Expression of hypoxia inducible factor 1α and 2α and its association with vitamin C level in thyroid lesions
Jóźwiak et al. Journal of Biomedical Science
Expression of hypoxia inducible factor 1α and 2α and its association with vitamin C level in thyroid lesions
Paweł Jóźwiak 0
Piotr Ciesielski 0
Agnieszka Zaczek 0
Anna Lipińska 0
Lech Pomorski 2
Marek Wieczorek 1
Magdalena Bryś 0
Ewa Forma 0
Anna Krześlak 0
0 Department of Cytobiochemistry, Faculty of Biology and Environmental Protection, University of Lodz , Pomorska 141/143, 90-236, Lodz , Poland
1 Department of Neurobiology, Faculty of Biology and Environmental Protection, University of Lodz , Pomorska 141/143, 90-236, Lodz , Poland
2 Department of General and Oncological Surgery, Medical University of Lodz , Pomorska 251, 92-213 Lodz , Poland
Background: Cells adapt to hypoxia by transcriptional induction of genes that participate in regulation of angiogenesis, glucose metabolism and cell proliferation. The primary factors mediating cell response to low oxygen tension are hypoxia inducible factors (HIFs), oxygen-dependent transcription activators. The stability and activity of the α subunits of HIFs are controlled by hydroxylation reactions that require ascorbate as a cofactor. Therefore, deficiency of intracellular vitamin C could contribute to HIFs overactivation. In this study, we investigated whether vitamin C content of human thyroid lesions is associated with HIF-1α and HIF-2α protein levels. Methods: Expression of HIF-1α and HIF-2α as well as vitamin C content was analyzed in thyroid lesions and cultured thyroid carcinoma cell lines (FTC-133 and 8305c) treated with hypoxia-mimetic agent (cobalt chloride) and ascorbic acid. The expression of HIFs and hypoxia-induced glucose transporters were determined by Western blots while quantitative real-time PCR (qRT-PCR) was performed to detect HIFs mRNA levels. Ascorbate and dehydroascorbate levels were measured by HPLC method. Results: We found an inverse correlation between vitamin C level and HIF-1α but not HIF-2α expression in thyroid lesions. These results agree with our in vitro study showing that vitamin C induced a dose - dependent decrease of HIF-1α but not HIF-2α protein level in thyroid cancer cells FTC-133 and 8305C. The decreased HIF-1α expression was correlated with reduced expression of hypoxia-related glucose transporter 1 (GLUT1) in thyroid cancer cells. Conclusion: The results demonstrate that HIF-1α activation is associated with vitamin C content in thyroid lesions. Our study suggests that high tumor tissue ascorbate level could limit the expression of HIF-1α and its targets in thyroid lesions.
HIF-1α/ HIF-2α/ vitamin C/GLUT1/ thyroid cancer
Rapidly proliferating cancer cells require adaptive metabolic
changes allowing them to continue biosynthesis and growth
under conditions of hypoxia and low nutrient availability
]. The capacity of living cells for adaptation to hypoxia
is mediated, in part, by hypoxia-inducible transcription
factors (HIFs). HIFs form heterodimers composed of oxygen
labile HIF-α subunit and constitutively expressed HIF-β
subunit. Three related α isoforms (HIF-1α, HIF-2α and
HIF-3α) and β-subunit are members of the basic
helixloop-helix (bHLH) proteins of the PER-ARNT-SIM (PAS)
family of transcription regulators . HIF
transcriptional response to lack of oxygen has been mostly
attributed to HIF-1α and HIF-2α. Under hypoxia
conditions, HIF-α subunits enter the nucleus, where they
dimerize with HIF-1β and then bind to a hypoxia response
element (HRE) within the promoter and enhancer regions
of the target genes [
]. HIF-1α preferentially drives the
transcription of genes that control glycolysis, angiogenesis
and glucose transport pathways, while HIF-2α is involved
in the regulation of genes important for tumor growth
and cell cycle progression [
]. Increased HIFs activity
has been shown to promote tumor progression and
resistance to chemo- and radiotherapy and is associated with
poor patient prognosis [
]. Therefore, there is a great
interest in potential inhibitors of HIFs for use in cancer
Activity and stability of HIFs are controlled by
posttranslational modification of the α subunits mediated by
specific prolyl hydroxylases (PHDs). Proline
hydroxylation is a signal for the recruitment of ubiquitin ligase
complex that leads to polyubiquitynation and rapid
proteosomal degradation of HIF-α subunit. Thus, optimal
PHDs activity directly suppress HIFs-dependent
transcriptional regulation. These enzymes have a non-heme
iron in the catalytic site and their activities depend on
the supply of substrates (oxygen and 2-oxoglutarate) and
a cofactor (ascorbate) that stabilizes the active-site Fe in
the reduced state [
]. Although ascorbate is a specific
reducer of PHDs and its deficiency limits HIF
hydroxylases activity, this cofactor has received relatively little
attention as a regulator of HIFs [
The role of vitamin C in cancer has been investigated for
a long time but the results of the studies are conflicting.
