Biomedicine & Pharmacotherapy 149 (2022) 112864

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Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha

Impact of anti-diabetic sodium-glucose cotransporter 2 inhibitors on tumor
growth of intractable hematological malignancy in humans
Sawako Nakachi a, Shiki Okamoto a, *, Keita Tamaki a, Ikumi Nomura a, Mamiko Tomihama a,
Yukiko Nishi a, b, f, Takuya Fukushima c, Yuetsu Tanaka c, Satoko Morishima a,
Minako Imamura b, f, Shiro Maeda b, f, Masato Tsutsui d, Masayuki Matsushita e,
Hiroaki Masuzaki a, *aDivision of Endocrinology, Diabetes and Metabolism, Hematology and Rheumatology, Second Department of Internal Medicine, Graduate School of Medicine, Universityof the Ryukyus, Okinawa, Japanb
Department of Advanced Genomic and Laboratory Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japanc
Laboratory of Immunohematology, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, Okinawa, Japand
Department of Pharmacology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japane
Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japanf
Division of Clinical Laboratory and Blood Transfusion, University of the Ryukyus Hospital, Okinawa, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
Adult T cell leukemia (ATL)
Hematological malignancy
Metabolic oncology
Sodium-glucose cotransporter-2 (SGLT2)
Warburg effect

Under the dysfunction of mitochondria, cancer cells preferentially utilize both glycolytic and pentose phosphatepathways rather than electron transport chains to desperately generate adenosine triphosphate (ATP) andnicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), classically recognized as the Warburg
effect. Based on this background, the present study tested the hypothesis that anti-diabetic sodium-glucose
cotransporter 2 (SGLT2) inhibitors would exert a tumor-suppressive impact on intractable human hematologicalmalignancies via the modulation of glucose metabolism within cells and cell cycles. The level of mRNA for SGLT2
was remarkably elevated in leukemic cells from patients with adult T-cell leukemia (ATL), one of the mostintractable blood cancers in humans, and as well as in two kinds of ATL cell lines (MT-1 and MT-2). Two kinds of
SGLT2 inhibitors, Luseogliflozin and Tofogliflozin substantially suppressed the proliferation of MT-1 and MT-2
cells in both adherent and anchorage-independent culture conditions. Such a suppressive effect on tumor cell
growth was reproduced by Luseogliflozin in leukemic cells in peripheral blood from patients with ATL. In MT-2
cells, both of SGLT2 inhibitors considerably attenuated glucose uptake, intracellular ATP levels, and NADPH
production, resultantly enhancing cell cycle arrest at the G0/G1 phase. From the standpoint of metabolic
oncology, the present study suggests that SGLT2 inhibitors would be a promising adjunctive option for thetreatment of the most intractable human hematological malignancies like ATL.

1. Introduction
Cancer cells robustly modify their circumstance advantageous topromote growth and proliferation, thereby surviving severe environ­ments including hypoxia and malnutrition [1]. Under the severe

dysfunction of mitochondria, cancer cells preferentially utilize glyco­
lytic and pentose phosphate pathways (PPPs) rather than electrontransport chains to desperately generate adenosine triphosphate (ATP)[2] and nicotinamide adenine dinucleotide phosphate (reduced form)(NADPH) [3,4], recognized as the Warburg effect [5]. Due to theAbbreviations: ATL, adult T-cell leukemia; ATP, adenosine triphosphate; CADM1, cell adhesion molecule 1; FDG-PET/CT, fluorodeoxyglucose-positron emission
tomography/computed tomography; Gem, gemcitabine; GLUT1, glucose transporter 1; GLUT3, glucose transporter 3; HK1, hexokinase 1; HK2, hexokinase 2; HTLV1, human T cell lymphotropic virus type 1; Luse, Luseogliflozin; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); PBMC, peripheral bloodmononuclear cells; PFK, phosphofructokinase; PK, pyruvate kinase; PPPs, pentose phosphate pathways; SGLT1, sodium-glucose cotransporter 1; SGLT2, sodiumglucose cotransporter 2; Tofo, Tofogliflozin.
* Correspondence to: Second Department of Internal Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa 903–0215, Japan.
E-mail addresses: shiki@med.u-ryukyu.ac.jp (S. Okamoto), hiroaki@med.u-ryukyu.ac.jp (H. Masuzaki).
https://doi.org/10.1016/j.biopha.2022.112864
Received 19 February 2022; Received in revised form 20 March 2022; Accepted 23 March 2022
0753-3322/© 2022 The Authors.
Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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2.3. Cell culture

inconvenient substrate shift to glycolysis with poor efficiency for
generating ATP, glucose uptake reciprocally increases in cancer cells[2]. Based on this notion, fluorodeoxyglucose-positron emission
tomography/computed tomography (FDG-PET/CT) is widely employedfor detecting malignancies in clinics [6–9].
We recently reported that FDG-PET/CT is advantageous for clinically
grading human adult T-cell leukemia (ATL) [10], an extremely intrac­
table hematologic malignancy caused by human T cell lymphotropic
virus type 1 (HTLV-1) [11]. Our studies support the notion that diseaseactivity of ATL is highly dependent on its potency of glucose uptake,exemplifying the Warburg effect in its glucose metabolism. Globally,
HTLV-1 is endemic predominantly in southwestern part of Japan,
sub-Saharan Africa, the Caribbean Basin and South America [12,13].
Cumulative incidence of clinically overt ATL is approximately 5%
among healthy carriers with HTLV-1 [12]. In most cases, clinicalmanifestation of ATL develops after 40~ 60 years of latent period oftime [12,13]. Considering the notion that T cells represent high demand
for glucose by nature [14,15], we employed ATL cell lines as anexperimental model to explore the impact of the Warburg effect inhuman hematological malignancies [16].
So far, a line of anti-cancer agents targeting exaggerated glucose
demand in malignancies have been developed including 2-deoxyglucose(2-DG), lonidamine, 3-bromopyruvate (3-BrPA) and oxythiamine [17].
However, these agents are not yet successfully applied to clinical prac­
tice. On the other hand, it is well characterized that sodium-glucose
cotransporter 2 (SGLT2) inhibitors potently prevent glucose reabsorp­
tion in renal tubule, increase glucose excretion to urine, thereby effec­
tively lowering blood glucose level in patients with diabetes mellitus[18]. Recently, it has been demonstrated that SGLT2 inhibitors consid­
erably improve cardiovascular and renal outcomes in patients with type2 diabetes [19]. Furthermore, A couple of recent studies in rodents re­ported that pharmacological inhibition of SGLT2 was shown to exert abeneficial potential for solid malignancies in pancreas, colon, liver andlung [20–23].
Of note, Scafoglio et al. demonstrated that SGLT2 inhibitors sub­stantially reduced the growth of human-derived pancreatic cancer in a
xenograft murine model [21], and also provided convincing evidence
that SGLT2 may play a pivotal role in growth of early staged lungadenocarcinoma in humans [23]. In this context, we aimed to examine
the expression of SGLT2 in cancer cells from peripheral blood of patientswith ATL as well as its cell lines, and explored whether pharmacologicalinhibition of SGLT2 would exert anti-cancer impact on ATL.

