Nimbolide Induces ROS-Regulated Apoptosis and Inhibits Cell Migration in Osteosarcoma

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1 Int. J. Mol. Sci. 2015, 16, ; doi: /ijms Article OPEN ACCESS International Journal of Molecular Sciences ISSN Nimbolide Induces ROS-Regulated Apoptosis and Inibits Cell Migration in Osteosarcoma Ju-Fang Liu 1, Cun-Han Hou 2, Feng-Ling Lin 3, Ya-Ting Tsao 2 and Seng-Mou Hou 4, * 1 Central Laboratory, Sin-Kong Wu Ho-Su Memorial Hospital, Taipei 11101, Taiwan; anti0822@otmail.com 2 Department of Ortopedic Surgery, National Taiwan University Hospital, Taipei 10617, Taiwan; s: cou@ntu.edu.tw (C.-H.H.); m @tmu.edu.tw (Y.-T.T.) 3 Department of Dermatology, Siji Catay General Hospital, Taipei 22174, Taiwan; lavande213@gmail.com 4 Department of Ortopedic Surgery, Sin-Kong Wu Ho-Su Memorial Hospital, No. 95, Wencang Road, Silin District, Taipei 11101, Taiwan * Autor to wom correspondence sould be addressed; sengmou@ms.sk.org.tw; Tel.: (ext. 9420); Fax: Academic Editor: William Ci-sing Co Received: 22 June 2015 / Accepted: 21 September 2015 / Publised: 29 September 2015 Abstract: Osteosarcoma (OS) is a primary malignant tumor of bone and is most prevalent in cildren and adolescents. OS is frequently associated wit pulmonary metastasis, wic is te main cause of OS-related mortality. OS as a poor prognosis and is often unresponsive to conventional cemoterapy. In tis study, we determined tat Nimbolide, a novel anti-cancer terapy, acts by modulating multiple mecanisms in osteosarcoma cells. Nimbolide induces apoptosis by increasing endoplasmic reticulum (ER) stress, mitocondrial dysfunction, accumulation of reactive oxygen species (ROS), and finally, caspase activation. We also determined tat Nimbolide inibits cell migration, wic is crucial for metastasis, by reducing te expression of integrin αvβ5. In addition, our results demonstrate tat integrin αvβ5 expression is modulated by te PI3K/Akt and NF-κB signaling cascade. Nimbolide as potential as an anti-tumor drug given its multifunctional effects in OS. Collectively, tese results elp us to understand te mecanisms of action of Nimbolide and will aid in te development of effective terapies for OS.

2 Int. J. Mol. Sci. 2015, Keywords: osteosarcoma; apoptosis; endoplasmic reticulum stress; reactive oxygen species; migration 1. Introduction Osteosarcoma (OS) is te most common primary malignant bone tumor among cildren and adolescents. Surgery and systemic cemoterapy are te mainstay treatment for OS, and te cure rate is in te range of 58% 76% [1]. However, te survival rate as sown limited improvement. Today, te five-year survival rate is approximately 20% after surgical treatment alone. Cemoterapy is usually employed in an adjuvant situation to improve prognosis and long-term survival. However, recurrence usually manifests, sometimes as a local recurrence or metastasis to distant bones. More frequently, recurrence occurs as pulmonary metastasis, wic results in a poor prognosis for many patients wit OS [2]. Tus, a novel strategy tat effectively inibits metastasis, especially to te lung, from te primary site of OS is igly desirable. Azadiracta indica is a plant used in traditional medicine. Its extracts are reported to ave anti-malarial [3] and anti-cancer properties [4,5]. Nimbolide, a constituent of A. indica, is a tetranortriterpenoid tat consists of a classic limonoid skeleton wit an α,β-unsaturated ketone system and a δ-lactone ring (Figure 1A). Previous studies ave indicated tat triterpenoids exibit anti-proliferative activity against cancers suc as breast cancer, lung cancer, neuroblastoma, OS, coriocarcinoma, and melanoma [6 8]. However, te mecanisms involved in te anti-tumor effects of Nimbolide ave not been investigated. In normal cells, reactive oxygen species (ROS) including superoxide anions (O2 ), ydrogen peroxide (H2O2), and te igly reactive ydroxyl radical ( OH), are generated as by-products of cellular metabolism and are in cellular redox balance wit biocemical antioxidants [9]. However, in many cancers, tis vital balance is disrupted, resulting in oxidative stress and accumulation of ROS [10]. Because ROS can cause damage of DNA, RNA, and proteins, tey contribute to te development of uman diseases including insulin resistance, diabetes mellitus, and cancer [11 13]. In cancer cells, low levels of oxidative stress can promote survival and proliferation, but iger levels can induce apoptosis and cell cycle arrest [14 16]. Te accumulated evidence indicates tat apoptosis is associated wit an increase in mitocondrial oxidative stress, release of cytocrome C, and te activation of caspases [17]. Traditional cemoterapeutic and radioterapeutic agents are toxic to cancer cells by increasing ROS production [18]. Terefore, increasing production of ROS may be an important strategy for cancer terapies. Te endoplasmic reticulum (ER) is a crucial organelle in protein folding, modification, and secretion. A variety of toxic conditions suc as ypoxia, failure of protein syntesis, protein misfolding, and Ca 2+ overload result in ER stress-related events [19,20]. Wen ER stress occurs, unfolded proteins accumulate and cause te unfolded protein response (UPR) [21]. UPR initiates a signal transduction cascade tat is regulated by tree conserved ER transmembrane proteins tat serve as sensors of ER stress: inositol-requiring kinase 1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) [22]. Moreover, UPR induces te expression of ER-resident caperones, suc as glucose-regulated protein (GRP) 78 and GRP94 [23]. A large body of

