Effects of metformin on sirt1 nrf2 and ahr expression in cancer cells

Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression --- 29 1.1.. Metformin suppresses endogenous AhR-ligand-

A Dissertation for the Degree of Doctor of Philosophy

Effects of metformin on Sirt1, Nrf2 and AhR

expression in cancer cells  

Department of Pharmacy Graduate School Chungnam National University

By

Minh Truong Do

Advisor Hye Gwang Jeong

August 2014

Effects of metformin on Sirt1, Nrf2 and AhR expression

Advisor Hye Gwang Jeong

Submitted to the Graduate School

in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy

April, 2014

Department of Pharmacy Graduate School

Chungnam National University

By

Minh Truong Do

Contents

Contents - i

List of Figures - vi

List of Abbreviations - x

Abstract - 1

I Introduction - 6

1 Metformin and reduced risk of cancer - 6

2 Role of the AhR, CYP1A1 and CYP1B1 in carcinogenesis and mechanisms of regulation of gene expression - 6

3 Regulation of gene expression and role of Nrf2 and HO-1 in tumorigenesis and chemoresistance - 9

4 Role of Sirt1 in tumorigenesis and chemoresistance - 11

II Materials & Methods - 15

1 Materials - 15

2 Cell culture and treatment - 16

3 Measurement of cell viability and cytotoxicity - 17

4 BrdU incorporation assay - 18

5 RNA preparation and reverse transcription-polymerase

chain reaction (RT-PCR) - 19

6 Quantitative real-time RT-PCR (qRT-PCR) - 20

7 Luciferase and β-galactosidase assays - 22

8 Western blotting - 23

9 Preparation of nuclear and cytosolic extracts - 24

10 Immunoprecipitation (IP) - 24

11 Chromatin immunoprecipitation (ChIP) - 25

12 Small interfering RNA transfection - 26

13 Sp1, HO-1, Sirt1, Pgc-1 and PPAR overexpression - 26

14 miR-34a inhibitor and mimic transfection - 27

15 Measurement of intracellular reactive oxygen species (ROS) - 27

16 Statistical analysis - 28

III Results - 29

1 Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression - 29

1.1 Metformin inhibits CYP1A1 and CYP1B1 expression in breast cancer cells - 29

1.2 Down-regulation of AhR expression is required for the

suppression of CYP1A1 and CYP1B1 by metformin - 33

1.3 Down-regulation of Sp1 by metformin inhibits AhR

transcriptional activity in breast cancer cells - 38

1.4 Inhibition of CYP1A1 and CYP1B1 expression by

metformin is independent of ER - 41 1.5 Metformin suppresses endogenous AhR-ligand-induced

CYP1A1 and CYP1B1 expression by reducing TDO

expression in breast cancer cells - 43

1.6 Metformin suppresses TDO expression by down-regulating

the Sp1/glucocorticoid receptor (GR) signaling pathway - 47

2 Metformin inhibits heme oxygenase-1 expression in cancer cells

through inactivation of Raf/ERK/Nrf2 signaling

and AMPK-independent pathways - 52 2.1 Metformin suppresses HO-1 expression in cancer cells - 52

2.2 Metformin suppresses Nrf2 expression through a Keap1-

independent mechanism in cancer cells - 54

2.3 Metformin suppresses Nrf2 expression in cancer cells via

Raf-ERK inactivation - 58

2.4 Down-regulation of HO-1 expression by metformin

is independent of AMPK - 61

2.5 Reduction of HO-1 contributes to anti-proliferative effects

of metformin in cancer cells - 65

3 Metformin induces microRNA-34a to down-regulate Sirt1/Pgc-1/Nrf2 pathway leading to increased susceptibility of cancer cells to

oxidative stress and therapeutic agents - 71

3.1 Metformin suppresses Sirt1 expression in p53 wild-type

cancer cells - 71

3.2 p53-dependent induction of miR-34a is required for the

reduction of Sirt1 by metformin - 73

3.3 Down-regulation of Sirt1 by metformin inhibits Nrf2 expression and increases susceptibility of wild-type p53 cancer cells

