Metformin Induces Cytotoxicity by Down-Regulating Thymidine Phosphorylase and Excision Repair Cross-Complementation 1 Expression in Non-Small Cell ...
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Basic & Clinical Pharmacology & Toxicology, 2013, 113, 56–65 Doi: 10.1111/bcpt.12052
Metformin Induces Cytotoxicity by Down-Regulating Thymidine
Phosphorylase and Excision Repair Cross-Complementation 1
Expression in Non-Small Cell Lung Cancer Cells
Jen-Chung Ko1, Yu-Ching Huang2*, Huang-Jen Chen2*, Sheng-Chieh Tseng2*, Hsien-Chun Chiu2, Ting-Yu Wo2, Yi-Jhen Huang2,
Shao-Hsing Weng2, Robin Y. Y. Chiou3 and Yun-Wei Lin2
1
Department of Internal Medicine, National Taiwan University Hospital, Hsin-Chu Branch, Taiwan, 2Molecular Oncology Laboratory,
Department of Biochemical Science and Technology, National Chiayi University, Chiayi, Taiwan and 3Department of Food Science, College of
Life Sciences, National Chiayi University, Chiayi, Taiwan
(Received 13 November 2013; Accepted 2 January 2013)
Abstract: Metformin is an antidiabetic drug recently shown to inhibit cancer cell proliferation and growth, although the involved
molecular mechanisms have not been elucidated. In many cancer cells, high expression of thymidine phosphorylase (TP) and
Excision repair cross-complementation 1 (ERCC1) is associated with poor prognosis. We used A549 and H1975 human non-
small cell lung cancer (NSCLC) cell lines to investigate the role of TP and ERCC1 expression in metformin-induced cytotoxic-
ity. Metformin treatment decreased cellular TP and ERCC1 protein and mRNA levels by down-regulating phosphorylated
MEK1/2-ERK1/2 protein levels in a dose- and time-dependent manner. The enforced expression of the constitutively active
MEK1 (MEK1-CA) vectors significantly restored cellular TP and ERCC1 protein levels and cell viability. Specific inhibition of
TP and ERCC1 expression by siRNA enhanced the metformin-induced cytotoxicity and growth inhibition. Arachidin-1, an anti-
oxidant stilbenoid, further decreased TP and ERCC1 expression and augmented metformin’s cytotoxic effect, which was abro-
gated in lung cancer cells transfected with MEK1/2-CA expression vector. In conclusion, metformin induces cytotoxicity by
down-regulating TP and ERCC1 expression in NSCLC cells.
Metformin (1,1-dimethylbiguanide hydrochloride) is the most platinum-based chemotherapeutic agents [17–20]. A previous
frequently used first-line drug for type 2 diabetes mellitus. study indicated that epidermal growth factor enhances ERCC1
Recently, its anticancer properties have been noted. For exam- expression in DU145 and LNCaP prostate carcinoma cells
ple, it improves survival in patients with breast cancer [1,2] through MAPK signalling [21]. Inactivation of vascular endo-
and decreases breast cancer risk [3,4]. Moreover, it exhibits an thelial growth factor receptors can enhance cisplatin-induced
antiproliferative action on prostate and breast cancer cell lines cytotoxicity by decreasing ERCC1 protein expression in
[5,6]. Furthermore, metformin decreases the occurrence and human ovarian cancer cells [22]. However, whether or not
mass of mammary adenocarcinomas in Her2/Neu transgenic metformin can affect ERCC1 expression through the MEK1/2-
mice [7]. However, its effect on lung cancer, particularly on ERK1/2 signal remains to be determined.
non-small cell lung cancer (NSCLC) viability, and the detailed This study attempted to verify the influence of metformin
molecular mechanism involved are still unknown. on TP and ERCC1 expression in two NSCLC cell lines, A549
Thymidine phosphorylase (TP) is an enzyme of the pyrimi- and H1975, and to identify the pathways involved. The study
dine salvage pathway that is highly expressed in cancers [8,9]. also aimed to evaluate the role of TP and ERCC1 expression
Its expression protects cells from apoptosis induced by chemo- in metformin-induced cytotoxicity in NSCLC cell lines.