HTLV-1-infected cell lines, MT-1and MT-2 [27] were cultured in a
RPMI 1640 medium (Sigma-Aldrich, St. Louis, MI, USA) supplemented
with 10% heat-inactivated fetal bovine serum (Biological Industries
USA, Cromwell, CT, USA), 100 U/ml of penicillin and 100 µg/ml of
streptomycin.
2.4. Assays for cell proliferation
MT-1 and MT-2 cells (5 × 104 cells) were seeded in triplicates into
96-well plates. Cells were treated with different concentrations of
Luseogliflozin or Tofogliflozin for 24, 48, and 72 h. Cell proliferation
was determined by use of Cell Counting Kit-8 (Dojindo MolecularTechnologies Inc., Kumamoto, Japan) [28]. The relative ratio of cellproliferation, calculated by dividing by the number of untreated cells
without reagents, was shown as the mean ± standard error of the meanof triplicate experiments.
ATL cells obtained from peripheral blood of patients (1 ×105 cells)
were seeded with RPMI-1640 medium containing 20 U/ml of recombi­
nant human IL-2 (Peprotech, Rockey Hill, NJ, USA) as a robust mitogen.
After 1 day-incubation, all proliferated cells were collected and the
population rate of CD4+CADM1+ (cell adhesion molecule 1) doublepositive cells was assessed by the flow cytometry. CADM1 iscommonly utilized as a valid cell surface marker for HTLV-1-infected
cells. It has been shown that CD4+CADM1+ double-positive cells are
significantly correlated with pro-viral load (PVL) in both ATLs and
HTLV-1 asymptomatic carriers [29]. The relative ratio of cell prolifer­ation was also calculated.
2.5. Assays for colony formation
For colony formation assay, anchorage-independent tumor growth
was assessed in a soft agar. It is well known that malignantly trans­
formed cells require less extracellular growth factors, are not inhibitedby cell-cell contact, and are often immortalized. Importantly, malig­
nantly transformed cells exhibit scaffold-independent proliferation. The
colony formation assay is therefore essential to accurately evaluatemalignantly transformed cells in vitro. Fifty microliters of base agar
matrix (CytoSelect 96-well In Vitro Tumor Sensitivity Assay kit; Cell
Biolabs, Inc., CA, USA) were dispensed into each well of a 96-well plate.
When the agar had solidified on the bottom surface of each well, 75 μl ofa cell suspension / soft agar matrix containing 5 × 104 cells were layeredon top of the agar, following by the addition of 50 μl of medium withdifferent concentrations of each SGLT2 inhibitor (0, 5, 10, 25, 50, 100µM). After 6 days of incubation, the agar matrix was solubilized and 3(4, 5-dimethylthiazol-2-yl)− 2, 5-diphenyl tetrazolium bromide wasreplenished to each well. The absorbance produced by the formation of
insoluble formazan product by viable cells was recorded at 570 nm.

2. Materials and methods
2.1. Reagents
Clinically-applied SGLT2 inhibitors in Japan, Luseogliflozin (Taisho
Pharmaceutical Co., Ltd.) [24] and Tofogliflozin (Kowa PharmaceuticalCo., Ltd.) [25] were dissolved in dimethyl sulfoxide (DMSO). Gemcita­bine, an inhibitor against nucleic acid synthesis (Wako Pure Chemical
Industries, Ltd.) were used as inducers for apoptosis.

2.6. Analyses of flow cytometry

2.2. Patients studied

Flow cytometry was performed with the BD FACSCalibur cell
analyzer and data were analyzed with CellQuest Pro software (BD Bio­sciences, San Jose, CA, USA). Cryopreserved PBMCs were retrieved by
rapid thawing of cryotubes in a 37 ℃-water bath. Cell surface staining
was performed on ice for 30 min using the following antibody combi­
nations: fluorescein isothiocyanate-conjugated rabbit anti- Synaptic Cell
Adhesion Molecule (SynCAM), tumor suppressor in lung cancer-1(TLSC1)/ CADM1 monoclonal antibody (MBL Co., Ltd, Nagoya,Japan), PE-cyanine7 mouse anti-human CD7 (BD Biosciences), Alexa
Fluor 647 anti-human CD4 antibody (Biolegend, San Diego, CA, USA). It
is well known that the expression of CADM1 is a valid marker for
aggressive ATL and stepwise downregulation of CD7 is associated withclonal expansion of HTLV-1-infected cells [29].

Using Ficoll-Paque density gradient centrifugation (GE Healthcare
Biosciences, Uppsala, Sweden), mononuclear cells in peripheral blood(PBMCs) were isolated from 9 healthy volunteers, 5 clinically occult
HTLV-1 carriers and 12 patients with ATL in university of the Ryukyus in
Okinawa, Japan. Clinical subtype of ATL in each patient was determinedaccording to the Shimoyama Criteria [26]. PBMCs from 12 patients with
ATL (5 of chronic type and 7 of acute type) were subjected to in vitro
experiments prior to a series of chemotherapies. The present study wasapproved by the institutional review board at University of the Ryukyus
hospital (approval number 1030) and all participants gave written
informed consent.
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2.7. Assays for glucose uptake

Table 1
Human PCR primer sequences.

