Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original treatment, and provide evidence that metformin may function through a mechanism involving post-translational histone modifications via an indirect histone deacetylase inhibitor (HDACi) activity. We find that metformin, at low physiological concentrations, reduces the expression of multiple classic protein markers of MDR in vitro and in preliminary in vivo models. Our demonstration that metformin can prevent MDR development and resensitize MDR cells to chemotherapy in vitro, provides important medical relevance towards metformin’s potential clinical use against MDR cancers.

Funding: Funded by Canadian Breast Cancer Foundation (TH and TA), Saskatchewan Health Research Foundation (TH, TA, JRG, GG), Canadian Foundation for Innovation (TH and TA), and Department of Medicine, University of Saskatchewan (TA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2017 Davies et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To study the underlying pathways necessary for the antiproliferative effect of metformin, as well as a direct test of the utility of metformin in preventing acquired drug resistance, we used the widely studied MCF7 breast cancer cell line and selected them for Doxorubicin (DOX) resistance. Our accelerated selection protocol occurs over ~2 weeks, generating cell populations that exhibits enhanced cell viability upon pulse exposure to normally toxic doses of the selected drug, exhibits resistance to previously unexposed drug classes, and expresses high levels of one or more of BCRP, MDR-1, or HIF1α [ 14 ]. The following details our studies testing our hypothesis that metformin can potentially reverse and prevent MDR development, and offer a means to elucidate molecular pathways impacted by metformin antiproliferative activity.

Early diagnosis, screening and advances in combination therapy of breast cancer has resulted in longer disease-free survival times and cure rates [ 5 , 6 ]. Nonetheless, many of these patients return years later with recrudescent tumors [ 2 ]. Providing hope for the identification of nontoxic therapies and a means to prevent MDR development in this important population of treatment resistant breast cancer survivors is our ultimate goal. To this end, we primarily utilize breast cancer cell lines selected in vitro for treatment resistance as our fundamental model of MDR (see Davies et al 2009 and 2014). Below we describe our investigations into the potential utility of metformin as adjunct therapy in the treatment of established MDR and in preventing the development of new treatment resistance. The oral insulin-sensitizing drug metformin is a first line therapeutic in the management of Type 2 diabetes (DM2), and has also been shown to have antiproliferative activity in vitro against multiple cancer cells lines [ 7 , 8 ]. An early meta-analysis performed on DM2 patients taking metformin with cancer reported a 31% reduction in the incidence of new cancers including pancreas, colorectal, breast and lung [ 9 ]. Recent meta-analyses confirm that individuals with DM2 who also have lung, colorectal and liver cancer derive significant survival benefits regarding clinical outcomes if also on metformin [ 10 – 12 ]. Patients with breast cancer benefited from metformin treatment in terms of all cause survival, but not in incidence [ 13 ]. To date, however, the molecular mechanisms facilitating metformin’s antiproliferative impact remains unclear. It also remains untested whether metformin pretreatment can provide a benefit to established MDR malignancy or interfere with the development of acquired drug resistance.

Resistance to therapies can be inherent at diagnosis, or acquired in subpopulations of silent surviving cells that reappear years later after an initial apparently successful treatment regime [ 2 ]. Resistance to one agent is frequently manifested as resistance to many, hence the term “multiple drug resistance” or MDR. This was recognized many years ago, and is reflected in our current chemotherapy cocktails that incorporate multiple therapeutic agents with unrelated modes of action to decrease the rate of new treatment resistance. Well-recognized molecular mechanisms of resistance are known and have been recently reviewed [ 3 ]. At the forefront is the ABC family of drug-efflux pumps whose over-activity has been consistently associated with drug resistance. Despite being novel drug therapy targets, clinical trials using ABC transporter inhibitors have been disappointing in part because of their toxicities [ 4 ], but also because they are unlikely to account for all mechanisms of resistance that are present. MDR appears to be a complex and multifactorial adaptation by cancer cells that enables survival of these cell population subsets, and it is clear that individual cancers appear to highjack more than one mechanism, without a single overriding molecular target accountable for all.