therapeutic agents, DNA-damaging agents, microtubule-inter-
fering agents, hypoxia and Fas ligands [10–13]. Elevated TP
levels are associated with poor prognosis and shorter survival Materials and Methods
[9,14,15]. However, whether metformin has the ability to reg- Chemicals and reagents. Metformin was purchased from Sigma
ulate TP expression warrants further investigation. Chemical (St. Louis, MO, USA), while U0126, N-acetyl-Leu-Leu-
Excision repair cross-complementation 1 (ERCC1) is a key norleucinal (ALLN) and MG132 were obtained from Calbiochem-
enzyme in the nucleotide excision repair (NER) pathway and Novabiochem (San Diego, CA, USA). Specific phospho-ERK1/2
(Thr202/Tyr204) and phospho-MEK1/2 (Ser217/Ser221) antibodies
is involved in DNA damage recognition and DNA strand inci-
were purchased from Cell Signaling (Beverly, MA, USA). Rabbit
sion [16]. Over-expression of the ERCC1 mRNA is found in polyclonal antibodies against the PD-ECGF(PGF-44C) (sc-47702),
several cancers and correlates negatively with the efficacy of ERCC1(FL-297) (sc-10785), ERK2(C-14) (sc-154), ubiquitin (P4D1)
(sc-8017), HA(F-7) (sc-7392) and actin (I-19) (sc-1616) were from
Author for correspondence: Yun-Wei Lin, Department of Biochemical Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Science and Technology, National Chiayi University, 300 Syuefu
Road, Chiayi 600, Taiwan (fax +886 5 271 7780, e-mail linyw@
mail.ncyu.edu.tw). Cell lines and plasmids. Human lung cell carcinoma A549 and H1975
*These authors contributed equally. were obtained from the American Type Culture Collection (Manassas,
Ó 2013 Nordic Pharmacological Society. Published by John Wiley & Sons LtdMETFORMIN DOWN-REGULATES THYMIDINE PHOSPHORYLASE AND EXCISION REPAIR CROSS-COMPLEMENTATION 1 EXPRESSION 57
VA, USA) and cultured at 37°C in a humidified atmosphere containing trypsinized to determine the cell numbers. The cells were plated at
5% CO2 in RPMI-1640 complete medium supplemented with sodium a density of 500 cells on a 60-mm-diameter Petri dish in triplicate
bicarbonate (2.2%, w/v), L-glutamine (0.03%, w/v), penicillin for each treatment and cultured for 14 days. The cell colonies
(100 units/mL), streptomycin (100 lg/mL) and foetal calf serum (10%). were stained with 1% crystal violet solution in 30% ethanol.
Plasmid transfection of MEK1/2-CA, a constitutively active form of Cytotoxicity was determined by the number of colonies in the
MEK1/2, was achieved as previously described [23]. Exponentially treated cells divided by the number of colonies in the untreated
growing human lung cancer cells (106) were plated for 18 hr. The control.
MEK1/2-CA expression vectors were then transfected into the A549 or
H1975 cells using Lipofectamine (Invitrogen, Carlsbad, CA, USA). Statistical analyses. For each protocol, three or four independent
experiments were performed. Results were expressed as the
Western blot analysis. After different treatments, the cells were rinsed mean standard error of the mean (S.E.M.). Statistical calculations
twice with cold PBS and lysed in whole-cell extract buffer [20 mM were performed using the SigmaPlot 2000 (Systat Software, San Jose,
HEPES (pH 7.6), 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, CA, USA). Differences in measured variables between the
0.1% Triton X-100, 0.1 mM Na3VO4, 50 mM NaF, 1 lg/mL experimental and control groups were assessed by unpaired t-test. A
leupeptin, 1 lg/mL aprotinin, 1 lg/mL pepstatin and 1 mM p < 0.05 was considered statistically significant.