Howerver, more recent studies have shown that ascorbate
administration can significantly reduce tumor growth rate
in mice [
]. Clinical trials demonstrated that millimolar
concentration of vitamin C in plasma could be achieved
with intravenous infusion and may have therapeutic effect
or can enhance chemotherapy efficacy [
]. Most cells
accumulate ascorbate (AA) to low millimolar levels by
active transport from the plasma via Na+ − dependent
vitamin C transporters (SVCTs) or dehydroascorbic acid
(DHAA) absorption via Na+ −independent facilitative
glucose transporters (GLUTs) followed by intracellular
]. In physiological conditions the DHAA level in
serum is very low but it is suggested that GLUT
transporters may have significant impact on intracellular level of
vitamin C in regions of inflammation associated with tumor
where AA may be oxidized to DHAA [
facilitative glucose transporters GLUT1 and GLUT3
mediate DHAA transport with a similar efficiency to that of
glucose and expression of these two carriers is controlled
by hypoxia-inducible transcription factors . However,
the relationship between AA and DHAA levels and HIF
expression patterns in human tumors has been poorly
investigated. Therefore, the aim of this study was to estimate
HIF-1α and HIF-2α expression and determine whether the
HIFs level in human thyroid lesions is associated with the
ascorbate and dehydroascorbate content.
Patients and samples
The analyzed specimens were obtained from the
Department of General and Oncological Surgery of the
Medical University of Łódź. Samples of thyroid lesions
were obtained from 106 patients who underwent
surgical resection due to nodular thyroid diseases. The
tissue specimens collected in the operation room were
prepared and evaluated by an experienced pathologist.
Thyroid specimens were immediately frozen after
resection and stored at −80 °C until needed.
Clinicopathological characteristics of specimens are shown in
Table 1. Typing and staging of tumors were carried
out according to the system accepted by the
International Union Against Cancer (UICC, 2010). The
studies were performed with the approval of the
Bioethical Commission of the Medical University of Lodz
Cell cultures and treatment
The thyroid cancer cells lines FTC133 (follicular
thyroid cancer cells) and 8305c (anaplastic thyroid
cancer cells) were obtained from the European
Collection of Cell Cultures (ECACC), (Wiltshire, UK). Cells
were maintained in DMEM supplemented with 2 mM
glutamine and 10% fetal bovine serum (FBS) in a
humidified atmosphere containing 5% CO2 at 37 °C.
Cells were treated with 0.1–1 mM L-ascorbic acid.
Dithiothreitol (DTT) was added to culture media in final
concentration of 100 μM to stabilize vitamin C in
ascorbate form. To mimic hypoxia conditions, hypoxia agent
cobalt chloride (CoCl2) was added to the medium in final
concentration of 100 μM.
