Biomedicine & Pharmacotherapy

Volume 121, January 2020, 109648

Thiamine mimetics sulbutiamine and

benfotiamine as a nutraceutical approach
to anticancer therapy
Hunter C. Jonus a, Charnel C. Byrnes a, Jaeah Kim a, Maria L. Valle a, Michael G. Bartlett a,
Hamid M. Said b c, Jason A. Zastre a

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Highlights
• Lipophilic thiamine mimetics possess anti-cancer potential in vitro.

• In vitro toxicity associates with enhanced thiamine homeostasis and PDH

activation.

• TPP is the probable thiamine-related species capable of inhibiting PDK

activity.

• Benfotiamine demonstrates in vivo efficacy to reduce tumor proliferation.

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 Abstract
Malignant cells frequently demonstrate an oncogenic-driven reliance on glycolytic
metabolism to support their highly proliferative nature. Overexpression of pyruvate
dehydrogenase kinase (PDK) may promote this unique metabolic signature of tumor
cells by inhibiting mitochondrial function. PDKs function to phosphorylate and
inhibit pyruvate dehydrogenase (PDH) activity. Silencing of PDK expression has
previously been shown to restore mitochondrial function and reduce tumor cell
proliferation. High dose Vitamin B1, or thiamine, possesses antitumor properties
related to its capacity to reduce PDH phosphorylation and promote its enzymatic
activity, presumably through PDK inhibition. Though a promising nutraceutical
approach for cancer therapy, thiamine’s low bioavailability may limit clinical
effectiveness. Here, we have demonstrated exploiting the commercially available
lipophilic thiamine analogs sulbutiamine and benfotiamine increases thiamine’s anti-
cancer effect in vitro. Determined by crystal violet proliferation assays, both
sulbutiamine and benfotiamine reduced thiamine’s millimolar IC50 value to
micromolar equivalents. HPLC analysis revealed that sulbutiamine and benfotiamine
significantly increased intracellular thiamine and TPP concentrations in vitro,
corresponding with reduced levels of PDH phosphorylation. Through an ex vitro
kinase screen, thiamine’s activated cofactor form thiamine pyrophosphate (TPP) was
found to inhibit the function of multiple PDK isoforms. Attempts to maximize
intracellular TPP by exploiting thiamine homeostasis gene expression resulted in
enhanced apoptosis in tumor cells. Based on our in vitro evaluations, we conclude
that TPP serves as the active species mediating thiamine’s inhibitory effect on tumor
cell proliferation. Pharmacologic administration of benfotiamine, but not
sulbutiamine, reduced tumor growth in a subcutaneous xenograft mouse model. It
remains unclear if benfotiamine’s effects in vivo are associated with PDK inhibition or
through an alternative mechanism of action. Future work will aim to define the action
of lipophilic thiamine mimetics in vivo in order to translate their clinical usefulness as
anticancer strategies.

Graphical abstract
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Keywords
Cancer; Metabolism; Pyruvate dehydrogenase; Pyruvate dehydrogenase kinase;
Thiamine

1. Introduction
Cancer cells often portray a Warburgian metabolic signature, which promotes tumor
proliferation by allowing the rapid generation of ATP and biomass [1]. This enhanced
glycolytic flux in tumor cells may partially be facilitated by the restriction of
mitochondrial metabolism through overexpression of pyruvate dehydrogenase kinase
(PDK) [2,3]. Mechanistically, PDK overexpression reduces mitochondrial ATP
production through phosphorylation and inactivation of the rate-limiting enzyme
pyruvate dehydrogenase (PDH), which bridges the juncture between glycolysis and
the tricarboxylic acid cycle (TCA, Kreb’s) [3]. Inhibiting PDK activity using small
molecules such as dichloroacetate (DCA) and sodium phenylbutyrate has
demonstrated promising pre-clinical chemotherapeutic efficacy [4,5]. Cancer cells
treated with DCA exhibit restored mitochondrial-dependent metabolism resulting in
mitochondrial membrane depolarization and the accumulation of reactive oxygen
species (ROS) [6,7]. Ultimately, PDK inhibition results in apoptosis and an overall
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 reduction in tumor cell proliferation [[7], [8], [9]].

Vitamin B1, or thiamine, may be an effective nutraceutical alternative to DCA and

sodium phenylbutyrate for targeting cancer cell metabolism. We previously identified
that high-dose thiamine therapy decreased tumor cell proliferation with an IC50
value in the mM range similar to that found for DCA [10]. In conjunction with reduced
proliferation, thiamine supplementation corresponds with a reduction in PDH
phosphorylation in tumor cells [10]. Interestingly, supplemental thiamine has also
been demonstrated to reduce the glycolytic activity of tumor cells in other studies [11
]. High-dose thiamine supplementation (∼2500 times the regular daily allowance)
inhibits tumor proliferation in vivo [12]. Together, these results suggest that thiamine
may function to inhibit tumor growth by stimulating PDH activity through a
mechanism similar to DCA. However, the active species mediating thiamine’s
anticancer effectiveness remains unclear. Once inside the cell, thiamine is rapidly
phosphorylated into its active cofactor form thiamine pyrophosphate (TPP) by the
activity of thiamine pyrophosphokinase-1 (TPK1) (Fig. 1) [13]. Alternatively, thiamine
can exist intracellularly in the form of other phosphorylated derivatives including
thiamine monophosphate (TMP) (Fig. 1). In vivo administration of TPP in the form of
hydroxyethylthiamine diphosphate reduced tumor burden by ∼75% [14]. Therefore,
TPP may be the species capable of mediating the growth-inhibitory effects of high-
dose thiamine during malignancy.
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Fig. 1. Schematic diagram representing chemical structures of thiamine, thiamine

phosphorylates, and thiamine analogues. The rapid intracellular phosphorylation of
(A) thiamine produces its active cofactor form (B) thiamine pyrophosphate (TPP).
Dephosphorylation of TPP forms a single phosphate thiamine derivative (C) thiamine
monophosphate (TMP). Lipophilic thiamine analogues (D) benfotiamine and (E)
sulbutiamine.