4-(2-aminoethyl) benzenesulfonyl fluoride]. Equal amounts of proteins
from each set of experiments were subjected to Western blot analysis
as previously described [24]. Results
Transfection with small interfering RNA. The sense-strand sequences Metformin reduced TP and ERCC1 protein and mRNA in
of siRNA duplexes were as follows: TP: 5′-AUA GAC UCC AGC NSCLC cells lines.
UUA UCC A-3′, ERCC1: 5′-GGA GCU GGC UAA GAU GUG U-3′ The effect of metformin on the expression of TP and ERCC1
and scrambled (as control): 5′-GCG CGC UUU GUA GGA TTC G-3′
in A549 and H1975 was first examined. Real-time RT-PCR
(Dharmacon Research, Lafayette, CO, USA). The cells were
transfected with siRNA duplexes (200 nM) using Lipofectamine 2000 and Western blot analysis demonstrated a time- and dose-
(Invitrogen) for 24 hr. dependent decrease in mRNA and protein levels of cellular TP
and ERCC1 after exposure to metformin (fig. 1). Metformin
Quantitative real-time polymerase chain reaction (PCR). The PCRs also induced a decrease in MEK1/2-ERK1/2 phosphorylation
were performed using an ABI Prism 7900HT according to the in a time- and dose-dependent manner (fig. 1B).
manufacturer’s instructions. Amplification of specific PCR products was
detected using the SYBR Green PCR Master Mix (Applied Biosystems,
Carlsbad, CA, USA). For each sample, data were normalized to the Metformin down-regulated TP and ERCC1 protein levels via
housekeeping gene glyceraldehyde 3-phosphate dehydrogenase
an ubiquitin–26S proteasome-mediated degradation pathway.
(GAPDH). The designed forward and reverse primers were
5′-AGCTGGAGTCTATTCCTGGATT-3′ and 5′-GGCTGCATATAGGA To examine whether metformin could post-transcriptionally
TTCCGTC-3′; 5′-GGGTGACTGAATGTCTGACCA-3′ and 5′-GGGT regulate TP and ERCC1 transcripts in NSCLC cells, A549 or
ACTTTCAAGAAGGGCTC-3′; and 5′-CATGAGAAGTATGACAA H1975 cells were first treated with metformin for 9 hr and
CAGCCT-3′ and 5′-AGTCCTTCCACGA TACCAAAGT-3′ for TP, then treated with actinomycin D to block de novo RNA syn-
ERCC1 and GAPDH, respectively. Analysis was performed using the thesis, and then, the levels of existing TP and ERCC1
comparative Ct value method.
mRNA were measured using RT-PCR at 3, 6 and 9 hrs.
After actinomycin D co-exposure for 9 hr, metformin treat-
Reverse transcription-PCR (RT-PCR). The RNA was isolated from
ment showed lower levels of TP and ERCC1 mRNA com-
cultured cells using TRIzol (Invitrogen) according to the
manufacturer’s instructions. The RT-PCR was performed using 2 lg pared to untreated cells (fig. 2A). To investigate whether the
total RNA using random hexamers following the Moloney murine TP and ERCC1 protein expressions affected by metformin
leukaemia virus reverse transcriptase cDNA synthesis system were regulated at the post-translational level, the cell cultures
(Invitrogen). The final cDNA was used for subsequent PCRs [23]. were first treated with metformin for 9 hr and then treated
with cycloheximide (an inhibitor of de novo protein synthe-
Cell viability assay. Cells were cultured at 5000 per well in 96-well sis) for 3, 6 and 9 hrs. The TP and ERCC1 protein levels
tissue culture plates. To assess cell viability, drugs were added after plating.
were progressively reduced with time in the presence of
At the end of the culture period, 20 lL of 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution
cycloheximide, but metformin treatment significantly
(CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, improved TP and ERCC1 protein degradation after cyclohexi-
Madison, WI, USA) was added. The cells were incubated for another 2 hr, mide treatment (fig. 2B).
