Introduction
Metformin and reduced risk of cancer
Metformin, a commonly prescribed oral biguanide for managing type 2 diabetes, shows significant potential as an antitumor agent Research suggests that metformin treatment is linked to a reduced risk of developing breast cancer.
Metformin has been shown to have potential anti-cancer effects across various types of cancer, including prostate, colon, pancreatic, and liver cancer Retrospective studies indicate that it can down-regulate oncogenes like HER2 and mTOR/p70S6K signaling, while up-regulating tumor suppressors such as p53, thereby offering protection against cancer This study aims to clarify the molecular mechanisms by which metformin regulates Sirtuin 1 (Sirt1), Nrf2, and AhR expression, along with their downstream target genes in cancer cells, highlighting its relevant anti-cancer activities.
Role of the AhR, CYP1A1 and CYP1B1 in carcinogenesis
The majority of breast cancer tumours constitutively express CYP1A1 and
CYP1B1 plays a crucial role in breast cancer biology, as evidenced by its ability to generate the catechol metabolite 4-hydroxyestradiol (4-OHE2), which produces free radicals through reductive-oxidative cycling in human breast tissue This process leads to oxidative DNA damage and the formation of mutagenic adducts, contributing to breast cancer development (Vinothini and Nagini, 2010; Yager, 2012) Additionally, the expression of CYP1A1 is linked to the regulation of breast cancer cell proliferation and survival (Rodriguez and Potter, 2013) Both CYP1A1 and CYP1B1 are controlled by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that forms a heterodimer complex with aryl hydrocarbon nuclear translocator (ARNT) to bind to xenobiotic responsive elements (XRE).
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,
The activation of the aryl hydrocarbon receptor (AhR) can disrupt cell–cell contact, leading to unregulated cell proliferation, dedifferentiation, and increased motility, which enhances the clonogenicity and invasiveness of cancer cells Transgenic mice with continuously active AhR develop tumors spontaneously, while reducing AhR levels in human breast cancer cells decreases tumor growth and metastasis Additionally, the AhR repressor (AhRR) acts as a tumor suppressor in various human cancers.
Tryptophan-2,3-dioxygenase (TDO), encoded by the TDO2 gene, converts L-tryptophan into L-kynurenine, which serves as an endogenous ligand for the aryl hydrocarbon receptor (AhR) Elevated levels of L-kynurenine are produced during cancer progression and inflammation, effectively activating AhR and contributing to immune suppression TDO, alongside indoleamine-2,3-dioxygenases 1 and 2 (IDO1/2), is linked to poor prognoses in various cancers, including glioma, B-cell lymphoma, and breast cancer Furthermore, TDO expression is associated with the upregulation of AhR target genes like CYP1A1 and CYP1B1 The regulation of TDO2 in cancer cells is influenced by the specific protein 1 (Sp1)/glucocorticoid receptor (GR) signaling pathway.
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 is a crucial regulator of gene expression related to cell proliferation, differentiation, and apoptosis, interacting with GC-rich promoter elements via its Cys2His2-type zinc fingers Its increased transcriptional activity is linked to tumorigenesis by influencing oncogenes and tumor suppressor genes Additionally, Sp1 is significant in regulating AhR transcriptional expression under constitutive conditions through a specific Sp1-site in the AhR promoter.
Regulation of gene expression and role of Nrf2 and HO-1
Heme oxygenase-1 (HO-1), a crucial member of the heat shock protein family, acts as a sensor and regulator of oxidative stress by catalyzing the degradation of heme into biliverdin, carbon monoxide (CO), and free iron This enzyme is vital for tissue protection, as it mitigates oxidative injury, reduces inflammation, inhibits apoptosis, and regulates angiogenesis and cell proliferation However, emerging research indicates that HO-1 may also contribute to the activation of tumorigenic pathways.
Heme oxygenase-1 (HO-1) is frequently overexpressed in tumor tissues and its levels increase further in response to therapy This overexpression can inhibit apoptosis in tumor cells and enhance tumor angiogenesis, growth, and metastasis Targeting HO-1 expression may serve as a promising therapeutic strategy to sensitize tumors to chemotherapy and radiotherapy.
Nrf2 is a crucial transcriptional activator of the antioxidant response element (ARE), regulating the expression of phase II detoxifying enzymes Notably, the HO-1 gene promoter contains an ARE sequence The activation of Nrf2 involves complex mechanisms, primarily through two pathways: the first involves sulfhydryl modification of the cytosolic protein Keap1 by chemical inducers, leading to Nrf2 dissociation and nuclear translocation to activate ARE sequences The second pathway includes regulation by various upstream signaling kinases, such as MAPKs, PKC, and PI3K Recent research indicates that activated H-Ras enhances HO-1 transcription in human renal cancer cells, with H-Ras-induced HO-1 overexpression primarily mediated by Raf-ERK activation of Nrf2, promoting the survival of these cancer cells.
Role of Sirt1 in tumorigenesis and chemoresistance
Sirt1 is a crucial NAD+-dependent histone deacetylase in mammals, playing a significant role in various cellular processes, including metabolism, oxidative stress resistance, aging, and cancer development It regulates key transcription factors such as p53, Pgc-1α, and FOXO proteins, highlighting its importance in cellular function and disease progression.
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 (Radak et al., 2013)
Sirt1 is crucial in cancer development and drug resistance, as it inhibits senescence and apoptosis while promoting cancer cell growth and angiogenesis by suppressing tumor suppressors like p53 and FOXO1 Overexpression of Sirt1 is observed in various cancers, including breast, colon, and lung cancers, and its inhibition through specific inhibitors can induce growth arrest in these cells Additionally, down-regulation of Sirt1 has been shown to inhibit prostate cancer growth, induce apoptosis, and enhance the effects of radiation in lung cancer cells Pharmacological inhibition of Sirt1 also triggers apoptosis in leukemia stem cells and suppresses tumor growth While Sirt1 knockout studies indicate its role in facilitating tumor growth by suppressing p53, other research highlights its tumor-suppressive effects, particularly when activated by resveratrol, which inhibits the growth of certain tumor cells This dual role suggests that targeting Sirt1 may be a promising strategy for treating cancers with wild-type p53.
Nrf2 is a redox-sensitive transcription factor that regulates the expression of cytoprotective genes, including heme oxygenase-1 (HO-1) and superoxide dismutase 2 (SOD2), enhancing cell survival in tumors and resistance to anti-cancer drugs Its expression is positively regulated by Sirt1, and Nrf2 contributes to TRAIL resistance in cancer cells Reducing Sirt1 levels leads to increased death receptor 5 (DR5) expression, making cancer cells more susceptible to TRAIL-induced apoptosis.
