metformin inhibits age related centrosome amplification in drosophila midgut stem cells through akt tor pathway

G Model ARTICLE IN PRESS MAD 10767 1–11 Mechanisms of Ageing and Development xxx (2015) xxx–xxx Contents lists available at ScienceDirect Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Q2 Q3 Hyun-Jin Na a,1 , Joung-Sun Park a,1 , Jung-Hoon Pyo a , Ho-Jun Jeon a , Young-Shin Kim a , Robert Arking b , Mi-Ae Yoo a,∗ a b Department of Molecular Biology, Pusan National University, Busan 609-735, South Korea Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA 22 a r t i c l e i n f o a b s t r a c t 10 11 12 13 14 Article history: Received 14 January 2015 Received in revised form 23 April 2015 Accepted May 2015 Available online xxx 15 21 Keywords: Drosophila intestinal stem cells (ISCs) Metformin Centrosome amplification AKT/TOR pathway DNA damage 23 Introduction 16 17 18 19 20 24 25 26 27 We delineated the mechanism regulating the inhibition of centrosome amplification by metformin in Drosophila intestinal stem cells (ISCs) Age-related changes in tissue-resident stem cells may be closely associated with tissue aging and age-related diseases, such as cancer Centrosome amplification is a hallmark of cancers Our recent work showed that Drosophila ISCs are an excellent model for stem cell studies evaluating age-related increase in centrosome amplification Here, we showed that metformin, a recognized anti-cancer drug, inhibits age- and oxidative stress-induced centrosome amplification in ISCs Furthermore, we revealed that this effect is mediated via down-regulation of AKT/target of rapamycin (TOR) activity, suggesting that metformin prevents centrosome amplification by inhibiting the TOR signalling pathway Additionally, AKT/TOR signalling hyperactivation and metformin treatment indicated a strong correlation between DNA damage accumulation and centrosome amplification in ISCs, suggesting that DNA damage might mediate centrosome amplification Our study reveals the beneficial and protective effects of metformin on centrosome amplification via AKT/TOR signalling modulation We identified a new target for the inhibition of age- and oxidative stress-induced centrosome amplification We propose that the Drosophila ISCs may be an excellent model system for in vivo studies evaluating the effects of anti-cancer drugs on tissue-resident stem cell aging © 2015 Z Published by Elsevier Ireland Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Metformin, a biguanide drug, is clinically approved- and welltolerated for the treatment of type diabetes, and is of interest for cancer prevention and therapy (Aljada and Mousa, 2011; Baur et al., 2010; Landman et al., 2009) The direct molecular target of Abbreviations: 4E-BP, eukaryotic translation initiation factor 4E-binding protein; 8-oxo-dG, 8-oxo-2 -deoxyguanosine; AMPK, AMP-activated protein kinase; EBs, enteroblasts; ECs, enterocytes; EEs, enteroendocrine cells; EGFR, epidermal growth factor receptor; IGF1R, insulin-like growth factor-1 receptor; InR, insulin receptor; ISCs, intestinal stem cells; PBST, phosphate-buffered saline with 0.1% Triton X-100; PH3, phospho-histone H3; PQ, paraquat; PTEN, phosphatase and tensin homolog; Raptor, regulatory-associated protein of mTOR; Rheb, Ras homolog enriched in brain; ROS, reactive oxygen species; S6K, ribosomal protein S6 kinase; JAK/STAT, Janus kinase/signal transducers and activators of transcription; JNK, c-Jun N-terminal kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin Q4 ∗ Corresponding author at: Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735, South Korea Tel.: +82 51 510 2278; fax: +82 51 513 9258 E-mail address: mayoo@pusan.ac.kr (M.-A Yoo) These authors contributed equally to this article metformin is mitochondrial respiratory complex (Owen et al., 2000) Inhibition of this protein complex decreases ATP production, which increases the AMP/ATP ratio and leads to subsequent activation of AMP-activated protein kinase (AMPK), an inhibitor of mammalian target of rapamycin (mTOR) (Owen et al., 2000) Many studies support for the anti-tumor effects of metformin (Algire et al., 2010; Berstein, 2012; Kato et al., 2012; Landman et al., 2009; Saito et al., 2013) Metformin has been shown to inhibit human cancer cells proliferation, and it can be used to prevent and treat a variety of cancers (Alimova et al., 2009; Ashinuma et al., 2012; Cantrell et al., 2010) Metformin blocks are the production of endogenous reactive oxygen species (ROS) (Halicka et al., 2011), and it inhibits pro-inflammatory responses and nuclear factor kappa B in human vascular wall cells (Isoda et al., 2006) However, the molecular mechanisms underlying the anti-tumor effects of metformin remain unclear The centrosome is the major microtubule-organizing center and plays an important in key cellular processes, including cell division, cell migration, and cell polarity (Bettencourt-Dias and Glover, 2007) Centrosome aberrations, such as centrosome amplification, lead to genomic instability (Rao et al., 2009) Centrosome http://dx.doi.org/10.1016/j.mad.2015.05.004 0047-6374/© 2015 Z Published by Elsevier Ireland Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 G Model MAD 10767 1–11 ARTICLE IN PRESS H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx Q7 Fig Inhibitory effect of metformin on age-related centrosome amplification in midgut ISCs (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (A) Guts from 14-day-old wild type flies (a–a” and e–e”), 45-day-old wild type flies (b–b” and f-f”), and 14-day-old Catn1 mutant flies (c–c” and g–g”) without (a–c”) or with (e–g”) mM metformin feeding for days were stained with anti-␥-tubulin (red), anti-PH3 (green), and DAPI (blue) Fourteen-day-old wild type flies without (d–d”) or with (h–h”) mM metformin feeding for days were treated with 10 mM PQ in standard media for 20 h, after which their guts were stained with anti-␥-tubulin (red), anti-PH3 (green), and DAPI (blue) a’, b’, c’, d’, e’, f’, g’, and h’ indicates enlarged PH3 staining images a”, b”, c”, d”, e”, f”, g”, and h” indicates enlarged images of ␥-tubulin staining images Original magnification is 400× (B) The number of PH3-positive cells was counted in whole guts from 14-day-old wild type, 45-day-old wild type, 14-day-old Catn1 mutant, and 14-day-old PQ-treated wild type flies, with (black bars) or without (gray bars) metformin feeding for days N is the number of observed guts and n is the number of observed PH3-positive cells (C) The frequency of supernumerary centrosome (>2) per mitotic ISC in 14-day-old wild type, 45-day-old wild type, 14-day-old Catn1 mutant, and 14-day-old PQ-treated wild type flies with (black bars) or without (gray bars) metformin feeding for days guts The centrosome numbers were counted in mitotic ISCs (PH3-positive cell) in the midgut N is the number of observed guts and n is the number of observed ␥-tubulin-positive cells (D) The frequency of mitotic ISCs with supernumerary centrosome per gut in 14-day-old wild type, 45-day-old wild type, 14-day-old Catn1 mutant, and 14-day-old PQ-treated wild type flies with (black bars) or without (gray bars) metformin feeding for days guts N is the number of observed guts The error bar represents standard error p-values were calculated using Student’s t-test 49 50 51 52 amplification, which occurs when there is more than two centrosomes during mitosis, is a common feature of human cancers (D’Assoro et al., 2002; Nigg, 2002; Pihan et al., 2003) Centrosome amplification is involved in the initial stages of tumorigenesis (Leonard et al., 