluorescence signals in thecytoplasm. The number of cells incorporating aryl-substituted trehalose was higher at 48 h than at 24 hafter the addition. Aryl-substituted trehalose did not affect cell cycle progression in HeLa CD4+ cells. It
was suggested that aryl-substituted trehalose is taken up into cancer cells via glucotransporter in the same
way as glucose uptake. Aryl-substituted trehalose is not taken up by normal cells but is specifically taken
up by cancer cells, so it may be used for detection of cancer cells.

Keywords：Aryl-substituted trehalose, Cancer cells, Cell imaging material

Received October 5, 2021; accepted November 5, 2021

1) Department of Life and Environmental Sciences, Graduate School of Engineering, Chiba Institute ofTechnology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan
2) Department of Life Sciences, Faculty of Advanced Engineering, Chiba Institute of Technology 2-17-1,
Tsudanuma, Narashino, Chiba 275-0016, Japan.
*Corresponding author: Naoko Miyano-Kurosaki, Department of Life and Environmental Sciences, Graduate
School of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan
E-mail: kurosaki.naoko@p.chibakoudai.jp

64

1 Introduction
Bioimaging technology has become an important
tool

in

the

field

of

biomedicine

for

the

fluorescent molecules to label and visualize
intracellular complex sugars11). Barattucci et al.
reportedthat

dimethylamino-substituted

oligo

multidimensional visualization of biomolecules,(phenylene ethynylene) glucosides act as efficient

cells, tissues, and organisms1,2). The visualization of

biocompatible fluorescent probes12,13). Ribagorda etbiological phenomena is a focus of disease research

al. found that dimethylamino-oligo glucosideand is providing new insights into the etiology ofderivatives have photophysical properties, such as

diseases3,4). In particular, fluorescence bioimaging,high quantum yield, singlet oxygen production,in which fluorescence is imparted to the target

biocompatibility,

substance for visualization, is an indispensableinternalization, and very good responsiveness; theytechnique in biomedical research3,5). For the further

can be utilized as photosensitizers for photodynamic

development of fluorescence bioimaging technology,

therapy14). In addition, Park et al. monitored theit is important to clarify correlations between the

fluorescence

molecular structure of imaging materials and optical

glucosamine-modified

properties, toxicity, and bioactivity5).

efficiently distinguish between cancer cells andFluorescent molecules used in cell imaging

normalstability,

intensity

cells15).

of

easy

cells

silicone

Therefore,

intracellulartreatedwithrhodaminetocombinationsofinclude those based on small organic fluorescentfluorescent molecules and sugars are expected to bemolecules and those based on fluorescent proteins,promising fluorescent probes for cell imaging.

such as green fluorescent protein6,7). Imaging with

Trehalose(α-D-glucopyranosyl-α-D-

fluorescent proteins requires gene transfer for

glucopyranoside) is a disaccharide composed of two

expression in living cells8). Small organic fluorescentglucose molecules linked by an α,α-1,1 bond16).

substances benefit from their ability to be quickly

Trehalose is widely distributed in nature and is used

introduced into all cells simply via extracellular

as an energy source in a broad range of taxa,

fluid9).

including bacteria, fungi, insects, plants, andThe design conditions for organic fluorescent

invertebrates17). In addition, it is widely used insmall molecule-based imaging materials include afoods and biomaterials and is attracting attention anlack of cytotoxicity and high water solubility toexcipient for pharmaceuticals. Trehalose has moreensure in vivo compatibility and hydrophobicity forfavorable hydration properties compared with otherthe permeation of cell membranes10). Many organic

sugars such as glucose, galactose, and sucrose.

fluorescent molecules are hydrophobic, which canGiven the need for imaging materials with water

result in various issues, including aggregation andsolubility for use in living tissues, as well as its

precipitation in extracellular fluid, emphasizing thehydrophobicity for penetrating the cell membrane

importance of water solubility.

and its low cytotoxicity, trehalose is a promising

Against this background, multiple cell imaging
techniques in which sugars are complexed withsugar.
We have previously synthesized 6,6′-bis(O-4-

fluorescent molecules have been reported. Hsu et al.

pyren-1-yl-ethynylbenzoyl)-α,α-trehalose

reported

incorporating

substituted trehalose) by TMS protection and

and

N-

selective deprotection of the hydroxy group ofacetylmannosamine by azide-alkyne click chemistrytrehalose, esterification of the primary hydroxywithgroup, and the Sonogashira coupling reaction13). The

thathyperacetylated

cell

glycans

alkynylfucose3-azido-7-hydroxycoumarinfunctionas(aryl-

65

aryl-substituted trehalose showed an absorption

observed using a flow cytometer. HeLa CD4+ cells

maximum in the visible region and a highwere seeded on 24-well plates (105 cells/well) for 24

fluorescence quantum yield in diluted THF solutions.

