(6)-Gingerolinduced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression

Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Original Contribution

(6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression Namrata Rastogi a,1, Rishi Kumar Gara a,1, Rachana Trivedi a, Akanksha Singh b, Preety Dixit b, Rakesh Maurya b, Shivali Duggal c, M.L.B. Bhatt c, Sarika Singh d, Durga Prasad Mishra a,n a

Endocrinology Division, Central Drug Research Institute, Uttar Pradesh 226021, India Medicinal and Process Chemistry Division, Central Drug Research Institute, Uttar Pradesh 226021, India c Department of Radiotherapy, CSM Medical University, Lucknow, Uttar Pradesh 226003, India d Toxicology Division, Central Drug Research Institute, Lucknow, Uttar Pradesh 226001, India b

art ic l e i nf o

a b s t r a c t

Article history: Received 24 March 2013 Received in revised form 12 December 2013 Accepted 16 December 2013

The natural polyphenolic alkanone (6)-gingerol (6G) has established anti-inflammatory and antitumoral properties. However, its precise mechanism of action in myeloid leukemia cells is unclear. In this study, we investigated the effects of 6G on myeloid leukemia cells in vitro and in vivo. The results of this study showed that 6G inhibited proliferation of myeloid leukemia cell lines and primary myeloid leukemia cells while sparing the normal peripheral blood mononuclear cells, in a concentration- and time-dependent manner. Mechanistic studies using U937 and K562 cell lines revealed that 6G treatment induced reactive oxygen species (ROS) generation by inhibiting mitochondrial respiratory complex I (MRC I), which in turn increased the expression of the oxidative stress response-associated microRNA miR-27b and DNA damage. Elevated miR-27b expression inhibited PPARγ, with subsequent inhibition of the inflammatory cytokine gene expression associated with the oncogenic NF-κB pathway, whereas the increased DNA damage led to G2/M cell cycle arrest. The 6G-induced effects were abolished in the presence of anti-miR27b or the ROS scavenger N-acetylcysteine. In addition, the results of the in vivo xenograft experiments in mice indicated that 6G treatment inhibited tumor cell proliferation and induced apoptosis, in agreement with the in vitro studies. Our data provide new evidence that 6G-induced myeloid leukemia cell death is initiated by reactive oxygen species and mediated through an increase in miR-27b expression and DNA damage. The dual induction of increased miR-27b expression and DNA damageassociated cell cycle arrest by 6G may have implications for myeloid leukemia treatment. & 2014 Published by Elsevier Inc.

Keywords: (6)-Gingerol Myeloid leukemia ROS MiR-27b DNA damage G2/M arrest Apoptosis Free radicals

Myeloid leukemia is characterized by an impaired hematopoietic process with rapid proliferation of undifferentiated and immature blood cells of the myeloid lineage [1]. Chemotherapeutic treatment strategies for both acute and chronic myeloid leukemia are effective at the early stages of the disease. However, major limitations of standard chemotherapy in the clinical setting are

Abbreviations: 6G, (6)-gingerol; iNOS, inducible nitric oxide synthase; NF-κB , nuclear factor κB; miRNA, microRNA; MRC, mitochondrial respiratory complex; ETC, electron transport chain; PBMC, peripheral blood mononuclear cell; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; NHE-1, sodium/hydrogen exchanger 1; ROS, reactive oxygen species; PARP, poly(ADP ribose) polymerase; PPARγ, peroxisome proliferator-activated receptor γ; miR-27b, microRNA 27b; PI, propidium iodide; NAC, N-acetylcysteine; DMSO, dimethyl sulfoxide; PCNA, proliferating cell nuclear antigen; IL, interleukin; TUNEL, terminal deoxynucleotidyl transferase enzyme-mediated dUTP nick-end labeling n Corresponding author. Fax: þ 91 522 2623405. E-mail addresses: [email protected], [email protected] (D.P. Mishra). 1 These authors contributed equally to this study.

side effects, such as cardiac and renal toxicity, severe myelosuppression, and development of chemoresistance, leading to poor survival outcomes [2–5]. Therefore it is imperative to look for novel therapeutic agents with lesser side effects urgently to address the underlying causes of poor treatment outcomes associated with conventional therapeutics. In search of novel yet nontoxic chemotherapeutic agents, attention has been focused on natural agents in recent times [6–11]. Many natural polyphenolic compounds have been known to restrict cancer cell proliferation through distinct mechanisms [12]. These compounds are preferred either as single agents or as adjuvants for chemotherapy owing to their immense antioxidative potential, lesser side effects, and ease of metabolism [12,13]. (6)-Gingerol (1-(40 -hydroxy-30 -methoxyphenyl)-5-hydroxy-32 decanone; 6G) , is a polyphenolic alkanone present in the major pungent extracts of ginger (Zingiber officinale Roscoe, Zingiberaceae) with established antitumorigenic and proapoptotic activities [14]. Its potent anti-tumor activity has been reported in a variety of

0891-5849/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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cancer types, including breast, pancreatic, gastric, colon, and colorectal cancers, and certain hematological malignancies [14–23]. Apart from cell culture models its antitumorigenic effects have also been studied in in vivo animal models [24,25]. The potential mechanism of its action is presumably through the inhibition of inducible nitric oxide synthase (iNOS), suppression of IκBα phosphorylation, nuclear factor κB (NF-κB) nuclear translocation, release of cytochrome c, caspase activation, and increase in apoptotic protease-activating factor-1, oxidative stress, and DNA damage leading to apoptosis induction [20–25]. Despite the promising anti-cancer activity of 6G, its limited clinical application is attributed to its low bioavailability and efficacy at relatively high concentrations in vitro [14–23,26–28]. Although ginger and its constituents such as 6G at doses up to 2.0 g daily have shown very low levels of toxicity and high levels of tolerability in both animal and human studies [26], it is rapidly metabolized into glucuronides and sulfates [28]. 6G has a terminal half-life ranging from 7.23 to 8.5 min in rat plasma [27], whereas its elimination half-life varies between 75 and 120 min at the 2.0-g dose in human plasma [28]. Despite these pharmacokinetic limitations, 6G has emerged as a modulator of key oncogenic signaling pathways in a variety of cancer cells [27]. Therefore, cancer-specific preclinical studies are essentially required to validate the usefulness of 6G as a chemotherapeutic or chemopreventive agent [29]. Its development as a chemotherapeutic agent holds promise because of its nontoxic and inexpensive nature and ease of availability and metabolism [14]. Therefore, characterization of the cancer-specific molecular mechanism of 6G action along with identification of its molecular and cellular targets could be an essential way forward in the phytochemical-derived drug discovery process [29,30]. Recent studies have shown that natural agents, including isoflavone, curcumin, indole-3-carbinol, 3,30 -diindolylmethane, (  )-epigallocatechin-3-gallate, resveratrol, etc., exert their anticancer activities through changes in a group of endogenous small noncoding RNAs of 19–25 nucleotides (  22 nt) in length, known as microRNAs (miRNAs). MiRNAs regulate gene expression by binding to the 30 untranslated region (30 -UTR) of target mRNA, resulting in either mRNA degradation or inhibition of translation [31]. Natural agents have been reported to either inhibit miRNAs associated with the oncogenic signaling pathways or activate expression of miRNAs associated with the cell death pathways, leading to the inhibition of cancer cell proliferation, induction of apoptosis, reversal of epithelial–mesenchymal transition, or enhancement of efficacy of conventional cancer therapeutics [10,32]. However, there is no information available concerning the regulation of miRNA expression by 6G. Therefore in this study, as an initial step in the assessment of 6G as a chemotherapeutic agent against myeloid leukemia, we investigated the effects of 6G on myeloid leukemia cell lines, primary leukemia cells, and mouse xenografts and further examined the cell death mechanism. Our results unveil a novel mechanism of action of 6G involving ROS generation through the inhibition of mitochondrial respiratory complex I (MRC I) and ROS-associated increase in miR-27b expression and DNA damage, leading to G2/M cell cycle arrest and apoptosis in myeloid leukemia cells. These preclinical studies suggest that 6G could serve as a promising agent for myeloid leukemia treatment.