RNA isolation and cDNA synthesis
Total RNA was isolated using Fenozol reagent (A&A
Biotechnology, Gdynia, Poland). The RNA quality was
confirmed by electrophoresis on an agarose gel and
spectrophotometrically. RNA samples with a 260/280 nm ratio
in the range of 1.8–2.0 were used for further analysis.
cDNA was synthesized from 2 μg of total RNA using a
RevertAid™ First-Strand cDNA Synthesis Kit (Thermo
Fisher Scientific Inc., Waltham, MA, U.S.A.), following
the manufacturer’s instructions.
Quantitative real-time PCR
Quantitative real-time PCR with commercially available
primers and fluorescent probes (TaqMan® Gene
Expression Assay; Applied Biosystems™, Foster City, CA, USA)
was used to detect the expression of HIF1A, HIF2A,
SLC2A1 and SLC2A3 genes (coding for HIF-1α, HIF-2α,
GLUT1 and GLUT3 respectively) in different types of
thyroid lesions. The assay numbers for studied genes
were as follows: Hs00153153_m1, Hs01026149_m1,
Hs00892681_m1, Hs00359840_m1 and Hs99999905_m1
(reference gene: GAPDH).
The RT-qPCR reaction was carried out using the
Mastercycler ep realplex (Eppendorf ) as previously
]. The 2ΔCt (Ctgene–CtGAPDH) method was
used to calculate the expression levels of studied genes.
The 2-ΔCt values were re-calculated into relative copy
number values (number of HIF-1α or HIF-2α mRNA
copies per 1.000 copies of GAPDH mRNA).
Thyroid specimens were homogenized using a Potter’s
homogenizer in 10 volumes of ice-cold RIPA buffer
(50 mM Tris-HCl – 150 mM NaCl – 1% Triton X-100 –
0.5% DOC – 0.1% SDS – 2 mM EDTA, pH 7.4) with
1 mM phenylmethylsulfonyl fluoride (PMSF) to inhibit
protease activity. Cell pellets were solubilized for 30 min
on ice in RIPA buffer and then sonicated using Vibra Cell
™ (Sonics) sonicator. The efficiency of homogenization
was monitored by phase-contrast light microscopy. The
supernatants obtained after centrifugation of the tissue
and cell lysates at 10000 x g at 4 °C for 10 min were
collected and saved for further analysis.
Western blot analysis
Protein samples (40 μg protein/lane) were resolved by
8% SDS-PAGE and electrotransferred onto Immobilon-P
transfer membranes (Millipore, Bedford, MA, USA). In
order to verify the quality of transfer Ponceau S staining
was used prior to blocking the membrane. The blots
were incubated for 2 h at room temperature with the
rabbit anti-human HIF-1α polyclonal antibodies in a
1:400 dilution (Santa Cruz Biotechnology® Inc., Santa
Cruz, CA, USA), mouse anti-human HIF-2α monoclonal
antibodies in a 1:500 dilution (Santa Cruz Biotechnology®
Inc.), rabbit anti-human GLUT1 polyclonal antibodies in
a 1:1000 dilution (Abcam® Cambridge, UK) or mouse
anti-human GLUT3 monoclonal antibodies in a 1:500
dilution (Santa Cruz Biotechnology® Inc.). After washing
with TBS-T (0.1% Tween-20 in Tris-buffered saline,
TBS) the blots were incubated with horseradish
peroxidase-labeled goat anti-mouse or anti-rabbit IgG
antibodies (Santa Cruz Biotechnology® Inc.). The
proteins were visualized on X-ray film by the
chemiluminescence method. Gel-Pro Analyzer software version 3.0
(Media Cybernetics Inc., Bethesda, MD, USA) was used
for densitometry analysis of the protein bands on blots
or gels. To confirm that the same amounts of proteins
were loaded into each lane, the standard silver staining
method was used for total protein identification on the
gels or blots were re-probed with anti-β-actin antibody
following a stripping protocol. In case of tissue proteins
analyses, during the preliminary Western blot
experiment two samples with mean expression of studied
proteins were chosen (one for HIF-1α and one for HIF-2α)
and then these samples were applied as a reference
samples on each gel. Integrated optical density (IOD) of total
proteins after silver staining was divided by IOD of total
proteins of reference sample. For further analysis only
samples with similar IOD of total proteins were used.