h to allow adherence and were then incubated with

It generated strong fluorescent signals. Strong

aryl-substituted trehalose at concentrations of 0 (1%fluorescence was maintained even in a THF/H2O

DMSO) and 9.4 µmol/L for 24 and 48 h. After they

mixed solvent. We further confirmed that aryl-

were washed twice with PBS(-), the cells were

substituted trehalose could be introduced into HeLa

trypsinized and collected for the measurement of

CD4+ cells and used as a green fluorescent probe.

uptake efficiency. Fluorescence was monitored using

In this study, we evaluated the effects of aryl-

a flow cytometer (BD Biosciences). Ten thousand

substituted trehalose, as a candidate cell imaging

events were collected per sample, and the data werematerial, on HeLa CD4+ cells.

analyzed using CellQuest (BD Biosciences).

2.4 Cell proliferation and viability assays.
2 Experimental
2.1 Culture conditions and reagents. HeLa
CD4+cells and Vero cells were cultured as amonolayer in Dulbecco’s Modified Eagle Medium(DMEM; Sigma-Aldrich) supplemented with 10%
heat-inactivated fetal bovine serum (Cytiva), 100
units/mL penicillin G, and 100 μg/mL streptomycin(Fujifilm Wako Pure Chemical Corporation). The
aryl-substituted trehalose was dissolved in dimethyl
sulfoxide (DMSO) and the final concentration of
DMSO was 1%.

2.2 Observation of uptake of the arylsubstituted trehalose with a fluorescencemicroscope. The uptake of the aryl-substituted
trehalose by HeLa CD4+ cells and Vero cells wasobserved using a fluorescence microscope. HeLa
CD4+ cells and Vero cells were seeded on 96-wellplates (2×104 cells/well) for 24 h to allow adherence
and were then incubated with aryl-substituted
trehalose at concentrations of 0 (1% DMSO) and 9.4µmol/L for 24 or 48 h. After incubation, nuclear
staining was performed using Hoechst33342(ImmunoChemistry Technologies). Cells

wereviewed under a fluorescence microscope (KeyenceCo.).

2.3 Evaluation of the aryl-substituted
trehalose uptake efficiency. Uptake of the arylsubstituted trehalose by HeLa CD4+ cells was

The trypan blue method was used to evaluate theeffects of aryl-substituted trehalose on the viability
and proliferation of HeLa CD4+ cells and Vero cells.
The aryl-substituted trehalose was synthesized anddiluted in DMSO. For proliferation and viability
assays, 105 cells were plated in 24-well plates andtreated with aryl-substituted trehalose for 24 and 48
h. After treatment, cells were trypsinized andcounted using a TC20 Automated Cell Counter (BioRad Laboratories, Inc.).

2.5 Comparison of glut4 expression incancer cells and normal cells by adding arylsubstituted trehalose. The expression level ofglut4 present on the cell surface was compared toinvestigate the cell uptake pathway of arylsubstituted trehalose in HeLa CD4+cells and Verocells. HeLa CD4+ cells were seeded into 24-wellplates (105 cells/well) for 24 h to allow the cells togrow adherently, followed by incubation with arylsubstituted trehalose at 0 (1% DMSO) and 9.4µmol/L for 48 h. Cells were stained with PE-bindinganti-glut4 antibody (BioLegend) for 15 min, after
which glut4 expression levels were measured with aflow cytometer.

2.6 Confirmation of NF-κB and Bcl-X
expression by flow cytometry. Cells wereincubated with 1% DMSO and 9.4 µmol/L arylsubstituted trehalose for 24 h and 48 h. NF-κB and66

Bcl-XL were measured by flow cytometry using PE-

supplemented with 1% DMSO, which solubilizes

conjugatedantibodies

aryl-substituted trehalose, and cells supplemented(BioLegend) along with standard binding buffer.

with aryl-substituted trehalose. We observed that

Antibody staining was performed following thearyl-substituted trehalose was taken up by HeLa

manufacturer’s recommended protocol.