and absorbance was measured at 490 nm using an ELISA plate reader Ubiquitin–26S proteasome-mediated degradation of TP and
(Bio-Rad Technologies, Hercules, CA, USA). ERCC1 protein after metformin treatment in A549 or H1975
cells was then examined. The 26S proteasome inhibitors
Trypan blue dye exclusion assay. Cells were treated with metformin MG132 or ALLN prevented the down-regulation of TP and
and/or arachidin-1 for 24 hr. The trypan blue dye was excluded by
ERCC1 by metformin (fig. 2C). Metformin treatment also sig-
living cells but could penetrate dead cells. The proportion of dead
cells was determined by hemocytometer to count the number of cells nificantly increased the levels of ubiquitin-conjugated TP and
stained with trypan blue. ERCC1 in H1975 cells (fig. 2D). Together, these results
revealed that down-regulation of TP and ERCC1 protein level
Colony-forming ability assay. Immediately after drug treatment, the was through increased ubiquitin conjugation of TP and
A549 cells were washed with phosphate-buffered saline and ERCC1 in metformin-exposed NSCLC cells.
Ó 2013 Nordic Pharmacological Society. Published by John Wiley & Sons Ltd58 JEN-CHUNG KO ET AL.
A
B
Fig. 1. Metformin decreased phospho-MEK1/2-ERK1/2, thymidine phosphorylase (TP) and excision repair cross-complementation 1 (ERCC1) pro-
tein and mRNA levels in human non-small cell lung cancer cell lines. (A) A549 or H1975 cells were cultured in complete medium for 18 hr and
then exposed to metformin (25 lM) for 3–24 hr (left panel) or various concentrations of metformin for 24 hr (right panel). Total RNA was then
isolated and subjected to reverse transcription-PCR (RT-PCR) (upper panel) and real-time PCR (lower panel) for TP and ERCC1 mRNA expres-
sion. (B) Cell extracts were examined by Western blot for phospho-MEK1/2, phospho-ERK1/2, TP, ERCC1, actin and ERK1/2 protein levels.
A B
C D
Fig. 2. Metformin down-regulated mRNA and protein levels of thymidine phosphorylase (TP) and excision repair cross-complementation 1
(ERCC1) via increased mRNA and protein instability. (A) A549 and H1975 cells were first exposed to metformin (25 lM) for 9 hr, followed by
the addition of actinomycin D (2 lg/mL) for 3, 6 and 9 hrs. Total RNA was then isolated and subjected to reverse transcription-PCR (RT-PCR)
for TP and ERCC1. (B) Cells were pre-treated with metformin (25 lM) for 9 hr, followed by the addition of cycloheximide (50 lg/mL) for 3, 6
and 9 hrs. Whole-cell extracts were collected for Western blot analysis of TP and ERCC1 protein levels. (C) Metformin (50 lM) was added to
A549 or H1975 cells for 14 hr. The cells were then cotreated with MG132 (10 lM) or N-acetyl-Leu-Leu-norleucinal (ALLN) (10 lM) for 10 hr,
and whole-cell extracts were collected for Western blot analysis. (D) After treatment with various metformin concentrations, equal amounts of pro-
teins in each cell extract were subjected to immunoprecipitation (IP) by ubiquitin antibodies. The immunoprecipitates were analysed by immunoblot
(IB) using anti-TP or anti-ERCC1 antibodies.
Ó 2013 Nordic Pharmacological Society. Published by John Wiley & Sons LtdMETFORMIN DOWN-REGULATES THYMIDINE PHOSPHORYLASE AND EXCISION REPAIR CROSS-COMPLEMENTATION 1 EXPRESSION 59
Metformin down-regulated TP and ERCC1 by inactivating Metformin-induced cytotoxic effects were abrogated by MEK1/
MEK1/2-ERK1/2. 2-CA expression vector transfection.
To examine the role of MEK1/2-ERK1/2 in the metformin- The MEK1/2-CA vector expression in metformin-treated
induced decrease in TP and ERCC1, A549 or H1975 cells A549 or H1975 cells was further enforced. The MEK1/2-CA
were transiently transfected with a plasmid carrying MEK1- vector expression restored the cell viability that was decreased
CA, a constitutively active form of MEK1. Compared to trans- by metformin (fig. 4D). In contrast, U0126 combined with
fection with the control vector pcDNA3, transfection with metformin enhanced metformin-induced cytotoxicity (fig. 4E).