2013; Nakajima et al., 2004; Nam et al., 2010) Centrosome amplification is caused by DNA damage (Nigg, 2002; Xu et al., 1999) Interestingly, DNA damage incurred during G2 phase leads to greater centrosome amplification than G1 phase Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 53 54 55 56 G Model MAD 10767 1–11 ARTICLE IN PRESS H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx Fig Inhibitory effect of metformin on centrosome amplification in ISCs of midgut with ISC/EB-specific activation of AKT/TOR signaling pathway (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (A) Guts from 9-day-old esgts > GFP (a and i), esgts > GFP + InR (b and j), esgts > GFP + PTENRNAi (c and k), esgts > GFP+AKT (d and l), esgts > GFP + Rheb (e and m), esgts > GFP + Raptor (f and n), esgts > GFP + d4EBPRNAi (g and o), and esgts > GFP + S6KSTDE (h and p) flies without (a–h) or with (i–p) mM metformin feeding for days were stained with anti-␥tubulin (red), anti-PH3 (green), and DAPI (blue) Original magnification is 400× (B) The number of PH3-positive cells was counted in whole guts of 9-day-old esgts > GFP, Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 G Model MAD 10767 1–11 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 ARTICLE IN PRESS H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx damage (Inanc et al., 2010) Furthermore, centrosome amplification can switch asymmetric division of stem cells to symmetric division, leading to polyploid cells and hyperplasia in Drosophila (Basto et al., 2008) Therefore, centrosome amplification in adult stem cells, particularly those in high turnover tissues, could affect tissue homeostasis and regeneration The AKT/TOR pathway is a central signaling pathway regulating lifespan (Haigis and Yankner, 2010) The TOR pathway controls cell proliferation, survival, and metabolism (Kapuria et al., 2012; Laplante and Sabatini, 2012) Hyperactivation of AKT/TOR signaling is observed in many human cancers (Alliouachene et al., 2008) Furthermore, TOR signaling-related factors, including insulin receptor (InR), ras homolog enriched in brain (Rheb), phosphatase and tensin homolog (PTEN), regulatory-associated protein of mTOR (Raptor), ribosomal protein S6 kinase (S6K), and the eukaryotic translation initiation factor 4E-binding protein (4E-BP), are also implicated in tumorigenesis (Alliouachene et al., 2008) In particular, the loss of PTEN is associated with centrosome abnormalities (Leonard et al., 2013; Levine et al., 1998; Nam et al., 2010) Constitutive activation of AKT has also been shown to cause centrosome abnormalities and amplification (Leonard et al., 2013; Nam et al., 2010) However, the effect of metformin on centrosome abnormalities amplification and the modulation of molecular pathways have not been reported Adult stem cells maintain tissue homeostasis and repair during adulthood through their ability to self-renew and produce differentiated cells (Amcheslavsky et al., 2011) Age-related changes in stem cells are associated with tissue aging and age-related disease (Liu and Rando, 2011) Therefore, understanding age-related changes in stem cells may provide mechanistic insights into tissue homeostasis and stem cell-derived diseases, including cancer Directed modulation of stem cell aging could result in the prevention of age-related diseases and thus promote healthy aging The Drosophila midgut is an excellent model system to study adult stem cells (Andriatsilavo et al., 2013; Jiang and Edgar, 2011) The intestine is a sensitive organ and is exposed to various environmental stressors and damage during replication The Drosophila intestinal stem cells (ISCs) undergo self-renewal and produce two major types of cells, enterocytes (ECs) and enteroendocrine cells (EEs), from enteroblasts (EBs) (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006, 2007) ISCs are only mitotic cells (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006, 2007), and several conserved signaling pathways regulate proliferation of ISCs, such as the epidermal growth factor receptor (EGFR), Wnt Janus kinase (JAK)/signal transducer and activator of transcription (STAT), c-Jun N-terminal kinase (JNK), Hippo, PVR/PVF2 and InR/TOR pathways (Beebe et al., 2009; Biteau et al., 2008; Biteau et al., 2010; Choi et al., 2008; Jiang and Edgar, 2009; Jiang et al., 2011; Jiang et al., 2009; O’Brien et al., 2011; Ren et al., 2010; Xu et al., 2011) Previous studies have reported age-associated increases in ISC hyperproliferation and mis-differentiation, leading to intestinal hyperplasia (Biteau et al., 2008; Choi et al., 2008; Park et al., 2009) Recently, we reported that foci of the DNA double strand break marker Drosophila ortholog of ␥H2AX (␥H2AvD) and the oxidative DNA damage marker 8-oxo-dG, accumulated with age and in response to oxidative stress in ISCs (Park et al., 2012) We also reported that centrosome amplification is increased in ISCs that were aged or exposed to oxidative stress(Park et al., 2014) Based on these data, Drosophila midgut ISCs may be a suitable model to evaluate the effects of anticancer drugs on tissue-resident stem cell aging in vivo We recently reported that metformin inhibits age-related accumulation of DNA damage in Drosophila ISCs and hyperplasia (Na et al., 2013) In the present study, we investigated the effects of metformin on centrosome amplification in Drosophila ISCs and the mechanism of its anti-cancer effect Our results showed that metformin prevents age- and oxidative stress-induced centrosome amplification in Drosophila midgut ISCs, which is correlated with the inhibition of DNA damage accumulation via down-regulation of the AKT/TOR signaling pathway 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 Materials and methods 129 2.1 Fly stock 130 Fly stocks were maintained at 25 ◦ C on standard food under a ∼12 h/12 h light/dark cycle Food consisted of 79.2% water, 1% agar, 7% cornmeal, 2% yeast, 10% sucrose, 0.3% bokinin, and 0.5% propionic acid To avoid larval overpopulation, 50–60 adult flies per vial were transferred to new food vials every 2–3 days Oregon-R (OR) was used as wild type Catn1 /TM3 (#4014), UAS-InR (#8248), UASAKT (#8191), UAS-Rheb (#9688), UAS-Raptor (#53726) and UAS-S6KSTDE (#6913) were acquired from Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN, USA) UAS-PTENRNAi (#101475) and UAS-d4E-BPRNAi (#100739) were obtained from the Vienna Drosophila RNAi Center (Vienna, Austria) Temperature-inducible Su(H)GBElacZ; esg-Gal4:tub-Gal80ts ,UAS-GFP/CyO (esgts > GFP) was kindly provided by Benjamin Ohlstein (Ohlstein and Spradling, 2007) Catn1 /+ flies were obtained from a cross of OR males and Catn1 /TM3 females The esgts > GFP/+, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4E-BPRNAi , and esgts > GFP + S6KSTDE flies were obtained from a cross of esgts > GFP females and OR, UAS-InR, PTENRNAi , UAS-AKT, UAS-Rheb, UAS-Raptor, d4E-BPRNAi , and UAS-S6KSTDE males esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP+AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4EBPRNAi , and esgts > GFP + S6KSTDE flies with (black bars) or without (gray bars) metformin feeding for days Metformin treatment flies reduced mitotic ISCs in esgts > GFP + InR (21–9.7), esgts > GFP + PTENRNAi (38–20), esgts > GFP + AKT (35–15), esgts > GFP + Rheb (23–6), esgts > GFP + Raptor (16–8), esgts > GFP + d4E-BPRNAi (18–7), and esgts > GFP + S6KSTDE flies (16–5) The number in parentheses indicate PH3+ cell per gut N is the number of observed guts and n is the number of observed PH3-positive cells (C) The frequency of supernumerary centrosome (>2) per mitotic ISC in guts The centrosome numbers were counted in mitotic ISCs from 9-day-old esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4EBPRNAi , and esgts > GFP + S6KSTDE flies with (black bars) or without (gray bars) metformin feeding for days Metformin treatment flies reduced the number of mitotic ISCs with supernumerary centrosomes in esgts > GFP + InR (19–3%), esgts > GFP + PTENRNAi (17–8%), esgts > GFP + AKT (17–5%), esgts > GFP + Rheb (16–7%), esgts > GFP + Raptor (24–8%), esgts > GFP + d4E-BPRNAi (16–4%), and esgts > GFP + S6KSTDE flies (22–2%) The number in parentheses indicate abnormal ␥-tubulin+ cell (>2) per PH3+ cell (%) N is the number of observed guts and n is the number of observed ␥-tubulin-positive cells (D) The frequency of mitotic ISCs with supernumerary centrosomes per gut in 9-day-old esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4EBPRNAi , and esgts > GFP + S6KSTDE fly with (black bars) or without (gray bars) metformin feeding for days Metformin treatment also reduced the number of mitotic ISCs with supernumerary centrosomes per gut in esgts > GFP + InR (3.7–0.5), esgts > GFP + PTENRNAi (7–1.6), esgts > GFP + AKT (6–0.9), esgts > GFP + Rheb (3.5–0.7), esgts > GFP + Raptor (4.5–0.9), esgts > GFP + d4E-BPRNAi (3–0.6), and esgts > GFP + S6KSTDE flies (3–0.3) The number in parentheses indicate abnormal ␥-tubulin+ cell (>2) per gut N is the number of observed guts The error bar represents standard error p-values were calculated using Student’s t-test (E) Cryosectioned guts from 9-day-old esgts > GFP + InR (b and j), esgts > GFP + PTENRNAi (c and k), esgts > GFP + AKT (d and l), esgts > GFP + Rheb (e and m), esgts > GFP + Raptor (f and n), esgts > GFP + d4EBPRNAi (g and o), and esgts > GFP + S6KSTDE (h and p) flies with or without mM metformin feeding for days were stained with rhodamine-phalloidin (red, actin, indicating visceral muscle), anti-GFP (green), and DAPI (blue) (a–h, without metformin; i–p, with mM metformin) Original magnification is 400× Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 G Model MAD 10767 1–11 ARTICLE IN PRESS H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx Fig Inhibitory effect of metformin on DNA damage accumulation by activation of TOR signal pathway in midgut ISCs/EBs (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (A) Guts from 9-day-old esgts > GFP (a–a’ and i–i’), esgts > GFP + InR (b–b’ and j–j’), esgts > GFP + PTENRNAi (c–c’ and k–k’), esgts > GFP + AKT (d–d’ and l–l’), esgts > GFP + Rheb (e–e’ and m–m’), esgts > GFP + Raptor (f–f’ and n–n’), esgts > GFP + d4EBPRNAi (g–g’ and o–o’), and esgts > GFP + S6KSTDE (h–h’ and p–p’) flies without (a–h’) or with (i–p’) mM metformin feeding for days were stained with anti-␥-H2AvD (red), anti-GFP (green), and DAPI (blue) a’, b’, c’, d’, e’, f’, g’, h’, i’, j’, k’, l’, m’, n’, o’ and p’ indicates enlarged ␥H2AvD staining images Arrowheads indicate ␥H2AvD-positive and GFP-positive cells Original magnification is 400× (B) Guts from 9-day-old esgts > GFP (a–a’ and i–i’), esgts > GFP + InR (b–b’ and j–j’), esgts > GFP + PTENRNAi (c–c’ and k–k’), esgts > GFP + AKT (d–d’ and l–l’), esgts > GFP + Rheb (e–e’ and m–m’), esgts > GFP + Raptor (f–f’ and n–n’), esgts > GFP + d4EBPRNAi (g–g’ and o–o’), and esgts > GFP + S6KSTDE (h–h’ and p–p’) flies mM metformin feeding for days were stained with anti-8-oxo-dG (red), anti-Arm (green), and DAPI (blue) a’, b’, c’, Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 G Model MAD 10767 1–11 H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 153 ARTICLE IN PRESS 2.2 Fly genotypes GmbH, Wetzlar, Germany) at −20 ◦ C Sections were then blocked in 5% BSA for h and incubated overnight with primary antibody After treatment with secondary antibody conjugated to fluorescent dye, samples were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) on microscope slide (Thermo Fisher Scientific Inc., Waltham, MA, USA) and analyzed using a Zeiss Axioskop 2plus microscope (Carl Zeiss Inc., Gottingen, Germany) 169 Fig 1A–D: +; +; +/+ +; +; Catn1 /+ Fig 2A–E, Fig 3A–D, and Supplementary Fig S1: Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts ,UAS-GFP/+; +/+ Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts ,UAS-GFP/UAS-InR; +/+ esg-Gal4:tub-Gal80ts ,UAS-GFP/UAS-PTENRNAi ; Su(H)GBE-lacZ; +/+ Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts ,UAS-GFP/UAS-Akt1; +/+ Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts ,UAS-GFP/+; UAS-Rheb/+ Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts ,UAS-GFP/UAS-Raptor; +/+ Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts ,UAS-GFP/ UAS-d4E-BPRNAi ; +/+ esg-Gal4:tub-Gal80ts ,UAS-GFP/+; UASSu(H)GBE-lacZ; S6k.STDE/+ Fig A and B: +; +; +/+ +; +; Catn1 /+ Fig 4C and D: Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts , UAS-GFP/+; +/+ 170 2.3 Temperature-controlled expression 2.8 Antisera 182 For transgene expression at specific developmental stages, the Gal80ts technique was used (McGuire et al., 2004) The flies were set up and maintained at 22 ◦ C until adulthood After maintaining the flies at 29 ◦ C for days, the midguts were dissected and analyzed Genotype-by-environment (GxE) interactions have been known (Lachance et al., 2013; Lewontin, 2000) Although the G × E interactions could not to be controlled, to address a possibility of phenotypic effects due to differences in genetic background, flies were assayed without the change in temperature (at 22 ◦ C) as a partial control In the experiments at 22 ◦ C, we observed no phenotypic effect due to differences in genetic background (Supplementary Fig S1) 183 2.4 Metformin feeding assay Antibodies were diluted as follows: rabbit anti-phospho-histone H3 (PH3, mitotic ISC marker), 1:1000 (Millpore Corporation, Billerica, MA, USA); mouse anti-GFP, 1:1000 (Molecular Probes, Eugene, OR, USA); rat anti-GFP, 1:1000 (Nacalai Tesque Inc., Kyoto, Japan); rabbit anti-␥H2AvD, 1:1000 (Rockland Immunochemicals Inc., Gilbertsville, PA, USA); mouse anti-␥-tubulin, 1:500 (Sigma–Aldrich); mouse anti-8-oxo-dG, 1:100, (Trevigen, Gaithersburg, MD, USA); mouse anti-Delta, 1:100, mouse anti-␤gal, 1:100 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA); and rabbit anti-phospho-4E-BP1 (T37/46), 1:100 (Cell Signaling Technologies, Beverly, MA, USA) For the detection of visceral muscle, we used rhodamine-phalloidin 1:300, (Molecular Probes) Secondary antibodies included Cy3-conjugated goat antimouse, FITC-conjugated goat anti-rabbit, FITC-conjugated goat anti-mouse, FITC-conjugated goat anti-rat, Cy3-conjugated goat anti-rabbit and goat anti-rabbit Alexa Fluor® 647 (Jackson Immuno Research, West Grove, PA, USA); used at a 1:500 dilution 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 171 172 173 174 175 176 177 178 179 180 181 190 Seven-day-old wild type flies, 38-day-old wild type flies, and 7-day-old Catn1 mutant flies were fed mM metformin (Sigma–Aldrich, St Louis, MO, USA; working concentration) mixed in standard food for days at 25 ◦ C Two-day-old esgts > GFP flies were fed mM metformin mixed in standard food for days at 29 ◦ C Flies were transferred to new metformin-containing food vials every 2–3 days 191 2.5 Paraquat feeding assay 184 185 186 187 188 189 195 Seven-day-old wild type flies pre-treated with mM metformin for days were fed 10 mM paraquat (methyl viologen, PQ, Sigma–Aldrich) in standard media for 18–20 h at 25 ◦ C The midgut guts of the flies were analyzed by immunostaining 196 2.6 Cryosection 192 193 194 197 198 199 200 201 The midguts were dissected, fixed in 4% para-formaldehyde for h at room temperature and