to oxidative stress - 77

3.4 Metformin inhibits Nrf2 expression mediated by

suppression of Pgc-1 - 83 3.5 Metformin suppresses Nrf2 expression by inhibiting

PPAR transcriptional activity and attenuating PPAR

binding to the Nrf2 promoter - 86

3.6 Up-regulation of DR5 expression by metformin sensitizes

wild-type p53 cancer cells to TRAIL-induced apoptosis - 90

IV Discussion - 97

V Conclusion - 118

VI References - 120

Abstract in Korean - 147

Appendix - 150

List of Figures

1 Metformin suppresses CYP1A1 and CYP1B1 expression in breast

cancer cells by down-regulating aryl hydrocarbon

receptor expression

Fig 1 Metformin down-regulates CYP1A1 and CYP1B1 transcription

in MCF-7 breast cancer cells - 31

Fig 2 Metformin down-regulates AhR expression in MCF-7

breast cancer cells - 35

Fig 3 Down-regulation of AhR expression is required for the reduction

of CYP1A1 and CYP1B1 by metformin in MCF-7 cells - 37

Fig 4 The reduction in Sp1 protein levels mediated by metformin

suppresses AhR transcriptional activity in MCF-7

breast cancer cells - 39

Fig 5 Metformin down-regulates CYP1A1 and CYP1B1 expression

in ER-negative MDA-MB-231 breast cancer cells - 42 Fig 6 Metformin attenuates endogenous AhR ligand-induced CYP1A1

and CYP1B1 expression by reducing tryptophan-2,3-dioxygenase

expression in MCF-7 breast cancer cells - 45

Fig 7 The down-regulation of TDO expression by metformin

is mediated via down-regulation of Sp1 and GR proteins - 49

Fig 8 Proposed signaling pathways underlying the effects of

metformin on down-regulation of CYP1A1 and

CYP1B1 expression in breast cancer cells - 51

2 Metformin inhibits heme oxygenase-1 expression in cancer cells

through inactivation of Raf/ERK/Nrf2 signaling and

AMPK-independent pathways

Fig 9 Metformin down-regulates HO-1 expression

in various cancer cells - 53

Fig 10 Effects of metformin on Nrf2 and Keap1 expression

in cancer cells - 56

Fig 11 Inactivation of Raf-ERK signaling by metformin is required

for down-regulation of Nrf2 expression in cancer cells - 59

Fig 12 Metformin suppresses HO-1 expression in cancer cells

in an AMPK-independent manner - 63 Fig 13 Effects of metformin on proliferation of cancer cells - 67

Fig 14 Role of HO-1 suppression in anti-proliferative effect

of metformin in cancer cells - 69

Fig 15 Proposed signaling pathways underlying the effects of metformin

on down-regulation of HO-1 expression in cancer cells - 70

3 Metformin induces microRNA-34a to down-regulate Sirt1/Pgc-1/Nrf2

pathway leading to increased susceptibility of cancer cells to oxidative stress and therapeutic agents

Fig 16 Metformin down-regulates Sirt1 expression in wild-type p53

cancer cells - 72

Fig 17 Metformin increases induction of p53 protein and miR-34a

in wild-type p53 cancer cells - 75

Fig 18 p53-dependent induction of miR-34a is required for Sirt1

reduction by metformin - 76

Fig 19 Metformin-mediated down-regulation of Sirt1 inhibits Nrf2

expression in MCF-7 breast cancer cells - 79

Fig 20 Effects of metformin on intracellular ROS production,

H2O2-induced cytotoxicity and apoptosis - 81

Fig 21 Metformin inhibited Nrf2 expression is mediated

by Pgc-1 suppression - 84 Fig 22 Metformin inhibits Nrf2 expression mediated by suppressing

transcriptional activity of PPAR - 88 Fig 23 Effects of metformin on CHOP and DR5 expression

in breast cancer cells - 92

Fig 24 Metformin sensitizes wild-type p53 cancer cells to

TRAIL-induced apoptosis - 93

Fig 25 Proposed signaling pathways underlying the effects of

metformin on Sirt1/Pgc-1/Nrf2 pathway leads to increased susceptibility of cancer cells to oxidative stress and

therapeutic agents - 96

List of Abbreviations

4-OHE2 4-Hydroxyestradiol

ARNT Aryl hydrocarbon receptor nuclear translocator BrdU 5-Bromo-2’-deoxyuridine

DN-AMPK Dominant-negative form of AMPK

FOXO Forkhead homeobox type O

HO-1 Heme oxygenase-1

Nrf2 Nuclear erythroid factor 2 (NE-F2)-related factor 2

Pgc-1 Peroxisome proliferator-activated receptor gamma

coactivator-1 alpha PPAR Peroxisome proliferator-activated receptor gamma

PPRE PPAR-responsive elements

qRT-PCR Quantitative real-time RT-PCR

RT-PCR Reverse transcription-polymerase chain reaction

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel

TRAIL Tumor necrosis factor–related apoptosis-inducing ligand

(Supervised by Prof Hye Gwang Jeong)

Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis On the other hand, heme oxygenase-1 (HO-1) is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapies

* A dissertation submitted to the committee of the Graduate School,

Chungnam National University in a partial fulfilment of the requirements for the degree of Doctor of Philosophy conferred in August 2014