Cells were seeded 3 × 103 MT-2 cells/well into white-walled 96-wellculture plate, and treated them with 0, 50, 100 μM concentrations of
Luseogliflozin or Tofogliflozin for 24 h, respectively. To evaluate 2DG
uptake, cells were incubated with 0.4 mM 2DG for 20 min at the room
temperature. Luminescence from accumulated 2DG 6-phosphate was
detected by use of the Glucose Uptake-Glo Assay (Promega Corporation,
Madison, WI, USA).

Gene

Sequences (5′ → 3′ )Source

SGLT2

Forward, TCTACTTCGCCATTGTGCTG;
Reverse, ATCTCCATGGCACTCTCTGG
Forward,
GCCCAACACTCTGATTTGCATTTA;
Reverse,CTGGTTCTACTTCACCCTGAGCAC
Forward, GTCCCACCACGTAATCTGATG;
Reverse, CCACCTCCGATTTGCCTTTTA
Forward, GCTAACTTTTTCCGCGTGTT;
Reverse, GGTGGAAGGTGAGATCCAGA
Forward,GCACGCTTGCGTATGTCTGTTA;
Reverse,TGTGGGCTACACTGCACATTGA
Forward,
ATCAGGACACACTACGACAGACTGG;
Reverse,TTAAGATGTTGCGGACGATTTCAC
Forward,GGCCGAGCCAATAGACTGAAGA;
Reverse,GCCATGAGTCCATTTGAAACGA
Forward,CCATGACTACTACACCACGGAGTTC;
Reverse, TGGTCCCATAGTTCCGGTCA
Forward,
GCTTTACCGTGAACCTCCAGAA;
Reverse,TGTCACCACAATCACCAGGTCTC
Forward, ACTGTGTTTGCTGACGCAAC;
Reverse, CAACACCACGGAATTGTCAG

NM_003041

SGLT1

CADM1

2.8. Assays for NADPH

GLUT1

To evaluate the involvement of PPPs within cells after the treatmentof SGLT2 inhibitors, the content of NADPH in MT-2 cells was measured.
Cells were seeded at a density of 2.5 × 10 5 cells / well into 12-well plate,
and treated in triplicate with 50 μM concentration of Luseogliflozin or
Tofogliflozin for 24 h. Intracellular NADPH level was measured by use ofNADP / NADPH Assay Kit-WST (Dojindo Molecular Technologies).

GLUT3

Hexokinase 1 (HK1)

Hexokinase 2 (HK2)2.9. Measurement of intracellular ATP concentration
MT-2 cells were seeded at a density of 3.9 × 102 cells/well into
white-walled 96-well plates and treated in triplicate with 0, 50, 100 μM
concentrations of Luseogliflozin or Tofogliflozin for 24 h. Cellular ATP
level was measured using ATP luminescence assay, Cell Titer-Glo® 2.0
assay (Promega Corporation). We detected the luminescence using
0.3–1 s integration on a luminometer.

Phosphofructokinase(PFK)
Pyruvate kinase (PK)beta-actin

2.10. Assays for mitochondrial activity
To evaluate mitochondrial function after the 1 day-treatment of
SGLT2 inhibitors, mitochondrial membrane potential was analysed. MT2 cells were seeded at a density of 1.0 × 105 cells / well into 12-wellplates and treated with 50 μM concentration of Luseogliflozin or Tofo­
gliflozin for 24 h. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP, amitochondrial uncoupling agent, Sigma-Aldrich) at 50 μM concentration
was used as a positive control for membrane potential. Cells were loadedwith the ratiometric indicator for mitochondrial potential, JC-1 at 5 µM
for 30 min at 37ºC. Cells were illuminated at 488 nm and the emission
was collected between 515 / 545 nm and 575 / 625 nm by using the flow
cytometer.

NM_000343

NM_014333
NM_006516
NM_006931

NM_000188

NM_000189

M64784
NM_000298

NM_001101.3

Luseogliflozin or Tofogliflozin for 48 h, respectively. Similarly, trans­
fected cells were seeded 3.9 × 102 cells / well into white-walled 96-wellplates, and treated in triplicate with 50 μM concentration of Luseogli­
flozin or Tofogliflozin for 24 h before assessment of the 2DG uptake.
2.13. Assays for apoptosis
To evaluate the cellular apoptosis, the TdT-mediated dUTP nick endlabeling (TUNEL) assay was performed using Apoptotic DNA Fragments
Extraction Kit, ApopLadder Ex™ (TaKaRa Bio Inc., Shiga, Japan). Afterthe treatment with 50 μM Luseogliflozin, 50 μM Tofogliflozin or 0.4 μM
Gemcitabine, one of representative anti-cancer drugs acting as a potent
nucleoside analogue, respectively, 1 × 106 cells were collected bycentrifugation, and re-suspended in the lysis buffer in the kit. Frag­mented DNAs from lysate were then purified and were analyzed using
agarose gel electrophoresis.

2.11. Quantitative real-time PCR
Total RNA was extracted using Trizol reagent (Thermo Fisher Sci­
entific Inc., Walthtam, MA, USA) and cDNA was synthesized using an
iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative
real-time PCR was performed using a StepOnePlus™ Real-Time PCR
System, and Fast SYBR Green Master Mix (Thermo Fisher Scientific Inc).
The mRNA levels were normalized by β-actin. As a control for human
SGLT2 mRNA expression, kidney-derived mRNA from a healthy subject
was used (Bio Chain Institute Inc., Newark, CA, USA). As a control forhuman SGLT1 mRNA expression, small intestine-derived mRNA from ahealthy subject was analysed (Bio Chain Institute Inc.). All primer se­quences are shown in the Table 1 below.

2.14. Assays for caspase-3 activity
Activity of caspase-3 was measured by the absorbance at 405 nm
after the cleavage of the synthetic substrate Ac-Asp-Glu-Val-Asp-pnitroaniline (Ac-DEVD pNA) via the colorimetric assay kit (TaKaRa Bio
USA Inc., MountainView, CA, USA). Cells were plated at a density of 2 ×
106 cells / well. One hour after the treatment with each SGLT2 inhibitor(100 μM), cells were collected. The enzyme activity was calculated on
the basis of a standard curve prepared using p-nitroaniline (pNA). The
relative levels of pNA were normalized by the protein concentration ofeach extract.