3 Int. J. Mol. Sci. 2015, evidence as sown tat ER stress as a pivotal role in apoptosis. ER stress is tougt to activate mitocondria-dependent cell deat patways [24,25] tat utilize members of te Bcl-2 family including Bax and Bak [21]. ER stress also leads to release of Ca 2+ from te ER and tis Ca 2+ subsequently activates calpains, wic could induce apoptosis by promoting te cleavage and activation of apoptosis-related caspases [26]. Activation of te mitocondria-dependent cell deat patway is also accompanied by te accumulation of ROS tat can be sequentially converted into toxic ROS, suc as ydrogen peroxide, wic can independently induce cell deat [27,28]. Here, we investigate te anti-cancer activity of Nimbolide. Our data indicate tat Nimbolide induces apoptosis and reduces migration of uman osteosarcoma cells. 2. Results 2.1. Nimbolide Induces Apoptosis in Human Osteosarcoma Cells To investigate weter Nimbolide could induce cell deat in uman osteosarcoma cells, we first examined te effect of Nimbolide on cell survival by using an MTT assay. Treatment of cells wit Nimbolide reduced viability of osteosarcoma cells (MG63, U2OS and HOS cells) but not of non-cancerous osteoblast cells (FOB 1.19; Figure 1B). Te anti-cancer activities of Nimbolide were furter assessed wit a colony formation assay, and te results indicate tat treatment of osteosarcoma cell lines wit Nimbolide reduces colony formation in a dose-dependent manner (Figure 1C). To determine weter Nimbolide induces cell deat troug an apoptotic mecanism, we used DAPI staining and a DNA ladder assay. Nimbolide treatment dramatically increased te amount of condensed and degraded cromatin (Figure 1D,E). We also confirmed tat Nimbolide induces apoptosis as sown not only by an increase in te percentages of cells in te sub G1 pase and double-labeled wit Annexin V and propidium iodide (PI), but also by a TUNEL assay (Figure 1F H). Tese results indicate tat Nimbolide induces apoptosis in osteosarcoma cells ROS and Mitocondrial Dysfunction Are Involved in Nimbolide-Induced Apoptosis in Human Osteosarcoma Cells Te results of previous studies indicate tat te generation of ROS plays a pivotal role in apoptosis and tat ROS can function as anti-cancer agents [29,30]. Terefore, we investigated weter te accumulation of ROS is involved in Nimbolide-induced cell deat. FACS analysis indicates tat treatment of osteosarcoma cells wit Nimbolide induces te accumulation of H2O2 (Figure 2A). Pretreatment of cells wit te ROS scavenger N-acetyl cysteine (NAC), te NADPH oxidase inibitor dipenyleneiodonium cloride (DPI), and catalase (an H2O2 scavenging enzyme) reduced Nimbolide-induced apoptosis (Figure 2B). ROS production and accumulation in mitocondria ave previously been sown to decrease te mitocondrial membrane potential (MMP) and initiate mitocondrial apoptosis [31]. Our data sow tat treatment of osteosarcoma cells wit Nimbolide also decreases te MMP (Figure 2C) as well as alters te expression of members of te Bcl-2 family to increase te ratio of pro-apoptotic to anti-apoptotic Bcl-2 proteins (Figure 2D). Tese results demonstrate tat Nimbolide induces apoptosis in osteosarcoma cells via ROS and mitocondrial apoptosis.

4 Int. J. Mol. Sci. 2015, μm Figure 1. Nimbolide induces apoptosis in osteosarcoma cells. (A) Te structure of Nimbolide. (B,C) Osteosarcoma cells (MG63, U2OS, and HOS) and osteoblast cells (FOB 1.19) were incubated wit control solution or various concentrations of Nimbolide for 1 or 2 days; (B) Cell viability was examined by using an MTT assay; (C) For te colony-formation assay, te cells were stained wit crystal violet and potograped. Te quantitative data are sown in te lower rigt panel; (D) MG63 osteosarcoma cells were incubated wit control solution or various concentrations of Nimbolide for 24 and nuclear morpology was recorded by using a DAPI stain and potograpy. Scale bar: 50 μm; (E) MG63 osteosarcoma cell lysates were collected for DNA purification, and te DNA was analyzed by gel electroporesis; (F G) Osteosarcoma cells were incubated wit control solution or various concentrations of Nimbolide for 24, and te percentages of apoptotic cells were analyzed by flow cytometric analysis of (F) annexin V and PI double-labeling, (G) PI staining, and (H) TUNEL staining. Te data are expressed as te mean ± SEM. * p < 0.05 as compared wit te control group.