It has been suggested that inhibition of HO-1 expression is a potential therapeutic approach to sensitize tumors to chemotherapy and radiotherapy Additionally, Sirtuin 1 (Sirt1) exhibits oncogenic properties in wild-type p53 cancer cells, while Sirt1 acts as a tumor suppressor in p53-mutated cancer cells Metformin belongs to the biguanide class of oral hypoglycaemic agents commonly used for the treatment of type 2 diabetes Accumulating evidence suggests that metformin has antitumor activity The expression of Sirt1, Nrf2 and AhR relates to tumorigenesis, chemoresistance and carcinogenesis Therefore, the aim of this study was to investigate effects of metformin on expression of Sirt1, Nrf2, AhR and its downstream target genes in cancer cells Results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)–induced conditions Down-regulation

of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor  (ER) signaling Furthermore, metformin markedly down-regulated Sp1 protein levels in breast cancer cells The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein Metformin inhibited endogenous AhR

tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels These results demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells

by down-regulating AhR signaling Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer

Metformin strongly suppresses HO-1 mRNA and protein expression in

human hepatic carcinoma HepG2, cervical cancer HeLa, and non-small-cell

lung cancer A549 cells Metformin also markedly reduced Nrf2 mRNA and

protein levels in whole cell lysates and suppressed tert-butylhydroquinone

(tBHQ)-induced Nrf2 protein stability and antioxidant response element (ARE)-luciferase activity in HepG2 cells The regulation of Nrf2 expression

by metformin is mediated through a Keap1-independent mechanism and that metformin significantly attenuated Raf-ERK signaling to suppress Nrf2 expression in cancer cells Inhibition of Raf-ERK signaling by PD98059 decreased Nrf2 mRNA expression in HepG2 cells, confirming that the inhibition of Nrf2 expression is mediated by an attenuation of Raf-ERK signaling in cancer cells The inactivation of AMPK by siRNA, DN-AMPK

or the pharmacological AMPK inhibitor compound C, revealed that metformin reduced HO-1 expression in an AMPK-independent manner

These results highlight the Raf-ERK-Nrf2 axis as a new molecular target in anti-cancer therapy in response to metformin treatment

Using human cancer cell lines that exhibit differential expression of p53, results indicated that metformin reduced Sirt1 protein levels in cancer cells bearing wild-type p53, but did not affect Sirt1 protein levels in cancer cell lines harboring mutant forms of p53 Metformin-induced p53 protein levels

in wild-type p53 cancer cells resulted in up-regulation of microRNA 34a The use of a miR-34a inhibitor confirmed that metformin-induced miR-34a was required for Sirt1 down-regulation Metformin suppressed peroxisome proliferator-activated receptor gamma (PPAR) coactivator-1 alpha (Pgc-1) expression and its downstream target Nrf2 in MCF-7 cells Genetic tools demonstrated that the reduction of Sirt1 and Pgc-1 by

(miR)-metformin caused Nrf2 down-regulation via suppression of PPAR

transcriptional activity Metformin reduced heme oxygenase-1 (HO-1) and superoxide dismutase 2 (SOD2) but up-regulated catalase expression in MCF-7 cells Metformin-treated MCF-7 cells had no increase in basal levels

of reactive oxygen species (ROS) but were more susceptible to oxidative stress Furthermore, up-regulation of death receptor (DR) 5 by metformin-mediated Sirt1 down-regulation enhanced the sensitivity of wild-type p53 cancer cells to TRAIL-induced apoptosis These results demonstrated that

metformin induces miR-34a to suppress the Sirt1/Pgc-1/Nrf2 pathway and increases susceptibility of wild-type p53 cancer cells to oxidative stress and TRAIL-induced apoptosis

This study demonstrated that metformin down-regulates Sirt1, Nrf2 and AhR expression in cancer cells and exhibits several relevant anti-cancer activities Metformin is the most commonly prescribed drug for type 2 diabetes Therefore, metformin may be an effective therapy for the cancer prevention and treatment

I INTRODUCTION

1 Metformin and reduced risk of cancer

Metformin, an oral biguanide widely used to treat type 2 diabetes, is a very promising antitumor agent Population studies indicate that metformin

treatment is associated with a decreased incidence of breast (Bodmer et al., 2010), prostate (Wright and Stanford, 2009), colon (Currie et al., 2009), pancreatic (Li et al., 2009) and liver cancer (Lai et al., 2012) Retrospective

studies demonstrated that metformin down-regulated various oncogenes such

as human epidermal growth factor receptor 2 (HER2) and mTOR/p70S6K signaling or up-regulated tumor suppressors such as p53 for protecting

against cancer (Ben Sahra et al., 2010; Del Barco et al., 2011; Zakikhani et

al., 2006) The present study elucidates molecular mechanisms of metformin

action concerning regulation of Sirtuin 1 (Sirt1), nuclear erythroid factor 2 (NE-F2)-related factor 2 (Nrf2), aryl hydrocarbon receptor (AhR) expression and its downstream target genes in cancer cells together with relevant anti-cancer activities