MEK1-CA restored the ERK1/2 phosphorylation and the TP Taken together, inactivation of MEK1/2-ERK1/2 signal partic-
and ERCC1 protein levels in metformin-exposed A549 or ipated in the metformin-mediated cytotoxic effects on human
H1975 cells (fig. 3A). However, MEK1-CA transfection lung cancer cells.
restored only the ERCC1 mRNA level, but not the TP mRNA
level, under metformin exposure (fig. 3B). The use of a
Arachidin-1 promoted metformin-mediated TP and ERCC1
MEK1/2 specific inhibitor, U0126, further decreased the TP
protein and mRNA down-regulation.
and ERCC1 protein by metformin compared with metformin
Arachidin-1 [trans-4-(3-methyl-1-butenyl)- 3,5,3′,4′ -tetra-
alone (fig. 3C). However, U0126 pre-treatment lowered the
hydroxystilbene] could be biosynthesized and isolated from
ERCC1 mRNA level, but not the TP mRNA level, compared
germinated peanut sprouts and could exhibit potent antioxi-
with metformin treatment alone (fig. 3D). Thus, metformin-
dant and anti-inflammatory activities [25–27]. Moreover,
mediated TP and ERCC1 protein down-regulation depended
arachidin-1 was characterized as an anticancer agent in
on MEK1/2-ERK1/2 inactivation.
human leukaemia HL-60 cells [28]. The present study has
been suggested that arachidin-1 might enhance metformin-
Specific knockdown of TP and ERCC1 expression enhanced induced cytotoxic effect by enhancing the down-regulation
metformin-induced cytotoxicity. of MEK1/2-ERK1/2-mediated TP and ERCC1 expression in
To examine the role of TP and ERCC1 down-regulation in NSCLC cells. Arachidin-1 treatment enhanced the down-reg-
metformin-induced cytotoxicity, A549 and H1975 cells were ulation of TP and ERCC1 protein expression by metformin
transfected with specific si-TP and si-ERCC1 RNA and then in A549 and H1975 cells (fig. 5A). Results from real-time
treated with metformin. Cytotoxicity was determined by MTS PCR analysis showed that arachidin-1 combined with met-
assay. Suppression of TP and ERCC1 protein expression by formin decreased TP and ERCC1 mRNA levels (fig. 5B).
specific siRNA resulted in further increased sensitivity to met- Moreover, arachidin-1 treatment decreased cellular TP and
formin and enhanced growth inhibition compared with si-con- ERCC1 mRNA and protein levels in a dose-dependent man-
trol transfected NSCLC cells (fig. 4A–C). ner (fig. 5C,D).
A C
B D
Fig. 3. The MEK1/2-ERK1/2 signalling pathway was involved in decreasing thymidine phosphorylase (TP) and excision repair cross-complementa-
tion 1 (ERCC1) protein levels by metformin. (A) Cells were transfected with MEK1-CA expression vectors for 24 hr and then treated with metfor-
min (25 or 50 lM) for 24 hr. The cell extracts were then examined by Western blot. The HA antibody was used to recognize the HA-MEK1-CA
vector expression in A549 and H1975 cells. (B) MEK1-CA vectors-transfected H1975 cells were treated with metformin for 24 hr, and then, total
RNA was isolated and subjected to real-time PCR for TP and ERCC1 mRNA expression. **p < 0.01, by Student’s t-test for comparison between
the cells treated with metformin in pcDNA3-vector- or MEK1-CA vector-transfected cells. (C and D) A549 or H1975 cells were pre-treated with
U0126 (2.5, 5, 10 lM) for 1 hr and cotreated with metformin (10 lM) for 24 hr. The cell extracts were then examined by Western blot and real-
time PCR to determine the protein and mRNA levels of TP and ERCC1, respectively. **p < 0.01, by Student’s t-test for comparison between the
cells pre-treated with or without U0126 in metformin-exposed cells.