The current study demonstrates that metformin effectively reduces the expression of CYP1A1 and CYP1B1 in breast cancer cells by down-regulating AhR It inhibits cancer cell proliferation by suppressing HO-1 expression through the Raf/ERK/Nrf2 signaling pathway and AMPK-independent mechanisms Furthermore, metformin enhances the expression of miR-34a, which in turn down-regulates the Sirt1/Pgc-1α/Nrf2 pathway, increasing the vulnerability of wild-type p53 cancer cells to oxidative stress Additionally, metformin promotes the induction of C/EBP homology protein (CHOP) and DR5, thereby enhancing TRAIL-induced apoptosis in these cancer cells.
Materials & Methods
Materials
Metformin, mithramycin A, L-kynurenine, L-tryptophan, dexamethasone, mifepristone, paclitaxel, zinc protoporphyrin IX (ZnPPIX), tert-butylhydroquinone (tBHQ), hydrogen peroxide (H2O2), and TRAIL were sourced from Sigma Chemical Co (St Louis, MO, USA), while Compound C and PD98059 were obtained from Calbiochem (La Jolla, CA, USA) TCDD was acquired from Chemsyn Science Lab (Lenexa, KS, USA) MTT and a lactate dehydrogenase (LDH) release detection kit were provided by Roche Applied Science (Indianapolis, IN, USA) The plasmid pCMV-β-gal came from Clontech (Palo Alto, CA, USA), and Lipofectamine™ 2000, SYBR® Safe DNA Gel Stain kit, and nitrocellulose membranes were purchased from Invitrogen (Carlsbad, CA, USA) Custom-synthesized oligonucleotide PCR primers were obtained from Bioneer (Seoul, South Korea), while a protein assay kit was sourced from Bio-Rad Laboratories, Inc (Hercules, CA, USA) The enhanced chemiluminescence (ECL) system was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden), along with antibodies against CYP1A1, CYP1B1, and AhR.
Key antibodies and reagents used in the study include ARNT, GR, Hsp90, Sp1, Keap1, Sirt1, PPARγ coactivator-1 (Pgc-1), PPARγ, catalase, SOD2, DR5, Lamin B1, and β-actin from Santa Cruz Biotechnology Additionally, antibodies for p-AMPKα (Thr172), AMPKα, p-Raf (Ser259), p44/42 MAPK (ERK/2), p-p44/42 MAPK (T202/Y204), p38 MAPK, p-p38 MAPK (T180/Y182), SAPK/JNK, p-SAPK/JNK (T183/Y185), p53, acetyl-p53, and PARP, along with HRP-linked anti-rabbit and anti-mouse IgG secondary antibodies, were sourced from Cell Signaling Technologies The anti-HO-1 antibody was procured from Calbiochem, while antibodies against Nrf2 and NQO1 were obtained from Abcam All chemicals and reagents used were of analytical grade.
Cell culture and treatment
The human breast cancer cell lines MCF-7 and MDA-MB-231, along with the human hepatocellular carcinoma HepG2, human lung adenocarcinoma A549, human cervical carcinoma HeLa, and human ovarian cancer SKOV3, were sourced from the American Type Culture Collection (Rockville, MD, USA) Additionally, the wild-type p53 and p53 knockout HCT 116 colon cancer cell lines (HCT 116 p53 -/-) were generously provided by Dr [Name].
Cells were cultured in RPMI 1640, DMEM, or McCoy's 5A medium at 37°C in a humidified 5% CO2 incubator, supplemented with 10% heat-inactivated FBS to achieve 70–80% confluence Metformin was dissolved in water, while other compounds such as Mithramycin A, dexamethasone, and Paclitaxel were dissolved in dimethyl sulphoxide (DMSO) L-kynurenine and L-tryptophan were first dissolved in DMSO before dilution in fresh DMEM medium ZnPPIX was prepared in a binding buffer of 50 mM potassium phosphate and 100 mM NaCl at pH 7.5, and TRAIL was dissolved in phosphate-buffered saline with 0.1% BSA Working concentrations of these compounds were added directly to the culture medium, with control cells receiving only the vehicle.
Measurement of cell viability and cytotoxicity
MCF-7 cells were cultured in 10% FBS medium in 96-well plates at 37°C After 24 hours, the growth medium was replaced with serum-free medium, and cells were pretreated with varying concentrations of metformin for another 24 hours Subsequently, the cells were exposed to H2O2 or TRAIL for 24 to 48 hours at 37°C Following treatment, MTT solution (0.5 mg/mL) was added for 1 hour to assess cell viability, with dark blue formazan crystals formed in viable cells being solubilized in DMSO Absorbance was measured at 570 nm using a microplate reader, while cell supernatants were analyzed for LDH activity at 490 nm The percentage of cell viability or cytotoxicity was determined by comparing absorbance to that of vehicle-treated control cells.
BrdU incorporation assay
The BrdU incorporation assay was conducted using the Cell Proliferation ELISA BrdU (colorimetric) Kit from Roche Applied Science Cells were cultured in 96-well plates at a density of 5000 cells per 100 µl per well in complete growth media Following a 24-hour incubation, the cells were treated with metformin or ZnPPIX, and after an additional 36 hours, the cells were labeled for analysis.
Cells were incubated with 10 µM BrdU per well overnight at 37°C in a humidified environment The following day, the culture medium was discarded, and the cells were fixed and denatured using FixDenat Subsequently, the cells were treated with anti-BrdU-POD antibody for 90 minutes at room temperature After removing the antibody conjugate, the cells were washed, and a substrate solution was added The resulting reaction product was quantified by measuring absorbance with a microplate reader.
(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 at concentrations ranging from 1 to 5 mM for a duration of 24 hours Following treatment, total RNA was extracted using the RNAiso-plus reagent from Takara, Japan, in accordance with the manufacturer's instructions The concentration of total RNA was measured after extraction.
cDNA was synthesized using a concentration of 100 ng/μL The PCR protocol consisted of an initial denaturation at 94°C for 10 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 60 seconds, with a final extension at 72°C for 10 minutes The primers used included human HO-1 forward (5’- CAG CAT GCC CCA GGA TTT G-3’) and reverse (5’- AGC TGG ATG TTG AGC AGG A-3’), yielding a 618 bp product after 35 cycles, as well as human GAPDH forward (5’-GCG CTG AGT ACG TCG TGG AG-3’) and reverse (5’-CAG TTG GTG GTG CAG GAG G-3’), resulting in a 196 bp product after 30 cycles The PCR products were analyzed on a 1.5% agarose gel, and the amplified DNA bands were visualized using the SYBR® Safe DNA gel stain kit.
Quantitative real-time RT-PCR (qRT-PCR)
Total RNA was extracted using the RNAiso-plus reagent, and cDNA synthesis was performed with the ImProm-II TM Reverse Transcriptase system PCR product formation was continuously monitored through Sequence Detection System software, with fluorescence from SYBR® reporter dye indicating accumulated PCR products The expression levels of various mRNAs, including CYP1A1, CYP1B1, AhR, Sp1, TDO, GR, Nrf2, Pgc-1α, CHOP, and DR5, in metformin-treated cells were compared to control cells using the comparative cycle threshold (Ct) method Specific primer sequences were utilized for each target gene, ensuring accurate amplification during the analysis.
This article provides a list of forward and reverse primers for various human genes, including Sp1, TDO, Pgc-1α, Nrf2, CHOP, and DR5, alongside the primers for the housekeeping genes β-actin and 18S rRNA The quantities of each transcript were measured according to the instrument manual and normalized to the levels of β-actin or 18S rRNA to ensure accurate expression analysis.
To detect miR-34a expression, total RNA was extracted from cells using the miRNeasy mini kit (Qiagen, Hilden, Germany) following the manufacturer's guidelines The extracted RNA was then reverse transcribed with the QuantiTect Reverse Transcription kit (Qiagen) miR-34a expression levels were quantified using the miScript SYBR Green PCR kit (Qiagen) and specific primers for miR-34a (Bioneer, Seoul, South Korea) To ensure accuracy, miR-34a expression values were normalized against RNU6B, using specific primers as an endogenous reference RNA (Bioneer, Seoul, South Korea).
Cells were transfected with 0.5 µg of human CYP1B1-Luc vector and other reporter vectors using Lipofectamine™ 2000 After 6 hours, fresh medium was added, and cells were pretreated with metformin (1–5 mM) for 1 hour before exposure to 10 nM TCDD or 30 µM tBHQ for 24 hours Following treatment, cells were lysed, and the preparations were centrifuged to obtain supernatants for luciferase and β-galactosidase activity assays Luciferase activity was measured using the Promega luciferase assay system, following the manufacturer's guidelines.
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 control
Whole-cell lysates were created using a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA, with protein concentrations assessed at 595 nm via the Bio-Rad protein assay kit Denatured protein samples were separated using SDS-PAGE on a 10% polyacrylamide gel and subsequently transferred to nitrocellulose membranes After blocking with 5% skim milk, membranes were incubated with primary antibodies and HRP-conjugated secondary antibodies, with visualization achieved through an ECL Western blot kit following the manufacturer's instructions To analyze multiple protein targets under identical treatment conditions, the blots were stripped and reused Equal sample loading was verified by measuring β-actin levels for whole-cell lysates and lamin B1 for nuclear fractions, while the integrated optical density of protein bands was calculated using Image-J software and normalized to the housekeeping genes β-actin or lamin B1.
9 Preparation of nuclear and cytosolic extracts
Nuclear extracts were prepared using a commercial kit from Active Motif, following the manufacturer's guidelines All procedures were conducted on ice or at 4°C, except where indicated, and included the use of protease inhibitors.
To prepare cellular extracts, 10 µg/mL of protease inhibitors (aprotinin and leupeptin) and reducing agents (1 mM dithiothreitol and 1 mM phenylmethylsulphonyl fluoride) were added to each buffer prior to use Cells were incubated in hypotonic Buffer A (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl) on ice for 15 minutes and then homogenized Nuclei were isolated by centrifugation at 900 × g for 15 minutes, and the supernatant was collected as the cytoplasmic extract The nuclei were washed with a nuclei wash buffer (10 mM HEPES, pH 7.9, 0.2 mM MgCl2, 10 mM KCl) before extraction with Buffer C.
To prepare the nuclear extract, incubate the solution containing 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, and 1.5 mM MgCl2 on ice for 30 minutes Following this, centrifuge the mixture at 14,000 × g for 10 minutes to remove insoluble material, and utilize the resulting supernatant as the nuclear extract.
MCF-7 cells were cultured to 70% confluence and treated with metformin for 24 hours Following treatment, nuclear extracts were prepared using a commercial kit The nuclear fraction was pre-cleared with protein G plus/protein A agarose at 4°C for one hour Immunoprecipitation (IP) was conducted at 4°C for three hours using 1 µg of anti-Pgc-1 antibody or normal IgG, with cell extracts containing 2 mg of protein After adding protein G plus/protein A agarose for an additional two hours, immunoprecipitates were collected, washed five times with lysis buffer, and then resuspended in SDS sample buffer before being boiled for five minutes at 95°C PPARγ was subsequently detected in the bound proteins using Western blotting.
A ChIP assay was conducted using the EZ ChIP kit (Milipore, Billerica, MA, USA) following the manufacturer's instructions Cells were cross-linked with formaldehyde, and chromatin was sheared by sonication to obtain DNA fragments averaging 300–500 bp An anti-PPARγ antibody was added to pre-cleared chromatin and incubated overnight, while input samples were treated with negative-control IgG Immune complexes were captured by incubating with protein G-agarose for 1 hour at 4°C After reversing crosslinks, DNA samples from immunoprecipitates were isolated, and RT-PCR was performed with specific primers flanking the PPRE of Nrf2, involving 35 cycles of amplification.
Nrf2, forward, 5'-CGA GAG CGC TGC CCT TAT TT-3'; and reverse, 5'- GGG GGA CCT AGA GGA GGT CT-3', and the control GAPDH primers (Millipore)
Small interfering RNA (siRNA) targeting AhR, AMPKα1/2, and Sirt1, along with the siRNA transfection reagent, were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while DR5 siRNA was procured from Bioneer Co (Seoul, South Korea) Cells were transfected with either specific siRNA or a non-specific control siRNA at 50% confluence, following the manufacturer's instructions, and allowed to incubate for 48 hours prior to experimentation.
13 Sp1, HO-1, Sirt1, Pgc-1 and PPAR overexpression
The Sp1 and Sirt1 expression vectors were generously provided by Dr Kwang Youl Lee from Chonnam National University in South Korea Additionally, the full-length cDNA of human HO-1, which is cloned in a mammalian expression plasmid vector, along with the pCMV6-Neo empty vector, were obtained from OriGene Technologies in Rockville, MD, USA The Pgc-1α expression vector was sourced from Addgene in Cambridge, MA, USA, while the PPARγ expression vector was also purchased from OriGene Technologies.
MD, USA) Cells were transfected with Sp1, HO-1, Sirt1, Pgc-1, PPAR or the empty vectors using Lipofectamine™ 2000 in antibiotic-free medium and cultured for 48 h prior to experiments
14 miR-34a inhibitor and mimic transfection miR-34a inhibitor and miR-34a mimic oligonucleotides were chemically synthesized by Bioneer (Seoul, South Korea) MCF-7 cells were transfected with the 200 nM oligonucleotides using Lipofectamine™ 2000, according to the manufacturer’s protocol One day after transfection, the cells were treated with 5 mM metformin or left untreated for an additional 24 h The cells were harvested for subsequent experiments 48 h post-transfection
15 Measurement of intracellular reactive oxygen species (ROS)
The fluorescent probe H2DCFDA was utilized to assess intracellular ROS generation in MCF-7 cells following treatment with metformin or H2O2 MCF-7 cells were pre-incubated in 96-well plates before the experiments.
Cells were treated with 10 μM H2DCFDA for 30 minutes at 37°C, followed by exposure to metformin (1–5 mM) or H2O2 (100 μM) for 1 hour Fluorescence intensity was then measured at excitation and emission wavelengths of 485 and 530 nm, respectively, using a fluorescence spectrophotometer to assess the generation of reactive oxygen species (ROS).
Western blotting
Whole-cell lysates were prepared using a lysis buffer consisting of 50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA, with protein concentrations measured at 595 nm via the Bio-Rad protein assay kit Denatured protein samples underwent separation through SDS-PAGE on a 10% polyacrylamide gel, followed by transfer to nitrocellulose membranes After blocking with 5% skim milk, the membranes were incubated with specific primary antibodies and HRP-conjugated secondary antibodies, with visualization achieved using an ECL Western blot kit To analyze multiple protein targets under identical treatment conditions, the blots were stripped and reused Sample loading was validated by assessing β-actin levels in whole-cell lysates and lamin B1 levels in nuclear fractions, with the integrated optical density of protein bands calculated using Image-J software and normalized to the housekeeping genes β-actin or lamin B1.
Preparation of nuclear and cytosolic extracts
Nuclear extracts were prepared using a commercial kit from Active Motif, following the manufacturer's guidelines All procedures were conducted on ice or at 4°C, except where noted, and included the use of protease inhibitors to ensure sample integrity.
To prepare the cell extracts, 10 μg/mL of protease inhibitors (aprotinin and leupeptin) and reducing agents (1 mM dithiothreitol and 1 mM phenylmethylsulphonyl fluoride) were added to each buffer immediately before use Cells were incubated in hypotonic Buffer A (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl) on ice for 15 minutes and then homogenized Nuclei were isolated by centrifugation at 900 × g for 15 minutes, and the supernatant was collected as the cytoplasmic extract The nuclei were subsequently washed with a nuclei wash buffer (10 mM HEPES, pH 7.9, 0.2 mM MgCl2, 10 mM KCl) before extraction with Buffer C.
To prepare the nuclear extract, mix 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, and 1.5 mM MgCl2, and incubate on ice for 30 minutes Subsequently, centrifuge the mixture at 14,000 × g for 10 minutes to remove insoluble material, and collect the supernatant as the nuclear extract.
Immunoprecipitation (IP)
In this study, MCF-7 cells were cultured to 70% confluence and treated with metformin for 24 hours Following treatment, nuclear extracts were prepared using a commercial kit, and the nuclear fraction was pre-cleared with protein G plus/protein A agarose Immunoprecipitation (IP) was conducted at 4°C for 3 hours, utilizing 1 µg of anti-Pgc-1 antibody or normal IgG with 2 mg of protein cell extracts, followed by the addition of agarose for an additional 2 hours The resulting immunoprecipitates were washed five times with lysis buffer, resuspended in SDS sample buffer, and boiled for 5 minutes at 95°C Finally, PPARγ was detected in the bound proteins through Western blotting.
Chromatin immunoprecipitation (ChIP)
A ChIP assay was conducted using the EZ ChIP kit (Milipore, Billerica, MA, USA) following the manufacturer's instructions Cells were cross-linked with formaldehyde, and chromatin was sheared by sonication to obtain DNA fragments of approximately 300–500 bp An anti-PPARγ antibody was added to pre-cleared chromatin and incubated overnight, while input samples were treated with negative-control IgG Immune complexes were captured using protein G-agarose for 1 hour at 4°C After reversing crosslinks, DNA samples from immunoprecipitates were isolated, and RT-PCR was performed with specific primers flanking the PPRE of Nrf2, involving 35 cycles of PCR amplification.
Nrf2, forward, 5'-CGA GAG CGC TGC CCT TAT TT-3'; and reverse, 5'-GGG GGA CCT AGA GGA GGT CT-3', and the control GAPDH primers (Millipore).
Small interfering RNA transfection
Small interfering RNA (siRNA) targeting AhR, AMPKα1/2, and Sirt1, along with the siRNA transfection reagent, were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while DR5 siRNA was procured from Bioneer Co (Seoul, South Korea) Cells were cultured to 50% confluence and transfected with either specific siRNA or a non-specific control siRNA, following the manufacturer's instructions, for a duration of 48 hours before conducting experiments.
Sp1, HO-1, Sirt1, Pgc-1 and PPAR overexpression
The Sp1 and Sirt1 expression vectors were generously provided by Dr Kwang Youl Lee from Chonnam National University in South Korea Additionally, the full-length cDNA of human HO-1, cloned into a mammalian expression plasmid vector, along with the pCMV6-Neo empty vector, were acquired from OriGene Technologies in Rockville, MD, USA Furthermore, the Pgc-1α expression vector was sourced from Addgene in Cambridge, MA, USA, while the PPARγ expression vector was also obtained from OriGene Technologies.
MD, USA) Cells were transfected with Sp1, HO-1, Sirt1, Pgc-1, PPAR or the empty vectors using Lipofectamine™ 2000 in antibiotic-free medium and cultured for 48 h prior to experiments.
miR-34a inhibitor and mimic transfection
The miR-34a inhibitor and mimic oligonucleotides were chemically synthesized by Bioneer in Seoul, South Korea MCF-7 cells underwent transfection with 200 nM oligonucleotides using Lipofectamine™ 2000, following the manufacturer's guidelines After a day of transfection, the cells were either treated with 5 mM metformin or left untreated for an additional 24 hours Finally, the cells were harvested for further experiments 48 hours post-transfection.
Measurement of intracellular reactive oxygen species (ROS)
The fluorescent probe H2DCFDA was utilized to assess intracellular ROS generation in MCF-7 cells following treatment with metformin or H2O2 MCF-7 cells were pre-incubated in 96-well plates before the experiment.
Cells were incubated with 10 μM H2DCFDA at 37°C for 30 minutes, then treated with metformin (1–5 mM) or H2O2 (100 μM) for 1 hour Fluorescence intensity was measured at excitation and emission wavelengths of 485 nm and 530 nm, respectively, using a fluorescence spectrophotometer to assess the generation of reactive oxygen species (ROS).
Statistical analysis
All experiments were conducted a minimum of three times, with data presented as means ± SD To assess the significance of differences among treatment groups, one-way analysis of variance (ANOVA) was employed, followed by the Newman-Keuls test for multi-group comparisons A p-value of less than 0.05 was considered statistically significant.
Results
Metformin suppresses CYP1A1 and CYP1B1 expression in breast
An experiment was conducted to explore the effects of metformin on the expression of CYP1A1 and CYP1B1 in breast cancer cells (MCF-7) Treatment with metformin (1–5 mM) for 24 hours resulted in a dose-dependent down-regulation of CYP1A1 and CYP1B1 protein levels, as demonstrated by Western blotting Further investigation into the time-course effects revealed that metformin inhibited the expression of these proteins in a time-dependent manner when cells were treated with 2.5 mM metformin for 6, 12, and 24 hours To assess whether metformin's inhibitory effects were at the transcriptional level, mRNA expression was analyzed using qRT-PCR, which confirmed that metformin significantly suppressed CYP1A1 and CYP1B1 mRNA levels Additionally, transfection of MCF-7 cells with CYP1B1-Luc promoter constructs followed by metformin treatment further supported the conclusion that metformin inhibits the transcription of CYP1B1 mRNA.
Metformin significantly reduced CYP1B1 promoter luciferase activity after 24 hours of treatment Additionally, TCDD exposure led to a marked increase in CYP1A1 and CYP1B1 protein levels; however, this induction was completely inhibited by prior treatment with metformin in MCF-7 breast cancer cells These findings suggest that metformin effectively down-regulates the expression of CYP1A1 and CYP1B1 in breast cancer cells, irrespective of the presence of xenobiotics.
R e la tiv e C Y P 1 B 1 l u c ife ra s e a c ti v ity ( fo ld o f c o n tro l)
Re la ti ve CY P1 A1 o r CY P1 B 1 mR N A to a c ti n (f o ld of c ont ro l)
Re la ti v e C Y P1A1 o r C Y P 1 B1 mRN A to act in (fold of cont ro l)
Fig 1 Metformin down-regulates CYP1A1 and CYP1B1 transcription in
In a study on MCF-7 breast cancer cells, Western blot analysis revealed that metformin significantly suppressed the protein levels of CYP1A1 and CYP1B1 in a dose- and time-dependent manner Additionally, quantitative real-time PCR (qRT-PCR) demonstrated that metformin repressed the mRNA levels of CYP1A1 and CYP1B1 similarly in a dose- and time-dependent fashion All experiments were conducted in triplicate, with results showing a statistically significant difference (P < 0.05) compared to the control Furthermore, metformin was found to inhibit CYP1B1 luciferase promoter activity, reinforcing its role in modulating these key proteins.
SD *P < 0.05 vs the control (F) Western blotting analysis showed that metformin attenuated the TCDD-induced CYP1A1 and CYP1B1 protein levels
1.2 Down-regulation of AhR expression is required for the suppression of CYP1A1 and CYP1B1 by metformin
Recent studies indicate that the aryl hydrocarbon receptor (AhR) is vital for the regulation of CYP1A1 and CYP1B1 transcription To investigate the impact of metformin on AhR expression in breast cancer cells, MCF-7 cells were treated with metformin (1–5 mM) for 24 hours The results showed that metformin significantly suppressed AhR protein levels in a dose- and time-dependent manner, as evidenced by Western blot analysis Furthermore, metformin inhibited nuclear AhR accumulation, while the stability partners of AhR, such as heat-shock protein 90 (Hsp90) and ARNT proteins, remained unaffected These findings suggest that metformin may down-regulate AhR expression through transcriptional modulation rather than degradation, as it also markedly reduced AhR mRNA levels in a similar dose- and time-dependent manner.
Metformin treatment significantly down-regulated AhR expression and suppressed CYP1B1 promoter luciferase activity in breast cancer cells In MCF-7 cells, metformin (1–5 mM) treatment for 24 hours notably reduced XRE luciferase activity, confirming the role of the transcription factor AhR in the down-regulation of CYP1A1 and CYP1B1 Additionally, pre-treatment with metformin for 1 hour effectively diminished TCDD-induced XRE luciferase activity Further evidence from siRNA-mediated knockdown of AhR expression showed a marked reduction in constitutive levels of AhR, CYP1A1, and CYP1B1 proteins These findings underscore the critical role of AhR down-regulation in the metformin-mediated suppression of CYP1A1 and CYP1B1 expression in breast cancer cells.
R e la ti ve A h R m R N A t o a ctin (f ol d of con tr o l)
R e la tiv e A h R m R N A to a c tin (f ol d o f c ont ro l)
Metformin significantly down-regulates AhR expression in MCF-7 breast cancer cells, as demonstrated by Western blotting analysis of AhR, Hsp90, ARNT, and β-actin protein levels in cell lysates The time course study revealed that treatment with 2.5 mM metformin affects AhR protein expression over time, with further analysis showing reduced AhR mRNA levels in a dose- and time-dependent manner All experiments were conducted in triplicate, with results indicating a statistically significant difference (P < 0.05) compared to the control group.
Fig 3 Down-regulation of AhR expression is required for the reduction of
Metformin effectively inhibits both constitutive and TCDD-induced XRE luciferase promoter activity in MCF-7 cells, as demonstrated by transient transfection with a XRE promoter luciferase construct and pre-treatment with metformin (1–5 mM) for 1 hour prior to 24-hour TCDD exposure The results, presented as mean ± SD, indicate significant differences with *P < 0.05 compared to the control and #P < 0.05 compared to cells treated with TCDD alone Additionally, AhR siRNA transfection for 48 hours significantly reduces the protein levels of CYP1A1 and CYP1B1, as confirmed by Western blot analysis of AhR, CYP1A1, CYP1B1, and β-actin.
R e la ti v e X R E l u c ife ra s e a c ti v ity (f ol d of c o nt ro l)
1.3 Down-regulation of Sp1 by metformin inhibits AhR transcriptional activity in breast cancer cells
Metformin significantly reduces Sp1 protein levels and its nuclear accumulation in MCF-7 cells without affecting Sp1 mRNA levels This suppression of Sp1 correlates with a decrease in AhR protein levels, as demonstrated by experiments where MCF-7 cells were transfected with a Sp1 expression vector and treated with metformin Additionally, Mithramycin A, an Sp1 inhibitor, further decreased AhR, CYP1A1, and CYP1B1 protein levels, confirming that metformin's down-regulation of Sp1 plays a crucial role in modulating AhR expression under constitutive conditions.
R e la tiv e S p 1 m R N A t o a cti n (f ol d o f co nt ro l)
Metformin reduces Sp1 protein levels, leading to the suppression of AhR transcriptional activity in MCF-7 breast cancer cells Western blot analysis confirmed that metformin down-regulates both total and nuclear Sp1 protein levels Additionally, qRT-PCR results indicated that metformin affects Sp1 mRNA levels The role of Sp1 in regulating AhR and its target gene expression was examined by transfecting MCF-7 cells with Sp1 or a control vector, followed by metformin treatment Western blotting was used to analyze protein levels of Sp1, AhR, CYP1B1, and β-actin Furthermore, the use of a competitive Sp1 inhibitor, mithramycin A, demonstrated its effects on the expression of AhR and its target genes, with protein levels assessed via Western blotting.
1.4 Inhibition of CYP1A1 and CYP1B1 expression by metformin is independent of ER
Metformin significantly decreased the expression of CYP1A1 and CYP1B1 in estrogen receptor (ER) α-positive MCF-7 cells To investigate the role of ERα in this down-regulation, ERα-negative MDA-MB-231 cells were treated with metformin at concentrations ranging from 1 to 5 mM for 24 hours The results showed a dose-dependent reduction in CYP1A1 and CYP1B1 protein levels in MDA-MB-231 cells, similar to the findings in MCF-7 cells Additionally, metformin treatment did not affect the levels of Hsp90 and ARNT proteins in MDA-MB-231 cells Further studies were conducted to assess the impact of metformin on CYP1A1 and CYP1B1 expression in the presence of TCDD.
231 cells were pre-treated with metformin (1–5 mM) for 1 h, followed by a
Exposure to 10 nM TCDD for 24 hours resulted in increased levels of CYP1A1 and CYP1B1 proteins in MDA-MB-231 cells, as shown by Western blotting However, pre-treatment with metformin significantly reduced these protein levels, demonstrating that metformin effectively inhibits both constitutive and TCDD-induced expression of CYP1A1 and CYP1B1 in breast cancer cells, independent of estrogen receptor alpha (ERα).
Fig 5 Metformin down-regulates CYP1A1 and CYP1B1 expression in
Metformin effectively inhibits the constitutive expression of CYP1A1 and CYP1B1 in ERα-negative MDA-MB-231 breast cancer cells, as shown by Western blotting analysis of protein levels in whole-cell lysates Additionally, pre-treatment with metformin significantly reduces the TCDD-induced expression of CYP1A1 and CYP1B1 proteins.
1.5 Metformin suppresses endogenous AhR-ligand-induced CYP1A1 and CYP1B1 expression by reducing TDO expression in breast cancer cells
TDO catalyzes the conversion of L-tryptophan to L-kynurenine, which activates the AhR in various cancer cell types, including MCF-7 breast cancer cells (Opitz et al., 2011; Pilotte et al., 2012) This study explores TDO's influence on metformin's effects on CYP1A1 and CYP1B1 expression Notably, L-kynurenine treatment significantly increased XRE luciferase activity 24 hours post-treatment, and prior metformin administration enhanced this effect.
Metformin significantly reduced L-kynurenine-induced XRE luciferase activity and CYP1A1 and CYP1B1 protein levels in MCF-7 cells after 24 hours of treatment Following pre-treatment with metformin, Western blotting revealed that L-kynurenine increased CYP1A1 and CYP1B1 levels, while metformin effectively attenuated this effect Additionally, metformin treatment (1–5 mM) for 48 hours led to a notable suppression of TDO protein and mRNA levels in MCF-7 cells, indicating its potential role in regulating TDO expression in breast cancer.
L-tryptophan, a specific substrate of TDO, significantly increased the protein levels of CYP1A1 and CYP1B1 in MCF-7 breast cancer cells However, pre-treatment with metformin inhibited this induction These findings indicate that the down-regulation of TDO expression plays a role in the reduced suppression of CYP1A1 and CYP1B1 in breast cancer cells.
Re la tiv e XRE l u c ife ra s e ac tivi ty (fol d o f c o ntr o l)
Re la ti v e TDO mR NA to a c ti n (f ol d of c ontrol)
Fig 6 Metformin attenuates endogenous AhR ligand-induced CYP1A1 and
Metformin inhibits heme oxygenase-1 expression in cancer cells
2.1 Metformin suppresses HO-1 expression in cancer cells
This study examined the hypothesis that metformin exerts antitumor effects in cancer cells by reducing HO-1 expression Human cancer cell lines, including HepG2 (hepatocellular carcinoma), A549 (lung adenocarcinoma), and HeLa (cervical carcinoma), were treated with metformin at concentrations ranging from 1 to 5 mM for 24 hours at 37°C The levels of HO-1 protein were subsequently analyzed using Western blotting.
Metformin significantly reduces HO-1 protein expression in a dose-dependent manner across various cancer cell lines, including A549 cells, which exhibit high levels of HO-1 Subsequent RT-PCR analysis confirmed that metformin treatment also suppresses HO-1 mRNA levels Additionally, the drug decreases both HO-1 and NAD(P)H: quinone oxidoreductase (NQO1) protein expression in A549 cells over time These findings demonstrate that metformin effectively down-regulates HO-1 mRNA and protein expression in multiple cancer cell types.
Fig 9 Metformin down-regulates HO-1 expression in various cancer cells
This study investigates the impact of metformin on the expression of HO-1 protein and mRNA levels, utilizing Western blotting and RT-PCR techniques Additionally, it examines the time-dependent effects of metformin on the protein expression of HO-1 and NQO1 in A549 cells through Western blot analysis.
2.2 Metformin suppresses Nrf2 expression through a Keap1- independent mechanism in cancer cells
Recent studies indicate that Nrf2 is crucial for regulating ARE-mediated target genes, including HO-1 and NQO1 To investigate the impact of metformin on Nrf2 protein expression and stability in HepG2 cells, researchers treated the cells with metformin (1–5 mM) for 24 hours The results showed a significant reduction in Nrf2 protein levels in the whole cell lysate, as confirmed by Western blotting Additionally, metformin pretreatment for one hour, followed by stimulation with 30, further elucidated its effects on Nrf2 stability.
Metformin significantly suppresses tBHQ-induced Nrf2 protein stability and its nuclear release in HepG2 cells, as demonstrated by a reduction in Nrf2 mRNA expression and ARE-luciferase activity Following a 1-hour pretreatment with metformin, HepG2 cells exhibited a marked decrease in tBHQ-induced ARE-luciferase activity after 24 hours Additionally, a dose-dependent decrease in Nrf2 protein levels was observed in HeLa and A549 cells, while metformin did not affect Keap1 protein levels.
These findings suggested that metformin down-regulates Nrf2 expression in several cancer cells by a Keap1-independent mechanism
Re la ti v e Nrf2 m R N A to Acti n (f old of c o n trol)
Re la ti ve ARE l u ci fera s e a c ti vi ty (f ol d o f c o ntro l)
Fig 10 Effects of metformin on Nrf2 and Keap1 expression in cancer cells
This study investigates the impact of metformin on Nrf2 and Keap1 proteins in whole cell lysates, as well as its effects on Nrf2 protein stability and nuclear translocation induced by tBHQ in HepG2 cells, assessed through Western blotting Additionally, the research evaluates the influence of metformin on Nrf2 mRNA expression in HepG2 cells using qRT-PCR, with all experiments conducted in triplicate, and results presented as mean ± SD.
Metformin significantly suppressed tBHQ-induced ARE-Luc activity in HepG2 cells (P < 0.05 vs control), with all experiments conducted in triplicate, showing mean ± SD Additionally, metformin's effects on Nrf2 and Keap1 protein expression were analyzed in HeLa and A549 cells through Western blotting, revealing significant differences (P < 0.05 vs control; P < 0.05 vs tBHQ-treated cells).
2.3 Metformin suppresses Nrf2 expression in cancer cells via Raf-ERK inactivation
In a study evaluating the impact of metformin on Nrf2 activation in cancer cell lines, HepG2, HeLa, and A549 cells were treated with 1–5 mM metformin for 24 hours, followed by Western blot analysis to assess MAPK phosphorylation The results demonstrated that metformin significantly inhibited the phosphorylation of Raf and ERK1/2 in a dose-dependent manner, while other MAPKs, such as p-SAPK/JNK and p-p38, showed no significant changes Further experiments with a MEK1/2 inhibitor, PD98059, revealed a strong suppression of Nrf2 mRNA expression in HepG2 cells, indicating that metformin's reduction of Nrf2 activation is linked to the inactivation of Raf-ERK signaling Notably, the combination of PD98059 and metformin exhibited synergistic effects in inhibiting HO-1 protein expression These findings suggest that metformin mediates the inhibition of Nrf2 expression in cancer cells through the disruption of Raf-ERK signaling pathways.
Rela ti v e N rf 2 mR NA to A c ti n (fo ld of c ontr o l)
Metformin effectively inactivates Raf-ERK signaling, which is essential for the down-regulation of Nrf2 expression in various cancer cells, including HepG2, HeLa, and A549, as demonstrated through Western blotting The impact of the MEK1/2 inhibitor PD98059 on Nrf2 mRNA expression in HepG2 cells was assessed using qRT-PCR, with results indicating significant changes (P < 0.05 vs control) Furthermore, the combination of metformin and PD98059 synergistically decreases HO-1 protein expression in HepG2 cells, with treatments lasting 24 hours and analyzed via Western blotting for HO-1 and β-actin.
2.4 Down-regulation of HO-1 expression by metformin is independent of AMPK
Metformin significantly reduced HO-1 expression in HeLa cells, which do not have the endogenous LKB1 necessary for AMPK activation In contrast, metformin effectively activated AMPK in HepG2 cells after 24 hours of treatment However, it did not induce AMPKα phosphorylation in HeLa cells, aligning with previous research findings This suggests that the regulation of HO-1 expression by metformin occurs through mechanisms that do not depend on AMPK activation.
Metformin treatment significantly reduced HO-1 protein levels in AMPK-depleted HepG2 cells, similar to control siRNA-transfected cells Further experiments showed that transfection with a dominant-negative form of AMPK (DN-AMPK) did not prevent metformin-induced HO-1 inhibition Additionally, pretreatment with the specific AMPK inhibitor compound C also failed to reverse the suppressive effects of metformin on HO-1 expression These findings indicate that metformin suppresses HO-1 expression in cancer cells through an alternative, AMPK-independent signaling mechanism.
Metformin suppresses HO-1 expression in cancer cells independently of AMPK activation In a study, HepG2 and HeLa cells were analyzed using Western blotting to assess the effects of metformin on AMPK activation Additionally, HepG2 cells were transfected with AMPK siRNA or control siRNA for 48 hours, followed by treatment with 5 mM metformin for 24 hours, revealing the impact on HO-1 protein expression and AMPK phosphorylation Furthermore, the study evaluated the effects of metformin on HO-1 expression in HepG2 cells transfected with dominant-negative AMPK (DN-AMPK) or a control vector, demonstrating that metformin's action on HO-1 is not reliant on AMPK signaling.
The expression levels of HO-1 and AMPKα were analyzed using Western blotting after a 24-hour treatment Additionally, the effects of metformin and the AMPK inhibitor compound C on HO-1 expression were evaluated HepG2 cells were pretreated with 10 µM of compound C for 1 hour, followed by a 24-hour treatment with 5 mM metformin, with HO-1 protein expression also assessed through Western blotting.
2.5 Reduction of HO-1 contributes to anti-proliferative effects of metformin in cancer cells
Previous research has demonstrated that metformin (3–10 mM) significantly reduces the proliferation of multidrug-resistant MCF-7/adr cells (Kim et al., 2011) This study investigates the hypothesis that the anti-proliferative effects of metformin on cancer cells are partially mediated by the inhibition of HO-1 To test this, HepG2, A549, and HeLa cells were treated with increasing doses of metformin (1–10 mM) alongside the specific HO-1 inhibitor ZnPPIX (5 μM).
The 48-hour 5-Bromo-2’-deoxyuridine (BrdU) incorporation assay was conducted to assess BrdU incorporation during DNA synthesis in drug-treated cells, as detailed in the Materials and Methods section The results indicated that both Metformin and ZnPPIX notably reduced proliferation in HepG2 and A549 cell lines.
Metformin induces microRNA-34a to down-regulate Sirt1/Pgc-1/Nrf2
3.1 Metformin suppresses Sirt1 expression in p53 wild-type cancer cells
The study investigated the impact of metformin on Sirt1 expression across various cancer cell lines with different p53 statuses, including MCF-7, HCT 116, and A549 cells with wild-type p53, as well as MDA-MB-231 cells with mutated p53 and p53 knockout HCT 116 and null-p53 SKOV3 cells Treatment with metformin (1–5 mM) for 24 hours led to a notable reduction in Sirt1 protein levels in the wild-type p53 cell lines, while no significant changes were observed in the cell lines with altered p53 expression These findings indicate that metformin's effect on Sirt1 expression is contingent upon the p53 status in cancer cells.
Fig 16 Metformin down-regulates Sirt1 expression in wild-type p53 cancer cells (A, C and E) Effects of metformin on Sirt1 protein expression in MCF-
This study investigates the effects of metformin on Sirt1 protein expression in various cancer cell lines, including MDA-MB-231 with mutant p53, p53 knockout HCT 116, and null-p53 SKOV3 cells The expression levels of Sirt1 and β-actin were analyzed through Western blotting in cell lysates.
Metformin (mM) - 1 2.5 5 wild-type p53 MCF-7
Metformin (mM) - 1 2.5 5 mutant p53 MDA-MB-231
Metformin (mM) - 1 2.5 5 wild-type p53 HCT 116
3.2 p53-dependent induction of miR-34a is required for the reduction of Sirt1 by metformin
Sirt1 plays a crucial role in regulating apoptosis by deacetylating targets such as p53, which in turn can enhance Sirt1 expression through a positive-feedback loop involving miR-34a A study investigated the impact of metformin on miR-34a expression in both wild-type p53 MCF-7 and p53-mutant MDA-MB-231 breast cancer cells Results showed that metformin significantly increased miR-34a levels in wild-type p53 MCF-7 cells, while having no effect on p53-mutant MDA-MB-231 cells Additionally, metformin treatment elevated acetylated p53 protein levels in wild-type MCF-7 cells but did not alter p53 expression in the mutant cells In wild-type p53 A549 lung cancer cells, metformin also induced p53 protein levels within 2 hours, with notable increases at 14 and 24 hours Further analysis using p53 wild-type and knockout HCT 116 colon cancer cells confirmed that metformin significantly raised miR-34a levels only in p53 wild-type cells, underscoring the essential role of p53 in this regulatory mechanism.