2 Role of the AhR, CYP1A1 and CYP1B1 in carcinogenesis and mechanisms of regulation of gene expression

CYP1B1 (Vinothini and Nagini, 2010) A recent study indicated that the expression of CYP1A1 regulates breast cancer cell proliferation and survival (Rodriguez and Potter, 2013) In human breast tissue, the catechol metabolite, 4-hydroxyestradiol (4-OHE2), formed by CYP1B1, generates free radicals from reductive-oxidative cycling Estrogenic quinones cause oxidative DNA damage as well as form mutagenic depurinating adenine and guanine adducts and lead to the development and evolution of breast cancer (Yager, 2012) CYP1A1 and CYP1B1 are regulated by the aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor The active transcriptional AhR/aryl hydrocarbon nuclear translocator (ARNT) heterodimer complex binds to xenobiotic responsive elements (XRE) in the

CYP1A1 and CYP1B1 promoter, resulting in increased transcription

Emerging evidence demonstrates a tumour-promoting role of the AhR, CYP1A1 and CYP1B1 in breast, lung, and hepatocellular carcinoma

(Androutsopoulos et al., 2009; Feng et al., 2013; Rodriguez and Potter,

2013) The activation of AhR may lead to deregulation of cell–cell contact, thereby inducing unbalanced proliferation, dedifferentiation and enhanced motility (Dietrich and Kaina, 2010) AhR activation also enhances the

clonogenicity and invasiveness of cancer cells (Gramatzki et al., 2009)

Transgenic mice with a constitutively active AhR spontaneously develop

tumours (Moennikes et al., 2004) Knockdown of AhR reduced tumor

growth and metastasis of human breast cancer cells (Goode et al., 2013), and

the repressor of the AhR (AhRR) is a tumour suppressor in multiple human

cancers (Zudaire et al., 2008)

Tryptophan-2,3-dioxygenase (TDO), encoded by TDO2, degrades

tryptophan to kynurenine to act as an endogenous ligand of AhR kynurenine is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient to activate AhR under constitutive conditions TDO, together with indoleamine-2,3-dioxygenases 1 and 2 (IDO1/2), plays a crucial role in suppression of anti-tumour immune

L-responses (Uyttenhove et al., 2003) and is associated with a poor prognosis

in various malignancies (Lob et al., 2009) Interestingly, TDO expression

correlates with the expression of AhR target genes such as CYP1A1 and CYP1B1 in glioma, as well as B-cell lymphoma, Ewing sarcoma, bladder carcinoma, cervix carcinoma, colorectal carcinoma, breast cancer, lung

carcinoma and ovarian carcinoma (Opitz et al., 2011) The specific protein 1 (Sp1)/glucocorticoid receptor (GR) signaling pathway regulates TDO2 in cancer cells (Kolla and Litwack, 1999; Soichot et al., 2013; Suehiro et al.,

2004) There is increasing evidence suggesting that down-regulation of TDO expression or inhibition of TDO activity may have a therapeutic application

in cancer treatment (Munn and Mellor, 2004; Opitz et al., 2011)

Sp1 regulates the expression of numerous genes related to cell proliferation, differentiation and apoptosis by binding to GC-rich promoter elements through three Cys2His2-type zinc fingers present at the C-terminal

domain (Kadonaga et al., 1988) An increase in Sp1 transcriptional activity is

associated with tumorigenesis through modulation of oncogenes and tumour

suppressor genes (Castro-Rivera et al., 2001; Stoner et al., 2004) Sp1 plays

important role in regulation of the AhR transcriptional expression under constitutive conditions through a Sp1-site located in the AhR promoter

regulating angiogenesis and cell proliferation (Akagi et al., 2005; Wagener et

al., 2001) Although it is a cytoprotective enzyme, a growing body of

evidence clearly suggests that HO-1 may also play a significant role in the

induction of tumorigenic pathways (Jozkowicz et al., 2007; Miyake et al.,

2011; Sass et al., 2008) HO-1 is often highly up-regulated in tumor tissues,

and its expression is further increased in response to therapy HO-1

overexpression can inhibit tumor cell apoptosis (Liu et al., 2004) and promote tumor angiogenesis, growth and metastasis (Jozkowicz et al., 2007; Sunamura et al., 2003; Was et al., 2010) Inhibition of HO-1 expression has

been suggested as a potential therapeutic approach to sensitization of tumors

to chemotherapy and radiotherapy (Alaoui-Jamali et al., 2009; Berberat et

al., 2005; Fang et al., 2004)

There is accumulating evidence demonstrating that Nrf2 is a key transcriptional activator of the antioxidant response element (ARE) that regulates the expression of antioxidant phase II detoxifying enzymes