5 Int. J. Mol. Sci. 2015, ) Figure 2. Nimbolide induces te generation of ROS and mitocondrial dysfunction in uman osteosarcoma cells. (A) MG63 cells were treated wit control solution or various concentrations of Nimbolide for 24. Te concentration of H2O2 as a measure of ROS production was examined by using flow cytometry; (B) MG63 cells were pretreated for 30 min wit N-acetylcysteine (NAC), dipenyleneiodonium cloride (DPI), and catalase followed by treatment wit Nimbolide (10 μm) for 24. Te percentage of apoptotic cells was ten analyzed by flow cytometric analysis of PI-stained cells; (C) MG63 cells were treated as described in (A). Te mitocondrial membrane potential was examined by using flow cytometry and JC-1 staining; (D) MG63 cells were treated wit control solution or various concentrations of Nimbolide for 24. Te expression levels of Bcl-2, Bak, Bax, mitocondrial cytocrome c, and cytosolic cytocrome c were examined by using western blot analysis; actin and voltage-dependent anion cannels (VDAC) were used as internal controls. Te data are expressed as te mean ± SEM. * p < 0.05 compared wit controls. # p < 0.05 compared wit te Nimbolide treated groups Nimbolide Induces ER Stress in Human Osteosarcoma Cells Te accumulation of oxidative stress in te ER frequently results in initiation of te UPR [32]. Terefore, we investigated te effects of Nimbolide treatment on ER stress. First, we accessed calcium release, wic is te link between ER stress and mitocondrial dysfunction [33]. Our results indicate tat Nimbolide increases te accumulation of intracellular calcium (Figure 3A). Moreover, pretreatment of osteosarcoma cells wit BAPTA-AM, a cell-permeant calcium celator, reduced apoptosis and

6 Int. J. Mol. Sci. 2015, increased viability in cells treated wit Nimbolide (Figure 3B,C). ER stress is generally caracterized by up-regulation of GRP78, GRP94, and calpains 1 and 2, and te expression of all but calpain 1 were increased after Nimbolide treatment (Figure 3D). Tese results reveal tat Nimbolide induces te ER stress-related apoptotic patway in osteosarcoma cells Figure 3. Nimbolide induces Ca 2+ release and ER stress in uman osteosarcoma cells. (A) MG63 cells were treated wit control solution or various concentrations of Nimbolide for 24. Ca 2+ release was examined by using flow cytometry; (B) MG63 cells were pretreated for 30 min wit BAPTA-AM (1 or 5 μm) followed by treatment wit Nimbolide (10 μm) for 24. Te percentage of apoptotic cells was ten analyzed by flow cytometric analysis of PI-stained cells; (C) MG63 cells were treated as described in (B), ten cell viability was examined by using an MTT assay; (D) MG63 cells were treated wit control solution or various concentrations of Nimbolide for 24. Te expression levels of GRP78, GRP94, calpain 1, and calpain 2 were examined by using western blot analysis; actin was used as an internal control. Te data are expressed as te mean ± SEM. * p < 0.05 compared wit controls. # p < 0.05 compared wit te Nimbolide treated groups Nimbolide Induces te Activation of Caspases in Human Osteosarcoma Cells Te results presented above suggest tat Nimbolide triggers accumulation of ROS, ER stress, and mitocondrial dysfunction in osteosarcoma cells. However, te apoptotic signaling patway is complex,

7 Int. J. Mol. Sci. 2015, and te canonical patway usually involves activated caspases. We determined tat treatment wit Nimbolide activates caspase-3, caspase-9, and PARP (a downstream effector of caspase-3) (Figure 4A,B). In addition, pretreatment wit inibitors of bot caspases abolised Nimbolide-induced cell deat and increased viability (Figure 4C,D). Tese results confirm tat caspases are involved in Nimbolide-induced apoptosis. Figure 4. Nimbolide induces te activation of caspases in uman osteosarcoma cells. (A) MG63 cells were treated wit control solution or various concentrations of Nimbolide for 24. Te expression levels of PARP, caspase-3, and caspase-9 were examined by using western blot analysis; (B) MG63 cells were treated as described in (A). Te activities of caspase-3 and caspase-9 were examined by using a caspase ELISA kit; (C,D) MG63 cells were pretreated for 30 min wit inibitors of caspase-3 and caspase-9, followed by treatment wit Nimbolide (10 μm) for 24. Te percentages of apoptotic cells were ten analyzed by flow cytometric analysis of PI-stained cells (C). Cell viability was examined by using an MTT assay (D). Te data are expressed as te mean ± SEM. * p < 0.05 compared wit controls. # p < 0.05 compared wit te Nimbolide treated groups.