infiltrated with 20% sucrose overnight at ◦ C After flash-freezing in Tissue-Tek optimal cutting temperature (OCT) medium (SAKURA, Tokyo, Japan), sections (10 ␮M) were cut on a Cryostat (Leica CM1850, Leica Microsystems, 2.7 Immunochemistry Intact adult guts were dissected, fixed at room temperature for h in 4% para-formaldehyde (Sigma–Aldrich), washed with PBST [0.1% Triton X-100 in phosphate-buffered saline (PBS)], and incubated overnight with primary antibody at ◦ C after h of incubation at 25 ◦ C The samples were then incubated for h with secondary antibodies at 25 ◦ C, washed in PBST, mounted with Vectashield and analyzed using a Zeiss Axioskop 2plus microscope (Carl Zeiss Inc.) 2.9 Quantitative analysis To quantitatively analyze PH3-positive cells, the number of PH3positive cells in the whole gut was counted To quantitatively analyze centrosome amplification, the number of ␥-tubulin stained spots per PH3-positive cell in the whole midguts was determined To quantitatively analyze Delta-positive cells, Su(H)-positive cells and p4E-BP-positive cells, the numbers of cells were counted in the microscopic fields at a magnification of 400× of the posterior midgut Quantified data are expressed as the mean ± SE Significant differences were identified using the Student’s t test Sigma Plot 10.0 (Systat Software Inc., San Jose, CA, USA) was used for analysis of standard error 2.10 Quantification of H2AvD and 8-oxo-dG fluorescence The ␥H2AvD and 8-oxo-dG images were captured at the same exposure time in each experiment using a microscope with the Axio Vision Rel 4.8 program (Carl Zeiss Inc.) We used Adobe Photoshop d’, e’, f’, g’, h’, i’, j’, k’, l’, m’, n’, o’ and p’ indicates enlarged 8-oxo-dG staining images Original magnification is 400× (C) Graph showing the average fluorescence intensity of ␥H2AvD in GFP-positive cells in 9-day-old esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4EBPRNAi , and esgts > GFP + S6KSTDE flies without or with mM metformin feeding for days N is the number of observed guts and n is the number of observed cells The number of observed cells were 8–20 per gut (D) Graph showing average fluorescence intensity of 8-oxo-dG staining in small cells from 9-day-old esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4EBPRNAi , and esgts > GFP + S6KSTDE flies without or with mM metformin feeding for days N is the number of observed guts and n is the number of observed cells The number of observed cells were 7–21 per gut The error bar represents standard error p-values were calculated using Student’s t-test Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 G Model MAD 10767 1–11 ARTICLE IN PRESS H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx Fig Inhibitory effect of metformin on age-related TOR activity in age- and oxidative stress-related midguts (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (A) Guts from 14-day-old wild type flies (a–a” and d–d”), 45-day-old wild type (b–b” and e–e”) and 14-day-old Catn1 mutant flies (c–c” and f–f”) without (a–c”) or with (d–f”) mM metformin feeding for days were stained with anti-p4E-BP (green), anti-Delta (red) and DAPI (blue) a’, b’, c’, d’, e’ and f’ indicates enlarged Delta staining images a”, b”, c”, d”, e” and f” indicates enlarged p4E-BP staining images Arrowheads indicate p4E-BP-positive Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 G Model MAD 10767 1–11 H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 ARTICLE IN PRESS CS5.1 extended software (Adobe System Incorporated, San Jose, CA, USA) to measure the fluorescence level of ␥H2AvD and 8-oxo-dG foci The fluorescence level was measured within the nucleus based on the boundaries defined using quick selection tool of Photoshop CS5.1 from the DAPI channel The mean fluorescence was analyzed after exclusion of the mean of the background region (from two spot excluding the nucleus portion in the posterior midgut) The fluorescence mean of background region was not significantly different with secondary only control Significant differences were identified using the Student’s t test Sigma Plot 10.0 was used for analysis of standard error Results 3.1 Inhibitory effect of metformin on age-related centrosome amplification in midgut ISCs We investigated the effect of metformin on age- and oxidative stress-related centrosome amplification in ISCs Age- and oxidative stress-related centrosome amplification with supernumerary centrosomes (>2) has been reported in ISCs (Park et al., 2014) In the present study, we confirmed that centrosome amplification increased in midgut ISCs after aging and oxidative stress exposure We stained cells with anti-␥-tubulin, a centrosome marker, and anti-PH3, a marker of mitotic cells (ISCs), and counted the number of cells displaying centrosome amplification Supernumerary centrosomes were observed in 7% of mitotic ISCs in 45-day-old wild type flies and in 9.4% of mitotic ISCs in 14-day-old Catn1 mutant flies, a model of intrinsic oxidative stress (Choi et al., 2008; Griswold et al., 1993; Park et al., 2012), as compared to 1.2% in 14-day-old wild type flies (Fig 1A(a–c”) and C) The number of mitotic ISCs with supernumerary centrosome per gut was 1.63 in 45-day-old wild type flies and 3.1 in 14-day-old Catn1 mutant flies, as compared to 0.16 in 14-day-old wild type flies (Fig 1D) Interestingly, metformin treatment reduced PH3-positive cells and the number of ISCs with supernumerary centrosomes by 3.6% in 45-day-old wild type and 3.4% in 14-day-old Catn1 mutant flies (Fig 1A(e–g”), B and C) The number of mitotic ISCs with supernumerary centrosomes per gut was reduced by 0.56 in 45-day-old wild type flies and by 0.83 in 14-day-old Catn1 mutant flies, whereas there was no change in 14-day-old wild type flies (Fig 1D) To confirm that metformin inhibits oxidative stress-induced centrosome amplification in the midgut, we applied extrinsic oxidative stress Seven-day-old wild type flies with or without mM metformin treatment for days were treated with 10 mM PQ for 20 h Both mitotic ISCs and mitotic ISCs with supernumerary centrosomes increased in the PQ-treated wild type flies, as compared to control flies (1.2–12%; Fig 1A(d–d”), B and C) Furthermore, the number of mitotic ISCs with supernumerary centrosomes per gut increased in PQ-treated flies (0.16–2.6; Fig 1D) However, in metformin pre-treated wild type flies, the number of PH3-positive cell and mitotic ISCs with supernumerary centrosomes decreased after PQ treatment (1–4%; Fig 1A(h–h”), B and C) Furthermore, the number of mitotic ISCs with supernumerary centrosomes per gut was reduced after PQ treatment, as compared to PQ-treated flies without metformin pretreatment (2.6–0.5; Fig 1D) These results indicated that metformin partially suppresses age- and oxidative stress-induced centrosome amplification in Drosophila midgut ISCs 3.2 Inhibitory effect of metformin on centrosome amplification in ISCs of midgut with ISC/EB-specific activation of AKT/TOR signaling pathway To determine if the inhibitory effect of metformin on agerelated centrosome amplification is associated with the AKT/TOR pathway, we overexpressed components of the AKT/TOR pathway in ISCs and EBs using heat-inducible esg-Gal4; tub-Gal80ts system (McGuire et al., 2004; Ohlstein and Spradling, 2006, 2007) For ISC- and EB-specific expression, 2-day-old esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi, esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4E-BPRNAi, and esgts > GFP + S6KSTDE flies were cultured at 29 ◦ C for days In all cases, GFP-positive cells increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control