Western blot analysis confirmed successful knockout of p53 expression in p53 -/- HCT 116 cancer cells, with no p53 induction, while p53 wild-type HCT 116 cells showed increased p53 levels To investigate the role of miR-34a in metformin-induced Sirt1 down-regulation, MCF-7 cells were transfected with a miR-34a inhibitor for 24 hours and then treated with 5 mM metformin for an additional 24 hours, resulting in the reversal of metformin-mediated Sirt1 down-regulation Conversely, a miR-34a mimic significantly decreased Sirt1 protein levels after 24 hours of transfection in MCF-7 cells These findings indicate that p53-dependent induction of miR-34a is essential for metformin's reduction of Sirt1.
Metformin enhances the induction of p53 protein and miR-34a in wild-type p53 cancer cells, as demonstrated in a study analyzing the effects of metformin on miR-34a levels in both wild-type p53 MCF-7 and p53-mutated MDA-MB-231 breast cancer cells using qRT-PCR The results indicated a significant increase in miR-34a levels with a P-value of less than 0.05 compared to control Additionally, Western blot analysis revealed that metformin elevated p53 protein expression in wild-type p53 MCF-7 cells and also affected p53 levels in MDA-MB-231 cells with a p53 mutation Notably, metformin induced p53 protein levels in wild-type p53 A549 lung cancer cells in a time-dependent manner, highlighting its potential role in cancer therapy.
Re la ti v e m iR -3 4 a t o RN U 6 B (f o ld of co nt ro l)
Wild-type p53 MCF-7 mutant p53 MDA-MB-231
Metformin (mM) - 1 2.5 5 mutant p53 MDA-MB-231
Metformin (mM) - 1 2.5 5 Acetyl-p53 wild-type p53 MCF-7
Metformin induces the expression of miR-34a, which is essential for the reduction of Sirt1, as demonstrated in both wild-type p53 and p53 knockout HCT 116 colon cancer cells The levels of miR-34a were quantified using qRT-PCR, with all experiments conducted in triplicate, showing statistically significant results (P < 0.05) compared to the control Additionally, the study assessed the impact of metformin on p53 protein expression in the same cell lines.
In a study involving 116 colon cancer cells, Western blotting was utilized to assess the protein levels of p53, acetyl-p53, and β-actin in cell lysates The findings revealed that anti-miR-34a effectively counteracts the metformin-induced down-regulation of Sirt1 Additionally, the introduction of a miR-34a mimic resulted in a decreased expression of Sirt1 protein levels in MCF-7 breast cancer cells, further confirmed through Western blot analysis of Sirt1 and β-actin in the cell lysates.
-actin Sirt1 miR-34a mimic (nM) Metformin (mM)
Rela tive miR- 34 a to RNU6B (f o ld of co nt ro l)
Wild-type p53 HCT 116 p53 knockout HCT 116
Metformin (mM) - 1 2.5 5 wild-type p53 HCT 116
3.3 Down-regulation of Sirt1 by metformin inhibits Nrf2 expression and increases susceptibility of wild-type p53 cancer cells to oxidative stress
Recent research has highlighted the regulation of Nrf2 expression by Sirt1 in human cancer cells (Kulkarni et al., 2014), prompting an investigation into their roles in metformin's effects In this study, MCF-7 cells were treated with metformin (1–5 mM) for 24 hours, followed by Western blot analysis The results demonstrated that metformin significantly decreased Nrf2 protein levels in a dose-dependent manner, with further reductions observed after long-term treatment at 48 and 72 hours.
In our study, we utilized qRT-PCR to demonstrate that metformin significantly decreases Nrf2 mRNA levels in MCF-7 cells after 24 hours of treatment To investigate the role of Sirt1 in regulating Nrf2 expression, MCF-7 cells were transfected with Sirt1 siRNA for 48 hours, resulting in a notable reduction of Nrf2 protein levels Conversely, transfection with a Sirt1 expression vector led to a remarkable increase in Nrf2 protein levels These findings indicate that the down-regulation of Nrf2 expression by metformin is mediated through a decrease in Sirt1 levels in cancer cells.
Recent experiments examined the impact of metformin on the expression of cytoprotective enzyme HO-1 and antioxidant enzyme SOD2, both regulated by Nrf2 (Cherry et al., 2014) The results indicated that metformin treatment for 24 hours significantly decreased the protein levels of HO-1 and SOD2 in MCF-7 cells (Fig 19D).
Metformin significantly increased catalase protein levels in treated conditions, highlighting its role in regulating oxidative stress The expression of SOD2, catalase, and HO-1 is crucial for maintaining ROS balance and protecting cells from oxidative damage In experiments with MCF-7 breast cancer cells, metformin at concentrations of 1–5 mM did not alter basal ROS levels; however, H2O2 stimulation resulted in a five-fold increase in ROS production.
(100 M) compared with the control after 1 h of treatment Metformin also did not change ROS generation after 24 h of treatment (data not shown) Interestingly, pre-treatment with metformin for 24 h significantly enhanced
H2O2-induced cytotoxicity and apoptosis were observed in MCF-7 breast cancer cells, while metformin pre-treatment did not enhance H2O2-induced cell death in p53-mutated MDA-MB-231 cells These findings suggest that metformin increases the susceptibility of wild-type p53 cancer cells to oxidative stress.
R e la tiv e N rf 2 m R NA t o 18 S r R N A (f o ld of co ntr o l)
Metformin treatment leads to a down-regulation of Sirt1, which in turn inhibits Nrf2 expression in MCF-7 breast cancer cells After 24 hours of metformin exposure, a notable decrease in Nrf2 protein levels is observed in these cells.
The study examined the effects of metformin on Nrf2 expression in MCF-7 cells over 48 and 72 hours, with protein levels assessed through Western blotting Results indicated that metformin significantly influenced Nrf2 mRNA expression after 24 hours, as demonstrated by qRT-PCR analysis Furthermore, the role of Sirt1 in regulating Nrf2 was explored by transfecting MCF-7 cells with Sirt1 siRNA, leading to changes in Nrf2 expression levels Additionally, the impact of metformin on the expression of HO-1, SOD2, and catalase was evaluated, with protein levels measured via Western blotting All experiments were conducted in triplicate, showing statistically significant results (P < 0.05) compared to control.
R O S pr o duc ti o n (r el at iv e t o c ont ro l)
C e ll v ia b il it y ( % o f c o n tr o l)
M etfo rm in alo ne
Metformin significantly influences intracellular ROS production and mitigates H2O2-induced cytotoxicity and apoptosis in MCF-7 cells Using the fluorescent probe H2DCFDA, the study revealed that metformin treatment resulted in a marked reduction in ROS levels compared to control groups (P < 0.05, n=6) Additionally, the MTT assay demonstrated that metformin enhances cell survival in wild-type p53 MCF-7 cells exposed to H2O2, while the LDH assay confirmed its protective effects against H2O2-induced cytotoxicity All experiments were conducted in quadruplicate, reinforcing the statistical significance of the findings (P < 0.05 vs control).