8 Int. J. Mol. Sci. 2015, Nimbolide Reduces Cell Migration by Decreasing te Expression of Integrin αvβ5 in Human Osteosarcoma Cells Tumor metastasis is te cause of deat for many patients wit cancer. A critical step in tumor metastasis is migration and invasion of cancerous cells to nearby tissues [34]. Terefore, we accessed te effects of Nimbolide on te cell migration penotype of osteosarcoma cells. We found tat low doses of Nimbolide inibited cell migration and invasion but did not affect cell viability (Figure 5A F and Figure S1). Next, we investigated wic component of te migration macinery was inibited by Nimbolide. Integrins ave been proposed as pivotal proteins tat participate in tumor cell migration, invasion, and metastasis [35 37], and it as been proposed tat integrin αvβ5 plays a crucial role in te metastasis of OS [38]. As we expected, Nimbolide treatment decreased te expression of integrin αvβ5 mrna and protein in osteosarcoma cell lines (Figure 5G L). Tese results reveal tat Nimbolide may regulate migration of osteosarcoma cells troug integrin αvβ5. Future researc will confirm te role of integrin αvβ5 in Nimbolide regulation of migration of osteosarcoma cells Te PI3K/Akt and NF-κB Signaling Patway Mediates Nimbolide Inibition of Cell Migration in Osteosarcoma Cells To investigate te mecanism by wic Nimbolide inibits cell migration in osteosarcoma cells, we assessed wic signaling patways it regulates. Previous studies ave indicated tat te PI3K/Akt and NF-κB signaling patway is regulated by Nimbolide [5,39]. We examined te expression and posporylation status of Akt and te p85 subunit of PI3K in osteosarcoma cells, and found decreased posporylation of bot proteins (Figure 6A,B). NF-κB transcription factors are localized to te cytoplasm, were tey bind to te inibitory protein IκB, wic inibits NF-κB binding and prevents nuclear translocation. Upon stimulation of cells, IκB proteins promote rapid posporylation by te multisubunit IKK complex. Posporylation ten targets IκB for ubiquitination and subsequent degradation by te 26S proteasome. Posporylation of IKKα/β, IκBα and te p65 subunit of NF-κB were all decreased by Nimbolide treatment (Figure 6C). Pretreatment wit inibitors of PI3K (Ly294002), Akt (Akti), and NF-κB (TPCK) blocked te effects of Nimbolide in a migration assay (Figure 6D). Moreover, we used a κb-luciferase activity assay to confirm tat NF-κB activity was decreased by treatment of cells wit Nimbolide (Figure 6E). Finally, we confirmed tat pretreatment wit Ly294002, Akti, and TPCK restored activity of te NF-κB luciferase reporter (Figure 6F). Tese results illustrate te pivotal role of te PI3K/Akt and NF-κB signaling cascade in Nimbolide function.

9 Int. J. Mol. Sci. 2015, Figure 5. Nimbolide inibits cell migration of uman osteosarcoma cells by modulating te expression of integrin αvβ5. (A D) Osteosarcoma cells (MG63, U2OS, and HOS) and osteoblast cells (FOB 1.19) were incubated wit control solution or various concentrations of Nimbolide for 1 or 2 days, ten cell viability was examined by using an MTT assay; (E,F) Osteosarcoma cells (MG63, U2OS, and HOS) were incubated wit control solution or various concentrations of Nimbolide for 24, ten migration and invasion were measured in vitro using Transwells; (G I) Osteosarcoma cells (MG63, U2OS, and HOS) were incubated wit control solution or various concentrations of Nimbolide for 24 ; Te expression of integrin αv and β5 mrnas was examined by qpcr (J L). Te expression of integrin αvβ5 protein was examined by flow cytometric analysis. Te data are expressed as te mean ± SEM. * p < 0.05 compared wit controls.