2.12. Small interfering RNA
To specifically repress the mRNA expression of SGLT2, predesigned
single-stranded small interfering RNAs (siRNA; Silencer Select siRNAs,
Thermo Fisher Scientific Inc.) were used. As a negative control, scram­bled siRNA (Thermo Fisher Scientific Inc.) was used. All siRNA trans­
fections were performed by using a Microporator MP-100 (Digital Bio
Technology, Seoul, Korea), pulsed twice at 1140 V (pulse voltage) for 30
msec (pulse width). For cell proliferation assay, transfected cells wereseeded (5 ×104 cells) and treated with 50 μM concentration of

2.15. Assays for cell cycle
The phase of cell cycle in MT-2 was analyzed by staining DNA withthe flow cytometer. Cells were seeded at a density of 5 × 105 cells / wellinto 6-well plates and treated in triplicate with 50 µM Luseogliflozin or
Tofogliflozin for 24 h. One day after the treatment of SGLT2 inhibitors,
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cells were stained by using Cell Cycle Assay Solution Blue at 37ºC for 15
min (Dojindo Molecular Technologies Inc.) and detected blue fluores­
cence (Excitation length at 405 nm and Emission length at 450 nm) todetermine the ration of the G0/1 - S phase and G2/M phase.

medium (11 mM) and high glucose medium (25 mM). It was reported
that inoculation of SCID mice with MT-2 cells elicited considerably highmortality, whereas that of MT-1 cells provoked subtle tumorigenesis[32]. We therefore preferentially utilized MT-2 cells henceforth in thepresent study. Expectedly, the cell growth of MT-2 was markedly
exaggerated in a high glucose medium compared to standard one in atime dependent fashion (Fig. 2A).

2.16. Statistical analyses
Data are expressed as the mean ± standard error of the mean (SEM)
in independent experiments. One-way analysis of variance (ANOVA),
Two-way ANOVA followed by multiple comparison tests (Tukey’s posthoc method) and Kruskal-Wallis analysis were used where applicable.

3.5. SGLT2 inhibitors substantially attenuated glucose uptake,intracellular level of ATP and NADPH in MT-2 cells

As shown in Fig. 1A left, upper panel, comparing to PBMCs fromhealthy subjects, mRNA level of SGLT2 was substantially elevated innormal human kidney as well as in ATL cells from patients with acutetype and MT-2 cells. There were no appreciable differences among
HTLV-1 carriers, chronic-type, MT-1 cells as compared to PBMC fromhealthy subjects. No significant differences were observed in the mRNAlevel of SGLT1 among PBMCs from healthy subjects, ATL, MT-1 and MT2 cells except HTLV-1 carriers (Fig. 1A, right, upper panel). As previ­
ously reported [21], level of SGLT1 mRNA in normal human intestine
was substantially elevated.
It has been shown that CADM1 was ectopically and highly expressedin HTLV-1 infected T cells and ATL cells [30]. CADM1 has been used as
one of the representative molecular markers in ATL and other T-cell
lymphoma [31]. As reported, mRNA level of CADM1 was markedly
elevated in ATL cells of acute-type and MT-2 cells (Fig. 1A, lower panel).

We examined whether glucose uptake as well as intracellular level of
ATP and NADPH level would be suppressed by SGLT2 inhibitors in MT-2
cells. There was no appreciable difference in glucose uptake between
normal and high glucose media for 24 h (Fig. 2B, left). Notably,
Luseogliflozin dose-dependently attenuated glucose uptake in a stan­
dard glucose medium as well as high glucose medium for 24 h (Fig. 2B,left). Based on our finding that there was no statistical difference inglucose uptake between standard and high glucose media with Luseo­
gliflozin (Fig. 2B, left), we next performed glucose uptake assays with
Tofogliflozin only in a standard glucose medium. Expectedly, Tofogli­
flozin also dose-dependently attenuated glucose uptake (Fig. 2C, left).
Importantly, both inhibitors dose-dependently reduced intracellular
ATP concentration (Fig. 2B, right and 2 C, right).
Of note, value of glucose uptake as well as intracellular level of ATP
and NADPH was substantially elevated in MT-2 cells compared to
PBMCs, respectively (Fig. 2D, E, F and G). On the other hand, Luseo­
gliflozin significantly attenuated each value as compared to vehicle(Figs. 2D and 2F). Tofogliflozin also significantly attenuated intracel­lular ATP concentration as compared to vehicle (Fig. 2E, right), andshowed a noticeable trend to reduce glucose uptake and NADPH level(p = 0.054, Fig. 2E left and p = 0.126, Fig. 2G).

3.2. Luseogliflozin substantially suppressed the growth of leukemic cells
from patients with acute-type ATL

3.6. Expression profile of mRNAs for glycolysis-related genes and GLUTs
in ATL cell lines

To examine whether pharmacological inhibition of SGLT2 would
suppress the proliferation of ATL cells from patients with chronic-type oracute-type of ATL as well as HTLV-1 infected T cells from clinicallyoccult HTLV-1 carriers, Luseogliflozin was replenished in the culture
media with 20 U/ml of recombinant human IL-2. Proliferation wasassessed at 24 h after the replenishment by the flow cytometry. Conse­
quently, Luseogliflozin markedly suppressed the proliferation of ATL
cells from patients with acute-type as compared to chronic-type(p = 0.022, Fig. 1B) at the concentration of 100 μM.

To gain further insight into the facet of glucose metabolism within
ATL cells, we assessed mRNA levels for glycolysis-related genes andglucose transporters (GLUTs) in MT-1 and MT-2 cells. Regarding genes
involved in glycolytic metabolic pathways, the level of a rate-limiting
enzyme, phosphofructokinase (PFK) was significantly elevated in MT2 cells compared to PBMCs (Fig. 3A upper, left). The level of pyruvatekinase (PK), another rate-limiting enzyme in glycolytic metabolic
pathways also showed a trend to increase in MT-2 cells (p = 0.380,
Fig. 3A lower, left). The value of hexokinase (HK) 1 was significantly
higher in MT-2 cells as compared to PBMCs, but not of HK2 (p = 0.020,
Fig. 3A upper, middle and p = 0.792, right). Regarding the glucose
transporters responsible for glucose uptake independent of insulin,mRNA levels for GLUT1 in MT-1 and MT-2 cells were markedly elevatedcompared to that of PBMCs (Fig. 3A lower, middle), whereas that of
GLUT3 was inexplicably reduced in both MT-1 and MT-2 cells (Fig. 3A
lower, right).