Supporting its margin of safety as a clinical therapeutic, thiamine administration in

doses greater than 1 g/day have been reported to be well-tolerated with only minor
side-effects including nausea and indigestion [15,16]. However, a major limitation for
thiamine’s clinical effectiveness will be its poor overall bioavailability, which is
estimated to be as low as 3.7–5.3% [17,18]. The quaternary nitrogen located within
thiamine’s thiazole ring and overall hydrophilicity of the molecule necessitates
carrier mediated transport across the plasma membrane for absorption and cellular
accumulation. Therefore, this therapy may be capacity limited as high-dose thiamine
therapy would saturate and limit thiamine accumulation within tumor cells. To
circumvent this, lipophilic thiamine analogs that do not require facilitative transport
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 may be exploited. Sulbutiamine and benfotiamine are two synthetic analogs of
thiamine that are commercially available as thiamine mimetics. Sulbutiamine
(isobutyrylthiamine disulfide) is a lipophilic precursor of thiamine that possesses the
ability to cross biological membranes [19]. After opening of their respective thiazole
rings and attachment of isobutyryl functional groups at each alcohol moiety, two
molecules of thiamine are joined by a disulfide bridge to form O-isobutyrylthiamine
disulfide, or sulbutiamine (Fig. 1) [20]. Sulbutiamine undergoes rapid conversion into
individual thiamine molecules by thioesterase activity inside the cell [21].
Benfotiamine (S-benzoylthiamine-O-Monophosphate, Fig. 1) is dephosphorylated in
the intestine and absorbed by diffusion. Due to its lipophilicity benfotiamine passes
the cell membrane and undergoes subsequent conversion into thiamine after thiazole
ring closure [22]. Both sulbutiamine and benfotiamine build up higher concentrations
of intracellular TPP compared with thiamine, suggesting they may enhance thiamine-
related toxicity in cancer cells [22,23]. Therefore, the present study was undertaken
to determine the sensitivity of cancer cells to lipophilic analogs of thiamine and
identify the active species mediating a reduction in PDH phosphorylation.

2. Materials and methods

Cell culture reagents including RPMI 1640 and DMEM:F12 media,
penicillin/streptomycin, and trypsin/EDTA were purchased from Corning (Manassas,
VA). Fetal bovine serum (FBS) was purchased from Seradigm (Radnor, PA). All flasks
and dishes used for routine maintenance of cultures were purchased from Greiner
Bio-One (Monroe, NC). Thiamine, HEPES, cholera toxin, insulin, transferrin,
hydrocortisone, and human recombinant EGF were purchased from Sigma (St. Louis,
MO). Both sulbutiamine and benfotiamine were purchased from Toronto Research
Chemicals (North York, ON).

2.1. Cell culture

Human cancer cell lines including HCT 116 (CVCL_0291), U-87 MG (CVCL_0022), and
MDA-MB-231 (CVCL_0062) cells were obtained from ATCC (Manassas, VA).
Spontaneously immortalized Fetal Human Colon (FHC, CVCL_3688) cells were also
purchased from ATCC and used as a non-cancerous comparison [24,25]. Cancer cell
lines were routinely cultured at 37 °C with 5% CO2 in complete RPMI 1640 medium,
which contained 10% FBS and 1% penicillin/streptomycin. Following ATCC protocol,
FHC cells were cultured in DMEM:F12 medium supplemented with 10 mM HEPES,
10 ng/mL cholera toxin, 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 100 ng/mL
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 hydrocortisone, 20 ng/mL human recombinant EGF, 10% FBS, and 1%
penicillin/streptomycin. All culture media was also supplemented with 0.1% Mycozap
(Lonza, Verviers, Belgium) as a prophylactic treatment for prevention of mycoplasma
contamination. Cells used for experimentation ranged from passage 4-25.

2.2. Quantitation of cellular proliferation

Cellular proliferation was determined using a crystal violet assay as previously
described [10]. HCT 116 (1000 cells/well), U-87 MG (1700 cells/well), and MDA-MB-
231 (1000 cells/well) were seeded into 96-well plates. Cells were allowed to attach
∼12 h in complete growth medium. Media was then aspirated and replaced with
complete growth medium containing serial dilutions of either thiamine,
sulbutiamine, benfotiamine, or mannitol. Plates were incubated under normal growth
conditions for 5 d in the presence of each compound. The treatment media was
aspirated from each well and cells were washed with ice-cold phosphate buffered
saline (PBS). PBS was replaced by buffered formalin (EMD Millipore, Darmstadt,
Germany) and plates were incubated at 4 °C for 30 min to fix cells. Formalin was
removed and plates were washed two times with diH2O. Fixed cells were
immediately stained with 0.1% crystal violet solution for 30 min at room temperature.
Crystal violet stain was then removed and plates were washed with diH2O. Plates
were allowed to dry in a biosafety cabinet under laminar air flow. When completely
dry, 1% Triton X-100 was used to lyse and destain fixed cells. Absorbance was
determined using a SpectraMax M2e (Molecular Devices, Sunnyvale, CA) microplate
reader at 550 nm. Proliferation was normalized by comparing the absorbance of
treated wells to untreated control wells. A non-linear regression ([inhibitor] vs.
normalized response) was fitted using GraphPad Prism 6® (GraphPad Software, La
Jolla, CA) and used to determine IC50 values.

2.3. Detection of apoptotic cell death

The Cell Death Detection ELISAPLUS (Roche LifeScience, Indianapolis, IN) was used to
quantitate cell death induced by various treatments through the analysis of
cytoplasmic histone-associated DNA fragments as previously described [26]. Briefly,
the provided lysis buffer was applied to treated cells and incubated at room
temperature for 30 min with gentle shaking. The resulting lysate was centrifuged at
200xg for 10 min at 15 °C. Supernatant was combined with immunoreagent in the
supplied microplate for 2 h at room temperature with gentle shaking at 300 rpm.
After 2 h, individual wells were aspirated and washed 3 times with incubation buffer.
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 ABTS developing solution was immediately added to each well. The plate was
incubated for 10 min at room temperature with gentle shaking at 250 rpm. Following
development, stop solution was added and the plate’s absorbance was read at 405 nm
using a SpectraMax M2e microplate reader (Molecular Devices, Sunnyvale, CA). Cell
death was calculated as the fold change comparing treated sample to untreated
control.

2.4. Quantitation of intracellular thiamine, TPP, sulbutiamine, and

benfotiamine
The effect of thiamine, sulbutiamine, and benfotiamine treatment on intracellular
thiamine and TPP concentration was established using ion-paired reversed phase
high-performance liquid chromatography (HPLC) as previously described [27]. In
addition, cells treated with sulbutiamine and benfotiamine were assessed for the
presence of the parent compounds as previously described [28]. Cells were collected
by scraping and immediately placed on ice, washed with ice-cold PBS, and then
pelleted by centrifugation at 600xg for 5 min at 4 °C in an Allegra X-22R centrifuge
(Beckman Coulter, Brea, CA). Subsequently, pellets were washed two additional times
with ice-cold PBS. Prior to pelleting following the final wash, suspended cells were
counted using a TC-20 automated cell counter (BioRad, Hercules, CA). The
intracellular level of thiamine or TPP (pmol) determined by HPLC was normalized to
total cell count.