Ó 2013 Nordic Pharmacological Society. Published by John Wiley & Sons Ltd60 JEN-CHUNG KO ET AL.
A C
B D E
Fig. 4. The role of thymidine phosphorylase (TP) and excision repair cross-complementation 1 (ERCC1) in metformin-induced cytotoxicity in non-
small cell lung cancer (NSCLC) cell lines. (A) H1975 cells were transfected with siRNA duplexes (200 nM) specific to TP, ERCC1 or scramble
(control) in complete medium for 24 hr prior to metformin treatment (5 and 10 lM) in complete medium for 24 hr. Whole-cell extracts were
collected for Western blot analysis. (B) After treatment, cytotoxicity was determined by MTS assay. (C) After transfection with si-TP/si-ERCC1 or
si-scrambled RNA, the cells were treated with metformin (5 lM) for 24, 48 and 72 hr, and the surviving alive cells were determined by MTS
assay. The results (mean S.E.M.) were from three independent experiments. **p < 0.01, by Student’s t-test for comparison between cells treated
with metformin in si-TP/si-ERCC1 RNA or si-scrambled RNA-transfected cells. The role of ERK1/2 inactivation in metformin-induced cytotoxicity
in A549 or H1975 cells. (D) MEK1/2-CA vector (1, 3 and 5 lg)-transfected or pcDNA3 (5 lg) expression vector–transfected NSCLC cells were
treated with metformin (50 lM) for 24 hr. Cytotoxicity was determined by MTS assay. **p < 0.01, *p < 0.05, by Student’s t-test for comparison
between cells treated with metformin in MEK1/2-CA- or pcDNA3-transfected cells. (E) A549 or H1975 cells were pre-treated with U0126 (1, 2
and 5 lM) for 1 hr and cotreated with metformin (5 lM) for 24 hr. Cytotoxicity was determined by MTS assay. **p < 0.01 (Student’s t-test), to
compare results from different cell lines treated with metformin alone or cotreated with U0126.
Arachidin-1 enhanced the cytotoxic and antiproliferative effect arachidin-1 or the 2-drug combination for 24 hr. Cotreatment
induced by metformin. of metformin and arachidin-1 resulted in greater loss of cell
To determine whether arachidin-1 could increase the cytotox- viability than those treated by either metformin or arachidin-1
icity of metformin on NSCLC cells using MTS and trypan alone in the A549 and H1975 cells (fig. 6A,B). Moreover,
blue exclusion assays, cells were treated with metformin, after the A549 and H1975 cells were exposed to metformin
Ó 2013 Nordic Pharmacological Society. Published by John Wiley & Sons LtdMETFORMIN DOWN-REGULATES THYMIDINE PHOSPHORYLASE AND EXCISION REPAIR CROSS-COMPLEMENTATION 1 EXPRESSION 61
and/or arachidin-1 and cell proliferation was determined by metformin and arachidin-1 cotreatment (fig. 6D,E). Thus, the
trypan blue dye exclusion assay during the 4-day incubation inhibition of MEK1/2-ERK1/2-mediated TP and ERCC1
period, the combination of metformin and arachidin-1 more expression in NSCLC was associated with metformin-induced
effectively inhibited cell growth than either treatment alone and arachidin-1-induced cytotoxicity.
(data not shown). Thus, arachidin-1 could sensitize human
lung tumour cells to metformin and enhance the metformin-
induced growth inhibition. Discussion
Given such effect of arachidin-1, colony-forming assays
Metformin is an extensively used and well-tolerated drug for
were conducted to investigate whether arachidin-1 affected
treating type 2 diabetes, obesity and polycystic ovarian syn-
long-term clonogenic cell survival in metformin-exposed lung
drome. Diabetic patients treated with metformin have reduced
cancer cells. The inhibition of colony formation by treatment
cancer risk [4,29], although it is unclear whether metformin
with metformin (49.4%) or arachidin-1 (10.63%) alone in
directly affects cancer or indirectly by inhibiting the diabetic
A549 cells was less compared to the effect of combining the
state. At the cellular level, metformin displays growth and pro-
two (86.42%) (fig. 6C). Therefore, the longer times were
liferation inhibitory effects in several cancer cells through
employed; the cytotoxic effects of metformin alone or combi-
reducing the EGFR, Src and MAPK activation, as well as
nation with arachidin-1 were more impressive.