The level of HIF-1α and HIF-2α expression in thyroid
specimens was normalized by the reference sample
(patient number 54 and 84, respectively) resolved by
electrophoresis that made possible comparison of intensity
of the bands from different membranes. The results of
HIF proteins expression analyses are shown as a relative
protein level which is a ratio of IOD of bands
corresponding to HIF-1α or HIF-2α in each sample and IOD
of HIFs in the reference samples.
Ascorbate and dehydroascorbate content
Tissue samples or cell pellets were homogenized in ice
cold 5% meta-phosphoric acid (MPA) containing 1 mM
EDTA. Ten microliters of MPA buffer was used per one
milligram of wet tissue. Cell suspensions (106 cells/ml)
were added to an equal volume of 10% cold MPA
containing 2 mM EDTA. The formed precipitates were
spun down by centrifugation (16.000 x g, 2 min) and
then supernatants were transferred to new vials.
Ascorbate and dehydroascorabate levels were
determined as previously described [
] by high-performance
liquid chromatography using modified Lykkesfeld’s
subtraction method with Tris[2-carboxyethyl]phosphine
(TCEP) as a reducing agent [
Statistical evaluation was performed using STATISTICA
version 12.0 (StatSoft Inc. 2014), data analysis software
system, www. statsoft.com). The non-parametric
MannWhitney U test was used when two groups were
compared. Comparisons between more than two groups were
done using Kruskal-Wallis test. For pairwise multiple
comparisons Dunn’s post hoc test was used. Spearman
correlation coefficient was calculated for correlation
analysis. The Student’s paired t-test was used to compare the
differences between treated and untreated cells. A p value
of <0.05 was considered significant.
Expression of HIF-1α and HIF-2α in thyroid lesions and
their association with clinicopathological characteristics
mRNA levels of HIF-1α and HIF-2α were analyzed by
real-time PCR in a sample set containing
nonneoplastic lesions (nodular goiters, NG), follicular
adenomas (FA), papillary carcinomas (PC) and follicular
carcinomas (FC). The results of HIF1A and HIF2A gene
expression analysis in different types of thyroid lesions
are graphed in Fig. 1. The results indicated that HIF1A
gene expression was significantly higher in PCs than in
non-neoplastic NG lesions (p < 0.05) (Fig. 1a). A lower
abundance of HIF-1α mRNA was observed in FCs in
comparison with PC cases (p < 0.05). There were no
statistically significant differences in HIF-2α mRNA levels
between different thyroid lesions (Fig. 1b). In the PC
group, HIF1A and HIF2A expression were higher in tissue
samples of patients with more advanced disease stages
(Fig. 1c and d). No significant differences were noted in
the expression of both genes between PC cases with and
without metastases to lymph nodes (Fig. 1e and f ).
To determine expression of HIF-1α and HIF-2α
proteins in the thyroid lesions, homogenized tissues were
examined by Western blotting. The samples of thyroid
lesions homogenates were resolved by SDS-PAGE. To
confirm that the same amounts of proteins were loaded
into each lane, the standard silver staining method was
used for total protein identification on the gels (Fig. 2a).
The representative blots after HIF-1α and HIF-2α
immunodetection are shown in Fig. 2b. HIF-1α and
HIF-2α protein expression was detected in all types of
thyroid lesions. Densitometric analysis of the bands
corresponding to HIF-1α and HIF-2α (Fig. 2c and d,
respectively) from all samples studied showed
significantly higher immunoreactivity in papillary cancers in
comparison with non-neoplastic cases (NG). No
statistically significant differences were noted in the HIF-1α
and HIF-2α protein levels between non-neoplastic
lesions and the benign neoplasm cases (Fig. 2c and d,
A tendency towards an increased expression of HIF-1α
was observed in the PC group with an increased tumor
stage, although this was not statistically significant (Fig.