2.5. Evaluation of protein expression and phosphorylation

To assess the extent of PDK protein expression and PDH phosphorylation at its E1α
subunit, Western blot analysis was performed on whole cell lysates (WCL). To prepare
WCLs, treated cells were washed with ice-cold PBS followed by immediate lysis using
1% Nonidet P-40 (NP40), 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium
deoxycholate, 0.01% sodium azide, 50 mM tris, 250 mM NaCl, and 1 mM
ethylenediaminetetraacetic acid (EDTA) at pH = 8.5 supplemented with
phenylmethanesulfonylfluoride (Calbiochem, La Jolla, CA) and protease/phosphatase
inhibitors (G-Biosciences, St. Louis, MO). Lysates were collected and cellular debris
was pelleted by centrifugation at 17,000xg using a Allegra X-22R centrifuge (Beckman
Coulter, Brea, CA) for 20 min at 4 °C. The supernatant was collected and total protein
content was determined using a BCA protein assay (Thermo Scientific, Rockford, IL).
WCLs (50 µg) from each treatment were resolved by electrophoresis using a 10% SDS-
PAGE gel. Separated proteins were then transferred to polyvinylidene difluoride
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 membranes. Membranes for detection of total PDH (E1α subunit) and PDK1–4
expression were blocked with 5% non-fat milk in tris buffered saline-tween 20 (TBS-
T) for 1 h at room temperature. Alternatively, membranes used to detect PDH
phosphorylation were blocked in 3% bovine serum albumin (Sigma, St. Louis, MO) for
1 h at room temperature. Membranes were then immunoblotted with primary
antibody for 12 h at 4 °C. Table 1 provides all information regarding manufacturer and
dilution (in TBS-T) for individual antibodies. After the primary antibody incubation,
blots were washed three times with TBS-T (10 min each) and then exposed to
horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit
secondary antibody (Bethyl Laboratories, Montgomery, TX) at a 1:10,000 dilution in
TBS-T for 1 h at room temperature. Blots were washed three additional times with
TBS-T. Protein expression was visualized using Supersignal-PLUS West Pico Solution
(Thermo Scientific, Rockford, IL) according to manufacturer’s instruction. Signal was
imaged using a Fluorchem SP digital imager (Alpha Innotech, San Leandro, CA).
Densitometry analysis was performed using Fluorchem SP Software (Alpha Innotech,
San Leandro, CA).

Table 1. Primary antibodies used for Western blot analysis.

Protein of Manufacturer (Catalog Dilution Secondary Antibody

Interest Number) Antibody Registry ID

PDK1 Genetex (GTX107405) 1:1000 Rabbit AB_11168810

PDK2 ProteinTech (15647-1-AP) 1:1000 Rabbit AB_2268006

PDK3 Genetex (GTX104286) 1:1000 Rabbit AB_1951161

PDK4 ProteinTech (12949-1-AP) 1:1000 Rabbit AB_2161499

PDHE1α Genetex (GTX104015) 1:1000 Rabbit AB_1951155

PDH p232 CalBiochem (AP1063) 1:1000 Rabbit AB_10616070

PDH p293 CalBiochem (AP1062) 1:1000 Rabbit AB_10616069

PDH p300 CalBiochem (AP1064) 1:1000 Rabbit AB_10618090

β-Actin Sigma-Aldrich (A2228) 1:1000 Mouse AB_476697

2.6. Quantitation of PDH activity

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 The activity of PDH following treatment with thiamine, sulbutiamine, benfotiamine,
or DCA was determined using the commercially available PDH Enzyme Activity
Microplate Assay Kit (Abcam) following manufacturer’s provided protocol. Adherent
cells were collected following treatment by scraping, suspended in ice-cold PBS, and
pelleted at 1000xg for 5 min at 4 °C in an Allegra X-22R centrifuge (Beckman Coulter,
Brea, CA). Cells were washed two additional times using ice-cold PBS. Following final
wash, cells were suspended in ice-cold PBS containing phosphatase inhibitor and
protease inhibitor (G-Biosciences, St. Louis, MO). The kit’s provided detergent solution
was added to each sample at a final dilution of 1:10. Samples were incubated with
detergent on ice for 10 min to allow solubilization and then centrifuged at 1000xg for
10 min at 4 °C in a Allegra X-22R centrifuge (Beckman Coulter, Brea, CA). The resulting
supernatant (1 mg protein/well) was loaded into the provided ELISA microplate to
capture PDH present within samples. The plate was covered with foil and incubated
on a rotating shaker (200 rpm) for 3 h at room temperature. After 3 h, wells were
aspirated and washed three times with 1X Stabilizer. Following washes, developing
solution was added to each well and the plate’s absorbance was measured kinetically
at 450 nm over 15 min using a SpectraMax M2e spectrophotometer. Based on the
kinetic read, the maximal velocity (Vmax) of PDH activity was determined for each
sample through SoftMax Pro Software (Molecular Devices, Sunnyvale, CA).

2.7. Assessment of gene expression

Gene expression of PDK isoforms 1–4 and SLC44A4 was determined by quantitative
real-time PCR. For analysis, RNA was extracted using the E.Z.N.A Total RNA Kit I
(Omega Bio-Tek, Norcross, GA) following the manufacturer’s protocol. The RNA
concentration was determined using a Nanodrop 2000c Spectrophotometer (Thermo
Scientific, Rockford, IL) and 1 µg of isolated RNA was reverse transcribed using the
qScript cDNA Synthesis Kit (Quanta BioSciences, Gaithersburg, MD) Gene detection
was performed using the Light-Cycler 480 II (Roche Applied Science, Indianapolis, IN)
For each gene of interest, specific primers were designed using the Roche Universal
Probe Library assay design center. Primer sets are listed in Table 2. Each primer set
corresponded with the Roche hydrolysis probe #5 labeled with fluorescein (FAM).
The expression of ACTIN (ACTB) was determined using the Universal ProbeLibrary
Human ACTB Gene Assay and used as a reference gene to calculate relative gene
expression based on the 2−∆Ct method with an assumed efficiency of 2.