flies (Fig 2A(a–h)) The number of PH3-positive cells increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control flies (Fig 2B) The number of mitotic ISCs with supernumerary centrosomes increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control (Fig 2C) The number of mitotic ISCs with abnormal centrosomes per gut increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control (Fig 2D) We also observed intestinal hyperplasia with a multilayered epithelium in cross-sectioned guts of the flies in which the AKT/TOR pathway was activated under esgts > GFP (Fig 2Ea-h) Compared to the non-treated group, metformin treatment reduced mitotic ISCs and the number of mitotic ISCs with supernumerary centrosomes in the gut of the flies in which the AKT/TOR pathway was activated under esg> GFP (Fig 2A(i–p) and B–C) Metformin treatment also reduced the number of mitotic ISCs with supernumerary centrosomes per gut in the gut of the flies in which the AKT/TOR pathway was activated under esg> GFP (Fig 2D) Furthermore, metformin treatment reduced the multilayered epithelium in the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to untreated flies (Fig 2E(i–p)) In contrast, metformin treatment did not affect the intestinal morphology of 9-day-old esgts > GFP flies (Fig 2E(a) and (i)) These results indicate that centrosome amplification and ISC hyperproliferation increased in ISCs by AKT/TOR pathway activation Furthermore, metformin reduced TOR pathway-induced increases in ISC proliferation, mitotic ISCs with supernumerary centrosomes, and multilayered epithelium 3.3 Inhibitory effect of metformin on DNA damage accumulation by activation of TOR signal pathway in midgut ISCs/EBs Centrosome amplification is associated with cell cycle arrest, particularly during G2-M phase, due to DNA damage (Nigg, 2002; and Delta-positive cells (B) Graph showing the ratio of p4E-BP-positive cells to Delta-positive cells in 14-day-old wild type flies, 45-day-old wild type and 14-day-old Catn1 mutant flies without (gray bars) or with (black bars) metformin (met) feeding The error bar is standard error p-values were calculated using Student’s t-test N is the number of observed guts n is the number of observed Delta-positive cells The number of observed cells were 10–45 per gut (C) Guts of 10-day-old Su(H)GBE-lacZ; esgts flies (a–a” and c–c”) and 40-day-old Su(H)GBE-lacZ; esgts flies (b–b” and d–d”) without (a–b”) or with (c–d”) mM metformin feeding were stained with anti-p4E-BP (green), anti-␤-gal (red) and DAPI (blue) a’, b’, c’ and d’ indicates enlarged ␤-gal staining images a”, b”, c” and d” indicates enlarged p4E-BP staining images Arrowhead indicate p4E-BP-positive and Su(H)-positive cells Original magnification is 400× (D) Graph showing the ratio of p4E-BP-positive cells to Su(H)-positive cells The numbers of p4E-BP-positive cells to Su(H)-positive cells were counted in 10-day-old Su(H)GBE-lacZ; esgts flies and 40-day-old Su(H)GBE-lacZ; esgts flies without (gray bars) or with (black bars) metformin (met) feeding N is the number of observed guts n is the number of observed Su(H)-positive cells The number of observed cells were 7–111 per gut The error bar is standard error p-values were calculated using Student’s t-test Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 G Model MAD 10767 1–11 ARTICLE IN PRESS H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 Xu et al., 1999) The correlation between DNA damage and centrosome amplification was assessed, considering the inhibitory effects of metformin Metformin reduced the accumulation of ␥H2AvD, as a marker of DNA damage, in the midguts of aging, oxidative stressinduced, and AKT overexpressing flies (Na et al., 2013) Based on the above observation, we assessed whether activation of the AKT/TOR signal pathway induced DNA damage, and ascertained the effect of metformin on the enhancement of DNA damage caused by TOR signaling pathway activation in midgut ISCs/EBs The ␥H2AvD fluorescence level of esg-positive cells increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control flies (Fig 3A(a–h’) and gray bars in 3C) The 8-oxo-dG fluorescence level also increased up to 2–4.5 fold in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to control flies (Fig 3B(a–h’) and gray bars in 3D) However, metformin reduced ␥H2AvD fluorescence in esgpositive cells by 20–60% in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to non-treated flies (Fig 3A(i–p’) and black bars in 3C) In addition, compared to the untreated flies, metformin also reduced 8-oxo-dG fluorescence by 15–37% in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP (Fig 3B(i–p’) and black bars in 3D) These results show that metformin can reduce the DNA damage accumulation that results from enhanced TOR signaling in the midgut 3.4 Inhibitory effect of metformin on age-related TOR activity in age- and oxidative stress-related midguts We recently reported that metformin inhibits age- and oxidative stress-induced increase of AKT activity in the Drosophila midgut ISCs/EBs (Na et al., 2013) To confirm the effect of metformin on TOR signaling in midgut ISCs/EBs, p4E-BP, a TOR activity marker in adult midgut, was evaluated by immunostaining The p4E-BP expression increased in 89% of Delta-positive cells (ISCs) in 45-oldday wild type and 85% of Delta-positive cells in 14-day-old Catn1 mutant flies, and in 84% of Su(H)-positive cells (EBs) in 40-oldday Su(H)GBE-lacZ flies, as compared to control flies (Fig 4A(a–c”), B, C(a–b”) and D) Metformin reduced the age-related increase of p4E-BP by 16% of Delta-positive cells in 45-old-day wild type and 23% of Delta-positive cells in 14-day-old Catn1 mutant flies, as well as in 37% of Su(H)-positive cells (progenitor cells) in 40old-day Su(H)GBE-lacZ flies, as compared to non-treated groups (Fig 4A(d–f”), B, C(c–d”) and D) This result indicates that the age- or oxidative stress-related activation of the TOR pathway in ISCs/EBs can be inhibited by metformin Discussion We recently reported that centrosome amplification increases in Drosophila ISCs with age (Park et al., 2014) In the present study, we report that the inhibitory effect of metformin on centrosome amplification in Drosophila ISCs as the underlying mechanism of its anti-cancer effect The major findings of the current study are: (1) metformin inhibits age-related centrosome amplification in midgut ISCs, which is a common feature of many cancer cells (2) Metformin inhibits AKT/TOR signal-induced centrosome amplification in midgut ISCs, and (3) metformin inhibits AKT/TOR signal-induced DNA damage accumulation in midgut ISCs We showed that metformin reduced centrosome amplification in midgut ISCs from flies undergoing aging and intrinsic (Catn1 mutant) and extrinsic (PQ) oxidative stresses The Catn1 heterozygous mutant flies as a model of intrinsic oxidative stress is based on previous reports showing a gene dosage-dependent effect on catalase activity (Griswold et al., 1993), higher ROS generation (Choi et al., 2008) and increased 8-oxo-dG and ␥H2AvD levels in the midguts (Park et al., 2012) Metformin has emerged as a promising anti-cancer drug (Algire et al., 2012; Emami Riedmaier et al., 2013) However, the molecular mechanisms underlying its anti-cancer effects remain unclear Centrosome amplification leads to genomic instability, a hallmark of cancer cells and is involved in the initial stages of tumorigenesis (Leonard et al., 2013; Nakajima et al., 2004; Nam et al., 2010) Metformin activates AMPK in adult Drosophila (Slack et al., 2012) Metformin has been reported to suppress