3. Results
3.1. Levels of SGLT2 and CADM1 mRNAs were substantially elevated inleukemic cells from patients with acute-type ATL and ATL cell lines

3.3. SGLT2 inhibitors substantially suppressed the growth of ATL celllines
To test whether pharmacological inhibition of SGLT2 would suppress
the proliferation and colony formation of ATL cell lines, Luseogliflozin
and Tofogliflozin, were replenished at various pharmacological con­centrations in the media of cells. Proliferation was assessed at 24, 48 and
72 h after the replenishment of each inhibitor. Colony formation wasassessed at 6 days after the replenishment of each inhibitor. Conse­
quently, both of inhibitors significantly suppressed the proliferation of
MT-1 and MT-2 cells in a dose-dependent manner as well as in a timedependent manner (Figs. 1C, 1D). In a similar fashion, Tofogliflozin
significantly suppressed the colony forming ability of MT-1 in a dosedependent manner, and Luseoglifrozin also showed a noticeable trend
to suppress the colony forming abilities in both MT-1 and MT-2 (Fig. 1E).

3.7. SGLT2 inhibitors did not affect the membrane potential of
mitochondria in MT-2 cells
To evaluate mitochondrial function after the 1 day-treatment of
SGLT2 inhibitors, we assessed the mitochondrial membrane potential by
the flow cytometer. One day after the treatment with SGLT2 inhibitors,
MT-2 cells were loaded with the ratiometric indicator for mitochondrial
potential, JC-1. JC-1 is a fluorescent cationic carbocyanine dye andrepresents potential-dependent accumulation in mitochondria which
form J aggregates (red), and then diffuses across mitochondria to form amonomeric state (green) upon depolarization. Subsequently, JC-1 redcolored cells containing high mitochondrial membrane potential weredetected in normally-functioned mitochondria. In our assays among

3.4. MT-2 cells grow more rapidly in high glucose media
To evaluate the glucose requirement for cell proliferation in ATL
cells, we compared the cell growth of MT-2 between standard glucose
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Fig. 1. SGLT2 inhibitors substantially suppressed the growth of ATL cell lines and leukemic cells from peripheral blood in patients with acute-type ATL.
(A) Level of mRNAs for SGLT2, SGLT1 and CADM1 in peripheral blood mononuclear cells (PBMCs) from healthy subjects (n = 9), HTLV-1 infected T-cells fromclinically occult carriers (n = 5), malignant cells from peripheral blood of ATL patients (chronic-type: n = 5, acute-type: n = 7), ATL cell lines (MT-1 and MT-2)(n = 5–6 in each group), and human kidney or intestine from healthy subjects (n = 5–6 in each), respectively. Data are expressed as the mean ± SEM, * ** :p < 0.001, * ** *: p < 0.0001 vs. PBMCs by Kruskal-Wallis analysis and Tukey’s post-hoc analyses. (B) Luseogliflozin substantially suppressed the growth of5

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malignant cells in peripheral blood from patients with acute-type ATL. Proliferation of ATL cells from patients with chronic-type or acute-type and HTLV-1 infected Tcells from clinically occult carriers under the treatment with Luseogliflozin for 24 h at a dose of 100 µM. After 1 day-incubation, all proliferated cells were collected
and the population rate of CD4+CADM1+ double-positive cells was assessed by the flow cytometry. The relative ratio of cell proliferation was calculated. Data areexpressed as mean ± SEM (n = 3–5/group). * : p < 0.05 vs. between line-connected values by One-way ANOVA and Tukey’s post-hoc analyses. (C)(D) SGLT2 in­hibitors suppressed the growth of ATL cell lines in adherent conditions. Proliferation of MT-1 or MT-2 cells treated with (C) Luseogliflozin or (D) Tofogliflozin,
respectively. Data are expressed as mean ± SEM (n = 8). * : p < 0.05, * *: p < 0.01, * ** : p < 0.001, * ** *: p < 0.0001 vs. 0 μM at the same time. †: p < 0.05, ‡:p < 0.001 vs. 24 h in the same concentration of each inhibitor by Two-way ANOVA and Tukey’s post-hoc analyses. (E) SGLT2 inhibitors suppressed the anchorageindependent growth of ATL cell lines. The growth of MT-1and MT-2 cells treated with Luseogliflozin or Tofogliflozin for 5 or 6 days at a dose of 0, 5, 10, 25, 50,
100 µM, respectively. Data are expressed as mean ± SEM (n = 3). * *: p < 0.01, * ** : p < 0.001, * ** *: p < 0.0001 vs. 0 μM in the same inhibitor by Two-way
ANOVA and Tukey’s post-hoc analyses.

Fig. 1. (continued).

vehicle and two of SGLT2 inhibitors, there was no statistical differencein mitochondrial depolarization rate as demonstrated by the change ofcolor after JC-1 staining (Fig. 3B, upper panel). The decrease in the red /green fluorescence intensity ratio indicates the depolarization of themitochondrial membrane, resulting in apoptosis due to mitochondrial
collapse. Fig. 3B, lower panel shows representative examples of color
sorting of typical JC-1 positive cells after the addition of SGLT2 in­hibitors. No significant increase in JC-1 green cells was detected after
the replenishment of Luseogliflozin or Tofogliflozin. In contrast, most ofcells turned a green color when CCCP, a potently depolarizer in the
mitochondrial membrane potential, was replenished (Fig. 3B, lower
panel).

electroporation methods. Consequently, replenishment of specificsiRNA against SGLT2 markedly decreased the mRNA level of SGLT2(Fig. 4A), where cell proliferation was considerably attenuatedcompared to those treated with negative-control (Scrambled) siRNA(Fig. 4B, left and 4C, left, respectively). Of note, effects of both of SGLT2
inhibitors on glucose uptake and the cell proliferation were disappearedin cells under the treatment of specific siRNA (Fig. 4B, 4C), further
supporting a crucial role of SGLT2 for growth of MT-2 cells.
3.9. Decrease in growth of MT-2 cells by SGLT2 inhibitors was
independent of apoptosis
To further explore mechanisms of SGLT2 inhibitors on growth sup­pression of MT-2 cells, we examined whether SGLT2 inhibitors would
augment apoptosis. Contrary to the apparent DNA ladder in gel elec­
trophoresis by the treatment with Gemcitabine [33], no appreciablechanges were observed in MT-2 cells treated with either Luseogliflozin
or Tofogliflozin (Fig. 4D). In contrast to the augmented activity of
caspase-3 in MT-2 cells treated with Gemcitabine, no appreciablechanges were also observed in MT-2 cells treated with either Luseogli­
flozin or Tofogliflozin (Fig. 4E). These data suggest that decrease in thegrowth of MT-2 cells by SGLT2 inhibitors is unlikely to relate directlywith apoptosis.