Table 2. Primer sequences and Roche UniversalProbe Library probe pairs for Real Time-
PCR analysis.
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 Gene Forward and Reverse Primer Sequences

F: 5’-GAGTCTTCAGGAGCT-3’
PDK1
R: 5’-TGCAACCATGTTCTTCTACCG-3’

F: 5’-TGAAGCAGTTTCTGGACTTCG-3’
PDK2
R: 5’-AGGTTGATCTCTTTCATGATGTTG-3’

F: 5’-TGTGTGAACAGTATTACCTGGTAGC-3’
PDK3
R: 5’-GTTTGTCTGCTTTGG-3’

F: 5’-CAGTGCAATTGGTTAAAAGCTG -3’
PDK4
R: 5’-GGTCATCTGGGCTTTTCTCA -3’

F: 5′-TGCTGATGCTCATCTTCCTGCG-3′
SLC44A4
R: 5′-GGACAAAGGTGACCAGTGGGTA - 3′

2.8. Determination of PDK inhibition

To determine the species of thiamine capable of inhibiting PDK activity, the
commercially available PDH Enzyme Activity Microplate Assay Kit (Abcam) was
adapted to screen kinase activity. Recombinant human protein for PDK1–4 was
obtained from Abcam. To carry out PDK inhibition screening assay, 25 µg of bovine
heart mitochondria (Abcam) was added to each well of the provided antibody-coated
microplate as a source of PDH protein. The plate was covered with foil and placed on
rotating shaker (200 rpm) for 2.5 h at room temperature to allow protein binding.
Following incubation, wells were washed four times using 1X stabilizer. PDKs
(1 µg/mL) were incubated with serial dilutions of inhibitor (thiamine, TMP, TPP,
sulbutiamine, or benfotiamine) at 37 °C for 15 min. To initiate the kinase reaction,
50 µM ATP (Cayman Chemical, Ann Arbor, MI) was added and samples were placed
into microplate with bound PDH. Plate was covered with foil and placed on rotating
shaker (200 rpm) for 20 min at room temperature. After 20 min, each well was
aspirated and washed four times with 1X stabilizer. Developing solution was
immediately added to each well and the plate’s absorbance was measured kinetically
at 450 nm over 15 min. Based on the kinetic read, the maximal velocity (Vmax) of PDH
activity was determined for each PDK with/without inhibitor through SoftMax Pro
Software (Molecular Devices, Sunnyvale, CA). As a negative control, PDH activity from
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 isolated bovine heart mitochondrial extract (Abcam) that had not been treated with
PDK was used. Activity for each PDK +/- inhibitor was normalized to the negative
control to determine fold change of PDK inhibition.

2.9. Overexpression of thiamine homeostasis genes

HCT 116 cells were used to determine the sensitivity of tumor cells to thiamine and
TPP following the transient overexpression of critical thiamine homeostasis genes.
THTR1 (thiamine transporter) overexpression was mediated through the introduction
of SLC19A2 using the expression plasmid pEGFP-N3 [29]. TPPT (TPP transporter)
overexpression was mediated through the introduction of SLC44A4 using the
expression plasmid pFLAG-CMV-2 previously developed [30]. Transfection of SLC19A2
and SLC44A4 was verified using qRTPCR (Supplemental Fig. 5). TPK1 (TPP producing
enzyme) overexpression was mediated through the introduction of TPK1 using the
expression plasmid pcDNA3.1(+) previously developed [31]. For all transfections, cells
were seeded at 6000 cells/well into 96-well plates and allowed to attach ∼12 h.
Normal culture media was then replaced with transfection media (100 µL) containing
500 ng plasmid DNA and 2.5% lipofectamine. Cells were transfected for 24 h, after
which transfection media was removed and replaced with complete growth medium
for a 24 h recovery period. After recovery, cells were dosed with thiamine or TPP for
24 h and cell death was determined by Cell Death Detection ELISAPLUS as described
above.

2.10. Xenograft studies

The animal use protocol for this study was approved by the University of Georgia
Institutional Animal Care and Use Committee (IACUC) in compliance with Guidelines
for the Use and Care of Laboratory Animals set by the National Institutes of Health.
Six-week old female athymic nude (Nu/Nu) mice were purchased from Taconic
Biosciences (Rensselaer, NY). Following acclimatization, xenografts were established
by injecting 100 µL containing 1 × 106 HCT 116 cells in a 4X dilution of BD Matrigel
Matrix (Corning, Manassas, VA) with PBS subcutaneously into the flank of each
mouse. Approximately 12 days following tumor implant mice demonstrated palpable
tumor volumes (W2xL/2) of ∼150 mm3.

To test the efficacy of sulbutiamine (n = 5) and benfotiamine (n = 5) in inhibiting in

vivo tumor growth, each compound was administered through bolus intraperitoneal
(IP) injection (250 mg/kg every second day). Due to the low solubility of sulbutiamine
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 and benfotiamine, 40 mg/ml suspensions were prepared using 15% Arabic gum in
normal saline as previously described [32]. As a vehicle control (n = 4), 15% Arabic
gum in normal saline was administered to control animals. All injections were
adjusted to pH = 6 to avoid irritation to the abdominal cavity. Other than lethargy, no
adverse symptoms were noted following injections. Throughout the course of the
study, two benfotiamine treatment animals were lost due to AUP established humane
endpoints unrelated to treatment. These animals were excluded from analysis
resulting in n = 3 for the benfotiamine treatment. Tumor volume, animal weights, and
food consumption were tracked over the course of the study. Lactate levels in tumors
isolated at the conclusion of the study were measured using methodology previously
reported [10].

2.11. Statistical analysis

A minimum of three independent replicates was performed for each experimental
data set. Statistical significance (p < 0.05) was established using either an unpaired
student’s t-test or a one-way analysis of variance (ANOVA) with Tukey’s post hoc test
using GraphPad Prism 6® (GraphPad Software, La Jolla, CA).

3. Results

3.1. Tumor cells demonstrate increased susceptibility to lipophilic

thiamine analogs
The sensitivity of tumor cell lines including HCT 116 (colorectal carcinoma), U-87 MG
(glioblastoma), and MDA-MB-231 (metastatic breast adenocarcinoma) to thiamine,
sulbutiamine, and benfotiamine was determined by crystal violet proliferation (Fig. 2
A) assay. All three cell lines demonstrated a decrease in proliferation with increasing
concentration of thiamine, sulbutiamine, or benfotiamine. IC50 values (Table 3) for
each compound were calculated and thiamine analogs reduced tumor cell
proliferation with approximately 30–50 fold lower concentrations than thiamine. To
rule out hypertonic effects in the dose-response profiles for each compound, HCT 116,
U-87 MG, and MDA-MB-231 were treated with similar concentrations of the cell
impermeable compound mannitol. No toxicity was found for mannitol treatment up
to 10 mM (Fig. 2B). Due to the extremely slow proliferation rate of the non-cancerous
FHC cell line, the Cell Death Detection ELISAPLUS was used to quantitate differences in
the sensitivity of non-cancerous and cancerous cells to thiamine, sulbutiamine, or
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 benfotiamine treatment. HCT 116 cells treated with 0.5 mM sulbutiamine or 1 mM
benfotiamine for 24 h demonstrated an ∼5- and 4-fold increase in cell death,
respectively (Fig. 2C). Though there was no significant effect for treatment with
25 mM thiamine, the fold change in cell death for HCT 116 cells trended higher (Fig. 2
C). Unlike their cancerous counterparts, FHC cells demonstrated no significant
increase in cell death markers following treatment with 25 mM thiamine, 0.5 mM
sulbutiamine, or 1 mM benfotiamine for 24 h (Fig. 2D).