2e). Results also showed significantly higher HIF-2α
protein expression in papillary carcinomas of stage II/III
compared with less advanced tumors (stage I) (Fig. 2f ).
There were no significant differences in HIF-1α and
HIF-2α expression between papillary carcinomas with or
without metastases to lymph nodes (Fig. 2g and h,
respectively). Due to the low number of FC cases, HIFs
expression data were not analyzed via clinicopathological
The Spearman’s analysis revealed a positive correlation
between HIF1A and HIF2A expression in thyroid lesions
(r = 0.507, p < 0.0001) (Fig. 3a). As expected there was
no correlation between HIF-1α or HIF-2α protein level
and their mRNA expression (Fig. 3b and c). It is well
known that the protein stability of HIF-α subunits is
controlled by posttranslational hydroxylation.
Nevertheless, we performed the correlation analysis to exclude
the possibility that the level of HIF proteins depends
mostly on gene expression regulation in thyroid lesions.
Effect of vitamin C on activation of hypoxia-inducible
factor 1α and 2α in thyroid cancer cells
As the activity of hydroxylases depends on ascorbate as a
cofactor, we set out to determine whether vitamin C
impacts HIF-1α and HIF-2α levels in thyroid cells. Thus,
FTC-133 and 8305c cells were grown in medium
containing hypoxia-mimetic agent (cobalt chloride) and were
treated for 24 h with increased concentration of L-AA
ranged from 0.1 to 1 mM. To prevent AA oxidation DTT
was added to culture media in a final concentration of
100 μM. Results showed that in both thyroid cancer cell
lines vitamin C induced a dose-dependent decrease of
HIF-1α protein level. This decrease was correlated with a
reduced expression of its target hypoxia associated
glucose transporter GLUT1 (Fig. 4a and b). There was no
impact of ascorbate on HIF-2α expression. Similarly,
vitamin C did not affect the level of hypoxia-related
GLUT3 glucose transporter. Quantification of vitamin
C in cells 24 h after L-AA treatment was performed by
HPLC-ECD using TCEP as a reducing agent according
to modified method of Lykkesfeldt [
]. The results of
vitamin C quantification in FTC-133 and 8305c cells
after ascorbate treatment are presented in Fig. 4c. The
data indicated that the dose-dependent impact of AA on
HIF-1α and its target GLUT1 protein expression was
associated with intracellular vitamin C content.
Vitamin C content and its relation to hypoxia activation markers in thyroid lesions
Total amount of vitamin C (TAA) as well as its reduced
form (AA) were measured by HPLC. The concentration
of oxidized form of ascorbate (DHAA) was calculated by
subtraction of the measured AA concentration from that
of TAA. Before the analyses, the stability of ascorbate in
banked specimens as well as our protocol for ascorbate
extraction was verified. There was no loss of ascorbate in
deproteinized samples up to 12 h at room temperature
and in long-term storage of intact tissue at -80 °C. The
ascorbate measurements were expressed as nmoles
ascorbate per 100 mg tissue. The results indicated that vitamin
C was stored mainly in its reduced state in each of
analyzed samples. There were no significant differences in AA
and TAA levels between different types of analyzed
thyroid lesions (Fig. 5a). In addition, we have observed
considerable variation in the total ascorbate levels in all
analyzed groups. To estimate whether vitamin C content
in the thyroid relates to activation of hypoxia pathway, the
total ascorbate in the samples was compared with the
levels of HIF-1α, HIF-2α and hypoxia-induced glucose
transporters which up-regulation in thyroid tumors we
had previously reported for (Fig. 5b-d) [
]. There was a
statistically significant negative correlation (r = − 0.288,
p = 0.025) between ascorbate content and HIF-1α protein
level. Most of specimens with high ascorbate levels had
low HIF-1α expression, whereas those with highest
HIF1α immunoreactivity were ascorbate deficient (Fig. 5b). A
similar trend was seen for GLUT1 protein (Fig. 5d),
however the results were not statistically significant.