CD4+ cells and Vero cells and then labeled the

NF-κB

and

Bcl-X

2.7 Statistical analysis. All data werecytoplasmic compartment (Fig. 1). We haveprocessed using Microsoft Office Excel

previously(Microsoft). Each experiment was repeatedconcentration of 37.5 µmol/L is taken up by cells; inindependently at least 3 times, and all

this study, we used the minimum concentration forresults were expressed as mean ± standard

cellular uptake13). Aryl-substituted trehalose was

deviation (SD). In addition, statisticaltaken up by HeLa CD4+ cells but not by Vero cells.

differences were analyzed by Student’s t-

This suggests that aryl-substituted trehalose is taken

tests. A p-value of less than 0.05 was

up by cancer cells but not by normal cells.

considered statistically significant.

confirmed

thattrehaloseata

3.2 Evaluation of aryl-substituted trehalose
uptake in HeLa CD4+cells by flow cytometry.

3 Result
3.1 Aryl-substituted trehalose uptake by
HeLa

CD4+

fluorescence

cells.
were

Nuclearstaining

andcompared

betweencells

As determined by flow cytometry, the fluorescenceintensities of aryl-substituted trehalose in HeLa
CD4+ cells at 9.4 µmol/L for 24 and 48 h were

Fig. 1 Analysis of aryl-substituted trehalose uptake by HeLa CD4+cells and Vero cells by fluorescencemicroscopy. HeLa CD4+cells were incubated with aryl-substituted trehalose or 1% DMSO for 24 h (A) or
48 h (B). Vero cells were incubated with aryl-substituted trehalose or 1% DMSO for 24 h (C) or 48 h (D).
From left to right, Phase, Hoechst, and Fluorescence images are shown. Scale bar represents 50 µm.

67significantly greater than that in the control by 13%
and 22%, respectively (Fig. 2). The fluorescenceintensity of aryl-substituted trehalose incorporated
into HeLa CD4+ cells increased in a time-dependent
manner.

Fig. 2 Flow cytometer analysis of thefluorescence intensity of aryl-substituted
trehalose incorporated into HeLa CD4+ cells.
Cells were incubated with aryl-substituted
trehalose for 24 h ( □ ) or 48 h ( ■ ) atconcentrations of 9.4 µmol/L and 1% DMSO.
Compared with the control, p < 0.001 (***). Alldata are representative of three experiments(±SD).

3.3 Cytotoxicity of aryl-substituted trehalose.
The trypan blue method was used to evaluate theeffect of aryl-substituted trehalose on the viability
and proliferation of HeLa CD4+ cells and Vero cells.
After treatment with aryl-substituted trehalose, cell
viability was greater than 90% at both 24 and 48 h.
In addition, viability did not differ significantly
between treated cells and 1% DMSO (data notshown). There was no difference in cell proliferation
between cells that were and were not supplemented
with aryl-substituted trehalose after 24 and 48 h (Fig.
3). It was suggested that aryl-substituted trehalose
does not affect the proliferative potential of cancer
cells and normal cells.

Fig. 3 Proliferation analysis of HeLa CD4+ cells
and Vero cells. HeLa CD4+ cells were treatedwith 1% DMSO and aryl-substituted trehalose
for 24 h (A) or 48 h (B). Vero cells were treatedwith 1% DMSO and aryl-substituted trehalose
for 24 h (C) or 48 h (D). All data arerepresentative of three experiments (±SD).

68

3.4 Intracellular uptake of aryl-substituted

aryl-substituted trehalose, but not in Vero cells (Fig.

trehalose. Cellular glucose uptake is mediated by

4). This result suggests that aryl-substituted

membrane-boundtrehalose is taken up into cells via glut4.

glucosetransporters.

The

expression level of glut4 present on the cell surface
was compared to investigate the cell uptake pathway

3.5 Expression of factors related to the NFκB

signaltransduction

pathway.

NF-κB

HeLa CD4+ cells,

expression levels were 14% higher than those in thewhich are cancer cells, and Vero cells, which arecontrol for concentrations of 9.4 µmol/L after 24 hnormal cells. The expression level of glut4 wasand 10% higher after 48 h (Fig. 5A, B). Furthermore,increased in HeLa CD4+ cells supplemented with

Bcl-XL expression levels were 15% higher thanof aryl-substituted trehalose of

Fig. 4 Comparison of glut4 expression by addition of aryl-substituted trehalose in cancer cells and normal
cells. The aryl-substituted trehalose was added to HeLa CD4+ cells (A,B) and Vero cells (C,D). Populationsof glut4-positive cells were analyzed using a flow cytometer. Compared to 1% DMSO, p < 0.05 (*) or p
< 0.001 (***). All data are means ± SD of three samples from three independent experiments.

69

Fig. 5 NF-κB and Bcl-XL expression in response to aryl-substituted trehalose. HeLa CD4+ cells were
stained with NF-κB-PE (A,B) or Bcl-XL-PE (C,D) for flow cytometry. (A,C) Representative profile. The
solid line and dashed line represent the aryl-substituted trehalose and 1% DMSO, respectively. (B,D) Eachbar represents the mean ± SD of three independent experiments (p < 0.01 (**) or p < 0.001 (***) compared
to 1% DMSO).
those in the control for concentrations of 9.4 µmol/Lmaterials is a lack of cytotoxicity. Anotherafter 24 h and 4% higher after 48 h (Fig. 5C, D). The

requirement for the design of an imaging material isaddition of aryl-substituted trehalose increased thegood water solubility for adapting to the biologicalexpression of NF-κB signaling pathway-related

environment and hydrophobicity for penetrating thefactors (i.e., NF-κB and Bcl-XL), thereby promoting

cell membrane. Therefore, we focused on trehalose,

the survival of HeLa CD4+ cells.

which is a water-soluble sugar. Trehalose has
beneficial characteristics for applications as a raw

4 Discussion

material for processed foods and as an excipient for

Cell imaging is expected to contribute to clinicalpharmaceuticals19). We successfully synthesized

diagnosis, treatment, drug discovery, and life sciencearyl-substituted trehalose, in which trehalose withresearch as a methodology for exploring andhigh hydration ability was combined with ainterpreting biological phenomena from a newhydrophobic organic fluorescent molecule, for cell

perspective18). One of the design requirements forimaging. Aryl-substituted trehalose showed efficientorganic fluorescent small molecule-based imaging

time-dependent cellular uptake (Fig. 1).
70

We verified that aryl-substituted trehalose wastrehalose is easily taken up by cells via glut4 andtaken up by HeLa CD4+ cells and verified its

does not affect cell viability and proliferation, so weeffectiveness as a cell imaging probe. In addition, a

expect it to be useful as a new cell imaging

quantitative flow cytometry analysis revealed thattechnology.

thefluorescenceintensityof aryl-substituted

In addition, aryl-substituted trehalose did notcells depends on theinfluence cell proliferation in HeLa CD4+ cells andtreatment duration. HeLa CD4+ cells supplemented

Vero cells. In this assay, we used HeLa CD4+ cells aswith aryl-substituted trehalose did not show

a representative cancer cell line and Vero cells as amorphological changes typical of apoptosis, as

representative normal cell line. Aryl-substituted

evaluated by light microscopy, even 48 h aftertrehalose was not taken up by Vero cells but was

treatment. During the early stages of apoptosis, cell

specifically taken up by HeLa CD4+ cells,

contraction and enrichment can be observed using asuggesting that it may be used for cancer celllight microscope. Cells show a reduced cell size,

detection and drug discovery. We believe that aryl-dense cytoplasm, densely packed organelles, andsubstituted trehalose will serve as a valuable tool inpyknosis as a result of chromatin condensation, athe treatment of cancer.

trehalose in HeLa

CD4+morphological feature of apoptosis20).
NF-κB is a nuclear transcription factor involved

Acknowledgments.

We would like to thankin the response to stimuli, such as stress21). NF-κB

all of our colleagues for their assistance,

has important roles in inflammatory and immune

especially Ms. K. Kobayashi and Dr. T.

responses22). It also regulates cytokines, which are

Shimasaki,

whogrowth factors in tumor cells, thereby contributing tosubstituted

trehalosecell proliferation23). In addition, NF-κB activation

Technology).

synthesized(Chibathearyl-Instituteofcontrols the anti-apoptotic cascade and antiapoptotic genes, such as genes in the Bcl-2 family24).

Conflicts of interest

The anti-apoptotic Bcl-2 protein family prevents the

The authors have no potential conflicts of interest

physiological destruction of mitochondria, prevents

to disclose.

the release of cytochrome c from mitochondria in
p53-regulated

apoptosis,

andsuppresses

apoptosis25,26).
Our experimental results showed that the
introductionofaryl-substituted

trehalosesignificantly increases the expression levels of NFκB and Bcl-XL, which act as anti-apoptotic proteinsin HeLa CD4+ cells (Fig. 5). These results suggestthat the NF-κB signaling pathway is activated in
HeLa CD4+ cells treated with aryl-substituted
trehalose.