10 Int. J. Mol. Sci. 2015, Figure 6. PI3K/Akt and NF-κB patways function downstream of Nimbolide to inibit cell migration in uman osteosarcoma cells. (A C) Osteosarcoma cells (MG63, U2OS, and HOS) were treated wit control solution or various concentrations of Nimbolide for 24. Te expression levels of posporylated (p)-p85, p85, p-akt, Akt, p-iκbα, IκBα, p-ikkα/β, IKKα/β, p-p65 and p65 were examined by using western blot analysis; (D) Osteosarcoma cells (MG63, U2OS, and HOS) were pretreated wit control solution or various concentrations of Nimbolide for 24 ; (E) Osteosarcoma cells (MG63, U2OS, and HOS) were transfected wit an NF-κB promoter reporter plasmid for 24. Te cells were ten treated as described in (A) and luciferase activity was measured; (F) Osteosarcoma cells (MG63, U2OS, and HOS) were pretreated wit PI3K inibitors (LY (5 μm) and Wortmannin (0.5 μm)), Akt inibitor (Akti (5 μm)), or NF-κB inibitors (TPCK (5 μm)) for 30 min before exposure to AREG. NF-κB luciferase activity was measured, and te results were normalized to te β-galactosidase activity. Te data are expressed as te mean ± SEM. * p < 0.05 compared wit controls. # p < 0.05 compared wit te Nimbolide treated groups. 3. Discussion OS is an infrequent malignancy tat always occurs in te adolescent. Te lung is te most common site of metastasis for OS. Accordingly, it is critical tat we develop novel drugs tat will reduce OS metastasis and improve prognosis. Tumor metastasis is a complicated process tat is mediated by a large number of cellular components. Terefore, cocktail terapies or multi-functional drugs would be more

11 Int. J. Mol. Sci. 2015, ideal candidates tan are drugs tat inibit a single target. In tis study, we found tat te anti-tumoral effects of Nimbolide target multiple cellular processes of osteosarcoma cells. Nimbolide activated te apoptotic signaling patway troug eliciting production of ROS, ER stress, mitocondrial dysfunction, and finally, caspase activation. Te details of te signaling cascade tat mediates Nimbolide-induced apoptosis will be examined in te future. We also determined tat Nimbolide inibits cell migration, an activity critical to metastasis, troug reducing te expression of integrin αvβ5. In addition, we found tat integrin αvβ5 expression is regulated by te PI3K/Akt and NF-κB signaling cascades. In conclusion, tese results indicate tat Nimbolide could serve as an anti-tumor drug for OS via its multifunctional effects. Cancer progression is possible only if te tumor cells evade apoptosis. Tis is accomplised by modulating te balance between pro- and anti-apoptotic proteins and down-regulation of deat receptors [40]. Triggering apoptosis in cancer cells as been recognized as an effective strategy to inibit cancer progression [41]. A large number of studies ave sown tat te triggering of apoptosis by ROS as an anti-tumoral effect in many cancers [42]. Apoptosis can be induced by ROS troug many different mecanisms, suc as cell cycle arrest, and te activation of several signaling patways including caspase-dependent, caspase-independent, deat receptor, mitogen-activated protein kinase, extracellular signal-regulated kinase, and pospoinositide 3-kinase patways [43 46]. Furtermore, levels of ROS are lower and te levels of antioxidants are iger in resistant cancer cells tan in non-resistant cancer cells [47]. Terefore, in bot types of cancer cells, ROS plays vital roles as cemoterapeutic agents. Several studies ave reported tat Nimbolide induces apoptosis via ROS production in colon cancer cells and uman coriocarcinoma cells [6,8]. Te ER is responsible for protein folding and syntesis of secretory proteins. Accumulation of misfolded proteins, ypoxia, Ca 2+ depletion, nutrient starvation, oxidative stress, and oter metabolic dysregulation can all cause ER stress [48]. ER stress induces apoptosis via activation of IRE1, posporylation of eif2a, and release of Ca 2+ from te ER [49]. Te increase in cytosolic calcium can activate calpains, wic activate caspase-dependent patways [50]. Calpain belongs to te calcium-activated non-lysosomal cysteine protease family, wic comprises 14 members in mammals. Calpain 1 (μ-calpain) and calpain 2 (m-calpain) are te best-caracterized isoforms of calpain [51]. Examining te effect of Nimbolide treatment on calpain expression, we observed an acute increase in te level of calpain 2 protein after cells were treated wit 5 and 10 μm Nimbolide for 24. We propose tat increase in calpain 2 was not due to increased transcription and te levels of calpain 2 protein are increased due to decreased degradation. Terefore, in cells exposed to a iger dose of Nimbolide, we observed te opposite effect. Te rapid increase in calpain 2 expression after Nimbolide treatment suggests tat tis protein is important in cell apoptosis and is directly regulated by Nimbolide. Few studies ave examined te potential of Nimbolide for treatment of OS. Te results presented in tis report are te first to sow tat Nimbolide induces apoptosis in osteosarcoma cells troug ROS, ER stress, mitocondrial dysfunction, and caspase activation. Moreover, Nimbolide as been reported to regulate expression of te Bcl-2 family of proteins, disrupt te MMP, activate caspases, and trigger apoptosis in breast cancer [52]. Our findings in osteosarcoma cells are in agreement wit tose of previous studies. Interestingly, we found tat Nimbolide induces ER stress in osteosarcoma cells. Tis effect is novel and as not yet been documented for oter types of cancer. Cancer metastasis occurs in multiple steps by wic cancer cells migrate from te primary site and form a secondary tumor at a distant site [53]. Te expression and tumor-promoting functions of integrins