Material and methods Reagents 6G was extracted and purified from the rhizomes of ginger (Z. officinale) at the medicinal process chemistry division of the

CSIR–Central Drug Research Institute, India, as per a standardized procedure (CDRI Plant Code 4735) [33]. The purified compound was dissolved in dimethyl sulfoxide (DMSO) to a final stock concentration of 10 mM. The stock solution was aliquotted and stored at  20 1C until further use. Primary antibodies for cleaved PARP (No. 9541), β-actin (No. 4970), cyclin B1 (No. 4138), Cdk1 (No. 9112), Cdc25B (No. 9525), Cdc25C (No. 4688), PPARγ (No. 2430), Bak (No. 6947), caspase-3 (No. 9661) (Cell Signaling Technologies, Boston, MA, USA); Bcl2 (No. 610538), Bad (No. 610391), Bax (No. 610982), XIAP (No. 610716), BclXL (No. 610746) (BD Biosciences, San Jose, CA, USA); and PCNA (No. sc25280) and p-H2AX (No. sc-101696) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used in this study. The secondary antibodies used in the experiments were from Chemicon (Temecula, CA, USA). All other chemicals were from Sigma (St. Louis, MO, USA).

Cell culture Chronic myeloid leukemia (CML) cell lines (K562, LAMA-84, JURL-MK1) and acute myeloid leukemia (AML) cell lines (U937, HL-60, NB4) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), both from Gibco (Carlsbad, CA, USA), along with 1% penicillin and streptomycin from Sigma in a humidified incubator at 37 1C with 5% CO2. Peripheral blood samples were obtained from normal healthy donors and myeloid leukemia patients of various clinically defined stages at the CSM Medical University (Lucknow, India), after written informed consent in compliance with the Declaration of Helsinki 2002. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll–Hypaque density gradient (1.0 g/ml) centrifugation. Subsequently, the isolated cells (106/ml) were cultured in complete RPMI 1640 medium supplemented with 10% FBS and subjected to 6G treatment for various time periods. Transfections were carried out in these cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At the end of the treatment period the samples either were processed for flow cytometry-based cell viability analysis or were resuspended in Trizol reagent or cell lysis buffer and processed for RT-PCR or Western blotting analysis, respectively.

Cell viability assay The cell viability assay was performed using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA) according to the manufacturer’s instructions.

Apoptosis and TUNEL assays 6G-induced apoptosis was measured by using the Apoptosis Detection Kit (Invitrogen) according to the manufacturer’s instructions. Cells were seeded in six-well culture plates at a density of 1  106 cells per well and were treated with 6G for 24, 48, and 72 h. At the respective time points cells were harvested and washed twice with phosphate-buffered saline (PBS). Subsequently the cell pellet was resuspended in 1  binding buffer containing annexin V–FITC and propidium iodide and incubated for 10 min in the dark. Both control and treated samples were analyzed for live, necrotic, and early and late apoptotic cells using a FACSCalibur (BD Biosciences) flow cytometer. The TUNEL assay was done using a flow cytometry-based kit (Roche, Mannheim, Germany) as per the manufacturer’s instructions.

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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Caspase activity assay Caspase-3 activity was measured using a Caspase-3 Colorimetric Assay Kit from Millipore (Billerica, MA, USA) as per the manufacturer’s instructions. Measurement of ROS We used the cell-permeative fluorogenic sensor dyes Cell ROX Deep Red (Invitrogen) and MitoPY1 for quantification of the cellular oxidant status and mitochondrial ROS, respectively. The cells (1  106) were incubated with 5 mM Cell ROX Deep Red reagent in the culture medium for 30 min at 37 1C and subsequently the indicated treatments were initiated. Fluorescence was measured at an excitation wavelength of 640 nm and an emission wavelength of 665 nm using the Fluostar Omega spectrofluorimeter (BMG Technologies, Offenburg, Germany). For quantification of the mitochondrial H2O2, the cells (1  106) were loaded with 10 μM MitoPY1 (No. SML0734) for 45 min at 37 1C and subsequently the indicated treatments were initiated. Fluorescence was measured at an excitation wavelength of 514 nm and an emission wavelength of 530 nm using the Fluostar Omega spectrofluorimeter (BMG Technologies). Measurement of MRC complex activity The activity of MRC complexes was determined with Mitochondrial Respiratory Chain Complexes Activity Assay Kits (Novagen, Merck, Darmstadt, Germany) as per the manufacturer’s instructions. Immunocytochemistry and immunohistochemistry Cells were seeded in 12-well plates and treated with the indicated doses of 6G for 6 h. At the end of the treatment period the cells were harvested and fixed with 4% paraformaldehyde for 30 min at 4 1C. Fixed cells were then washed twice with PBS and pellets were resuspended in 20 ml of PBS. This cell suspension was smeared on poly-L-lysine-coated slides and air dried for 30 min. The cells were subsequently washed with PBS and dipped in 0.3% Triton X-100 in PBS for 5 min at room temperature for permeabilization. Blocking was done in 5% bovine serum albumin (BSA) for 1 h at room temperature followed by incubation with primary antibody overnight at 4 1C. The cells were washed and incubated with Alexa Fluor-488-conjugated secondary antibody for 1 h at room temperature, washed, and counterstained with DAPI. The immunohistochemistry was performed on the mouse tumor xenograft tissue samples. Paraffin-embedded tumor tissue was immunostained with the indicated primary antibodies (1:2500) or with secondary antibodies (1:2000). Staining was quantified under 40  by calculating the ratio of the positively stained tumor cells to the total number of tumor cells in the nonnecrotic tumor area using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA). Comet assay Briefly, 1  105 cells were seeded in six-well culture plates and treated with the indicated doses of 6G for 12 h. Thereafter the cells were harvested and washed twice with ice-cold PBS. Pellets were resuspended in 50 ml of PBS and mixed with 100 ml of 0.5% lowmelting-point agar maintained at 37 1C. This cell suspension was layered over frosted slides with the help of a micropipette tip and incubated on ice for 15 min in the dark. Subsequently the slides were placed in prechilled lysis buffer (10 mM Tris, 100 mM EDTA, 2.5 M NaCl, 1% Triton X-100, 10% DMSO, pH 10.0) and incubated at 4 1C for 1 h in the dark. The slides were then placed in an