Hypoxia is a common condition found in a wide range of
solid tumors and has been accepted to play an important
role in cancer development and progression. Tumor
adaptation to hypoxia depends mostly on HIF-1α and HIF-2α.
Both isoforms are regulated by oxygen-dependent
hydroxylation that results in intracellular degradation of proteins
by the proteasome pathway.
HIF-1α and HIF-2α have a similar structure and
function but they have unique tissue and cell type distributions
2, 3, 25
]. Recent data indicate that regulation of HIF-2
target genes depends on tissue type, tumor type and
coexpression with HIF-1 [
]. Studies concerning the
role of HIFs in thyroid carcinoma are very limited.
Moreover, there is no study to date which looked at both
isoforms expressions in the same specimens. Therefore, in
this study, we investigated whether there is any
difference in the expression of HIF-1α and HIF-2α between
malignant and benign neoplasms, as well as
nonneoplastic thyroid lesions. Our results showed a higher
intensity of bands corresponding to HIF-1α in majority of
malignant carcinoma cases in comparison with nodular
goiters (Fig. 2b and c). An association between HIF-1α
expression and tumor type was previously noted by Burrows
et al. [
] who observed significantly higher HIF-1α level
in all types of thyroid malignancy, especially in follicular
and anaplastic carcinomas, than in normal tissue. Another
immunohistochemical study also showed higher HIF-1α
immunoreactivity in most of PC cases in comparison with
paired normal tissue samples [
]. Authors observed
increased HIF-1α staining intensity in higher stage and
metastatic cancers. Similarly, we noted a higher tendency
for HIF-1α protein expression in more advanced
papillary cancers, although it was not statistically significant
HIF-1α and HIF-2α showed some overlap of target
genes, however both of them have also distinct
downstream targets. Therefore, hypoxia-induced metabolic
reprogramming varies depending on the relation between
HIF-1α and HIF-2α expressions. There is little
information in the literature regarding the HIF-2α expression in
thyroid cancer. In this study, we observed significantly
higher HIF-2α immunoreactivity in the PC group than
in non-neoplastic thyroid lesions (Fig. 2b and d). The
expression of HIF-2α protein was elevated in PC cases
with higher tumor stage (Fig. 2d). Our results agree with
the previously reported data by Wang et al. [
showed statistically significant differences in HIF-2α
protein expression levels between PCs and normal thyroid
tissues as well as nodular hyperplasia tissues.
Our study demonstrated pathophysiological relevance of
HIF-1α and HIF-2α proteins in thyroid lesions, however
the studied groups characterized wide variations in both
subunits level. Similar observation was made by Burrows
et al. [
], therefore the authors suggested regulation of
HIF proteins via a combination of tumor genotype and
microenvironment. We have observed significant positive
correlation between HIF1A and HIF2A expression which
may suggest similar regulation of both genes in the
thyroid. Our results showed that HIFs mRNAs expression
did not correlate with HIF proteins levels (Fig. 3b and c)
which is not surprising taking into account
posttranslational regulation of HIFs. It is well known that HIF-α
proteins are controlled by 2-oxoglutarate dependent
dioxygenases that require intracellular ascorbate for
optimal activity [
]. Several in vitro studies showed
that ascorbate can suppress HIF-α protein stability and
transcriptional activity [
]. Our in vitro studies
also demonstrated a dose-dependent decrease in the
expression of HIF-1α and GLUT1 in thyroid cancer
cells after vitamin C treatment (Fig.4). This effect was
associated with ascorbate uptake into treated cells. We
have analyzed, for the first time, vitamin C content in
thyroid samples and compared the obtained results
with HIFs-α expression. Our results showed an inverse
correlation between vitamin C content and HIF-1α
level (r = −0.288, p = 0.025). The data suggests that
high tumor tissue ascorbate level may decrease HIF-1
transcriptional activity in thyroid lesions. The ascorbate
content of tumor tissue has been measured in some
earlier studies with variable results. Brain and colorectal
tumors contained significantly less ascorbate than
normal tissue, whereas oral, lung and breast cancers had
significantly more ascorbate than corresponding normal
]. More recent studies showed that
endometrial and colorectal tumors of high histological grade
had less ascorbate than matched, adjacent normal
]. In our study, we did not observe any
differences in vitamin C levels between thyroid lesions.