other responses including AMP-activated protein kinase
(AMPK) activation, and reduces mammalian target of rapamy-
Metformin-induced and arachidin-1-induced cytotoxic effects cin (mTOR) signalling [30–32]. In addition, metformin
were abrogated by MEK1/2-CA expression vector transfection. decreases phorbol-12-myristate-13-acetate (PMA) induced
To determine whether the MEK1/2-ERK1/2 pathway was Ca2+-dependent protein kinase C(PKC)a/ERK and JNK/AP-1-
directly affected by arachidin-1 in terms of the cellular signalling pathways in human fibrosarcoma cells [33]. In con-
response to metformin, A549 or H1975 cells were transfected trast, treatment of two osteoblast-like cells (UMR106 and
with MEK1/2-CA plasmids, treated with metformin and MC3T3E1) with metformin led to induce activation and redis-
arachidin-1 and assessed using the MTS assay. Transfection tribution of phosphorylated ERK1/2 in a transient manner
with MEK1/2-CA restored the TP and ERCC1 expression and [34]. The present study showed that metformin attenuates
enhanced cell survival, which was previously suppressed by MEK1/2-ERK1/2 signalling and inhibits the growth of human
A B
C D
Fig. 5. Arachidin-1 decreased phospho-ERK1/2, thymidine phosphorylase (TP) and excision repair cross-complementation 1 (ERCC1) protein
levels in metformin-treated A549 or H1975 cells. (A) Cells were treated with various concentrations of metformin and/or arachidin-1 (1 lM) for 24
h. Whole-cell extracts were collected for Western blot analysis. **p < 0.01 by Student’s t-test for comparison between cells treated with metformin
alone or metformin and arachidin-1 combination. (B) After treatment, total RNA was isolated and subjected to real-time PCR for TP and ERCC1
mRNA expression. (C) The cells were exposed to various concentration of arachidin-1 for 24 h. Total RNA was then isolated and subjected to RT-
PCR and real-time PCR for TP and ERCC1. **p < 0.01, *p < 0.05, by Student’s t-test for comparison between cells treated with or without arachi-
din-1. (D) After treatment with arachidin-1 for 24 h, whole-cell extracts were collected for Western blot analysis.
Ó 2013 Nordic Pharmacological Society. Published by John Wiley & Sons Ltd62 JEN-CHUNG KO ET AL.
A D
E
C
B
Fig. 6. Arachidin-1 cotreatment with metformin synergistically enhanced cytotoxicity. (A) Left panel, arachidin-1 (0.5, 1, 5 and 10 lM) and met-
formin (25 lM) were added to A549 or H1975 cells for 24 hr. Right panel, metformin (5, 10, 25 and 50 lM) and arachidin-1 (0.5 lM) were
added to the cells for 24 hr. Cytotoxicity was determined by MTS assay. (B) After treatment, unattached and attached cells were collected and
stained with trypan blue dye, and the numbers of stained cells (dead) were manually counted. Columns, percentage of trypan blue-positive cells
representing the population of dead cells; bar, S.D. from three independent experiments; **p < 0.01, by Student’s t-test for comparison between
the cells treated with metformin/arachidin-1 alone or arachidin-1 and metformin combined. (C) A549 cells were treated with arachidin-1 (1 lM)
and/or metformin (10 lM) for 24 hr, and cytotoxicity was determined by colony-forming ability assay. MEK1/2-CA over-expression restored met-
formin- and arachidin-1-suppressed phospho-ERK1/2, thymidine phosphorylase and ERCC1 protein levels and cell viability. (D) MEK1/2-CA
expression plasmids were transfected into cells using lipofectamine. After expression for 24 hr, the cells were treated with metformin (25 lM) and
arachidin-1 (5 lM) for another 24 hr, and whole-cell extracts were collected for Western blot analysis. (E) After treatment, cytotoxicity was deter-
mined by MTS assay. **p < 0.01, by Student’s t-test for comparison between the metformin–arachidin-1 cotreated cells transfected with MEK1/2-
CA or pcDNA3 vectors.