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Fig. 2. Lipophilic thiamine analogues inhibit tumor cell proliferation (A) Proliferation
of HCT 116, U-87 MG, and MDA-MB-231 cells determined by crystal violet assay
demonstrating the effect of thiamine (●), sulbutiamine (◼), and benfotiamine (▲)
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 treatment following 5 days of exposure. Results are normalized as mean percent
proliferation +/- standard deviation (SD) comparing treated cells to untreated control.
(B) Proliferation of HCT 116 (▲), U-87 MG (◼), and MDA-MB-231 (●) cells determined
by crystal violet assay demonstrating the effect of mannitol treatment after 5 days of
exposure. Results are normalized as mean percent proliferation +/- SD comparing
treated cells to untreated control. (C) Apoptotic cell death demonstrated as fold
change +/- SD in HCT 116 cells seeded at 6000 cells/cm2 and treated with thiamine (T,
25 mM), sulbutiamine (SLBT, 0.5 mM), or benfotiamine (BNFO, 1 mM) for 24 h relative
to untreated control (CTL). Treatment of HCT 116 cells with 15 µM camptothecin
serves as positive control (+ CTL) for detection of apoptosis. (D) Apoptotic cell death
demonstrated as fold change +/- SD in FHC cells treated with thiamine (T, 25 mM),
sulbutiamine (SLBT, 0.5 mM), or benfotiamine (BNFO, 1 mM) for 24 h relative to
untreated control (CTL). Due to slow growth rate of FHC cell line, cells were seeded at
30,000 cells/cm2 and cultured 1 week prior to initiating treatments. Treatment of FHC
cells with 15 µM camptothecin serves as positive control (+ CTL) for apoptosis. (*)
Represents statistically significant difference (p < 0.05) based on results of one-way
ANOVA with Tukey’s post-hoc test.

Table 3. IC50 values with 95% confidence intervals for thiamine, sulbutiamine, and
benfotiamine determined by crystal violet proliferation assay.

Thiamine (mM) Sulbutiamine (µM) Benfotiamine (µM)

HCT 116 4.83 (3.44, 6.81) 153 (117, 202) 91 (71, 118)

U-87 MG 4.85 (3.45, 6.83) 157 (135, 182) 90 (75, 107)

MDA-MB-231 5.43 (3.71, 7.98) 162 (135, 194) 72 (60, 87)

3.2. Thiamine and its lipophilic analogs increase intracellular

thiamine and TPP concentration
To determine how dosing with thiamine, sulbutiamine, or benfotiamine impacts
intracellular thiamine and TPP levels, HCT 116 cells were treated with each compound
for 24 and 48 h. Based on 5-day proliferation profiles, concentrations of 25 mM
thiamine and 250 µM sulbutiamine and benfotiamine were chosen for short-term
studies at 24 h and 48 h to minimize the impact of cellular toxicity. At these doses,
there was no significant change in cell count compared to control cells after 24 h and
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 only a moderate reduction after 48 h (Fig. 3A). Treatment with each compound
significantly increased intracellular thiamine levels after 24 h and 48 h of treatment (
Fig. 3B). Thiamine treatment at 100X greater concentration of both sulbutiamine and
benfotiamine resulted in exaggerated increases for intracellular thiamine (Fig. 3B). No
intracellular sulbutiamine or benfotiamine was detected following treatment with
either analog (Supplemental Fig. 1). Treatment with thiamine, sulbutiamine, or
benfotiamine also significantly increased intracellular TPP levels after 24 h and 48 h
by nearly 2-fold (Fig. 3C). Although thiamine concentration was 100X compared to
sulbutiamine and benfotiamine, the level of TPP induction was comparable (Fig. 3C).

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Fig. 3. Enhanced intracellular thiamine status following pharmacologic treatment

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 with thiamine and its lipophilic analogues (A) Effect of thiamine (T, 25 mM),
sulbutiamine (SLBT, 250 µM), or benfotiamine (BNFO, 250 µM) on tumor cell
proliferation demonstrated by mean cell count +/- SD of HCT 116 cells seeded at
20,000 cells/cm2 and treated for 24 or 48 h compared to untreated control (CTL). (B)
HPLC analysis demonstrating fold change in intracellular thiamine levels +/- SD
determined in HCT 116 cells seeded at 20,000 cells/cm2 and treated with 25 mM
thiamine (T), 250 µM sulbutiamine (SLBT), or 250 µM benfotiamine (BNFO) compared
to untreated control (CTL) for 24 h or 48 h. (C) HPLC analysis demonstrating fold
change in intracellular TPP levels +/- SD determined in HCT 116 cells seeded at 20,000
cells/cm2 and treated with 25 mM thiamine (T), 250 µM sulbutiamine (SLBT), or
250 µM benfotiamine (BNFO) compared to untreated control (CTL) for 24 h or 48 h. (*)
Represents statistically significant difference (p < 0.05) based on results of (A) one-
way ANOVA with Tukey’s post-hoc test or (B,C) an unpaired student’s t-test for each
individual treatment with CTL.

3.3. Activation of PDH by thiamine, sulbutiamine, and benfotiamine

The expression of all four PDK isoenzymes in HCT 116 cells was confirmed at the gene
and protein level. In 7 different passages, HCT 116 cells demonstrated consistent gene
expression of PDK1, PDK2, PDK3, and PDK4 (Supplemental Fig. 2A). Of the four
isomeric forms, PDK2 demonstrated the highest relative gene expression followed by
PDK3 > PDK4 = PDK1 (Supplemental Fig. 2A). Protein expression for each PDK isoform
was also detectable and stable over the course of several passages (Supplemental Fig.
2B).

Changes in PDH phosphorylation and activity due to treatment with thiamine,

sulbutiamine, or benfotiamine, was assessed using DCA as a positive control. DCA has
previously been shown to enhance PDH activity by decreasing phosphorylation
through inhibiting PDK activity [33]. Three phosphorylation residues (Ser232, Ser293,
Ser300) are involved in PDK-mediated inhibition of PDH [34]. Fig. 4A, B, and
Supplemental Fig. 3. demonstrate that treatment 25 mM DCA, as well 25 mM
thiamine, 250 µM sulbutiamine, and 250 µM benfotiamine for 48 h significantly
reduced PDH phosphorylation by approximately half at all three regulatory residues.
Despite changes in phosphorylation, there was no significant change for the total
expression of the PDH complex (E1α subunit) following DCA, thiamine, sulbutiamine,
or benfotiamine treatment (Fig. 4A, B, Supplemental Fig. 3). Decreased
phosphorylation was associated with a significant increase in PDH activity following
:
 treatment with 25 mM DCA, 25 mM thiamine, 250 µM sulbutiamine, and 250 µM
benfotiamine (Fig. 4C).