Interestingly, the promoter region of the HO-1 gene contains an ARE sequence (Kobayashi and Yamamoto, 2005; Kobayashi et al., 2006; Lee and Surh, 2005; Martin et al., 2004) The mechanisms underlying Nrf2 activation

are complex, but the available evidence points to two key pathways The first

is a sulfhydryl modification of its cytosolic sequestering protein Keap1 by chemical inducers, which leads to Nrf2 dissociation from Keap1 and subsequent translocation into the nucleus, thereby activating ARE sequences

(Kobayashi et al., 2006) In the second pathway, several upstream signaling

kinases, including mitogen-activated protein kinases (MAPKs; ERK, p38,

regulate Nrf2/ARE activity (Kobayashi and Yamamoto, 2005; Lee and Surh,

2005; Martin et al., 2004) A recent study showed that activated H-Ras

promotes transcriptional activation of HO-1 in human renal cancer cells; and H-Ras-induced HO-1 overexpression is mediated primarily through the Raf-ERK activation of Nrf2, which leads to the survival of renal cancer cells

(Banerjee et al., 2011)

4 Role of Sirt1 in tumorigenesis and chemoresistance

Sirt1, a mammalian NAD+-dependent histone deacetylase, is involved in diverse cellular processes such as metabolism, cellular redox balance, resistance to oxidative stress, aging, oncogenesis, and cancer development

(Brooks and Gu, 2009; Radak et al., 2013) Sirt1 regulates important transcription factors such as p53 (Luo et al., 2001; Vaziri et al., 2001),

peroxisome proliferator-activated receptor  coactivator 1 (Pgc-1) (Amat

et al., 2009), forkhead homeobox type O (FOXO) proteins (Frazzi et al.,

2013), and Nrf2 (Kulkarni et al., 2014) which regulates the transcription of

pro- and anti-oxidant enzymes, by which the cellular redox state is affected

inhibition of the tumor suppressor p53, FOXO1, and Ku70-mediated

functions (Jung-Hynes et al., 2009; Kim et al., 2013; Luo et al., 2001; Vaziri

et al., 2001) Sirt1 is overexpressed in human breast, colon, non-small-cell

lung, prostate cancer cells (Huffman et al., 2007; Jung-Hynes et al., 2009)

and a sirtinol- or nicotinamide-specific inhibitor of Sirt1 increased senescence-like growth arrest in human breast, lung and prostate cancer cells

(Jung-Hynes et al., 2009; Ota et al., 2006) Down-regulation of Sirt1 by

antisense oligonucleotides inhibited the growth and viability of human

prostate cancer (Jung-Hynes et al., 2009), induced apoptosis, and enhanced radiation-induced anti-proliferative effects in human lung cancer cells (Sun

et al., 2007) Moreover, pharmacological inhibition of Sirt1 or Sirt1

knockdown induced apoptosis in leukemia stem cells and suppressed growth

in vitro and in vivo (Audrito et al., 2011; Li et al., 2012) Sirt1-knockout

mice exhibited p53 hyperacetylation, and Sirt1-deficient cells enhanced radiation-induced thymocyte apoptosis, suggesting that Sirt1 can facilitate

tumor growth by suppressing p53 function (Cheng et al., 2003) However,

several studies have shown that Sirt1 has tumor-suppressive effects Activation of Sirt1 by resveratrol inhibited growth of BRCA1-deficient and p53+/- tumor cells (Wang et al., 2008b) and reduced tumorigenesis in p53+/-mice (Wang et al., 2008a) Paradoxically, a recent study reported that

which was related to Sirt1 inhibition, p53 and FOXO3a hyperacetylation

(Frazzi et al., 2013) These results suggest that Sirt1 has an oncogenic effect

in cells expressing wild-type p53 but has a tumor-suppressive effect in mutated p53 cells Therefore, down-regulation of Sirt1 expression or inhibition of Sirt1 activity might be an effective approach to the treatment of cancers harboring wild-type p53

Nrf2 is a redox-sensitive transcription factor regulating the expression of

a battery of cytoprotective genes, including heme oxygenase-1 (HO-1) and

anti-oxidative enzymes such as superoxide dismutase 2 (SOD2) (Cherry et

al., 2014) Constitutive Nrf2 activation in many tumors enhances cell

survival and resistance to anti-cancer drugs (Wang et al., 2008c) Nrf2 expression is positively regulated by Sirt1 (Kulkarni et al., 2014), and Nrf2

was shown to be involved in tumor necrosis factor-related apoptosis inducing

ligand (TRAIL) resistance in cancer cells (Arlt et al., 2009)

Down-regulation of Sirt1 resulted in induction of death receptor 5 (DR5) and

sensitized cancer cells to TRAIL-induced apoptosis (Kim et al., 2013)