An understanding of how and why tumor cells develop multiple drug resistance (MDR) has remained a significant question in cancer research, and its elucidation may identify future targets to prevent, or reverse, treatment resistance. For many common malignancies, the delayed emergence of MDR following initial successful responses is a devastating event. Breast, colon, lung and hematological cancers are common cancers that have high rates of acquired treatment resistance [ 1 ]. Minimally effective therapies, and poorly tolerated side effects from the necessary rescue therapies raises the importance of understanding the underlying biology within this patient population to overcome this important clinical development. Discovering non-toxic, effective treatment options to overcome treatment resistance, or perhaps prevent MDR development in the first place, is of great interest.

K562 Doxorubicin-resistant cells (1 x 10 6 cells/mouse in 100 μL of PBS) were injected subcutaneously into the sacral region. Metformin (130 mg/kg) was administered by intraperitoneal (i.p.) injection on alternating days, starting when tumors were ~25 mm 3 , as measured by calipers every other day (~11 days after seeding tumor cells). Control mice received vehicle only (200 μL PBS). At day 28, tumors were surgically excised and analyzed (n = 2 per treatment arm). The 4–28 tumor was obtained from a patient with triple negative (TN) breast cancer with written informed consent from the donor, and compliant with the Research Ethic board approved protocol at the University of Saskatchewan. A fragment from the original tumor was passaged 8 times in NSG mice; the resultant tumor was excised and ~2 mm fragments were grafted subcutaneously into 4 separate NSG mice. On day 37 metformin was delivered to each mouse as 0, 50, 100 or 200 mg/kg per i.p. injection. The mice were sacrificed after 72 hours of receiving metformin, with tumors surgically removed and analyzed (n = 1 per treatment arm).

Female NOD/SCID/common gamma-chain knock-out (NSG: NOD/Prkdc SCID /IL2RN -/- ) 8 to 14 week-old mice (Jackson Laboratory, USA) were used, and fed ad libitum. All experiments were approved by the University of Saskatchewan animal ethics office, in accord with the guidelines of the Canadian Council on Animal Care.

MCF7 cells were transfected with 1 μg of duplex human AMPKα1/2, NFκB or scrambled siRNA according to the manufacturers’ instructions (Santa Cruz Biotechnology). 1 μg (80 pmol) of targeted siRNA was delivered to 50 μl of the cationic delivery agent Lipofectamine RNAiMAX (Gibco) for 30 minutes at RT, then incubated for 72 hours followed by drug treatments.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and a non-radioactive thymidine analogue 5-ethynyl-2’-deoxyuridine (EdU) kit were used to measure cell viability and proliferation. For thymidine, the Click-IT kit (Life Technologies) labeled cells with EdU for 24 hours. For trypan blue assays (TB), MCF7 cells were cultured to 70% confluency in 6-well multiwell tissue culture dishes and treated in triplicate with metformin at the dosages indicated for 48 hours, trypsinized and centrifuged to pellet in microtubes. The pellet was treated with 0.25% TB solution in PBS (Hyclone) and cell viability was assessed using a TC20 automated cell counter (Biorad).

Adherent MCF7 cells were scraped, and centrifuged with sterile PBS for collection and resuspended in RIPA buffer followed by pulse sonication. Westerns were performed as described [ 14 ]. Antibodies against the following proteins were used, typically at 1:2000 dilution: MDR-1 (Sigma), BCRP (Santa Cruz Biotechnology; SCBt), HIF1α (Abcam), S6K total (SCBt), S6K S411phos (SCBt), p53 (SCBt), p53 S392phos (Abcam), TFPI1 (Abcam), AMPKα1/2 total (SCBt), AMPKα1 T183 /2 T172phos (Abcam), AKT total (SCBt), AKT S473phos (SCBt), PARP (Sigma), ERα (SCBt), histone H3 total (Millipore), H3 K9Ac (Millipore), H2B total (Abcam), H4 K12Ac (Abcam), NFκB (SCBt), NREL (SCBt), tubulin (Sigma), actin (Sigma), and GAPDH (Millipore). Luminescence was captured on film (Kodak) with subsequent chemical development. Collection and semiquantitation of Western blots densitometry was done using ImageJ Version 1.51 from scans of the original film.

MCF7 and T47D ER + , and BT-20 and MDA-MB-231 ER - human breast cancer, and K562 leukemia cells were obtained from commercial sources; the American Type Culture Collection (ATCC), USA. The chemicals Doxorubicin hydrochloride (DOX; Pfizer), Tamoxifen (TAM; Cayman Chemical), phenformin (Sigma), Trichostatin A (TSA; Sigma), estradiol (Cayman Chemical), Apicidin (Sigma), and Troglitazone (TRG; Calbiochem) were acquired from the indicated providers. All treatment compounds were reconstituted in dimethylsulfoxide (DMSO) except metformin (Sigma), which was reconstituted in molecular-grade water (Hyclone). The HDACi assay and in vitro hypoxia experiments were conducted as previously described [ 14 ]. MCF7 and K562 parental cells were selected for drug resistance according to our published methods [ 14 , 15 ].

Results

Metformin has antiproliferative potential against drug resistant cell populations in vitro, and works additively with Doxorubicin Our in vitro biological model of drug resistance made use of the well characterized and widely utilized estrogen receptor positive (ERα+) MCF7 breast cancer cell line (parental), and its paired cell line selected for Doxorubicin resistance (DOXRes), using our previously published in vitro protocol [14, 15]. Cells selected against DOX using our protocol are considered MDR due to resistance against unrelated drugs, including metformin [16] (Fig 1A). The dosing of metformin in vitro is a contentious issue with concerns that supra-physiological doses result in off-target effects and are not reflective of in vivo events. Our in vitro metformin dosing ranges were from 0.1 mM to 5 mM; in comparison, the in vivo trough human serum levels of metformin (average 1500 mg oral daily dose) has been reported at 2–6 μM [17], with peak levels of 38 μM and steady state ranges of 15.5 μM [18]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Metformin possesses antiproliferative activity. (A) MCF7 parental and DOXRes cells were treated with three doses of metformin over 48 hours, with cell proliferation measured using MTT assays on three biological repeats. (B) MCF7 parental and DOXRes cells were treated with metformin and/or DOX for 48 hours, in triplicate, with cell proliferation measured using MTT assays from three biological repeats. (C) Protein extracts were prepared from the MCF7 parental cells used in (A) and analyzed using Western analysis with the antibodies shown. The blot shown is representative of three reproducible experiments. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant. https://doi.org/10.1371/journal.pone.0187191.g001 We measured the proliferation of treatment sensitive (MCF7 parental) versus resistant (DOXRes) MCF7 cell populations following a 48-hour metformin exposure. Metformin impaired growth in a dose-dependent manner in both populations of MCF7, but to a significantly lesser degree in DOXRes cells (Fig 1A). To determine whether metformin could enhance DOX cytotoxicity as combination therapy in MCF7 DOXRes cells, we treated MCF7 parental and DOXRes cells with DOX (1 μM) and metformin (1 mM), individually and in combination. The results show that there is an additive increase in killing when metformin is combined with DOX in both sensitive and resistant cell populations (Fig 1B). Metformin activity was confirmed, as AMPKα1T183/2T172phos and AKTS473phos was relatively increased, and decreased, respectively, as expected [19, 20] (Fig 1C). Furthermore, the reduced proliferation observed with metformin exposure was correlated with enhanced apoptosis (Fig 1C) (measured as poly(ADP-ribose) polymerase (PARP) cleavage), as has been previously reported [21, 22].

Metformin has enhanced antiproliferative potential in ERα+ cells and decreases total ERα protein levels The estrogen receptor α (ERα+) status of breast cancer cells is utilized as both a prognostic and predictive marker of cancer outcomes; triple negative (TN) breast cancers lack the ERα, progesterone (PR) and HER2 receptors and are inherently more difficult to treat, whereas ERα+ tumors respond well to therapies and are uniquely suited to adjunct use of anti-estrogen therapy [23]. We showed above that metformin had antiproliferative activity against ERα+ cells (Fig 1), and we next investigated whether metformin had a similar, or different, impact on ERα+ versus ERα− cell lines. We exposed ERα+ (T47D and MCF7) and TN (BT-20 and MD-MBA-231) breast cancer cells to metformin with cell proliferation measured by MTT analyses (Fig 2A). The results show that metformin had antiproliferative potential regardless of ERα status, but had a noticeably greater impact against ERα+ cells. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Metformin is effective against ERα- and ERα+ breast cancer cells, and decreases ERα levels. (A) ERα− (BT-20 and MDA-MB-231) cells, and ERα+ (T47D and MCF7) cells were treated, in triplicate, with 5 mM metformin (+), or left untreated (−), for 48 hours, with cell proliferation measured by MTT from two biological repeats. p < 0.001 for all cell lines comparing 0 mM to 5 mM metformin, n = 6 samples analysed. (B) Protein lysates were prepared from MCF7 cells treated with 5 mM metformin, or left untreated, for 48 hrs. The lysates were then tested by Western analysis using the antibodies shown. These results are reproducible over three separate experiments. (C) MCF7 cells were treated with metformin and/or Tamoxifen (TAM), for 48 hours and analyzed as described above for (A). Values normalized to control. n = 3 samples analyzed. * = p < 0.05. https://doi.org/10.1371/journal.pone.0187191.g002 Malignancies of the breast can be hormone-responsive, including acting through estrogen binding to ERα [24], with ER modulators such as tamoxifen typically used as adjuvant therapy [25]. To determine whether metformins’ greater influence on ERα+ cells reflects modulation of the ERα protein, we determined ERα total protein levels compared to untreated controls, and noted that 5 mM metformin markedly reduced ERα abundance (Fig 2B). Western analyses of AMPK phosphorylation confirmed the expected AMPK phosphorylation/activation with metformin exposure (Fig 2B). Tamoxifen interferes with ERα function, and we asked whether Tamoxifen and metformin have similar antiproliferative impacts on ERα. We found that the combination of metformin in combination with Tamoxifen is more effective than either compound alone in halting the growth of MCF7 parental cells (Fig 2C). Metformin may, therefore, have additional functions that are independent of the ERα effects mediated by Tamoxifen.