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electrophoresis tank and incubated in freshly prepared alkaline buffer (300 mM NaOH, 1 mM EDTA, pH 13.0) at room temperature in the dark; thereafter the slides were run at 15 V and 0.30 A for 30 min. Subsequently the slides were placed in the neutralizing Tris buffer (0.4 M Tris–HCl, pH 7.5) for 15 min and stored at 4 1C overnight. Finally the slides were stained with propidium iodide and observed under an E600 upright fluorescence microscope (Nikon, Japan) and analyzed for the comet tail lengths using Casp software (version 1.2.3, Comet Assay Software Project, CASPlab. com). Cell cycle analysis For the cell cycle analysis cells were seeded at a density of 1  106 cells per well in six-well plates with serum-free medium and synchronized through serum starvation for 6 h. Thereafter, serum was added and the cells were treated with the indicated doses of 6G for 24 h. After incubation, the cells were harvested and washed twice with PBS and fixed in 70% ethanol at  20 1C overnight. Fixed cells were washed with PBS and resuspended in propidium iodide solution containing PI (50 mg/ml) and RNase A (50 mg/ml) diluted in PBS and incubated for 30 min at room temperature. Stained cells were analyzed for specific cell cycle phase arrest in a FACSCalibur (BD Biosciences) and data were analyzed using Modfit LT 3.0 software. MiRNA quantitative PCR array analysis MiRNA expression in the cell lines was measured using the RT2 miRNA PCR array system (Qiagen, Hilden, Germany). Expression analysis of 376 miRNA sequences was performed as per the manufacturer’s instructions in a LightCycler 480 II (Roche). The PCR conditions were set according to the manufacturer’s instructions. Data analysis was performed using the RT2 Profiler PCR Array Data Analysis template (Qiagen). Normalization of the data was done using four miRNAs (hsa-SNORD-44, hsa-SNORD47, hsaSNORD48, and hsa-U6) and the relative miRNA expression levels ΔΔ were calculated with 2  Ct. Three samples each were analyzed for each of the experimental groups (K562 and U937 cell lines with and without 6G treatment). MiRNA Northern blot analysis MiRNAs were extracted from K562 and U937 cells using the Nucleospin miRNA kit (Macherey–Nagel, Duren, Germany). MiRNA Northern blot was performed using an miRNA Northern Blot Assay Kit (Signosis, Sunnyvale, CA, USA) according to the manufacturer’s instructions. MiRNA target gene identification To identify the candidate target genes of identified miRNAs, bioinformatics guided analysis using miRecords (http://mirecords. biolead.org/; September 2009) was employed. miRecords integrates predicted targets of 11 target prediction tools such as DIANA-microT, MicroInspector, miRanda, MirTarget2, miTarget, NBmiRTar, PicTar, PITA, RNA22, RNAhybrid, and Target Scan/ Target ScanS. Real-time PCR The mRNA from the samples was extracted using Trizol and total miRNA extraction was achieved using the Nucleospin miRNA kit (Macherey–Nagel). To evaluate the level of gene expression, real-time PCR with SYBR green dye was used in an LC480 II LightCycler real-time PCR machine. The real-time PCR mixture

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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contained 10 μl SYBR Green Super Mix, 100 nM each primer (Supplementary Table 2), and 1 μl cDNA. All samples were run in triplicate and each experiment was repeated at least three times independently. Each sample was normalized on the basis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Luciferase reporter assays The firefly luciferase reporter plasmids containing the entire wild-type 30 -UTR of PPARγ were commercially procured (Genecopeia, Rockville, MD, USA). The mutated derivative deleted for the 8-bp seed sequence was generated by inverse-PCR using standard procedures. The Renilla plasmids (0.5 mg) were cotransfected into K562 or U937 cells with 50 nM anti-miR-27b (Ambion, Austin, TX, USA), miR-27b (Dharmacon, Lafayette, CO, USA), or nontargeting control (Ambion) using Lipofectamine 2000 (Invitrogen). The PPARγ luciferase activity of the luciferase vector construct was normalized to 1 and the other transfection combinations were compared with it. Cells were harvested 48 h after transfection and assayed using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA). NF-κB activity assay The NF-κB activity was measured using the ActivELISA kit (Imgenex, San Diego, CA, USA) as per the manufacturer’s instructions. Western blotting Both cells and tissue lysates were prepared in cell lysis buffer (50 mmol/L Tris–HCl, 150 mmol/L NaCl, 1% NP-40, 0.5% SDS, and 1% deoxycholic acid). Briefly the lysates were heated at 95 1C for 5 min and subsequently centrifuged at 14,000 g for 10 min and supernatants were collected in fresh tubes and stored at  80 1C until use. A total of 30 mg of estimated protein per sample was loaded in 10% SDS–PAGE gels. Blocking was done with 2% BSA and blots were incubated in primary antibodies (1:5000) overnight at 4 1C. Thereafter the blots were washed in 0.1% Tween 20 in PBS three times and incubated with horseradish peroxidaseconjugated secondary antibody (1:2000) for 1 h at room temperature. Blots were developed using the chemiluminescence substrate (Millipore). Xenograft experiments All the animal experiments were performed in accordance with the Institutional Animal Care and Use Committee procedures and guidelines of the CSIR–Central Drug Research Institute. Male (nu/ nu) nude mice (5 to 6 weeks of age) were maintained under pathogen-free conditions. Exponentially growing K562 cells (3  106) were injected subcutaneously into each mouse for tumor induction. Once the mice attained a tumor volume of 90 to 100 mm3, they were divided into various groups of six mice each for the indicated treatments. Treatments of vehicle, 6G (5 mg/kg body wt), anti-miR-27b (1 mg/kg body wt), and NAC (1 mg/kg body wt) were administered through the intraperitoneal route every alternate day for 45 days. The anti-miR-NC (control) and anti-miR-27b were dissolved in 100 μl of saline and administered through the intraperitoneal route in the vicinity of the tumor at a dose of 1 mg/kg body wt in a similar manner. The kinetics of tumor formation was estimated by measuring tumor size and volume at 3-day intervals. Tumor size was measured with calipers, and tumor volume was determined using the formula volume ¼ 0.5  width2  length. At the end of the experiment, the animals were sacrificed through cervical dislocation and tumors were

dissected, weighed, and processed for RT-PCR, immunohistochemical, and immunoblotting analysis. Statistical analysis All the values are represented as means 7 SEM from at least three independent experiments. Data were analyzed using oneway ANOVA followed by Newman–Keuls comparison test. Values of p o 0.05 were considered significant.