the phosphorylation of insulin-like growth factor receptor (IGF1R)/InR, AKT, mTOR and ribosomal protein (rp S6) (Aljada and Mousa, 2011; Owen et al., 2000) Upregulation of AKT/TOR signaling is associated with age-related diseases, such as cancer (Feng et al., 2005; Foster, 2009; Yang and Ming, 2012) We recently reported that metformin inhibits age- and oxidative stress-associated increase of AKT activity in the Drosophila midgut ISCs and progenitor cells (Na et al., 2013) In the present study, we showed that p4E-BP expression, a TOR activity marker, was increased in the midgut of aged and oxidative stress-treated flies, and that metformin reduced this effect We further showed that centrosome amplification is increased by hyperactivation of the TOR signal-pathway in midgut ISCs, and that metformin reduces AKT/TOR signal-induced centrosome amplification In mammals, constitutive activation of AKT has been shown to cause centrosome abnormalities and amplification (Nam et al., 2010), and loss of PTEN, an inhibitor of the AKT pathway, is associated with centrosome abnormalities (Leonard et al., 2013; Levine et al., 1998; Nam et al., 2010) Therefore, our data suggest that the inhibitory effect of metformin on age-related centrosome amplification in midgut ISCs is mediated by down-regulation of the AKT/TOR pathway in the midgut Based on these observations, we propose that metformin exerts its anti-cancer effect by suppressing centrosome amplification through an inhibition of AKT/TOR signaling Metformin has been also reported in mammalian cultured cells to inhibit TOR kinase in Rag GTpases-dependent manner which is TSC1/2-independent TOR activation pathway (Kalender et al., 2010) Rag GTpases-mediated TOR activation is conserved in Drosophila and Rag proteins regulate starvation responses (Efeyan et al., 2012) Therefore, we should note a possibility that metformin inhibits centrosome amplification in Rag GTpases-dependent manner Metformin has a beneficial effect in pathogenic mold infection (Shirazi et al., 2014) Metformin modulates metabolism of microbiota in C elegans (Cabreiro et al., 2013) It has been known that microbiota accelerate dysplasia in the gut (Broderick et al., 2014; Buchon et al., 2009) Whether the inhibitory effect is dependent on altered microbiota caused by metformin is not addressed in the present study Therefore, we cannot exclude a possibility that the inhibitory effect of metformin on centrosome amplification may be associated with altered microbiota by administration of metformin Metformin has been shown to have physiological effects similar to those of dietary restriction (Shirazi et al., 2014) We observed that in agreement with the previous report (Slack et al., 2012), concentration of mM metformin used in our experiments did not disrupt intestinal physiology in analyses of fly excreta (Supplementary Fig S2) However, in the present study, we cannot completely exclude the possible dietary restriction effect of metformin Whether or not dietary restriction can inhibits centrosome amplification would be an important question that warrants further exploration How does metformin actually suppress the AKT/TOR pathway? Metformin is known to activate AMPK, which is an inhibitor of mTOR (Owen et al., 2000) However, in a previous study, we showed Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 G Model MAD 10767 1–11 H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 10 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 Q5 536 537 538 539 540 541 542 543 544 ARTICLE IN PRESS that pAMPK is localized in enteroendocrine cells but not in the ISCs/EBs (Na et al., 2013) In contrast, pAKT activity increases in ISCs/EBs with age and oxidative stress, suggesting that the suppressive effect of metformin on the AKT/TOR pathway is not directly associated with AMPK activation Metformin reduces intracellular ROS levels (Halicka et al., 2011) It was reported that metformin reduces the PQ-induced ROS production in an AMPK-independent manner (Algire et al., 2012) The AKT/TOR pathway promotes protein synthesis and cell growth, leading to ROS production, thereby increasing mitochondrial ROS production (Wellen and Thompson, 2011) Therefore, metformin may suppress the AKT/TOR pathway by reducing intracellular ROS levels in the Drosophila midgut Furthermore, we showed that metformin inhibits AKT/TOR signal-induced DNA damage accumulation in midgut ISCs Several recent studies have reported a relationship between the level of AKT/TOR signaling and that of DNA damage accumulation PTEN-deficient cells have been reported to show increased DNA damage (Ming and He, 2012) The AKT hyperactivation leads to DNA damage accumulation in midgut ISCs via ␥H2AvD/8-oxo-dG accumulation (Na et al., 2013) The rpS6 mutants are reported to show increased staining of ␥H2AX in mammals (Khalaileh et al., 2013) The AKT/TOR signaling pathway is a negative regulator of autophagy It was reported that allelic loss of beclin 1, the essential autophagy regulator, increases DNA damage accumulation (␥H2AX foci) (Karantza-Wadsworth et al., 2007; Mathew et al., 2007) and induces supernumerary centrosomes (Mathew et al., 2007), and beclin is proposed as a biomarker of good health, based on its high levels in healthy centenarians (Emanuele et al., 2014) It was also reported that mice with a deletion of Atg5 and Atg7 displayed increased 8-oxo-dG and ␥H2AX foci (Takamura et al., 2011) Therefore, AKT/TOR signaling could increase DNA damage accumulation via inhibiting autophagy Centrosome amplification can be induced by DNA damaging agents, including ionizing radiation (Inanc and Morrison, 2011; Levis and Marin, 1963; Saladino et al., 2009; Yih et al., 2006) We also reported centrosome amplification in Drosophila ISCs by Gy irradiation (Pyo et al., 2014) Centrosome amplification is associated with cell cycle arrest, especially during G2-M phase, due to DNA damage (Nigg, 2002; Xu et al., 1999) Although several studies have reported that AKT/TOR signaling factors are localized in the centrosome (Goshima et al., 2007; Rossi et al., 2007; Wakefield et al., 2003), AKT/TOR signaling-induced DNA damage accumulation may also be a major cause of centrosome amplification due to the altered AKT/TOR signal present in such cases In summary, the present data demonstrate that metformin reduces age-, and oxidative stress-, and TOR signaling-induced centrosome amplification in Drosophila ISCs Our data suggest that metformin may be a beneficial anti-cancer drug via its inhibition of DNA damage-induced centrosome amplification by hyperactivation of AKT/TOR signaling Our study suggests that the Drosophila midgut is a suitable model system for in vivo evaluation of anticancer drugs in aging tissue-resident stem cells Uncited references Cabreiro and Gems (2013) and Cognigni et al (2011) Acknowledgements We would like to thank Benjamin Ohlstein for his generous gift of the fly strains We would also like to thank the Developmental Studies Hybridoma Bank for the antibodies and the Bloomington Stock Center and Vienna Drosophila RNAi Center for the Drosophila stocks We thank Prof Byung P Yu (University of Texas Health Science Center at San Antonio, Texas) for invaluable comments on the manuscript This work was supported by the Basic Science Q6 Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2012029) and the JSPS Core-to Core Program, B Asia-Africa Science Platforms Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mad.2015.05.004 References Algire, C., Amrein, L., Zakikhani, M., Panasci, L., Pollak, M., 2010 Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase Endocr Related Cancer 17, 351–360 Algire, C., Moiseeva, O., Deschenes-Simard, X., Amrein, L., Petruccelli, L., Birman, E., Viollet, B., Ferbeyre, G., Pollak, M.N., 2012 Metformin reduces endogenous reactive oxygen species and associated DNA damage Cancer Prev Res 5, 536–543 Alimova, I.N., Liu, B., Fan, Z., Edgerton, S.M., Dillon, T., Lind, S.E., Thor, A.D., 2009 Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro Cell Cycle 8, 909–915 Aljada, A., Mousa, S.A., 2011 Metformin and neoplasia: implications and indications Pharmacol Ther 133, 108–115 Alliouachene, S., Tuttle, R.L., Boumard, S., Lapointe, T., Berissi, S., Germain, S., Jaubert, F., Tosh, D., Birnbaum, M.J., Pende, M., 2008 Constitutively active Akt1 expression in mouse pancreas requires S6 kinase for insulinoma formation J Clin Invest 118, 3629–3638 Amcheslavsky, A., Ito, N., Jiang, J., Ip, Y.T., 2011 Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells J Cell Biol 193, 695–710 Andriatsilavo, M., Gervais, L., Fons, C., Bardin, A.J., 2013 [The Drosophila midgut as a model to study adult stem cells] Int J Med Sci 29, 75–81 Ashinuma, H., Takiguchi, Y., Kitazono, S., Kitazono-Saitoh, M., Kitamura, A., Chiba, T., Tada, Y., Kurosu, K., Sakaida, E., Sekine, I., Tanabe, N., Iwama, A., Yokosuka, O., Tatsumi, K., 2012 Antiproliferative action of metformin in human lung cancer cell lines Oncol Rep 28, 8–14 Basto, R., Brunk, K., Vinadogrova, T., Peel, N., Franz, A., Khodjakov, A., Raff, J.W., 2008 Centrosome amplification can initiate tumorigenesis in flies Cell 133, 1032–1042 Baur, D.M., Klotsche, J., Hamnvik, O.P., Sievers, C., Pieper, L., Wittchen, H.U., Stalla, G.K., Schmid, R.M., Kales, S.N., Mantzoros, C.S., 2010 Type diabetes mellitus and medications for type diabetes mellitus are associated with risk for and mortality from cancer in a German primary care cohort Metab Clin Exp 60, 1363–1371 Beebe, K., Lee, W.C., Micchelli, C.A., 2009 JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage Dev Biol 338, 28–37 Berstein, L.M., 2012 Metformin in obesity, cancer and aging: addressing controversies Aging 4, 320–329 Bettencourt-Dias, M., Glover, D.M., 2007 Centrosome biogenesis and function: centrosomics brings new understanding Nat Rev 8, 451–463 Biteau, B., Hochmuth, C.E., Jasper, H., 2008 JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut Cell Stem Cell 3, 442–455 Biteau, B., Karpac, J., Supoyo, S., Degennaro, M., Lehmann, R., Jasper, H., 2010 Lifespan extension by preserving proliferative homeostasis in Drosophila PLoS Genet 6, e1001159 Broderick, N.A., Buchon, N., Lemaitre, B., 2014 Microbiota-induced changes in drosophila melanogaster host gene expression and gut morphology mBio 5, e01117–01114 Buchon, N., Broderick, N.A., Chakrabarti, S., Lemaitre, B., 2009 Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila Genes Dev 23, 2333–2344 Cabreiro, F., Au, C., Leung, K.Y., Vergara-Irigaray, N., Cocheme, H.M., Noori, T., Weinkove, D., Schuster, E., Greene, N.D., Gems, D., 2013 Metformin retards aging in C elegans by altering microbial folate and methionine metabolism Cell 153, 228–239 Cabreiro, F., Gems, D., 2013 Worms need microbes too: microbiota, health and aging in Caenorhabditis elegans EMBO Mol Med 5, 1300–1310 Cantrell, L.A., Zhou, C., Mendivil, A., Malloy, K.M., Gehrig, P.A., Bae-Jump, V.L., 2010 Metformin is a potent inhibitor of endometrial cancer cell proliferation – implications for a novel treatment strategy Gynecol Oncol 116, 92–98 Choi, N.H., Kim, J.G., Yang, D.J., Kim, Y.S., Yoo, M.A., 2008 Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor Aging Cell 7, 318–334 Cognigni, P., Bailey, A.P., Miguel-Aliaga, I., 2011 Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis Cell Metab 13, 92–104 D’Assoro, A.B., Lingle, W.L., Salisbury, J.L., 2002 Centrosome amplification and the development of cancer Oncogene 21, 6146–6153 Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 G Model MAD 10767 1–11 ARTICLE IN PRESS H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 Efeyan, A., Zoncu, R., Chang, S., Gumper, I., Snitkin, H., Wolfson, R.L., Kirak, O., Sabatini, D.D., Sabatini, D.M., 2012 Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival Nature 493, 679–683 Emami Riedmaier, A., Fisel, P., Nies, A.T., Schaeffeler, E., Schwab, M., 2013 Metformin and cancer: from the old medicine cabinet to pharmacological pitfalls and prospects Trends Pharmacol Sci 34, 126–135 Emanuele, E., Minoretti, P., Sanchis-Gomar, F., Pareja-Galeano, H., Yilmaz, Y., Garatachea, N., Lucia, A., 2014 Can enhanced autophagy be associated with human longevity? Serum levels of the autophagy biomarker beclin-1 are increased in healthy centenarians Rejuvenation Res 17, 518–524 Feng, Z., Zhang, H., Levine, A.J., Jin, S., 2005 The coordinate regulation of the p53 and mTOR pathways in cells Proc Natl Acad Sci U.S.A 102, 8204–8209 Foster, D.A., 2009 Phosphatidic acid signaling to mTOR: signals for the survival of human cancer cells Biochim Biophys Acta 1791, 949–955 Goshima, G., Wollman, R., Goodwin, S.S., Zhang, N., Scholey, J.M., Vale, R.D., Stuurman, N., 2007 Genes required for mitotic spindle assembly in Drosophila S2 cells Science 316, 417–421 Griswold, C.M., Matthews, A.L., Bewley, K.E., Mahaffey, J.W., 1993 Molecular characterization and rescue of acatalasemic mutants of Drosophila melanogaster Genetics 134, 781–788 Haigis, M.C., Yankner, B.A., 2010 The aging stress response Mol Cell 40, 333–344 Halicka, H.D., Zhao, H., Li, J., Traganos, F., Zhang, S., Lee, M., Darzynkiewicz, Z., 2011 Genome protective effect of metformin as revealed by reduced level of constitutive DNA damage signaling Aging 3, 1028–1038 Inanc, B., Dodson, H., Morrison, C.G., 2010 A centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage Mol Biol Cell 21, 3866–3877 Inanc, B., Morrison, C.G., 2011 Getting permission: how DNA damage causes centrosome amplification Cell Cycle 10, 1890–1891 Isoda, K., Young, J.L., Zirlik, A., MacFarlane, L.A., Tsuboi, N., Gerdes, N., Schonbeck, U., Libby, P., 2006 Metformin inhibits proinflammatory responses and nuclear factor-kappaB in human vascular wall cells Arterioscl Throm Vas Biol 26, 611–617 Jiang, H., Edgar, B.A., 2009 EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors Development 136, 483–493 Jiang, H., Edgar, B.A., 2011 Intestinal stem cells in the adult Drosophila midgut Exp Cell Res 317, 2780–2788 Jiang, H., Grenley, M.O., Bravo, M.J., Blumhagen, R.Z., Edgar, B.A., 2011 EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila Cell Stem Cell 8, 84–95 Jiang, H., Patel, P.H., Kohlmaier, A., Grenley, M.O., McEwen, D.G., Edgar, B.A., 2009 Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut Cell 137, 1343–1355 Kalender, A., Selvaraj, A., Kim, S.Y., Gulati, P., Brule, S., Viollet, B., Kemp, B.E., Bardeesy, N., Dennis, P., Schlager, J.J., Marette, A., Kozma, S.C., Thomas, G., 2010 Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner Cell Metab 11, 390–401 Kapuria, S., Karpac, J., Biteau, B., Hwangbo, D., Jasper, H., 2012 Notch-mediated suppression of TSC2 expression regulates cell differentiation in the Drosophila intestinal stem cell lineage PLoS Genet 8, e1003045 Karantza-Wadsworth, V., Patel, S., Kravchuk, O., Chen, G., Mathew, R., Jin, S., White, E., 2007 Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis Genes Dev 21, 1621–1635 Kato, K., Gong, J., Iwama, H., Kitanaka, A., Tani, J., Miyoshi, H., Nomura, K., Mimura, S., Kobayashi, M., Aritomo, Y., Kobara, H., Mori, H., Himoto, T., Okano, K., Suzuki, Y., Murao, K., Masaki, T., 2012 The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo Mol Cancer Ther 11, 549–560 Khalaileh, A., Dreazen, A., Khatib, A., Apel, R., Swisa, A., Kidess-Bassir, N., Maitra, A., Meyuhas, O., Dor, Y., Zamir, G., 2013 Phosphorylation of ribosomal protein S6 attenuates DNA damage and tumor suppression during development of pancreatic cancer Cancer Res 73, 1811–1820 Lachance, J., Jung, L., True, J.R., 2013 Genetic background and GxE interactions modulate the penetrance of a naturally occurring wing mutation in Drosophila melanogaster G3 3, 1893–1901 Landman, G.W., Kleefstra, N., van Hateren, K.J., Groenier, K.H., Gans, R.O., Bilo, H.J., 2009 Metformin associated with lower cancer mortality in type diabetes: ZODIAC-16 Diabetes Care 33, 322–326 Laplante, M., Sabatini, D.M., 2012 mTOR signaling in growth control and disease Cell 149, 274–293 Leonard, M.K., Hill, N.T., Bubulya, P.A., Kadakia, M.P., 2013 The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes Cell Cycle 12, 1406–1415 Levine, R.L., Cargile, C.B., Blazes, M.S., van Rees, B., Kurman, R.J., Ellenson, L.H., 1998 PTEN mutations and microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid carcinoma Cancer Res 58, 3254–3258 Levis, A.G., Marin, G., 1963 Induction of multipolar spindles by X-radiation in mammalian cells in Vitro Exp Cell Res 31, 448–451 Lewontin, R.C., 2000 The triple helix: gene, organism and environment Nat Med 6, 1206 Liu, L., Rando, T.A., 2011 Manifestations and mechanisms of stem cell aging J Cell Biol 193, 257–266 Mathew, R., Kongara, S., Beaudoin, B., Karp, C.M., Bray, K., Degenhardt, K., Chen, G., Jin, S., White, E., 2007 Autophagy suppresses tumor progression by limiting chromosomal instability Genes Dev 21, 1367–1381 11 McGuire, S.E., Roman, G., Davis, R.L., 2004 Gene expression systems in Drosophila: a synthesis of time and space Trends Genet 20, 384–391 Micchelli, C.A., Perrimon, N., 2006 Evidence that stem cells reside in the adult Drosophila midgut epithelium Nature 439, 475–479 Ming, M., He, Y.Y., 2012 PTEN in DNA damage repair Cancer Lett 319, 125–129 Na, H.J., Park, J.S., Pyo, J.H., Lee, S.H., Jeon, H.J., Kim, Y.S., Yoo, M.A., 2013 Mechanism of metformin: inhibition of DNA damage and proliferative activity in Drosophila midgut stem cell Mech Ageing Dev 134, 381–390 Nakajima, T., Moriguchi, M., Mitsumoto, Y., Sekoguchi, S., Nishikawa, T., Takashima, H., Watanabe, T., Katagishi, T., Kimura, H., Okanoue, T., Kagawa, K., 2004 Centrosome aberration accompanied with p53 mutation can induce genetic instability in hepatocellular carcinoma Mod Pathol 17, 722–727 Nam, H.J., Chae, S., Jang, S.H., Cho, H., Lee, J.H., 2010 The PI3K-Akt mediates oncogenic Met-induced centrosome amplification and chromosome instability Carcinogenesis 31, 1531–1540 Nigg, E.A., 2002 Centrosome aberrations: cause or consequence of cancer progression? Nat Rev Cancer 2, 815–825 O’Brien, L.E., Soliman, S.S., Li, X., Bilder, D., 2011 Altered modes of stem cell division drive adaptive intestinal growth Cell 147, 603–614 Ohlstein, B., Spradling, A., 2006 The adult Drosophila posterior midgut is maintained by pluripotent stem cells Nature 439, 470–474 Ohlstein, B., Spradling, A., 2007 Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling Science 315, 988–992 Owen, M.R., Doran, E., Halestrap, A.P., 2000 Evidence that metformin exerts its anti-diabetic effects through inhibition of complex of the mitochondrial respiratory chain Biochem J 348 (Pt 3), 607–614 Park, J.S., Kim, Y.S., Yoo, M.A., 2009 The role of p38b MAPK in age-related modulation of intestinal stem cell proliferation and differentiation in Drosophila Aging 1, 637–651 Park, J.S., Lee, S.H., Na, H.J., Pyo, J.H., Kim, Y.S., Yoo, M.A., 2012 Age- and oxidative stress-induced DNA damage in Drosophila intestinal stem cells as marked by Gamma-H2AX Exp Gerontol 47, 401–405 Park, J.S., Pyo, J.H., Na, H.J., Jeon, H.J., Kim, Y.S., Arking, R., Yoo, M.A., 2014 Increased centrosome amplification in aged stem cells of the Drosophila midgut Biochem Biophys Res Commun 450, 961–965 Pihan, G.A., Wallace, J., Zhou, Y., Doxsey, S.J., 2003 Centrosome abnormalities and chromosome instability occur together in pre-invasive carcinomas Cancer Res 63, 1398–1404 Pyo, J.H., Park, J.S., Na, H.J., Jeon, H.J., Lee, S.H., Kim, J.G., Park, S.Y., Jin, Y.W., Kim, Y.S., Yoo, M.A., 2014 Functional modification of Drosophila intestinal stem cells by ionizing radiation Radiat Res 181, 376–386 Rao, C.V., Yamada, H.Y., Yao, Y., Dai, W., 2009 Enhanced genomic instabilities caused by deregulated microtubule dynamics and chromosome segregation: a perspective from genetic studies in mice Carcinogenesis 30, 1469–1474 Ren, F., Wang, B., Yue, T., Yun, E.Y., Ip, Y.T., Jiang, J., 2010 Hippo signaling regulates Drosophila intestine stem cell proliferation through multiple pathways Proc Natl Acad Sci U.S.A 107, 21064–21069 Rossi, R., Pester, J.M., McDowell, M., Soza, S., Montecucco, A., Lee-Fruman, K.K., 2007 Identification of S6K2 as a centrosome-located kinase FEBS Lett 581, 4058–4064 Saito, T., Chiba, T., Yuki, K., Zen, Y., Oshima, M., Koide, S., Motoyama, T., Ogasawara, S., Suzuki, E., Ooka, Y., Tawada, A., Tada, M., Kanai, F., Takiguchi, Y., Iwama, A., Yokosuka, O., 2013 Metformin, a diabetes drug, eliminates tumor-initiating hepatocellular carcinoma cells PLoS One 8, e70010 Saladino, C., Bourke, E., Conroy, P.C., Morrison, C.G., 2009 Centriole separation in DNA damage-induced centrosome amplification Environ Mol Mutagen 50, 725–732 Shirazi, F., Farmakiotis, D., Yan, Y., Albert, N., Kim-Anh, D., Kontoyiannis, D.P., 2014 Diet modification and metformin have a beneficial effect in a fly model of obesity and mucormycosis PLoS One 9, e108635 Slack, C., Foley, A., Partridge, L., 2012 Activation of AMPK by the putative dietary restriction mimetic metformin is insufficient to extend lifespan in Drosophila PLoS One 7, e47699 Takamura, A., Komatsu, M., Hara, T., Sakamoto, A., Kishi, C., Waguri, S., Eishi, Y., Hino, O., Tanaka, K., Mizushima, N., 2011 Autophagy-deficient mice develop multiple liver tumors Genes Dev 25, 795–800 Wakefield, J.G., Stephens, D.J., Tavare, J.M., 2003 A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment J Cell Sci 116, 637–646 Wellen, K.E., Thompson, C.B., 2011 Cellular metabolic stress: considering how cells respond to nutrient excess Mol Cell 40, 323–332 Xu, N., Wang, S.Q., Tan, D., Gao, Y., Lin, G., Xi, R., 2011 EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells Dev Biol 354, 31–43 Xu, X., Weaver, Z., Linke, S.P., Li, C., Gotay, J., Wang, X.W., Harris, C.C., Ried, T., Deng, C.X., 1999 Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells Mol Cell 3, 389–395 Yang, Z., Ming, X.F., 2012 mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases Obes Rev 13 (Suppl 2), 58–68 Yih, L.H., Tseng, Y.Y., Wu, Y.C., Lee, T.C., 2006 Induction of centrosome amplification during arsenite-induced mitotic arrest in CGL-2 cells Cancer Res 66, 2098–2106 Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 ... Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age- related centrosome amplification in Drosophila midgut stem cells through AKT/ TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004... Please cite this article in press as: Na, H.-J., et al., Metformin inhibits age- related centrosome amplification in Drosophila midgut stem cells through AKT/ TOR pathway Mech Ageing Dev (2015), http://dx.doi.org/10.1016/j.mad.2015.05.004... (3) metformin inhibits AKT/ TOR signal-induced DNA damage accumulation in midgut ISCs We showed that metformin reduced centrosome amplification in midgut ISCs from flies undergoing aging and intrinsic