12 Int. J. Mol. Sci. 2015, ave been well studied [36]. Te present study sowed tat Nimbolide inibited migration of osteosarcoma cells by decreasing te expression of integrin αvβ5. Altoug te role of Nimbolide in inibiting cell migration in cancer as been caracterized, te mecanisms underlying tis regulation remain largely unknown. Here, we ave provided novel insigt regarding te inibitory effects of Nimbolide. Previous studies ave sown tat Nimbolide modulates cell signaling cascades including te PI3K/Akt and NF-κB patway [5,39]. We ave also found tat tese patways were inibited by Nimbolide treatment in osteosarcoma cells. Moreover, our data indicate tat Nimbolide regulates te expression of integrin αvβ5 via te PI3K/Akt and NF-κB patway. Future researc sould clarify if additional signaling cascades are modulated by Nimbolide. A better way to alt tumor progression may be to use a combination of several drugs or a drug wit multiple targets. Previous reviews ave discussed te anti-tumoral effects of Nimbolide [54,55]. In tis report, we present te first evidence tat Nimbolide may be able to prevent te progression of OS by inducing apoptosis and decreasing migration of osteosarcoma cells. Nimbolide as been reported as a potential cemopreventive agent tat is able to prevent cancer progression troug multiple mecanisms. Weter Nimbolide can suppress te progression of OS by oter mecanisms suc as inibiting proliferation and angiogenesis will be investigated in te future. 4. Experimental Section 4.1. Materials Nimbolide was obtained from Sigma-Aldric (St. Louis, MO, USA). Horseradis peroxidase-conjugated anti-mouse and anti-rabbit IgG, and rabbit polyclonal antibodies specific for VDAC, cytocrome c, Bcl-2, Bax, Bak, Bad, Bid, GRP78, GRP94, calpain 1, calpain 2, PI3-kinase (p85), Akt, and NF-κB (p65) were purcased from Santa Cruz Biotecnology (Santa Cruz, CA, USA). Rabbit polyclonal antibodies specific for IKKα/β posporylated at serine (Ser)180/181, IκBα posporylated at Ser32/36, p65 posporylated at Ser536, Akt posporylated at Ser473, p85 posporylated at tyrosine (Tyr)458, caspase-3, and caspase-9 were purcased from Cell Signaling (Danvers, MA, USA). An NF-κB luciferase plasmid was purcased from Stratagene (La Jolla, CA, USA). A psv-β-galactosidase vector and luciferase assay kit were purcased from Promega (Madison, WI, USA). All oter cemicals were obtained from Sigma-Aldric Cell Culture Te uman osteosarcoma cell lines (MG63, U2OS, HOS) and osteoblast cell line (FOB 1.19) were purcased from te American Type Culture Collection (BCRC; Hsincu, Taiwan). MG63, U2OS, HOS, and FOB 1.19 cells were maintained in DMEM, McCoy s 5A, DMEM, and DMEM/Ham s F-12 media, respectively. MG63, U2OS, and HOS cells were incubated at 37 C in an atmospere of 5% CO2 in media supplemented wit 20 mm of 4-(2-ydroxyetyl)-1-piperazineetanesulfonic acid (HEPES), 10% eat-inactivated fetal calf serum (FBS), 2 mm glutamine, 100 U/mL penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). FOB 1.19 cells were incubated at 34 C in an atmospere of 5% CO2 in medium supplemented wit 10% eat-inactivated FBS and 0.3% G418 (Sigma-Aldric). All media were canged every 48.