Results 6G inhibits proliferation of myeloid leukemia cell lines through apoptotic cell death 6G (purity data shown in Supplementary Fig. S1) has been reported to induce cell death in various cancer cell lines [15–23] and to exert anti-tumor activity in vivo [24,25]. However, its precise mechanism of action in myeloid leukemia cells has not been studied in detail. To explore the effects of 6G on myeloid leukemia cell viability, assessment of its dose- and timedependent effects was carried out using myeloid leukemia (CML cell types K562, LAMA-84, and JURL-MK1 and AML cell types U937, HL-60, and NB4) cell lines and nontransformed healthy donorderived PBMCs. Results of the CCK-8 cell viability assays showed that the IC50 dose of 6G was  85 mM in K562 cells, whereas it was  75 mM in LAMA-84, JURL-MK1, U937, HL-60, and NB4 cells. 6G induced significant (p o 0.05) cell death in both chronic and acute myeloid cell lines in a dose- (Fig. 1A) and time- (Supplementary Fig. S2A) dependent manner. Soft agar colony formation assays, performed as a surrogate for in vitro tumorigenicity [49] assay, further validated these results as an  35% colony inhibition was observed (Supplementary Fig. S2B) across the cell lines. As 6G is known to induce DNA fragmentation [14], we next assayed for DNA fragmentation in 6G-treated cells using a flow cytometrybased TUNEL assay. The results showed a significant (p o 0.01) increase in TUNEL-positive cells (Fig. 1B) after 6G treatment in all the cell types. Caspase-3 is a molecular target of 6G and the activation of caspase-3-like proteases is involved in apoptotic cell death [14]. Therefore we next determined the caspase-3 activity in 6G-treated cells. An  3-fold increase in caspase-3 activity over control cells was observed across the myeloid leukemia cell lines (Fig. 1C; p o 0.01). These results suggested that 6G inhibited cell proliferation and induced TUNEL positivity and caspase-3 activity in myeloid leukemia cells while sparing the PBMCs at the dose of 50 mM (Fig. 1A). Therefore we selected this dose of 6G for further mechanistic studies. As PARP cleavage is indicative of apoptotic cell death, we next assayed for PARP cleavage in cells treated with the dose of 50 mM 6G. Western blotting experiments clearly showed PARP cleavage, characterized by elevated levels of cleaved PARP fragment, in myeloid leukemia cells at 24 h of treatment. The cleaved PARP levels were elevated by  5.3-, 4.9-, 5.8-, 5.5-, 5.9-, and 5.4-fold in K562, LAMA-84, JURL-MK1, U937, NB4, and HL60 cells, respectively (Fig. 1D), whereas no such effect was observed in the PBMCs. These results collectively suggested that the 6G-induced inhibition of myeloid leukemia cell proliferation was due to apoptotic cell death. 6G inhibits proliferation of primary myeloid leukemia cells by apoptosis Recent studies have shown that natural compounds also induce inhibition of primary leukemia cells [34]. Therefore we next investigated the effects of 6G on PBMCs obtained from 40 AML and 7 CML patients (Supplementary Fig. S2C) along with the

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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Control PBMC K562 LAMA-84 JURL-MK1 U937 NB-4 HL60

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50μM 6G Cleaved β Actin Cleaved β Actin Cleaved β Actin Cleaved β Actin Cleaved β Actin Cleaved β Actin Cleaved β Actin

PARP PARP PARP PARP PARP PARP PARP

Fig. 1. 6G treatment inhibits myeloid leukemia cell proliferation through the induction of apoptotic cell death 24 h posttreatment. (A) 6G treatment inhibits proliferation of myeloid leukemia cells. Myeloid leukemia cells (chronic myeloid leukemia cell lines K562, LAMA-84, and JURL-MK1 and acute myeloid leukemia cell lines U937, HL60, and NB4) and normal PBMCs were treated with various doses (0, 10, 25, 50, 100, and 200 mM) of 6G for 24 h and CCK-8 assay was used to measure cell viability. (B) 6G treatment induces DNA fragmentation. Cells were treated with the indicated doses of 6G for 24 h and TUNEL assay was done using flow cytometry and the % TUNEL-positive cells was measured. (C) 6G treatment induces caspase-3 activation. Myeloid leukemia cells were treated with the indicated doses of 6G for 24 h and caspase-3 activity was measured using the Caspase-3 Colorimetric Activity Assay kit. (D) 6G treatment induces PARP cleavage. Cells were treated with 50 mM 6G for 24 h and the cell lysates from both treated and control cells were subjected to Western blotting for cleaved PARP. Representative images of at least three independent experiments are shown. All data are represented as means 7 SE, n ¼ 3; np o 0.05, nnp o 0.01, statistically different from control.

6 healthy donors. AML samples were categorized on the basis of the French–American–British (FAB) classification of AML. The FAB classification of AML, developed in the 1970s by a group of French, American, and British leukemia experts, categorizes acute myeloid leukemias into subtypes, M0 through M7, based upon morphology as determined by the degree of differentiation along different cell lines and the extent of cell maturation [35]. Each of these primary leukemia cell cultures was exposed to 50 mM 6G for 48 h and annexin V binding was measured by flow cytometry individually. The optimal effects of 6G in inducing apoptosis in responsive AML and CML cells in culture were achieved by 48 h posttreatment. 6G-mediated apoptosis was observed in 30 of the 40 AML samples and 6 of the 7 CML samples tested (Supplementary Fig. S2C; p o 0.01). Exposure to 6G significantly increased apoptosis (  3.5to 5.5-fold) in the 30 AML samples from FAB M0, M1, M2, M3, and M5 patients. However, the FAB M4 samples did not respond to 6G treatment. The 6 samples from CML patients also showed a significant ( 5.5-fold) increase in apoptosis after 6G treatment (Supplementary Fig. S2C). 6G treatment did not markedly affect the viability of normal PBMCs (Supplementary Fig. S2C; p o 0.01). These results collectively suggested that 6G was effective at inducing apoptosis in both primary AML and CML cells. We therefore sought to unravel its molecular mechanisms of action in detail, with an emphasis on critical events mediating its apoptotic actions in myeloid leukemia cells. We selected the chronic myeloid leukemia cell line K562 and the acute myeloid

leukemia cell line U937 as model cell lines for the detailed mechanistic studies. 6G treatment induces ROS production in myeloid leukemia cells ROS play a critical role in mediating the cytotoxicity induced by many natural chemotherapeutic agents [6,11,12] including 6G [23]. Therefore, we next assayed for the effects of 6G on ROS generation in K562 and U937 cells. For the estimation of the cellular oxidant status we used the cell-permeative fluorogenic ROS sensor CellROX Deep Red reagent. 6G treatment induced a dose-dependent increase in ROS generation (Fig. 2A). As there was no significant (p 4 0.01) difference between the doses of 50 and 100 mM with respect to ROS generation, we conducted the time-dependent studies using the dose of 50 mM 6G. The results showed that 50 mM 6G induced a time-dependent increase in ROS generation (Fig. 2B) in both K562 and U937 cells. ROS levels were increased by  5.7- and 6.4-fold at 12 h and 7.2- and 7.7-fold at 24 h of 6G treatment in K562 and U937 cells, respectively (Fig. 2B; p o 0.01). Subsequently our cellular fractionation studies indicated the mitochondrial localization of 6G (data not shown). Therefore, we examined the effects of 6G on mitochondrial ROS production using MitoPY1. 6G treatment induced a significant increase in mitochondrial ROS production, which was effectively attenuated by the mitochondrial ROS inhibitors MitoQ (Antipodean Pharmaceutical, San Francisco, CA, USA) and SkQ1 (Mitotech, Santa Fe, NM, USA)