3.8. Suppression of SGLT2 via the specific RNA interference completely
abolished the effect of SGLT2 inhibitors on glucose uptake andproliferation of ATL cells
To clarify whether the function of SGLT2 would directly influence
the proliferation of ATL cells, specific RNA interference against SGLT2
was introduced. Because mRNA level of SGLT2 in MT-2 cells wasapparently higher than that of MT-1 cells (Fig. 1A, left), we preferen­
tially utilized MT-2 cells in pertinent experiments. MT-2 cells weretransfected with either siRNA against SGLT2 or scramble siRNA via the6

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Biomedicine & Pharmacotherapy 149 (2022) 112864

Fig. 2. SGLT2 inhibitors substantially attenuated glucose uptake, intracellular level of ATP and NADPH in MT-2 cells.(A) MT-2 cells grow more rapidly in ahigh glucose medium as compared to regular glucose medium. Cells were cultured in a regular glucose medium (11 mM, open column) or high glucose medium
containing 25 mM glucose (closed column). * : p < 0.05 vs. 11 mM glucose at the same time. †: p < 0.05 vs. 0 h (hr) at the same glucose concentration. ‡: p < 0.001.
(n = 8/group).(B, C) SGLT2 inhibitors attenuated glucose uptake and intracellular ATP concentration in MT-2 cells. Effects of (B) Luseogliflozin and (C) Tofogliflozin
on glucose uptake for 24 h in MT-2 cells (left). MT-2 cells were cultured in a regular glucose medium (11 mM) under the treatment of Tofogliflozin. * *: p < 0.01,* ** : p < 0.001, * ** *: p < 0.0001 vs. 0 μM at the same concentrations of glucose. (n = 3/group). Effects of SGLT2 inhibitors on intracellular ATP concentration for
24 h in MT-2 cells (right).* : p < 0.05, * *: p < 0.01, * ** : p < 0.001 vs. 0 μM. (n = 3/group).(D, E) Glucose uptake and intracellular ATP concentration in MT-2 cells
were considerably elevated as compared to PBMCs, and SGLT2 inhibitors substantially attenuated glucose uptake and intracellular ATP concentration in MT-2 cells.
Effects of (D) Luseogliflozin and (E) Tofogliflozin on glucose uptake and intracellular ATP concentration for 24 h in MT-2 cells. * : p < 0.05, * *: p < 0.01, * ** :p < 0.001, vs. the same treatment in PBMCs. (n = 3/group).
(F, G) NADPH level in MT-2 cells was markedly elevated as compared to PBMCs, and SGLT2 inhibitors significantly attenuated NAPDH level in MT-2 cells. Effects of(F) Luseogliflozin and (G) Tofogliflozin on NADPH level for 1 day-treatment in MT-2 cells. * : p < 0.05, * ** : p < 0.001 vs. the same treatment in PBMCs. (n = 3 in
PBMCs, n = 4 in MT-2 cells). Statistical significance was calculated using Two-way ANOVA and Tukey’s post-hoc analyses (A), (B left), (D), (E), (F) and (G) or Oneway ANOVA and Tukey’s post-hoc analyses (B right) and (C). Data are expressed as the mean ± SEM.

3.10. SGLT2 inhibitors considerably modulated the cell cycle of MT-2
cells

primary lung cancer and metastatic lesions, while that of SGLT2 wasmarkedly elevated in metastatic lesions as compared to primary lesions
in lung cancer [35,36]. These findings tempt us to imagine that SGLT2
would be further involved in glucose uptake in glucose-requiring met­
astatic regions. In fact, SGLT1 is a high-affinity, but low-capacity
Na+ /glucose cotransporter for glucose, while SGLT2 is a low-affinity,
but high-capacity Na+ /glucose cotransporter [35]. In this context,
such a functional property may be a facet explaining the differential
expression of SGLT isoforms in a respective stage of cancer cells.
Importantly, MT-2 cells grew more rapidly in a high glucose medium
compared to a standard medium (Fig. 2A), consistent with recent reportsthat SGLT2 is highly expressed in a line of progressive cancer cells withrobust uptake of glucose [21,37–39], further supporting a pivotal role of
SGLT2 in a glucose-dependent growth of ATL cells.
The present study demonstrated that suppression of glucose uptake
via the SGLT2 was linked with the decreased proliferation of ATL cells(Fig. 1B-E). Luseogliflozin substantially suppressed the proliferation of
ATL cells from patients with acute-type as compared to those withchronic-type (p = 0.022, Fig. 1B) at the concentration of 100 μM. Our
results suggest that Luseogliflozin has a potential to suppress aggressive
ATL representing high level of SGLT2 expression. We also demonstrated
that glucose uptake as well as intracellular level of ATP and NADPH
were substantially elevated in MT-2 cells, and that SGLT2 inhibitors
significantly attenuated a line of value (Fig. 2 D-G). Furthermore, RNAinterference against SGLT2 resulted in a marked decrease in the mRNAlevel of SGLT2 (Fig. 4A) accompanied by a considerable decrease in cellproliferation and glucose uptake (Figs. 4B and 4C, open columns). Under
the interference, no statistical significance was observed in cell prolif­
eration (Fig. 4B, left and 4 C, left) and glucose uptake (Fig. 4B, right and
4 C, right) among vehicle, Luseogliflozin and Tofogliflozin, highly sug­gesting that SGLT2 contributes to the growth of MT-2 mainly throughthe supply of glucose into cancer cells. Furthermore, we demonstrated
that the decrease in cell proliferation of MT-2 by SGLT2 inhibitors wasunrelated with apoptosis (Figs. 4D and 4E), further supporting thatgrowth suppression of MT-2 is largely attributed to the attenuation of
glucose uptake by inhibiting SGLT2.
The mRNA level for the rate limiting enzymes in glycolytic metabolic
pathways, PFK and PK, showed a trend to elevate in MT-2 cells (Fig. 3A,left), in agreement with previous reports that glycolytic metabolic
pathways were augmented in glucose-requiring malignant cells [40,41].
Furthermore, mRNA level for the first-stage enzymes in glycolytic
metabolic pathways, HK1 and HK2, also showed a trend to elevate inMT-2 cells as previous reports (Fig. 3A, upper) [42,43]. In contrast, it
has been reported that GLUT3 plays a major role for glucose uptake inhuman B cell lymphomas [41]. Basically, the mRNA level for GLUT1 andGLUT3, high affinity glucose transporters, was reported to elevate inlymphocytes in healthy humans [44,45]. However, little has been
known about the expression of SGLTs and GLUTs exclusively in T cell
lymphoma in humans. In this context, we would speculate that there
seems a cancer-specific and stage-dependent manner of glucose uptake.
For example, a recent work by Scafoglio et al. demonstrated that SGLT2

To evaluate the effect of SGLT2 inhibitors on the proliferation of MT2 cells, after the treatment with 50 μM concentration of Luseogliflozin or
Tofogliflozin for 24 h, the cell cycle was analyzed by the flow cytometry.
Treatment with Luseogliflozin or Tofogliflozin significantly increased
the ratio of cells at G0/G1 phase (p = 2.23e-4 and p = 1.81e-5 vs. cor­
responding value for same phase of vehicle treatment), suggesting thatboth of compounds induce the G0/G1 arrest in MT-2 cells (Fig. 5, upper
panel).
As shown in representative examples (Fig. 5, lower panel), the ratioof cells at the G2/M phase showed an apparent decrease in cells treatedwith Luseogliflozin (12.23% vs. corresponding value for the same phaseof vehicle treatment, 18.78%) or a remarkable decrease in cells treatedwith Tofogliflozin (10.40% vs. corresponding value for the same phaseof vehicle treatment, 18.78%), in accordance with the notion that G0/
G1 arrest is exaggerated in MT-2 cells treated with SGLT2 inhibitors.
4. Discussion
To our knowledge, the present study is the first to demonstrate that
SGLT2 inhibitors exert potent tumor-suppressive effects in both ATL
cells from patients and ATL cell lines by attenuating cellular glucose
uptake. Recent works in mice suggested that pharmacological inhibitionof SGLT2 may provide cancer-reducing impact on solid tumors [20–23].
However, therapeutic potential of SGLT2 inhibitors on hematologicalmalignancies has not yet been elucidated. In this context, the present
study provides the first convincing evidence that SGLT2 inhibitors
would be beneficial as an adjunctive for hematological malignancies inhumans.
It is noteworthy that mRNA level of SGLT2 in both ATL cells frompatients and ATL cell lines was substantially elevated, while that of
SGLT1 was not increased except healthy carrier patients (Fig. 1A). On
the other hand, the mRNA level of CADM1 was also substantiallyelevated in ATL cells of acute-type and MT-2 cells (Fig. 1A, lower panel).
CADM1 was originally identified as a tumor suppressor gene in lungcancer, playing a pivotal role in the cell-cell adhesion among endothelial
cells. In line with our results, it has been reported that the mRNA level of
CADM1 was significantly elevated in patints with both chronic and acutetype ATL as compared to healthy volunteers [30]. In this context,
comparison of expression profile between SGLT2 and CADM1 mayprovide a clue to estimate the metabolic status of intractable hemato­
logical malignancy such as ATL.
The reason for the unexpectedly higher level of SGLT1 mRNA in
carrier patients still remains unclear. Since it has been reported thatmRNA expression of SGLT1 was upregulated predominantly in activatedT cells [34], it would be possible to speculate that the mRNA level of
SGLT1 in caririer patients is substantiated in activated T cells under thechronic infection of HTLV-1. Furthermore, Madunic et al. and Ishikawa
et al. recently reported that the level of SGLT1 mRNA was comparable in8

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Biomedicine & Pharmacotherapy 149 (2022) 112864

Fig. 3. SGLT2 inhibitors showed no effect on mitochondrial activity in ATL cell lines with enhanced expression of glycolysis-related genes.(A) Level ofmRNAs for a line of genes involved in glycolytic metabolic pathways (PFK, PK, HK1 and HK2) and glucose transporters (GLUT1 and GLUT3) in MT-1 and MT-2 cells.
Relative values against PBMCs are expressed as the means ± SEM (n = 3/group), * : p < 0.05, * *: p < 0.01, * ** : p < 0.001 by One-way ANOVA and Tukey’s posthoc analyses. PFK: phosphofructokinase, PK: pyruvate kinase, HK1: hexokinase 1,HK2: hexokinase 2, GLUT1: glucose transporter 1, GLUT3: glucose transporter 3. (B)
Mitochondrial viability and function as evaluated by JC-1 Red / Green ratio were unaffected by SGLT2 inhibitors in MT-2 cells as shown in the upper panel. After cells
were treated with Luseogliflozin or Tofogliflozin for 24 h, cells were loaded with the ratiometric indicator for mitochondrial potential, JC-1. Cells were illuminated at
488 nm and the emission was collected between 515 / 545 nm and 575 / 625 nm by use of the flow cytometer. Data are expressed as the mean ± SEM, P values were
calculated by One-way ANOVA and Tukey’s post-hoc analyses (n = 7/group). Representative data were shown in the lower panels. As compared to vehicle and two of
SGLT2 inhibitors, the ratio of JC-1 Green cells was apparently elevated in the treatment of CCCP at 50 μM concentration. CCCP: Carbonyl cyanide m-chlor­ophenyl hydrazone.