13 Int. J. Mol. Sci. 2015, Autentication of Cell Lines Te uman osteosarcoma cell lines MG63 (BCRC No.60279), U2OS (BCRC No.60187), and HOS (BCRC No.60308), and osteoblast cell line FOB 1.19 (BCRC No.60357) were obtained from te Biosource Collection and Researc Center (BCRC; Hsincu, Taiwan) in October Te cell lines were tested and autenticated by te BCRC. We purcased te cell lines from te BCRC and passaged it in our laboratory for fewer tan 6 monts after te cells were tawed MTT Assay Cell viability was determined by using a 3-(4,5-dimetyltiazol-2-yl)-2,5-dipenyltetrazolium bromide (MTT) assay. After treatment wit control solution or different concentrations of Nimbolide for 1 or 2 days, te cultured cells were wased wit PBS. MTT (0.5 mg/ml) was ten added to eac well and te mixture was incubated for 2 at 37 C. Culture media were ten replaced wit an equal volume of DMSO to dissolve formazan crystals. After te mixture was saken at room temperature for 10 min, te absorbance of te solution in eac well was determined at 550 nm by using a microplate reader (Bio-Tek, Winooski, VT, USA) Colony Formation Assay Cells ( ) were seeded into 6-well plates and cultured for 10 days. Media were replaced every 2 days. After incubation wit control solution or different concentrations of Nimbolide for 10 days, colonies were wased wit PBS, fixed for 15 min wit 4% paraformaldeyde and stained wit 0.1% crystal violet for 5 min. Te colony forming cells were potograped. After te colonies were wased 3 times wit ddh2o, acetic acid was added to a final concentration of 33% (v/v), wic was followed by measuring te absorbance at 550 nm wit a microplate reader. Te colony formation assay was repeated 3 times, eac of wic was performed in duplicate wells DAPI Staining 4-6-Diamidino-2-penylindole (DAPI), a DNA-binding fluorescent dye, was used to monitor apoptosis by observing nuclear morpology. After treatment wit Nimbolide for 48, te cells were wased 3 times wit PBS, fixed in a 3.7% formaldeyde solution for 10 min, fixed in 1 ml of metanol, and ten stained wit DAPI for 10 min. Apoptosis was determined by visual observation of nuclear morpology wit fluorescence microscopy DNA Ladder Assay MG63 cells ( ) were treated wit control solution or Nimbolide for 48, ten total DNA was extracted and analyzed wit agarose gel electroporesis (2%). Etidium bromide staining was used to assess DNA fragmentation as an indicator of apoptosis. Ultraviolet spectroscopy at 302 nm was used to visualize te DNA after electroporesis.

14 Int. J. Mol. Sci. 2015, Western Blot Analysis Te cell lysates were prepared as described previously [56]. Proteins were resolved wit SDS-PAGE and transferred to immobilon polyvinylidene difluoride (PVDF) membranes. Te membranes were blocked wit 4% BSA for 1 at room temperature and ten probed wit primary antibodies (1:1000) for 1 at room temperature. After 3 wases, te membranes were subsequently incubated wit peroxidase-conjugated secondary antibodies (1:1000) for 1 at room temperature. Te protein bands were visualized by enanced cemiluminescence wit Kodak X-OMAT LS film (Eastman Kodak, Rocester, NY, USA) Cytocemical Analysis wit Annexin V and Propidium Iodide (PI) Apoptosis was assessed by using annexin V, a protein tat binds to pospatidylserine residues, wic are exposed on te cell surface of apoptotic cells, as previously described [57]. Cells were treated wit control solution or Nimbolide for te indicated time intervals. After treatment, te cells were wased twice wit PBS, and ten resuspended in staining buffer containing 1 μg/ml PI and μg/ml FITC-conjugated annexin V. Double-labeling was performed at room temperature for 10 min in te dark, ten analyzed immediately by using FACScan and te Cellquest program (BD Biosciences, San Diego, CA, USA) Cell Cycle Analysis by PI Staining Quantitative assessment of apoptotic cells was also performed by examining te cell cycle. Cells were collected by centrifugation and te concentration was adjusted to cells/ml. Cilled etanol was added to 0.5 ml of cell suspension and te mixture was incubated at 4 C for 30 min. Etanol was ten removed by centrifugation, and cellular DNA was stained wit 100 μg/ml PI (in PBS containing 0.1% Triton-X 100 and 1 mm EDTA) in te presence of an equal volume of DNase-free RNase (200 μg/ml). After staining, te cells were analyzed immediately wit a FACScan and te Cellquest program. Te extent of apoptosis was determined by measuring te DNA content of cells below te sub G1 peak [58] Terminal Deoxynucleotidyl Transferase dutp Nick End Labeling (TUNEL) Assay Quantitative assessment of apoptotic cells was also performed by using a TUNEL assay (BD ApoAlert DNA Fragmentation Assay Kit, BD Biosciences Clontec, Palo Alto, CA, USA), wic examines DNA-strand breaks during apoptosis. Briefly, cells were incubated wit control solution or Nimbolide for te indicated times. Te cells were trypsinized, fixed wit 4% paraformaldeyde, and ten permeabilized wit 0.1% Triton-X 100 in 0.1% sodium citrate. After being wased, te cells were incubated wit te reaction mixture for 60 min at 37 C. Te stained cells were ten analyzed by using flow cytometry Determination of Mitocondrial Membrane Potential (MMP) Te MMP (ΔΨm) was assessed by using te fluorometric probe JC-1 (Calbiocem, CA, USA), a positively carged mitocondria-specific fluoropore tat indicates depolarization by a sift in

15 Int. J. Mol. Sci. 2015, fluorescence emission from green (525 nm) to red (610 nm) [59]. Briefly, te cells were plated in 6-well culture dises, grown to confluence, and ten treated wit control solution or Nimbolide. After incubation, te cells were stained wit JC-1 (5 μg/ml) for 15 min at 37 C and ten analyzed by FACScan using an argon laser (488 nm). Mitocondrial depolarization, wic is specifically indicated by a decrease in te intensity ratio of red to green fluorescence, was analyzed by using te Cellquest program Measurement of ROS Levels of intracellular H2O2 were assessed by using spectrofluorometry to monitor te oxidation of te probe H2DCFDA (Molecular Probes, Waltam, MA, USA). Cells were plated at a density of , allowed to attac overnigt, and exposed to control solution or Nimbolide for te specified time intervals. Te cells were incubated wit H2DCFDA (10 μm) for 10 min at 37 C and te fluorescence intensity in te cells was determined by using flow cytometry Detection of Ca 2+ Concentration To detect canges in Ca 2+ levels, cells were plated in 12-well plates and incubated wit control solution or Nimbolide for te specified time intervals. Te cells were arvested, wased twice, resuspended in Fluo 3/AM (3 μg/ml), and incubated at 37 C for 30 min before being analyzed by using flow cytometry Measurement of Caspase Activity Te caspase activity assay is based on te ability of te active caspase enzyme to cleave te cromopore p-nitroaniline from a peptide substrate: Ac-DEVD-pNA was te substrate for caspase-3 and Ac-LEHD-pNA was te substrate for caspase-9. Te cell lysates were prepared and incubated wit antibodies specific for caspase-3 and caspase-9. Te immunocomplexes were incubated wit te substrate in assay buffer (100 mm NaCl, 50 mm 4-[2-ydroxyetyl]-1-piperazine-etanesulponic acid [HEPES], 10 mm ditiotreitol, 1 mm EDTA, 10% glycerol, 0.1% 3-[(3-colamidopropyl) dimetyl ammonio]-1-propanesulfonate [CHAPS], ph 7.4) for 2 at 37 C. Te release of p-nitroaniline was monitored at 405 nm wit a microplate reader. Te results are presented as te percent cange of activity between te test sample and te untreated control In Vitro Cell Migration and Invasion Assays Te migration assay was performed wit Transwell inserts (Costar, NY, USA; 8-mm pore size) in 24-well dises. Approximately cells in 200 μl of serum-free medium were placed in te upper camber, and 300 μl of te same medium containing control solution or different concentrations of Nimbolide were placed in te lower camber. Te cells were incubated for 24, ten fixed in 3.7% formaldeyde for 5 min and stained wit 0.05% crystal violet in PBS for 15 min. Te cells on te upper side of te filters were removed and te filters were wased wit PBS. Te cells on te underside of te filters were examined and counted by using ligt microscopy. Eac experiment was performed in triplicate and repeated at least 3 times. Te number of migrated cells in eac experiment was corrected for te effects of proliferation by using a cell viability assay (corrected invading cell number = counted

16 Int. J. Mol. Sci. 2015, invading cell number/percentage of viable cells) [60]. For te invasion assay, Transwell inserts were coated wit BD Matrigel (BD Biosciences) before use. Te experimental protocol for te invasion assay was as described above for te cell migration assay Reporter Gene Assays Cells were co-transfected wit an NF-κB reporter plasmid (0.7 µg) and a p-sv-β-galactosidase plasmid (0.3 µg) by using Lipofectamine 2000 according to te manufacturer s recommendations. After transfection for 24, te cells were treated wit control solution or Nimbolide for 24. Cell extracts were ten prepared, and te activities of luciferase and β-galactosidase were measured Statistical Analyses Te values reported are means ± SEM. Statistical analyses of differences between 2 samples were performed by using te Student s t-test. Statistical comparisons of more tan 2 groups were performed by using 1-way analysis of variance (ANOVA) wit Bonferroni s post-oc test. In all cases, p < 0.05 was considered significant. 5. Conclusions Our data indicate tat Nimbolide induces apoptosis and inibits cell migration in uman osteosarcoma cells. Nimbolide-induced cell deat is mediated by increasing levels of ROS, mitocondrial dysfunction, ER stress, and Ca 2+ release, all of wic subsequently lead to increased activity of calpain, caspase-3, and caspase-9. Furtermore, we ave also sown tat Nimbolide inibits cell migration by modulating te expression of integrin αvβ5, wic is regulated by te PI3K/Akt and NF-κB signaling cascade. We ope tat our proposed working model for te molecular basis of Nimbolide function will provide valuable insigts for te development of an effective cemoterapy for OS by targeting te appropriate signal transduction proteins. Supplementary Materials Supplementary materials can be found at ttp:// Acknowledgments Tis work was supported by grants from te National Science Council of Taiwan (NSC B MY3; NSC B MY3) and Sin-Kong Wu Ho-Su Memorial Hospital (SKH ; SKH ). We tank te staff of te Eigt Core Lab, Department of Medical Researc, National Taiwan University Hospital for tecnical support during te study. Autor Contributions Ju-Fang Liu and Seng-Mou Hou conceived and designed te experiments. Cun-Han Hou, Feng-Ling Lin, Ya-Ting Tsao, and Ju-Fang Liu performed te experiments. Cun-Han Hou,

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