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Fig. 4. Activation of PDH activity through treatment with thiamine, sulbutiamine, and
benfotiamine (A) Representative Western blots demonstrating PDH expression (E1α
subunit) and the extent of phosphorylation at its three regulatory sites in WCLs
isolated from HCT 116 cells seeded at 10,000 cells/cm2 and treated with
dichloroacetate (DCA, 25 mM), thiamine (T, 25 mM), sulbutiamine (SLBT, 250 µM), or
benfotiamine (BNFO, 250 µM) for 48 h compared to untreated control (CTL). (B)
Densitometry analysis of the fold change in PDHE1α expression and its
phosphorylation at serine residues 232, 293, and 300 +/- SD in HCT 116 cells seeded
at 10,000 cells/cm2 treated with dichloroacetate (DCA, 25 mM), thiamine (T, 25 mM),
sulbutiamine (SLBT, 250 µM), or benfotiamine (BNFO, 250 µM) for 48 h compared to
untreated control (CTL). (C) Fold change +/- SD of PDH activity in lysates isolated from
HCT 116 cells following treatment with dichloroacetate (DCA, 25 mM), thiamine (T,
25 mM), sulbutiamine (SLBT, 250 µM), or benfotiamine (BNFO, 250 µM) for 48 h
compared to untreated control (CTL). (*) Represents statistically significant difference
(p < 0.05) based on results of (B) one-way ANOVA with Tukey’s post-hoc test or (C) of
an unpaired student’s t-test.
:
 3.4. TPP inhibits ex vitro PDK activity
To determine if thiamine, TPP, thiamine monophosphate (TMP), sulbutiamine, or
benfotiamine inhibit PDK activity, an ex vitro kinase assay was used. When screening
each kinase, uninhibited PDH activity from isolated bovine mitochondria provided a
negative control (Fig. 5A–E). The addition of PDK1, PDK2, and PDK3 significantly
reduced detectable PDH activity (Fig. 5A–E). PDK4 protein was unable to provide a
positive control for the inhibition of PDH activity and was excluded from analysis.
DCA increased PDH activity in the presence of PDK1, PDK2, and PDK3 demonstrating
the assay was able to detect kinase inhibition (Supplemental Fig. 4). Addition of TPP
inhibited the activity of PDK1 in a dose-dependent manner with moderate inhibition
at 1 µM and significant inhibition at higher doses of 10 µM, 100 µM, and 1 mM (Fig. 5
A). The greatest inhibitory effect for TPP on PDK1 was found between 10 and 100 µM (
Fig. 5A). TPP inhibited PDK2 in a similar profile found for PDK1. Moderate inhibition
was found at 1 µM and increased in a dose-dependent manner from 10 to 100 µM (
Fig. 5A). For PDK2, maximal inhibition occurred at 100 µM TPP (Fig. 5A). TPP
demonstrated PDK3 inhibition, but only at higher concentrations of 100 µM and
1 mM (Fig. 5A). No effect for thiamine, TMP, sulbutiamine or benfotiamine was
observed at any of the doses considered for inhibition of PDK1, PDK2, or PDK3 (Fig. 5
B–E).
:
 Download: Download high-res image (548KB)
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Fig. 5. TPP inhibits PDK function Ex vitro demonstration of PDK1, PDK2, and PDK3
activity in the presence and absence of (A) TPP, (B) thiamine (T), (C) TMP, (D)
benfotiamine (BNFO), or (E) sulbutiamine (SLBT) demonstrated by fold change in PDH
activity +/- SD. Uninhibited PDH activity (-PDK) from isolated bovine mitochondria
lysate determined as Vmax of reaction serves as the negative control for normalization
in each assay. The fold change +/- SD in PDH activity in the presence of each PDK
(+PDK) compared to uninhibited control (-PDK) serves as the positive control for
functional PDK activity. The ability of each test compound to inhibit PDK activity and
restore PDH function demonstrated as fold change +/- SD in PDH activity was tested
by introducing TPP, thiamine (T), TMP, sulbutiamine (SLBT) and benfotiamine (BNFO)
in logarithmic dilutions from 1000 µM. (*) Represents statistically significant
:
 difference (p < 0.05) based on results of one-way ANOVA with Tukey’s post-hoc test.

3.5. Targeted TPP accumulation demonstrates anticancer potential

The anticancer effect of TPP was analyzed based on results above suggesting an active
role for TPP in inhibiting PDK(s). To do so, various thiamine homeostasis genes were
overexpressed in HCT 116 cells to enhance intracellular TPP accumulation and the
extent of apoptosis was measured using the Cell Death Detection ELISAPLUS. SLC44A4
encodes the thiamine pyrophosphate transporter (TPPT), responsible for transporting
TPP across the plasma membrane [35]. HCT 116 cells transfected with SCL44A4
demonstrated an ∼3.5-fold increase in cell death in the presence of TPP compared to
vector control cells (Fig. 6A). There was no effect on cell death due to treatment with
thiamine, which is not known to be transported by TPPT (Fig. 6A). Overexpression of
SLC19A2, the gene encoding for the high-capacity thiamine transporter THTR1,
demonstrated enhanced cell death (∼2-fold) following thiamine treatment in HCT 116
cells (Fig. 6B). TPP treatment resulted in no increase in apoptotic cells following
overexpression of SLC19A2, which is a selective thiamine transporter (Fig. 6B). Fig. 6C
demonstrates that overexpression of TPK1 resulted in an ∼4-fold enhancement in
HCT 116 sensitivity to thiamine, but had no effect following TPP treatment.
:
 Download: Download high-res image (357KB)
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Fig. 6. Targeted TPP accumulation induces apoptotic tumor cell death Analysis of
apoptotic cell death in HCT 116 cells following overexpression (A) SLC44A4, (B)
SLC19A2, or (C) TPK1 and treatment with 1 mM thiamine (T) or TPP for 24 h relative to
untreated control (CTL). Fold change in cell death +/- SD compares treated or
untreated HCT 116 cells overexpressing each gene of interest (described in materials
and methods) to its treated or untreated vector control. (D) Relative mRNA expression
levels of SLC44A4 determined by qRT-PCR in RWPE-1 (non-cancerous) and LNCaP
−∆Ct
:
 (cancerous) prostate derived cell lines normalized by the 2−∆Ct method. (E) Apoptotic
cell death demonstrated as fold change +/- SD in RWPE-1 cells following treatment
with 1 mM thiamine (T) or TPP for 24 h compared to untreated control (CTL). (F)
Apoptotic cell death demonstrated as fold change +/- SD in LNCaP cells following
treatment with 1 mM thiamine (T) or TPP for 24 h compared to untreated control
(CTL). (*) Represents statistically significant difference (p < 0.05) based on results of
one-way ANOVA with Tukey’s post-hoc test.