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Biomedicine & Pharmacotherapy 149 (2022) 112864

Fig. 4. SGLT2 inhibitors suppressed the proliferation of MT-2 cells mainly via the blockade of glucose uptake without inducing apoptosis.(A) MT-2 cells
were treated with SGLT2 siRNA. Level of mRNA for SGLT2 was normalized against that of β-actin. Control means cells without transfection of siRNAs. Data areexpressed as mean ± SEM (n = 6). * *: p < 0.01 vs. scrambled siRNA-transfected cells by One-way ANOVA and Tukey’s post-hoc analyses.(B, C) After treating with
SGLT2 siRNA, proliferation and glucose uptake of cells replenished with (B) Luseogliflozin or (C) Tofogliflozin in a dose of 50 μM for 48 h were analyzed. Data areexpressed as mean ± SEM (n = 3–4). n.s.: no significant difference, * : p < 0.05, * *: p < 0.01, * ** : p < 0.001 vs. scrambled vehicle by Two-way ANOVA andTukey’s post-hoc analyses. (D, E) Decrease in growth of MT-2 cells by SGLT2 inhibitors was unrelated with apoptosis. DNA ladders of MT-2 cells treated with 50 μM
Luseogliflozin, 50 μM Tofogliflozin or 0.4 µM Gemcitabine by a conventional phenol-chloroform method (D). After treating with 100 μM concentration of SGLT2
inhibitors or 0.4 µM Gemcitabine for 1 h, caspase-3 activity was measured via the colorimetric assay kit (E). Data are expressed as mean ± SEM (n = 4). * *: p < 0.01
vs. vehicle by One-way ANOVA and Tukey’s post-hoc analyses. Gem: Gemcitabine; Tofo: Tofogliflozin; Luse: Luseogliflozin; 100 bp M: 100 bp DNA ladder, Roche,
11721933001; 1 kb M: 1 kb DNA ladder, NEB, N0468S; N: no loading lane.

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Biomedicine & Pharmacotherapy 149 (2022) 112864

Fig. 5. SGLT2 inhibitors potentiated the cell cycle arrest at the G0/G1 phase in MT-2 cells.Effects of SGLT2 inhibitors on the cell cycle and DNA replication in MT-2
cells.The cell cycle was analyzed by staining DNA by the flow cytometer. One day after the treatment with SGLT2 inhibitors, cells were stained by use of Cell Cycle
Assay Solution Blue, and blue fluorescence (Pacific Blue-A: Excitation length at 405 nm and Emission length at 450 nm, respectively) was detected to determine theratio of the G0/G1, S and G2/M phase. Data are expressed as mean ± SEM (n = 6). * ** : p < 0.001 vs. corresponding value for same phase of vehicle treatment by
Two-way ANOVA and Tukey’s post-hoc analyses. Representative data are shown in the lower panels. After 24 h of SGLT2 inhibitor treatment, double-stranded DNA
was stained with Pacific Blue-A, and the cell ratios of G0/G1 phase, S phase, and G2/M phase were calculated from the gating by the difference of fluorescenceintensity using the flow cytometer.

preferentially expressed in the early stage of human lung adenocarci­
noma and GLUT1 mainly expressed in the late stage, suggesting that
SGLT2 and GLUT1 may play a stage-related role for glucose uptake inlung adenocarcinoma [23]. Similarly, the mRNA level for GLUT1
showed a trend to elevate in both cell lines (MT-1 and MT-2) ascompared to that for GLUT3, suggesting the expression of glucose
transporter differ according to cell types or malignant grade (Fig. 3A,
lower).
In the present study, we demonstrated that NADPH level in MT-2
cells was remarkably increased as compared to that of PBMCs, andsignificantly decreased under the treatment of Luseogliflozin (Fig. 2 F)
and showed a trend to decrease under the treatment of Tofogliflozin（Fig. 2G). These results suggest that SGLT2 inhibitors may suppress
PPPs in MT-2 cells. Since the PPPs synthesize both nucleic acids andNADPH serving as fatty acid synthesis, it is reasonable to hypothesizethat inhibition for PPPs by SGLT2 inhibitors also contribute to growth
inhibition in MT-2. Furthermore, cell cycle analyses demonstrated thatboth Luseogliflozin and Tofogliflozin facilitated the G0/G1 arrest in MT2 cells (Fig. 5), which is also likely to suppress DNA synthesis in cancer
cells. In this context, further studies are required to clarify the stagedependent contribution of SGLT2 to glycolytic metabolic pathways
and PPPs in hematological malignancies in humans.
We do recognize that the present study has a couple of limitations. In
in vitro experiments, we used only two kinds of cell lines for ATL. Studies
using a variety of cell lines are required to strengthen our findings.
Second, we could not fully address the mechanism of discordance

between the suppression of cell proliferation and the inhibition of
glucose uptake by SGLT2 inhibitors. Because simultaneous assessmentof both phenomena in the same in vitro system seems technically aherculean task, further studies are warranted to clarify this issue.
5. Conclusion
Collectively, the present study is the first to demonstrate that phar­macological inhibition of SGLT2 has a potent ability to suppress thegrowth of ATL cells by attenuating glucose uptake and resultantly
decreasing intracellular levels of ATP and NADPH, all of which are likely
to potentiate cell cycle arrest at G0/G1 phase. Our findings may shedlight on the SGLT2 inhibitors-based novel adjunctive therapies against
intractable hematological malignancies such as ATL.
Funding
This work was supported in part by Grants-in-Aid from Japan Society
for the Promotion of Science (JSPS; KAKENHI, 20K08912, 21K08560),
Grant from Construction of the Okinawa Science & Technology Inno­
vation System (2020–2021).
Conflict of interest disclosure
The authors declare no competing interests.

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SN, SO, IN, YN, KT and MT contributed to the research design,acquisition, assembly, analysis and interpretation of data, and drafting
the manuscript. TF, YT, MI, SM, MT, MM and SM contributed to theconceptual design of experiments and discussion. HM conceived theidea, contributed to the overall experimental design, data analysis andinterpretation, and writing and critical revision of the manuscript. All
authors read and approved the final manuscript. HM and SO are
responsible for the integrity of the present study as a whole.
Conflict of interest statement
The authors declare no competing interests.
Acknowledgments
We thank Taisho Pharmaceutical Co., Ltd. and Kowa PharmaceuticalCo., Ltd. for the donated course for University of the Ryukyus. We thankY. Murayama, C. Horiguchi and T. Ikematsu, M. Shimoji, M. Hirata, T.
Uema and C. Noguchi for excellent assistance.
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