The present study shows that metformin reduces constitutive and inducible CYP1A1 and CYP1B1 expression through mechanisms involving AhR down-regulation in breast cancer cells Metformin inhibited cancer cell proliferation by suppressing HO-1 expression through inhibition of a Raf/ERK/Nrf2 signaling and AMPK-independent pathways Additionally,

metformin up-regulates miR-34a to down-regulate the Sirt1/Pgc-1/Nrf2 pathway and increases susceptibility of wild-type p53 cancer cells to oxidative stress Metformin induces C/EBP homology protein (CHOP) and DR5 expression, enhancing TRAIL-induced apoptosis in wild-type p53 cancer cells

II MATERIALS AND METHODS

1 Materials

Metformin, mithramycin A, L-kynurenine, L-tryptophan, dexamethasone,

mifepristone, paclitaxel, zinc protoporphyrin IX (ZnPPIX),

purchased from Sigma Chemical Co (St Louis, MO, USA) Compound C and PD98059 were purchased from Calbiochem (La Jolla, CA, USA) TCDD was purchased from Chemsyn Science Lab (Lenexa, KS, USA) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and a lactate dehydrogenase (LDH) release detection kit was obtained from Roche Applied Science (Indianapolis, IN, USA) The plasmid pCMV--gal was obtained from Clontech (Palo Alto, CA, USA) Lipofectamine™ 2000,

purchased from Invitrogen (Carlsbad, CA, USA) Oligonucleotide polymerase chain reaction (PCR) primers were custom-synthesised by Bioneer (Seoul, South Korea) A protein assay kit was purchased from Bio-Rad Laboratories, Inc (Hercules, CA, USA) The enhanced chemiluminescence (ECL) system was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden) Antibodies against CYP1A1, CYP1B1, AhR,

ARNT, GR, Hsp90, Sp1, Keap1, Sirt1, peroxisome proliferator-activated receptor gamma (PPAR) coactivator-1 (Pgc-1), PPAR, catalase, SOD2, DR5, Lamin B1, and -actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Antibodies against p-AMPK (Thr172), AMPK, p-Raf (Ser259), p44/42 MAPK (ERK/2), p-p44/42 MAPK (T202/Y204) (p-ERK1/2), p38 MAPK, p-p38 MAPK (T180/Y182), SAPK/JNK, p-SAPK/JNK (T183/Y185), p53, acetyl-p53, and poly(ADP-ribose) polymerase (PARP) and horseradish peroxidase (HRP)-linked anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Cell Signaling Technologies

(Beverly, MA, USA) Anti-HO-1 antibody was obtained from Calbiochem (San Diego, CA, USA) Antibodies against Nrf2 and NQO1 were obtained from Abcam (Cambridge, MA, USA) All other chemicals and reagents were

of analytical grade

2 Cell culture and treatment

The human breast cancer MCF-7, MDA-MB-231, human hepatocellular

carcinoma HepG2, human lung adenocarcinoma A549, and human cervical

carcinoma HeLa, human ovarian cancer line SKOV3 cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) The human colon cancer cell line HCT 116 containing wild-type p53 and p53

Vogelstein at Johns Hopkins University (Baltimore, MD, USA) All cells were cultured in the appropriate RPMI 1640, DMEM or McCoy's 5A medium in a humidified 5% CO2 incubator at 37°C in complete medium supplemented with 10% heat-inactivated FBS (Invitrogen) to 70–80% confluence Metformin was dissolved in water Mithramycin A, dexamethasone, mifepristone, TCDD, Paclitaxel, compound C, PD98059, and tBHQ were dissolved in dimethyl sulphoxide (DMSO) L-kynurenine and L-tryptophan were initially dissolved in DMSO, followed dilution into fresh DMEM medium ZnPPIX was dissolved in binding buffer (50 mM potassium phosphate; 100 mM NaCl, pH 7.5) TRAIL was dissolved in phosphate-buffered saline containing 0.1% BSA The working concentrations were added directly to culture medium Control cells were treated with vehicle only

3 Measurement of cell viability and cytotoxicity

MCF-7 cells were cultured in medium containing 10% FBS in 96-well plates

at 37°C After incubation for 24 h, the growth medium was renewed with serum-free medium and the cells were pretreated with different concentrations of metformin for 24 h, followed by treatment with H2O2 or TRAIL for an additional 24 h or 48 h at 37°C, respectively After treatment, cells were treated with MTT solution (final concentration, 0.5 mg/mL) for 1

h The dark blue formazan crystals formed in intact cells were solubilized with DMSO, and absorbance at 570 nm measured with a microplate reader (Varioskan; Thermo Electron, Waltham, MA) Cell supernatants were used in LDH assay, with measurement of absorbance at 490 nm using a microplate reader (Varioskan; Thermo Electron, Waltham, MA) Percentage cell viability or cytotoxicity was calculated based on absorbance relative to that

of vehicle-treated control cells

4 BrdU incorporation assay

BrdU incorporation assay was performed using Cell Proliferation ELISA, BrdU (colorimetric) Kit (Roche Applied Science, Indianapolis, IN, USA) Briefly, the cells were cultured in 96-well plates at a density of 5000 cells/100 l/well in complete growth media After 24 h, the cells were treated with metformin or ZnPPIX Thirty-six h later the cells were labeled using 10