lung cancer cells. Although the precise mechanism by which triple-negative breast cancer cell line that lacks the oestrogen,
metformin attenuates MEK1/2-ERK1/2 activation is currently progesterone and HER2 receptors [32]. These observations
not known, we do not rule out the possibility that this phe- suggest the possibility that metformin may be useful as an
nomenon may be complex and may also be cell type depen- anticancer drug even in non-diabetic patients [37,38].
dent. A recent report indicates that removing the ERK1 Thymidine phosphorylase catalyses the reversible conver-
protein using siRNA combined with metformin markedly pot- sion of thymidine to thymine and 2-deoxy-D-ribose-1-phos-
entiates metformin-mediated prostate cancer cell death [35]. phate. Its expression in various kinds of tumours is higher
Treatment of human aortic smooth muscle cell (HASMC) with than in adjacent non-neoplastic tissues, and numerous studies
metformin decreases leptin-induced activation of ERK1/2 and consistently report that the high TP expression in these tumour
inhibits the effect of leptin on HASMC proliferation [36]. sites is associated with poor prognosis [39–41]. It is thought
Consistent with these data, the present study reveals that the to promote tumorigenesis by inducing tumour cell growth
inhibition of ERK1/2 activation by MEK1/2 inhibitor, U0126, through the inhibition of the apoptosis pathway [11,13,42] and
enhances metformin-induced cytotoxicity. In nude mice, tumour angiogenesis via the PI3K-mTOR pathway [43].
metformin modestly inhibits tumour growth of xenografts of a Furthermore, TP expression in neoplastic cells can be used as
Ó 2013 Nordic Pharmacological Society. Published by John Wiley & Sons LtdMETFORMIN DOWN-REGULATES THYMIDINE PHOSPHORYLASE AND EXCISION REPAIR CROSS-COMPLEMENTATION 1 EXPRESSION 63
a biomarker to predict response to chemotherapy and survival of combined metformin and/or arachidin-1 cannot be ruled
in oesophageal squamous cell carcinoma and breast cancer out, the effect may at least partly contribute to the inhibition
[44,45]. Another recent study indicated that the TP inhibitor of TP and ERCC1 expression. Nonetheless, the role of metfor-
enhances the radio-therapeutic efficacy in the experimental min as a promising therapeutic regimen for NSCLC requires
colorectal cancer [46]. Moreover, up-regulation of ERK1/2- further studies in a clinical setting.
dependent TP expression contributes to cisplatin resistance in
human lung cancer cells [47]. In the present study, the TP is Acknowledgements
novel target of metformin action in NSCLC cells, and both We thank Dr. Jia-Ling Yang for providing expression plas-
the transcripts and the protein expression of TP are decreased mids for transfection and Dr. Chiou for providing arachidin-1.
by metformin. In addition, TP confers cytoprotection against This work was supported by the National Science Council of
metformin on human lung cancer cells, and the down-regula- Taiwan, grants no. [NSC 99-2320-B-415-001-MY3] and the
tion of TP-mediated cytotoxic effects is at least partly medi- research grant of HsinChu General Hospital (HCH101-05).
ated by the inactivation of the MEK1/2-ERK1/2 signalling.
Interestingly, the knockdown of TP expression by specific
Conflict of Interest
siRNA or arachidin-1 promotes the cytotoxic effect induced
None of the authors have a financial relationship with a
by metformin.
commercial entity that has an interest in the subject of this
Recently, a signal transducer and activator of transcription 3
manuscript.
(STAT3)-specific inhibitor acts synergistically with metformin
to reduce cell growth and induce apoptosis in triple-negative References
breast cancers cells [48]. Our unpublished data also indicated
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