Furthermore,thearyl-substituted

trehalose-induced increases in the expression of NFκB and Bcl-XL promoted the maintenance of HeLa
CD4+ cell survival. In other words, aryl-substituted

References
1) A. Shibata, Yakugaku Zasshi, 2017, 137, 13231337.
2) J. Cao, B. Zhu, K. Zheng, S. He, L. Meng,
J. Song, H. Yang, Front. Bioeng. Biotechnol.,
2020, 7, 487.

3) L. Lu, Z-Y. Wu, X. Li, F. Han, Acta.
Pharmacol. Sin., 2019, 40, 717-723.
4) Y. L. Pak, K. M. K. Swamy, J. Yoon, Sensors,
2015, 15, 24374-24396.

5) A. S. Boutorine, D. S. Novopashina, O.
A. Krasheninina,K.

Nozeret,A.

G. Venyaminova, Molecules, 2013, 18, 1535771

15397.

6) Y. Jung, J. Jung, Y. Huh, D. Kim, J. Anal.
Methods. Chem., 2018, 14, 5249765.
7) T. Ebe, Tanpakushitsu Kakusan Koso, 2007,
52, 1766-1767.
8) S. Subramanian, F. Srienc, J. Biotechnol., 1996,
49, 137-151.
9) Y. Shinden, H. Ueo, T. Tobo, A. Gamachi, M.
Utou, H. Komatsu, S. Nambara, T. Saito, M.
Ueda, H. Hirata, S. Sakimura, Y. Takano, R.
Uchi, J. Kurashige, S. Akiyoshi, T. Iguchi, H.
Eguchi, K. Sugimachi, Y. Kubota, Y. Kai, K.
Shibuta, Y. Kijima, H. Yoshinaka, S. Natsugoe,

14) E. Deni, A. Zamarrón, P. Bonaccorsi, M. C.
Carreño, Á. Juarranz, F. Puntoriero, M. T.
Sciortino, M. Ribagorda, A. Barattucci, Eur.

J. Med. Chem., 2016, 111, 58-71.
15) A. Jo, J. Sung, S. Lee, H. Nam, H. W. Lee, J.
Park, H. M. Kim, E. Kim, S. B. Park,
Bioconjug. Chem., 2018, 29, 3394-3401.

16) S. Ohtake, Y. J. Wang, J. Pharm. Sci., 2011,
100, 2020-2053.
17) A. Patist, H. Zoerb, Colloids Surf. B
Biointerfaces, 2005, 40, 107-113.
18) T. Haraguchi, Cell Struct. Funct., 2002, 27,
333-334.

M. Mori, Y. Maehara, M. Sakabe, M. Kamiya,19) C. Colaço, S. Sen, M. Thangavelu, S. Pinder,J. W. Kakareka, T. J. Pohida, P. L. Choyke, H.

B. Roser, Biotechnology (N Y), 1992, 10,

Kobayashi, H. Ueo, Y. Urano, K. Mimori, Sci.

1007-1011.

Rep., 2016, 6, 27525.
10) Y. Urano, M. Kamiya, K. Kanda, T. Ueno,
K. Hirose, T. Nagano, J. Am. Chem. Soc.,
2005, 127, 4888-4894.

11) T-L. Hsu, S. R. Hanson, K. Kishikawa, SK. Wang, M. Sawa, C. H. Wong, Proc. Natl.

20) S. Elmore, Toxicol. Pathol., 2007, 35, 495516.

21) J. F. R Kerr, Toxicology, 2002, 181-182, 471474.

22) S. Ghosh, M. J. May, E. B. Kopp, Annu. Rev.
Immunol., 1998, 16, 225-260.

Acad. Sci. U. S. A., 2007, 104, 2614-2619.

23) N. S. Chandel, W. C. Trzyna, D. S. McClintock,

12) A. Barattucci, E. Deni, P. Bonaccorsi, M. G.

P. T. Schumacker, J. Immunol., 2000, 165,

Ceraolo, T. Papalia, A. Santoro, M. T.
Sciortino, F. Puntoriero, J. Org. Chem., 2014,
79, 5113-5120.
13) K. Kobayashi, R. Saito, K. Udagawa, N.
Miyano-Kurosaki, N. Asano, T. Iwanaga, N.
Teramoto, T. Shimasaki, M. Shibata, Euro. J.
Org. Chem., 2018, 26, 3444-3453.

1013-1021.
24) B. B. Aggarwal, Cancer Cell, 2004, 6, 203208.

25) K. S. Ahn, G. Sethi, B. B. Aggarwal, J.
Immunol., 2007, 178, 2507-2516.
26) Y. Dogu, J. Díaz, Biophys. Chem., 2009, 143,
44-54.

72