Tumor cells have previously been demonstrated to upregulate the expression of

critical thiamine homeostasis genes [36]. Expression of SLC44A4 was compared
between the androgen-sensitive human prostate adenocarcinoma cell line LNCaP and
the normal prostate epithelial cell line RWPE-1. LNCaP cells demonstrated greater
than 200-fold enhancement in SLC44A4 expression compared to RWPE-1 cells (Fig. 6
D). Considering the endogenous expression difference of SLC44A4, the sensitivity of
each cell line to TPP was assessed. Fig. 6E demonstrates no significant change in cell
death when RWPE-1 cells were exposed to 1 mM TPP for 24 h. However, TPP
treatment resulted in a nearly 3-fold enhancement of cell death for th LNCaP cell line
(Fig. 6F). No increase in apoptotic index was observed in either cell line following
treatment with 1 mM thiamine for 24 h (Fig. 6E and F).

3.6. Benfotiamine reduces in vivo tumor growth

To determine the impact of pharmacologic treatment with sulbutiamine or
benfotiamine on in vivo tumor growth mice were administered each compound
through bolus IP injection every second day following tumor formation. Long-term
treatment with sulbutiamine and benfotiamine at 250 mg/kg every second day had
no effect on animal weight (Fig. 7A). Over the course of the study, tumor volume
tracked lower for mice administered sulbutiamine and benfotiamine compared with
control animals (Fig. 7B). The effect was more pronounced for benfotiamine
compared to sulbutiamine (Fig. 7B). At the time of final tumor measurement, there
was a significant reduction in the fold change for tumor growth following
benfotiamine treatment compared with control tumors (Fig. 7C). There was also a
trending reduction in the total weight of tumors isolated from benfotiamine treated
animals compared with control (p = 0.112, Fig. 7D). No effect on isolated tumor weight
was observed for sulbutiamine (Fig. 7D). HPLC analysis of treated tumor tissue
demonstrated trending increases for both thiamine and TPP concentration compared
to vehicle control for both sulbutiamine and benfotiamine treatment (Fig. 7E and F).
:
 Neither sulbutiamine nor benfotiamine were detected in tumor isolates of treated
animals (Supplemental Fig. 5). Tumor tissue isolated from benfotiamine treated
animals compared with vehicle control demonstrated a modest reduction in PDH
phosphorylation at serine residue 232 (Fig. 7G). No change in phosphorylation at
other regulatory residues or in total PDH protein (E1α subunit) was observed
following treatment with sulbutiamine or benfotiamine compared to isolated control
tissue (Fig. 7G).

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Fig. 7. Benfotiamine reduces in vivo tumor growth (A) Average animal weight +/- SEM
at study conclusion for vehicle control (CTL), sulbutiamine (SLBT), and benfotiamine
(BNFO) treated animals. (B) Quantitative analysis demonstrating the effect of
pharmacologic treatment with sulbutiamine (◼) and benfotiamine (▲) compared to
vehicle control (●) on HCT 116 tumor volume over the course of 38 days following
tumor implant (Day 0). Average tumor volume is presented as the mean +/- SEM of
n = 4 control animals and n = 5 animals for sulbutiamine and n = 3 animals for
:
 benfotiamine. (C) Fold change +/- SEM comparing final measured tumor volume
compared with measured volume prior to commencement of IP injections for control
(CTL), sulbutiamine (SLBT), and benfotiamine (BNFO) treated animals. (D) Average
tumor weight +/- SEM depicting effect of pharmacologic treatment with sulbutiamine
or benfotiamine compared to vehicle control (CTL) at the conclusion of 38-day
growth period. (E) HPLC analysis demonstrating average thiamine level +/- SD
determined in tumor tissue isolated from animals treated with sulbutiamine (SLBT)
or benfotiamine (BNFO) compared to vehicle control (CTL). (F) HPLC analysis
demonstrating average TPP level +/- SD determined in tumor tissue isolated from
animals treated with sulbutiamine (SLBT) or benfotiamine (BNFO) compared to
vehicle control (CTL). (G) Western blots demonstrating PDH expression (E1α Subunit)
and the extent of phosphorylation at its three regulatory sites in lysates isolated from
tumor tissue of animals treated with sulbutiamine (SLBT) or benfotiamine (BNFO)
compared to vehicle control (CTL) with n = 3 animals for each condition. (H) Average
lactate level +/- SD determined in tumor tissue isolated from animals treated with
sulbutiamine (SLBT) or benfotiamine (BNF)) compared to vehicle control (CTL) with
n = 3 animals for each condition. (*) Represents statistically significant difference
(p < 0.05) based on results of an unpaired student’s t-test.

4. Discussion
Considering their presumable safety and notable pre-clinical anticancer effects,
exploiting pharmacologic doses of nutraceutical compounds (i.e. vitamins) provides a
promising strategy for cancer therapy [37,38]. The early work of Linus Pauling and
colleagues, demonstrated high-dose vitamin C as a safe and effective therapy to
improve symptoms and prolong the lifespan of terminal cancer patients [39,40].
Pharmacologic treatment with vitamin C in low mM dosages has since been
demonstrated to selectively kill cancer cells in vitro [41,42]. These findings translate
in vivo where pharmacologic ascorbate treatment (4 g/kg, once or twice daily)
significantly reduced the growth rate of tumors [41]. This anticancer activity of
vitamin C has been attributed to its pro-oxidant properties and generation of an
ascorbate radical resulting in H2O2-dependent cytotoxicity [41,43]. Like vitamin C,
thiamine, has also been proposed to slow in vivo tumor growth at pharmacologic
concentrations [12]. Here, we provide evidence that exploiting lipophilic thiamine
mimetics potentiates the anticancer activity of thiamine by requiring lower dosing
equivalents for effectiveness.
:
 The sensitivity of three tumor cell lines from different tissue origins was increased
following treatment with sulbutiamine or benfotiamine compared with thiamine.
Reduction in tumor cell proliferation corresponded with increased apoptotic cell
death in the colorectal cancer cell line HCT 116 following independent exposures to
both sulbutiamine and benfotiamine. This is congruent with DCA treatment, which
has previously been shown to enhance apoptosis in colorectal cancer cells [44]. DCA-
mediated apoptosis results from the depolarization of the mitochondrial membrane
potential (MMP) triggering the release of pro-apoptotic factors [4,44]. We previously
demonstrated that like DCA, high-dose thiamine supplementation results in
reduction of MMP and subsequent cell death through an apoptotic associated
mechanism [10]. Following treatment with sulbutiamine and benfotiamine, total
intracellular levels of thiamine and TPP were increased but the accumulation of the
lipophilic derivatives was not detected. Therefore, the apoptotic response observed
following sulbutiamine and benfotiamine treatment in vitro may be due to the
accumulation of thiamine and/or its derivatives. Of note, the non-cancerous cell lines
HB2 and HEK-293 demonstrate no reduction in MMP following DCA treatment and a
lack of sensitivity to DCA at comparable concentrations that inhibit tumor cell
proliferation [44]. A similar effect was observed for sulbutiamine and benfotiamine,
which induced significant apoptotic cell death in HCT 116 cancer cells but not their
non-cancerous FHC counterparts.

Mechanistically it remains undefined how thiamine inhibits PDH phosphorylation to

reduce tumor cell proliferation. Pyruvate, the primary substrate of PDH, serves as a
metabolic inhibitor of PDK, functioning through direct binding [45]. DCA, a pyruvate
mimetic, also binds to inhibit PDK activity [33]. In addition to pyruvate, PDK activity
may be inhibited by other physiological molecules including ADP, NAD+, and CoA-SH [
3]. TPP possesses structural similarity to ADP and has been characterized to mimic
ADP binding ex vitro [46]. We demonstrate that TPP inhibits PDK activity in an ex vitro
reaction following a dose-dependent manner for PDK1 and PDK2. TPP also inhibited
PDK3 activity, but only at the highest concentrations assayed. Thiamine, TMP,
sulbutiamine, and benfotiamine demonstrated no ability to inhibit PDK activity in our
ex vitro assay system. These results suggest that TPP may be the active species
mediating a reduction in PDH phosphorylation by directly inhibiting PDK activity.
HPLC analysis of intracellular TPP levels demonstrated an ∼2-fold enhancement of
TPP following treatment with thiamine, sulbutiamine, or benfotiamine. Considering
intracellular TPP balance remains highly regulated by TPK1 and thiamine
pyrophosphatase activity, this marginal increase in TPP appears sufficient to reduce
:
 PDH phosphorylation in vitro. Supporting a role for a TPP-mediated anticancer effect,
attempts to maximize TPP production through increasing thiamine transport
(SLC19A2, THTR1) and conversion to TPP (TPK1) resulted in toxicity following only
thiamine treatment. TPP administration had no effect in the latter cases most likely
due to its requirement for carrier-mediated transport to cross the plasma membrane.
Alternatively, the exogenous overexpression of SLC44A4, which encodes the thiamine
pyrophosphate transporter (TPPT), resulted in increased apoptosis of HCT 116 cells
following TPP treatment but not thiamine treatment. Interestingly, SLC44A4
demonstrates strong up-regulation in malignant cells. In non-cancerous tissue, this
transporter demonstrates restricted expression within the colon associated with
uptake of microbiota produced TPP [35]. Therefore, the deregulation of SLC44A4
expression during cancer may allow therapeutic targeting of TPP therapy to tumor
cells.

The chemotherapeutic effectiveness of benfotiamine translated in vivo where its

administration reduced tumor growth in a subcutaneous xenograft model of HCT 116
cells. To our knowledge, this is the first in vivo evidence for the anticancer effect of a
commercially available thiamine analog. Highlighting the therapeutic safety of
benfotiamine, no systemic toxicity was observed following bolus pharmacologic
doses (250 mg/kg) administered via IP injection every second day. This supports the
tolerability of daily benfotiamine administration (600–900 mg/day) that has
previously been demonstrated in clinical trials for diabetic nephropathy [47,48].
Interestingly, no significant effect was observed for sulbutiamine administration
towards in vivo tumor growth, despite both compounds yielding similar intratumoral
accumulations of thiamine and TPP. Neither sulbutiamine nor benfotiamine
significantly changed PDH phosphorylation or lactate levels measured in tumor tissue
isolated at the conclusion of the study. Lack of changes to PDH phosphorylation may
have been impacted by the terminal end point used for analysis of tumor tissue and
not indicative of the initial response to treatment that would be more
complementary to our in vitro findings. Alternatively, the increase in TPP achieved by
our in vivo dosing paradigm may not have been substantial enough to sufficiently
inhibit PDK. However, the reduction in tumor burden by benfotiamine in the absence
of significant changes to PDH phosphorylation and lack of effect for sulbutiamine
suggest additional mechanisms may also have contributed to benfotiamine’s in vivo
chemotherapeutic activity. Benfotiamine has been shown to induce parapoptotic cell
death by inhibiting the activity of constitutively active ERK1/2 while concomitantly
increasing phosphorylation of JNK1/2 in leukemia cells [49]. Tapias et al. recently
:
 identified that active metabolites of benfotiamine (but not thiamine) can activate the
nuclear factor erythroid 2-related factor (NRF2)/antioxidant response element (ARE)
pathway in a model of Alzheimer’s Disease resulting in attenuated oxidative damage
and inflammation [50]. Considering cancer as a disease associated with deregulated
oxidative stress and inflammation, benfotiamine’s impact on NRF2 pathway may also
play an important role.

5. Conclusion
In conclusion, we have demonstrated that lipophilic thiamine analogs decrease in
vitro tumor cell proliferation following a mechanism similar to that of DCA and high-
dose thiamine supplementation. Like thiamine and DCA, sulbutiamine and
benfotiamine reduce PDH phosphorylation in vitro and increase its activity. Our
analysis has revealed that TPP may be the active species mediating the effects of
thiamine, sulbutiamine, and benfotiamine on PDH phosphorylation. Both
sulbutiamine and benfotiamine demonstrate enhanced potency requiring only
micromolar concentrations to elevate intracellular TPP. Surprisingly, only
benfotiamine administration reduced tumor growth in vivo by additional
mechanisms that may be independent of the PDH-PDK axis. Future work on the role
of TPP and benfotiamine metabolites on in vivo tumor growth will be required to fully
understand the chemotherapeutic potential of this lipophilic thiamine mimetic.

Funding
This work was supported by the American Cancer Society [Research Scholar Grant,
RSG-14-026-01-CNE].

Declaration of Competing Interest

None.

Acknowledgments
The authors would like to thank the American Cancer Society in support of this work.

Appendix A. Supplementary data

The following is Supplementary data to this article:
:
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