Metformin decreases the abundance of MDR markers in vitro We previously reported that troglitazone (TRG), an insulin sensitizer of the thiazolidinedione class used in the treatment of DM2 (metformin is an unrelated biguanide insulin sensitizer), reduced the protein levels of the ABC transporter class of drug efflux pumps elevated in multiple aggressive cancer cells (MDR-1 and BCRP in MCF7 DOXRes, and MDR-1 in K562 DOXRes cells) [15]. Additional recent reports by others indicate that metformin may also possess this ability in vitro [26, 27]. Considering that metformin reduced the abundance of ERα (Fig 2B), and decreased the proliferation of MCF7 parental and DOXRes cells, we asked if metformin could decrease the abundance of multiple markers of MDR as a means of explaining the enhanced treatment sensitivity of MDR cells upon metformin treatment. We also asked if metformin had effects across drug-resistant cell populations of unrelated cancer types. We first looked in vitro at our MCF7 cell line selected for resistance to DOX (Fig 3A). We directly compared the relative protein abundance of MDR markers between parental and MCF7 DOXRes cells across a wide concentration range of metformin. At baseline without metformin (Fig 3A, compare 0 lanes), the DOXRes population displayed enhanced protein abundance of these MDR protein markers compared to sensitive cell populations. In turn, there was a trend to decrease the abundance of MDR-1, HIF1α, p53S392phos (versus total p53) and S6KS411phos (versus total S6K) in a dose-dependent manner that was much more prevalent in the MCF7 DOXRes cell population. We included an assessment of p53 phos S392 since p53phosS392 in MCF7 parental cells is a beneficial activating and stabilizing signal that acts to target cancer cells for apoptosis [28], but in aggressive cancer cells, p53phos392 is often elevated, with this contributing to tumor progression [29, 30]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Metformin reverses the expression of cancer related proteins in vitro and in vivo. (A) MCF7 parental and DOXRes cells were treated with a range of metformin concentrations for 48 hours. Equal protein loads of the cell lysates were prepared for Western analyses using the antibodies shown. Results are consistent with three repeat experiments. (B) T47D cells were treated with the drugs shown while maintained in a 1% O 2 hypoxia chamber for 24 hours. Cell lysates were prepared and analyzed using the antibodies shown, representative of two consistent experimental repeats. (C) A preliminary study of 2 mice per treatment arm: NOD/SCID mice harboring palpable tumors derived from K562 DOXRes cells were treated with metformin i.p. (130 mg/kg) or saline (considered Day 1). Tumors were excised after 28 days and processed for Westerns using the antibodies shown. The same protein concentration of each lysates was used for all western analysis, with tubulin representing a protein loading control for all. (D) A preliminary study of acute metformin effects on tumor markers in vivo: NOD/SCID mice growing tumors derived from a human patient with triple negative breast cancer were treated with single specific doses of metformin by i.p (0, 50, 100 and 200 mg/kg body weight) as indicated. 72 hours later, the tumors were excised from each mouse, protein lysates were prepared, equal protein amounts separated by SDS PAGE and assessed by Western analysis using the antibodies indicated. Actin represents the protein loading control. PDX: patient-derived xenograft. https://doi.org/10.1371/journal.pone.0187191.g003 We observed several marked differences in the abundance of MDR protein markers between MCF7-sensitive and -resistant cell populations in response to metformin (Fig 3A). First, in parental/sensitive cells, HIF1α protein levels were consistently increased by metformin, even at the lowest concentration tested. Secondly, abundance of phospho-, but not total p53 and S6K were enhanced in parental cells in a dose-dependent manner (Fig 3A, left panels). Next, in resistant populations (MCF7 DOXRes), metformin reduced p53phos392 levels in a dose-dependent manner, which was also observed for the protein levels of HIF1α, MDR-1 and S6KS411pho (Fig 3A, right panels). Taken together, these results are consistent with metformin activity being context dependent, with metformin playing distinct roles in treatment sensitive versus resistant MCF7 cells. Metformin exposure decreased HIF1α protein levels under normal oxygenation levels (Fig 3A). Hypoxia is a powerful inducer of HIF1α expression, just as the hypoxia present in the intratumoral environment is considered a major driver of aggressive and treatment resistant tumors [31, 32]. We queried whether metformin was capable of blocking the accumulation of HIF1α under strong hypoxic induction conditions. We observed that hypoxia was sufficient to markedly induce the expression of HIF1α protein (Fig 3B), but that metformin was the most effective agent tested that blocked hypoxia-induced HIF1α protein abundance. Indeed, it was more effective than a second biguanide, phenformin, in inhibiting HIF1α protein expression. Unlike metformin however, phenformin is not in clinical use.

Metformin dependent reversal of MDR-associated protein markers occurs in vivo in breast and non-breast cancers We next asked if metformin demonstrated similar efficacy in lowering MDR markers in an in vivo model. In this preliminary study, we grew human tumors in mice from both leukemia cell lines (K562) and patient-derived (PDX) breast cancer cells. Specifically, we injected severely immunodeficient NOD-SCID-IL-2Rgamma-/- (NSG) mice with K562 human myelogenous leukemia cells selected for DOXRes [16], but also xenografted tumor fragments obtained from a patient-derived TN partially-resistant breast tumor (based on actual clinical response) into NSG mice (4–28). Once tumors formed (~25 mm3; day 11 for K562 DOXRes cells, and day 37 for 4–28 tumor slices), metformin was administered by i.p. injection. NSG mice growing K562 DOXRes cells were injected on alternating days (days 11–28) and sacrificed on day 28, whereas the 4–28 tumor-bearing mice were sacrificed 72 hours after a single metformin treatment. The excised tumor tissue was assessed for changes in MDR-1 and TFPI1 protein abundance in response to metformin exposure (we previously found TFPI1 involved in MDR development [14]) (Fig 3C), as well as HIF1α protein abundance (Fig 3D). Metformin monotherapy did not decrease the size, nor slow the growth of the K562 DOXRes tumors, as compared to the untreated control arm in vivo in this pilot test. Despite this lack of clinical response, the MDR-1 and TFPI1 protein levels did noticeably decrease in the metformin treated arms (Fig 3C). Metformin was physiologically active in the NSG mice, based on the ready detection of AMPK phosphorylation in the metformin treatment arm (Fig 3C). In our metformin dose response pilot study using 4–28 human patient-derived tumor fragments, HIF1α levels were reduced even at the lowest metformin concentration (50 mg/kg; Fig 3D). The above results suggest that metformin reduces MDR markers both in vitro and in vivo, in breast and non-breast cancers.

Metformin-dependent reversal of MDR markers in resistant cell populations is AMPK-independent We next investigated whether metformins’ antiproliferative activity is NFκB–or AMPK-dependent, and tested this in both sensitive and resistant cell populations. A previous report suggested that the metformin-dependent decline of MDR-1 protein levels in MDR cells is mediated through inhibition of NFκB activity [26]. However, we observed that NFκB is reduced in MCF7 DOXRes cells, and that metformin did not appreciably impact NFκB protein levels in the parental cell population in a dose dependent manner (S1 Fig). Consistent with this, we found that silencing NFκB did not impact metformin antiproliferative activity (S2 and S3 Figs). We conclude that NFκB is not required for the antiproliferative activity of metformin on treatment resistant MCF7 cells. The metabolic beneficial activity of metformin in DM2 is believed to occur in part via the phosphorylation and activation of AMPK [33]. Studies have shown that metformin facilitates the phosphorylation of AMPK indirectly through the action of the upstream kinase LKB1 [34,35]. In contrast to the known contribution of AMPK/LKB1 in metformin’s glucoregulatory activities, it remains unclear how metformins’ antiproliferative activity is regulated, and is still being debated [36–38]. A recent report indicated that metformin required AMPK, but not LKB1, to halt the proliferation of human non-small cell lung cancer cells [37]. To test the AMPK-dependence of metformin-induced decreases in MDR-associated proteins in breast cancer, we silenced AMPK in treatment sensitive and resistant MCF7 cell populations (Fig 4). As noted previously, there was an increase of MDR-1 and BCRP markers in the treatment resistant cell population, in the absence of treatment (Fig 4B, lane 1 vs. 5). In the resistant cell population, metformin tended to decrease both MDR-1 and BCRP protein levels regardless of AMPK silencing (Fig 4B, Lane 5 & 6 vs. Lane 7 & 8). In contrast, metformin treatment in parental MCF7 cells caused MDR-1 and BCRP levels to increase in controls cells (scrambled siRNA; scr), which was partially reversed in AMPK-silenced cells (Fig 4B, left panel). One interpretation of this is to suggest that in DOXRes cells, metformin acts in an AMPK-independent manner. This suggests that MDR-1 and BCRP, as members of a stress response system [39] are activated in the presence of metformin, and this appears to be AMPK-dependent. This supports the possibility that metformin uses different cellular machinery depending on sensitive versus drug resistant cell populations. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. AMPKα1/2 is not required for metformin activity on cancer protein abundance. (A) Scrambled (scr) and targeted siRNAs (si) against AMPKα1/2 were transfected into MCF7 parental cells. Lysates were prepared and Westerns were performed using the antibodies shown. Immunoblot results were consistent over three biological repeat experiments. (B) AMPKα1/2 was silenced in MCF7 parental and DOXRes cells, followed by metformin addition. Controls were left untreated. Cell lysates were prepared and assessed using the antibodies shown: the immunoblot represents consistent results observed for three biological repeats. https://doi.org/10.1371/journal.pone.0187191.g004

Metformin possesses indirect HDACi activity We had previously observed that the non-biguanide insulin sensitizer Troglitazone (TRG), a thiazolidinedione class of drugs used to treat DM2, increased histone posttranslational modifications (15, 16, 40),. We asked if metformin was also capable of modifying histones, given that this modification may alter gene expression and explain in part the altered MDR-marker protein levels we have seen with metformin exposure. We performed Western analyses of histone H3 Lys9 acetylation (H3K9Ac) in MCF7 cells with increasing durations of metformin, TRG and a second biguanide, phenformin, and observed that a fixed dose of metformin tended to increase H3K9Ac in a time dependent manner (Fig 5A) and that metformin and phenformin both exhibited a comparable temporal induction of histone acetylation to that of TRG. Metformin also induced H4 acetylation (H4K12Ac) in a dose dependent manner (Fig 5B), while total histone H2B remained unchanged with time and dose (Fig 5A and 5B). This may be due to an underlying histone deacetylase inhibitor (HDACi) activity. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Metformin possesses indirect HDACi activity. (A) MCF7 parental cells were treated with metformin (5 mM), TRG (100 μM), or phenformin (5 mM) for 24 hours. Cells were removed at the times shown and lysates assessed by Western analysis using the antibodies indicated. The immunoblot is representative of duplicate biological repeats. (B) Western analyses of MCF7 cells following 48-hour exposures to various metformin doses. Primary antibodies are indicated, and represent three consistent repeat experiments. (C) MCF7 cells were treated with doses of metformin (5 mM) and/or TRG (50 μM) for 48 hours. Proliferation was measured by MTT assays from four biological repeats. n = 4 samples analyzed (D) HDAC assays were performed on lysates obtained from T47D cells treated with the drugs shown for 48 hours. HDAC activity was measured on three biological repeats, and reported as % Activity using arbitrary units (AU) normalized to control. n = 6 samples analyzed. (E) 80 μg of protein prepared from parental T47D cells was treated with the indicated concentrations of TSA, metformin or phenformin for 30 minutes at 30°C. HDAC activity was measured in duplicate, on three biological repeats. n = 6 samples analyzed. * = p < 0.05, *** = p < 0.001, ns = not significant. https://doi.org/10.1371/journal.pone.0187191.g005 Although from unrelated drug classes, we found the insulin sensitizers TRG and metformin to induce global histone acetylation, and to exert cytotoxic effects on MCF7 cells. To determine if they are acting through separate or similar pathways, we compared the cytotoxic effects of the drugs when used alone or in combination. The results show that cell proliferation is most greatly impaired when both metformin and TRG are used in combination (Fig 5C), supporting the idea that separate pathways are utilized by TRG and metformin when it comes to inhibiting cancer cell proliferation. We had previously provided evidence that increased global histone acetylation in response to TRG exposure was due to an inherent and direct HDACi activity [40]. This is highly relevant, as HDACi’s possess antiproliferative activity in cancer cells and are presently in phase III clinical trials [41,42]. To test whether the increases in histone acetylation noted here with metformin are also due to HDACi activity, we performed in vitro quantitative assays to measure the deacetylation of an acetylated peptide, as done previously [40]. To test for the presence of indirect histone deacetylation activity, we measured HDAC activity in whole cell lysates collected after the cells were exposed to the classic HDACi Trichostatin A (TSA), as well as the biguanides metformin and phenformin. The lysates were incubated with acetylated peptides that fluoresce when deacetylated, allowing quantitative measurement. Compared to untreated controls, all chemical agents significantly inhibited HDAC activity (Fig 5D). Since this could occur via a cell signaling mechanism within whole cells (indirectly) and not by direct inhibition of HDAC enzymes, we repeated the assay in untreated cell lysates to determine if there is indirect HDACi activity. TSA again blocked the deacetylation of the peptide, whereas metformin and phenformin did not (Fig 5E). Together, these results are consistent with the biguanide compounds, metformin and phenformin, indirectly inhibiting HDAC activity, rather than by directly affecting HDAC enzymes.