However, in contrast to previously mentioned studies
we did not compare the ascorbate level between thyroid
lesions and normal tissue. The results showing the
association between HIF-1 level and ascorbate content in
thyroid lesions are in accordance with studies
concerning other tumors. Kuiper et al.  showed that
endometrial tumors with the highest HIF-1α protein content
were ascorbate deficient. The authors showed the same
results in case of colorectal cancer [
]. They found a
strong inverse relationship between the HIF-1 pathway
score and tumor ascorbate content. Protein levels of
VEGF and BNIP3 that are HIF-1-controlled
prosurvival target genes were inversely correlated to tumor
ascorbate content [
]. Intracellular accumulation of
ascorbate is mostly dependent on expression of the
SVCT transporters. On the other hand it has been
suggested that ascorbate is oxidized in the extracellular
environment and then GLUT transporters mediate
DHAA uptake into the tumors [
]. Our previous
data suggested that GLUT1 was the main DHAA
transporter in thyroid cancer cells . Intracellular DHAA
is rapidly reduced to ascorbate by a range of biological
reducers and enzyme systems [
]. In the present study,
we have shown that treatment of thyroid cancer cells
with vitamin C impacts the amount of GLUT1 protein.
There was also a tendency towards inverse association
between GLUT1 expression and vitamin C content in
thyroid tissue samples. Similarly, Kuiper et al. [
showed an association between ascorbate content and
GLUT1 expression in colorectal cancer. The ascorbate
ratio (tumor: normal) was inversely correlated to
GLUT-1 protein level.
Results found in this study demonstrate that HIF-1α
activation is associated with vitamin C content in thyroid
lesions. Our findings revealed that high tumor tissue
ascorbate level could limit the expression of HIF-1α and
its targets in thyroid lesions. Therefore, a better
understanding of ascorbate pharmacokinetics and mechanism
of action in tumors may improve vitamin C clinical
AA: Ascorbate; ARNT: Aryl hydrocarbon receptor nuclear translocator protein;
bHLH: Basic helix-loop-helix; DHAA: Dehydroascorbic acid; DTT: Dithiothreitol;
FA: Follicular adenoma; FC: Follicular carcinoma; GLUT: Glucose transporter;
HIF: Hypoxia inducible factor; HPLC: High-performance liquid
chromatography; HRE: Hypoxia response element; IOD: Integrated optical
density; NG: Nodular goiter; PC: Papillary carcinoma; PER: Period circadian
protein; PHDs: Prolyl hydroxylases; qRT-PCR: Quantitative real-time
polymerase chain reaction; SIM`: Single-minded protein; SLC2A1/3: Solute carrier
Family 2 Member 1/3, glucose transporter genes; SVCTs: Na + − dependent
vitamin C transporters; TAA: Total amount of vitamin C
This study was supported by statutory funds of University of Lodz.
Availability of data and materials
Data and materials related to this work are available upon request.
PJ designed the study, performed the experiments and participated in
manuscript drafting, PC and AZ carried out the Western blot analyses and
performed in vitro experiments; LP recruited patients, and collected the
clinicopathological data, AL and MB revised the manuscript, EF performed
qRT-PCR analyses, MW provided some theoretical and experimental guidance
for the design and performing the HPLC experiments, AK participated in
results discussion, data analyses and manuscript drafting. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
All procedures performed in studies involving human participants were in
accordance with the ethical standards of the institutional and/or national
research committee and with the 1964 Helsinki declaration and its later
amendments or comparable ethical standards. The studies were performed
with the approval of the Bioethical Commission of the Medical University of
Consent for publication
All authors read the final manuscript and approved manuscript for
The authors declare that they have no competing interests.
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