M BrdU per well and re-reincubated overnight at 37°C in a humidified atmosphere The next day, the culture medium was removed, the cells were fixed, and the DNA was denatured in one step by adding FixDenat Next, the cells were incubated with the anti-BrdU-POD antibody for 90 minutes at room temperature After the removal of the antibody conjugate, the cells were washed and the substrate solution was added The reaction product was

(Varioskan; Thermo Electron, Waltham, MA, USA) at 370nm with a reference wavelength of 492 nm Percentage BrdU incorporation was calculated based on absorbance relative to that of vehicle-treated control cells

5 RNA preparation and reverse transcription-polymerase chain reaction (RT-PCR)

Cells were treated with metformin (1–5 mM) for 24 h Total RNA was extracted from treated cells using the RNAiso-plus reagent (Takara, Shiga, Japan) according to the manufacturer’s protocol Total RNA concentration at

100 ng/L was used to make cDNA The following sequences were performed for PCR: 94°C for 10 min (one cycle); 94°C for 45 s, 58°C for 45

s, and 72°C for 60 s for numbers of cycles, see below; the final extension was at 72°C for 10 min Sequences of the primers, numbers of cycles performed, and length of resulting amplicons were as follows: human HO-1 forward, 5’- CAG CAT GCC CCA GGA TTT G-3’; human HO-1 reverse, 5’- AGC TGG ATG TTG AGC AGG A-3’, 35 cycles, 618 bp; human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5’-GCG CTG AGT ACG TCG TGG AG-3’; human GAPDH reverse, 5’-CAG TTG GTG GTG CAG GAG G-3’, 30 cycles, 196 bp PCR products were run on a 1.5% agarose gel, the band intensities of the amplified DNA were visualized

using the SYBR® Safe DNA gel stain kit

6 Quantitative real-time RT-PCR (qRT-PCR)

Total RNA was isolated with RNAiso-plus reagent (Takara, Shiga, Japan) and cDNA was synthesised with the ImProm-IITM Reverse Transcriptase system (Promega, Madison, WI, USA) Product formation was monitored continuously during PCR using Sequence Detection System software (ver 1.7; Applied Biosystems, Foster City, CA, USA) Accumulated PCR products were detected directly by monitoring an increase in SYBR®reporter dye fluorescence The expression levels of CYP1A1, CYP1B1,

AhR, Sp1, TDO, GR, Nrf2, Pgc-1, Nrf2, CHOP and DR5 mRNAs in

metformin-treated cells were compared to those in control cells at each time point using the comparative cycle threshold (Ct)-method The following primer sequences were used: human AhR forward, 5'-ACT CCA CTT CAG CCA CCA TC-3'; human AhR reverse, 5'-GTG CAC AGC TCT GCT TCA GT-3'; human CYP1A1 forward, 5'-CAA GAG GAG CTA GAC ACA GT-3'; human CYP1A1 reverse, 5'-AGC CTT TCA AAC TTG TGT CT-3'; human CYP1B1 forward, 5'-TTC GGC CAC TAC TCG GAG C-3'; human CYP1B1 reverse, 5'-AAG AAG TTG CGC ATC ATG CT-3'; human GR forward, 5'-GAA CTT CCC TGG TCG AAC AGT T-3'; human GR reverse, 5'-GAG

AAG ACC CAC CA-3'; human Sp1 reverse, 5'-ATA TTG GTG GTA ATA AGG GC-3'; human TDO forward, 5'-GGG AAC TAC CTG CAT TTG GA-3'; human TDO reverse, 5'-GTG CAT CCG AGA AAC AAC CT-3'; human Pgc-1, forward, 5'-AAC AGC AGC AGA GAC AAA TGC ACC-3'; reverse, 5'-TGC AGT TCC AGA GAG TTC CAC ACT-3'; human Nrf2, forward, 5'-TAC TCC CAG GTT GCC CAC A-3'; reverse, 5'-CAT CTA