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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Fig. 2. 6G treatment induces ROS generation through the inhibition of mitochondrial respiratory complex I. 6G induces dose- and time-dependent ROS generation. Normal PBMCs and myeloid leukemia (K562 and U937) cells were treated (A) with the indicated doses and (B) for the indicated time intervals with 6G. Cells were stained with CellROX dye and analyzed by fluorimetry. 6G-induced ROS generation was reversed by antioxidants. (C) K562 and (D) U937 cells were stained with CellROX dye. Thereafter, they were treated with or without Mito-Q or SkQ1 followed by treatment with 50 mM 6G. The cells were then analyzed for ROS kinetics by fluorimetry for 6 h. (E) Inhibition of MRC I activity is involved in mediating ROS generation induced by 6G. K562 and U937 cells were treated with or without 50 μM 6G for 30 and 60 min. The activity of MRCs was measured with a mitochondrial respiratory chain complex enzyme activity assay kit and is represented as a histogram. (F) 6G-induced MRC I activity was inhibited by NAC. K562 and U937 cells were treated with 50 μM 6G in the absence or presence of 2.5 mM NAC for 12 h. MRC activity was measured. All data are means 7 SE from three independent experiments. np o 0.05 and nnp o 0.01.

(Fig. 2C and D; p o 0.01). These data clearly indicated that 6G treatment altered the cellular oxidant status, inducing mitochondrial ROS generation.

collectively suggested that 6G treatment altered the oxidant status through inhibition of MRC I and generation of mitochondrial ROS. 6G-induced ROS induce DNA damage and G2/M arrest

6G induces ROS production through inhibition of MRC I We sought to identify the target for 6G-induced ROS generation. Inhibition of the MRCs is known to promote ROS production [37,38]. Therefore, we speculated that 6G may possibly induce ROS production by inhibiting MRC activity. Our results showed that 6G significantly inhibited MRC I activity as early as 30 min after treatment (Fig. 2E; p o 0.01), and pretreatment with NAC reversed this inhibition (Fig. 2F; p o 0.01). These results

6G-induced ROS generation is known to lead to DNA damage in cancer cells [14,39]. We therefore checked these effects of 6G in U937 and K562 cells. Phosphorylation of histone H2AX at serine 139 is a sensitive marker of DNA double-strand breaks [40] and an increase in comet tail length is indicative of DNA fragmentation [13,14]. We observed a substantial increase in p-H2AX staining, indicative of 6G-induced DNA damage (Fig. 3A). Similarly, 6G treatment also increased the comet tail length in both K562 and U937 cells (Fig. 3B), further confirming 6G-induced DNA damage.

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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Fig. 3. 6G treatment induces DNA damage and G2/M cell cycle arrest in myeloid leukemia cells. (A) K562 and U937 myeloid leukemia cells were treated with 6G for 12 h and subjected to immunofluorescence analysis for the detection of p-H2AX and comet assay. Cells treated with 6G showed increased expression of p-H2AX (green) in the nucleus (blue). Representative images of at least three independent experiments are shown. (B) Normal PBMCs, K562, and U937 cells were treated with 6G for 12 h and subjected to comet assay analysis. 6G-treated cells showed increased comet tail lengths (red). Representative images of at least three independent experiments are shown. Bar graph of comet analysis indicates increase in tail length of comets in treated K562 and U937 cells, whereas normal PBMCs remained unaffected. (C) 6G treatment induced G2/M cell cycle arrest in myeloid leukemia cells. Flow cytometry-based cell cycle analysis was done to analyze percentage of cells in G2/M phase after 6G treatment. Bar graph indicates increased accumulation of cells in the G2/M cell cycle phase. (D) 6G treatment reduces proteins associated with G2/M cell cycle progression. K562 and U937 cells were treated with 6G for 1, 4, 12, or 24 h and the cell lysates were subjected to Western blotting. Blots indicate reduced expression of cyclin B1, Cdk1, Cdc25B, and Cdc25C, key players in G2/M progression. Representative images of at least three independent experiments are shown. Data are represented as means 7 SE, n ¼ 3; np o 0.05 and nnp o 0.01, statistically different from control.

6G treatment is also known to induce G2/M cell cycle arrest in cancer cells [39] associated with DNA damage. Therefore, we checked the effects of 6G treatment on cell cycle dynamics in K562 and U937 cells. The results clearly showed that 6G treatment induced significant (p o 0.05) G2/M phase cell cycle arrest in both cell types (Fig. 3C). G2/M phase cell cycle arrest is often accompanied by inhibition of intracellular proteins, such as cyclin B1, Cdk1, Cdc25B, and Cdc25C, usually associated with the G2/M cell cycle progression [38]. Therefore we further analyzed for the expression of these proteins in the 6G-treated cell lysates at time points corresponding to the G2/M cell cycle arrest by Western blotting. 6G treatment reduced expression of proteins related to G2/M cell cycle phase transition (Fig. 3D). These results demonstrated that 6G treatment induced G2/M cell cycle arrest consistent with the appearance of DNA damage in myeloid leukemia cells.

6G treatment induces increased expression of miR-27b Oxidative stress due to accumulation of ROS is known to induce changes in miRNA expression in various cell types [41–45]. Therefore we further investigated the changes in miRNA expression in the myeloid leukemia cell lines K562 and U937 upon 6G-induced ROS accumulation. We conducted a miRNA array analysis using the RT2 miRNA PCR array system. Three samples from each of the four groups (two cell lines with and without 6G treatment) were analyzed in this experiment. The results showed that the miR27b expression was increased by  4.8- and  4.9-fold higher in the K562 and U937 cells treated with 6G compared to the untreated cells (Fig. 4A). The results from the miRNA array were further validated through miRNA Northern blot analysis (Fig. 4B). These results indicated that miR-27b may possibly be related to the proapoptotic effects of 6G. Therefore, we further checked the

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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miR-30c miR-29a miR-29b iR 106b miR-106b miR-27b miR-98 miR-101 miR-19a miR-19b miR-128b miR-130a miR-141 iR 141 miR-143 miR-146a miR-148a miR-148b miR-151 miR-199a miR-345 miR 200b miR-200b miR-33b miR-494 miR-573 miR-449 miR-220a miR-561 miR-663

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Fig. 4. 6G treatment increases expression of miR-27b. (A) Heat map analysis showing relative cellular miRNA expression after the indicated treatments in K562 and U937 cell lines. K562 and U937 cells were treated with 50 mM 6G for 24 h and the total miRNA was subjected to real-time miRNA PCR array analysis. The data are expressed as a microarray heat map with or without 6G treatment. The cutoff lines represent fivefold change between the control and the 6G-treated groups. The heat map was automatically generated by uploading the CT values to the Qiagen Web site. (B) The miRNA microarray data were further validated using Northern blotting. Normal PBMCs and K562 and U937 cells were treated with 50 mM 6G for 24 h and subjected to Northern blotting for detection of miR-27b. U6 was used as control. Representative images of at least three independent experiments are shown. (C) miR-27b plays a critical role in 6G-induced apoptosis. K562 and U937 cells were treated with 50 mM 6G for 24 h with or without anti-miR-27b and subsequently the cells were stained with annexin V–FITC/PI and analyzed by flow cytometry to assess the percentage of apoptotic cells. The bar graph indicates reversal of 6G-induced apoptotic effect in the anti-miR-27b-treated groups. Data are means 7 SE, n ¼ 3; np o 0.05, statistically different from control.

effect of miR-27b inhibition on 6G-induced effects on viability in K562 and U937 cells. Inhibition of miR-27b significantly (p o 0.05) reduced 6G-induced apoptotic effects in both myeloid leukemia cell lines (Fig. 4C). To determine whether the miR-27b expression in the myeloid leukemia cell lines is relevant to the human disease condition, we measured miR-27b levels in primary leukemia samples from human patients. In all 47 samples tested, miR-27b levels in patient-derived primary leukemia cells were 3.5- to  6.3-fold lower than in the normal PBMCs, indicating that the reduced levels of miR-27b are possibly associated with myeloid leukemia (Supplementary Fig. S4). These results suggested that 6G-induced activation of miR-27b expression is critical in mediating its proapoptotic effects in leukemia cells.

MiR-27b through PPARγ inhibits the inflammatory gene expression associated with the NF-κB pathway Studies have established that the miR-27 family members (miR-27a and miR-27b) directly target the 30 -UTR of PPARγ in normal [46,48,49] and cancer cells [47]. Our bioinformatics-guided in silico analysis also predicted that the PPARγ 30 -UTR is the target of miR-27b (Fig. 5A). Abundant PPARγ expression has been reported in myeloid cell lines and the primary leukemia cells used in this study (Supplementary Fig. S4), indicating its oncogenic nature [47]. Luciferase reporter plasmids containing the wild-type 30 -UTR sequence of PPARγ or a deletion mutant were transfected into the K562 and U937 cell lines with either miR-27b or anti-miR-27b.

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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Fig. 5. MiR-27b targets the 30 -UTR of PPARγ. (A) Sequence complementarity (vertical lines showing the seed sequence of positions 82–88) between miR-27b and the PPARγ 30 -UTR. MiR-27b expression regulates PPARγ activity in (B) K562 and (C) U937 cells. PPARγ luciferase activity of reporters containing the wild-type or 8-bp-deleted 30 -UTR of PPARγ is shown 24 h after transfection with miR-27b, anti-miR-27b, or miR negative control or untransfected cells. (D) 6G treatment reduces PPARγ mRNA levels. Real-time PCR analysis indicates the PPARγ mRNA levels in K562 and U937 cells and patient-derived primary PBMCs (AML, patients 1 and 2, and CML, patient 3) treated with 6G alone or with anti-miR-27b. (E) 6G treatment reduces PPARγ protein levels. Western blot analysis showing PPARγ protein levels in cells with the indicated treatments; levels of GAPDH served as a loading control. Representative images of at least three independent experiments are shown. nnp o 0.01 and nnnp o 0.001, statistically different from control.

The results clearly showed that PPARγ luciferase activity of the wildtype reporter was reduced by  5.5-fold upon miR-27b overexpression, whereas it increased by  58% upon miR-27b inhibition in K562 cells (Fig. 5B). In the U937 cells the PPARγ luciferase activity of the wild-type reporter was reduced by  5.2-fold upon miR-27b overexpression, whereas it increased by  55% upon miR-27b inhibition (Fig. 5C). In contrast, no change in the PPARγ luciferase activity was observed in the mutant reporter plasmid upon overexpression of miR-27b or inhibition with anti-miR-27b in either cell line. In both myeloid leukemia cell lines and patient-derived primary leukemia cells (AML, patients 1 and 2, and CML, patient 3), the PPARγ mRNA expression was significantly (p o 0.01) reduced ( 3.5-fold) upon 6G treatment (Fig. 5C). As expected, the antisense-mediated inhibition of miR-27b resulted in an increase in PPARγ mRNA levels in both the cell lines and the primary myeloid leukemia cells (Fig. 5D). In addition, PPARγ protein levels were also observed to be decreased upon 6G treatment and increased upon treatment with anti-miR-27b

(Fig. 5E). PPARγ is known to influence NHE-1 levels in cancer cells [47]; however, we did not observe any change in its levels after 6G treatment in our studies (data not shown). Thus, these results suggested that 6G-induced miR-27b inhibits PPARγ. The inflammatory transcription factor NF-κB physically interacts with PPARγ and upregulates inflammatory cytokines (such as IL-1A, IL-6, JAK2, and IL1B) to promote carcinogenesis [41]. Therefore we further examined the roles of miR-27b and PPARγ in the regulation of the oncogenic inflammatory response. Inhibition of miR-27b in K562 and U937 cells increased mRNA levels of the inflammatory cytokines IL-1A, IL-6, and IL-1B, whereas overexpression of miR-27b resulted in decreased expression (Fig. 6A and B), similar to the 6G-induced effects. In addition, the mRNA levels of these inflammatory factors were strongly reduced upon siRNA-mediated inhibition of PPARγ (Fig. 6A and B). Importantly, the increased expression of inflammatory cytokines upon reduction of miR-27b is blocked by simultaneous inhibition of PPARγ (Fig. 6A and B), suggesting that the effects of miR-27b are

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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Fig. 6. MiR-27b through PPARγ regulates the NF-κB pathway in myeloid leukemia cells. 6G inhibits the NF-κB pathway-associated inflammatory cytokine gene expression in myeloid leukemia cells. (A and B) mRNA levels of inflammatory cytokine genes with the indicated treatments in (A) K562 and (B) U937 cells. (C) 6G-induced miR-27b inhibits PPARγ in mouse xenografts. Tissues collected from mouse xenografts were analyzed for PPARγ level with or without anti-miR-27b (1 mg/kg body wt) treatment. (D) 6G treatment, expression of miR-27b, or inhibition of PPARγ inhibits NF-κB activity in vivo. Tissues collected from mouse xenografts treated with 50 mM 6G or control miR (1 mg/ kg body wt) or miR-27b (1 mg/kg body wt) or the PPARγ inhibitor GW9662 (1 mg/kg body wt) were analyzed for NF-κB activity. (E) 6G treatment, expression of miR-27b, or inhibition of PPARγ inhibits IL-6 levels in vivo. IL-6 mRNA levels were measured in tissues collected from mouse xenografts with the earlier indicated treatments. Data represent means 7 SE, n ¼ 3; nnp o 0.01 and nnnp o 0.001, statistically different from control.

mediated through PPARγ. Further, validation of these results using mouse tumor xenografts indicated that 6G induced miR-27b, inhibited PPARγ mRNA levels (Fig. 6C), reduced NF-κB activity (Fig. 6D), and reduced the levels of the inflammatory cytokine IL-6 in vivo (Fig. 6E). Collectively these results suggested that 6G treatment induces miR27b that targets PPARγ and results in the suppression of NF-κB pathway-associated inflammatory cytokine gene expression, highlighting its antileukemic effects. 6G inhibits myeloid leukemia cell proliferation and inhibits tumor growth in a murine xenograft model To further validate the results obtained in vitro, we determined whether 6G could inhibit tumor development in a murine xenograft tumor model in vivo. Twenty-four nude mice bearing K562 tumor xenografts (approximate volume 90 mm3) were randomly divided into four groups and were treated with vehicle (0.1% sodium carboxyl methyl cellulose in sterile water), 6G (5 mg/kg body wt), anti-miR-27b (1 mg/kg body wt) along with 6G, and NAC (1 mg/kg body wt) along with 6G for 45 days (through the intraperitoneal route on alternate days). The body weights and tumor sizes of all mice were measured every 3 days throughout the experimental period. The tumors in the vehicle group grew to an average volume of 497.5711.9 mm3, whereas the tumors in the anti-miR-27b þ 6G and the NAC þ 6G groups grew to 429.1 79.7

and 419.9 713.2 mm3, respectively, compared to the 6G-treated groups, which grew to 164.1 76.5 mm3 after 45 days. Statistical analysis of tumor size showed a robust reduction of 58% at day 30 and 51% at day 45, confirming the potent antileukemic effect of 6G in vivo. Significant 6G-induced inhibition of tumor growth was observed, validating the antileukemic activity of 6G (Fig. 7A). However, there was no difference in the anti-miR-27b þ 6G and the NAC þ 6G groups, further confirming the role of miR-27b and ROS in 6G-induced effects. 6G treatment also significantly (p o 0.001) reduced tumor weights in mouse xenografts (Fig. 7B), whereas no significant (p 4 0.01) difference was observed in the tumor weights in the anti-miR-27b þ 6G and the NAC þ 6G groups. To determine if the antileukemic activity of 6G in tumor xenografts is attributable to apoptosis induction, a TUNEL analysis and an immunohistochemical analysis of PCNA expression were carried out. Significantly increased TUNEL-positive cells and decreased PCNA-positive cells were observed in the 6G-treated tumors compared to the corresponding vehicle-treated control, anti-miR-27b þ 6G, and NAC þ 6G groups (Fig. 7C). Finally we carried out immunoblot analysis of the mouse xenograft tumors from the four treatment groups for pro- and antiapoptotic proteins. The results showed that 6G treatment significantly downregulated the antiapoptotic proteins (PCNA, Bcl2, BclXL, and XIAP) and upregulated the proapoptotic proteins (Bax, Bak, Bad, cleaved PARP, and activated caspase-3) compared to the corresponding

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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Fig. 7. 6G inhibits myeloid leukemia cell proliferation in vivo in a mouse xenograft model. (A) 6G treatment reduces tumor volume in vivo. K562 (3  106) cells were implanted subcutaneously on both flanks in nude mice. After tumor establishment, the animals received intraperitoneal injections of either vehicle control (n ¼ 6) or 6G alone (n ¼ 6 at a dose of 5 mg/kg body wt) or along with anti-miR-27b (n ¼ 6 at a dose of 1 mg/kg body wt) or NAC (n ¼ 6 at a dose of 1 mg/kg body wt) for a period of 45 days. (B) 6G treatment reduces tumor weights in vivo. Tumor weights were recorded in these groups. (C) 6G treatment increases tumor apoptosis in vivo. Tumor xenografts were analyzed for TUNEL, as a marker of apoptosis, and PCNA, as marker of cellular proliferation. (D) 6G treatment reduces antiapoptotic protein expression and increases proapoptotic protein expression in tumor xenografts in vivo. Protein was extracted from frozen tumor xenograft samples (control, n ¼ 6; 6G treated, n ¼ 6; 6G þ miR-27b, n ¼ 6; 6G þ miR-27b, n ¼ 6; and 6G þ NAC, n ¼ 6) and the tumor lysates were immunoblotted with the indicated antibodies. Representative images of at least three independent experiments are shown. Data are means 7 SE; nnnp o 0.001, significantly different from control.

control, anti-miR-27b þ 6G, and NAC þ 6G groups (Fig. 7D). 6G treatment did not adversely affect the hematological parameters (Supplementary Table 1A) or body weights (Supplementary Table 1B), indicating its chemotherapeutic potential. Collectively, these results clearly indicated that 6G inhibited leukemia cell proliferation in vivo without significant side effects.

Discussion The development of chemoresistance against conventional chemotherapeutic drugs, along with their systemic side effects, is a major hurdle in the management of myeloid leukemia [50].

Therefore in recent times use of nutrient-based phytochemicals for leukemia therapy has gained wide acceptance [6,8–12,34]. 6G, a polyphenol derived from ginger, a widely used dietary ingredient of the traditional Indian diet, has proven antitumorigenic effects [14–23]. In this study we demonstrated that 6G induces apoptosis in myeloid leukemia cells through the elevation of intracellular ROS, activation of miR-27b expression, and DNA damage-mediated G2/M phase cell cycle arrest. 6G induced apoptosis in myeloid leukemia cell lines (Fig. 1) and patient-derived primary cells (Supplementary Fig. S2C) while sparing the normal PBMCs, consistent with earlier reports [15– 23]. However, none of the samples of the FAB M4 subtype responded to the 6G treatment. The resistance to 6G effects may

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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possibly be due to the high expression of CD14 leading to chemoresistance in the FAB M4 subtype [51]. However, the impact of 6G on a larger number of AML samples from patients with the M4 subtype needs to be investigated. Mechanistic studies using the K562 and U937 lines showed that 6G induced ROS generation in myeloid leukemia cells (Fig. 2A and B) in agreement with earlier studies [14,25,37,38]. Intracellular ROS levels regulate cellular processes such as proliferation, differentiation, and apoptosis [52–56]. ROS accumulation is a critical mediator of the anti-cancer effects of several chemotherapeutic agents [38,40,54–56]. ROS generation in cancer cells is attributed to the regulation of iNOS, NADPH oxidases, and cytochrome P450 [57]; downregulation of antioxidative proteins [58–61]; or inhibition of MRCs [38,55,56]. 6G-induced hyperpolarization of the mitochondrial inner membrane (data not shown) was suggestive of a stalled mitochondrial ETC and an increased likelihood of stray electron transfer and increase in ROS [38]. Cotreatment with mitochondria-specific antioxidants led to lower cellular ROS levels (Fig. 2C and D) but did not affect the increase in ΔΨm triggered by 6G (data not shown). This finding suggested that the mitochondrial membrane hyperpolarization preceded ROS generation and further established mitochondria as one of the targets of 6G. Our results, consistent with an earlier study [38], showed that 6G-induced inhibition of MRC I activity was a source of ROS production (Fig. 2E and F). The results implied that 6G could be used to selectively target the deregulated redox environment within the myeloid leukemia cells. 6G-induced ROS accumulation is known to induce multiple signaling pathways [14,25]. In this study we observed 6G-induced DNA damage characterized by increased p-H2AX staining and increased comet tail length leading to G2/M cell cycle arrest in myeloid leukemia cells (Fig. 3), in agreement with earlier reports [16,23,25,39]. Defective cell cycling and apoptotic mechanisms are considered to play a role in both the development of myeloid leukemia and resistance to chemotherapeutic drugs [1,4,5,50]. Therefore to develop rational chemotherapeutic approaches [2,3] it is imperative to look for novel chemotherapeutic agents capable of targeting these cells. In this context these results highlight the cell cycle regulatory effect of 6G in myeloid leukemia cells. The in vitro and ex vivo cell-death-inducing effects of 6G observed in this study are in agreement with the naturally occurring polyphenols with documented effects on ROS production, cell cycle regulation, ROS production, and induction of apoptosis [13,14,17,20]. An exciting finding of this study is the elevation of miR-27b expression in myeloid leukemia cells upon 6G treatment (Fig. 4). MiR-27b levels are known to be regulated by oxidative stress [46– 48]. Therefore, it can be speculated that 6G-induced ROS levels induced activation of miR-27b expression. We observed significantly (p o 0.01) higher levels of miR-27b in normal PBMCs compared to the levels in primary myeloid leukemia samples and AML/CML cell lines (Supplementary Fig. S4), possibly indicating the association of low levels of its expression with oncogenic progression. Interestingly, miR-27b has opposite effects in cancer cells, functioning as either an activator [62,63] or an inhibitor [47,64] of oncogenic signaling, possibly depending on the cellular context. This dual effect of miR-27b is consistent with the other miRNAs, such as miR-17-92 [65] and miR-7 [66,67], with similar effects. Bioinformatics analysis showed that miR-27b targets the 30 -UTR of the PPARγ pathway (Fig. 5), in agreement with recent studies [46–49]. PPARγ is a nuclear transcription factor known to have oncogenic activity in vitro and in vivo [68–71], 72. High levels of PPARγ observed in the patient-derived primary leukemia cells in this study are consistent with the levels expressed in transformed B lymphocyte and myeloid cell lines [67–71], 72 (Supplementary Fig. S4), indicating

its cell-specific oncogenic activity. Our results clearly demonstrate that miR-27b functions as a tumor suppressor by directly inhibiting the expression of PPARγ in myeloid leukemia cells. Recent studies have indicated that deregulation of miRNAs with tumor-suppressive function often leads to cancer development and progression [10,31]. We further observed that 6G, through the inhibition of PPARγ, suppressed the NF-κB pathway-associated inflammatory cytokine gene expression (Fig. 6A and B). These results are in agreement with other studies, as oncogenic activity of PPARγ is known to be mediated through direct interaction with NF-κB, resulting in increased expression of inflammatory cytokines [68–70]. We further confirmed the inhibitory effect of miR-27b on PPARγ, NF-κB activity, and IL-6 levels in a mouse xenograft model (Fig. 7). These results are in agreement with a recent study in neuroblastoma cells [47]. These results highlight the regulation of miRNA expression by a natural compound [10] such as 6G and further confirms that its proapoptotic effects are mediated through the activation of miR-27b expression and inhibition of PPARγ–NF-κB signaling. As it was evident that miR-27b and ROS-mediated DNA damage are critical mediators of 6G-induced effects, we used specific antimiR-27b and the ROS scavenger NAC to validate these results (Supplementary Fig. S3). The results further confirmed the dual proapoptotic effect of 6G in myeloid leukemia cells and indicated the possible synergy between the two pathways, as direct or indirect inhibition of the NF-κB pathway is known to induce G2/M cell cycle arrest and apoptosis in cancer cells [71], similar to ROSinduced effects. In agreement with the in vitro data, the in vivo xenograft model also confirmed inhibition of myeloid leukemia cell proliferation and efficient tumor regression with 6G treatment. Administration of 6G significantly reduced tumor volume, reduced tumor weight, inhibited PCNA expression, and increased tumor cell death as indicated by increased TUNEL positivity in vivo (Fig. 7), without eliciting systemic side effects (Supplementary Tables 1A and 1B). It can be speculated that 6G may have multiple in vivo effects, i.e., growth inhibition, DNA damage, cell cycle arrest, and induction of apoptosis, consistent with the in vitro studies [14–23]. In conclusion, our present studies show that 6G could induce apoptosis in myeloid leukemia cells in vitro and in vivo. Taken together, the results of our study suggest that (i) 6G inhibits MRC I to increase ROS levels and (ii) increased ROS levels activate miR-27b expression and DNA damage, critical for myeloid leukemia cell death. These findings support the clinical potential of 6G as a component of therapeutic strategies for myeloid leukemia. However, its development as a chemotherapeutic agent would warrant further studies on lead optimization and targeted synthesis of 6G derivatives based on its metabolic signature [28] for improved pharmacokinetic profiles and bioavailability. To summarize, we report a previously unknown apoptotic property of 6G though the dual induction of increased miR-27b expression and DNA damage-associated cell cycle arrest, which may have implications for myeloid leukemia treatment.

Uncited reference [36].

Acknowledgments This work was supported by the grants from the Ministry of Health and CSIR–CDRI core funds to D.P. Mishra. We thank Dr. T. K. Chakraborty, for continued support, and Dr. N. Chattopadhyay, Dr. Sabyasachi Sanyal, and the members of the D.P. Mishra laboratory for helpful discussions. Namrata Rastogi, Rishi Kumar

Please cite this article as: Rastogi, N; et al. (6)-Gingerol-induced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.016i

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Gara, Rachana Trivedi, Akanksha Singh, and Preety Dixit acknowledge fellowships from the Council of Scientific and Industrial Research, New Delhi. This is CSIR–CDRI Manuscript No. 144/ 2012/DPM.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed. 2013.12.016.

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