CAA ACG GGA ATG TCT GC-3'; CHOP, forward, 5'-CAA CTG CAG AGA

TGG CAG CT-3'; reverse, 5'-CTG ATG CTC CCA ATT GTT CA-3'; DR5, forward, 5'-GCC CCA CAA CAA AAG AGG TC-3'; reverse, 5'-GGA GGT CAT TCC AGT GAG TG-3'; human -actin forward, 5'-TGG CAC CCA GCA CAA TGA A-3'; human -actin reverse, 5'-CTA AGT CAT AGT CCG CCT AGA AGC A-3' 18S rRNA, forward, 5'-GCT GGA ATT ACC GCG GCT-3'; and reverse, 5'-CGG CTA CCA CAT CCA AGG AA-3' The quantity of each transcript was calculated as described in the instrument manual and normalised to the amount of -actin or 18S rRNA as a housekeeping gene

For detection of miR-34a, total RNA was extracted from the cells using the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions Total RNA was reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen) Levels of miR-34a

expression were detected using the miScript SYBR Green PCR kit (Qiagen) with specific primers for miR-34a (Bioneer, Seoul, South Korea) Values for miR-34a expression were normalised using specific primers to RNU6B (Bioneer, Seoul, South Korea) as an endogenous reference RNA

7 Luciferase and -galactosidase assays

Cells were transfected with 0.5 g of human CYP1B1-Luc vector, Luc, pGL3-ARE-Luc, PPRE-Luc reporter vector and/or 0.2 g of pCMV--gal per well using Lipofectamine™ 2000 At 6 h after transfection, fresh medium was added Cells were pretreated with metformin (1–5 mM) for 1 h, followed by treating with 10 nM TCDD or 30 M tBHQ for 24 h and lysed The lysed cell preparations were then centrifuged (12,000 rpm, 10 min), and the supernatant was assayed for both luciferase and -galactosidase activity Luciferase activity was measured using the luciferase assay system (Promega, Madison, WI, USA) with a luminometer, according to the manufacturer’s instructions The -galactosidase assay was carried out in

pXRE-250 L of assay buffer containing 0.12 M Na2HPO4, 0.08 M NaH2PO4, 0.02

M KCl, 0.002 M MgCl2, 0.1 M -mercaptoethanol and 50 g

o-nitrophenyl--galactoside Luciferase activity was normalized to -galactosidase activity and expressed as the proportion of activity detected, relative to the vehicle

to the manufacturer’s protocol To investigate multiple protein targets under the same treatment conditions, the blot was stripped and re-used Equal sample loading was confirmed by measuring -actin levels for whole-cell lysates and lamin B1 for nuclear fractions The integrated optical density for the protein band was calculated by Image-J software, and the values were normalised to house-keeping gene -actin or Lamin B1

9 Preparation of nuclear and cytosolic extracts

Nuclear extracts were prepared with a commercial kit according to the manufacturer’s instructions (Active Motif, Carlsbad, CA, USA) All steps were performed on ice or at 4°C, unless stated otherwise Protease inhibitors (10 g/mLaprotinin and 10 g/mL leupeptin) and reducing agents (1 mM dithiothreitol and 1 mM phenylmethylsulphonyl fluoride) were added to each buffer just prior to use Briefly, cells were incubated in five volumes of hypotonic Buffer A (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl)

on ice for 15 min and homogenized Nuclei were recovered by centrifugation

at 900 × g for 15 min, and the supernatant was collected as the cytoplasmic

extract The nuclei were washed once using a nuclei wash buffer (10 mM HEPES, pH 7.9, 0.2 mM MgCl2, 10 mM KCl) and extracted using Buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1.5

centrifugation at 14 000 × g for 10 min, and the supernatant used as the

nuclear extract

10 Immunoprecipitation (IP)

For IP, MCF-7 cells were grown to 70% confluence and subsequently treated with metformin in fresh medium for 24 h The cells were harvested and

manufacturer’s instructions The nuclear fraction was pre-cleared with 30 L

of protein G plus/ protein A agarose for 1 h at 4C IP was performed at 4°C for 3 h by incubation of 1 g anti-Pgc-1 antibody or normal IgG with cell extracts containing 2-mg protein, followed by addition of 30 L of protein G plus/protein A agarose for another 2 h Immunoprecipitates were collected and washed five times with lysis buffer, resuspended in SDS sample buffer and boiled for 5 min at 95°C PPAR was detected in bound proteins by Western blotting

11 Chromatin immunoprecipitation (ChIP)

A ChIP assay was performed using the EZ ChIP kit (Milipore, Billerica, MA, USA) according to manufacturer’s protocol Briefly, cells were cross-linked with a formaldehyde solution, and the chromatin sheared by sonication to isolate DNA fragments that averaged 300–500 bp An anti-PPAR antibody was added to aliquots of pre-cleared chromatin and incubated overnight Input samples were incubated with the negative-control IgG The immune complexes were captured by incubation with protein G-agarose for 1 h at 4°C After reversing the crosslinks, DNA samples from immunoprecipitates were isolated, and RT-PCR were performed using the following specific

primers flanking the PPRE of Nrf2 with 35 cycles of PCR amplifications: