Pharmacological exploitation of the phenothiazine antipsychotics to develop novel antitumor agents-A drug repurposing strategy

Abstract

Phenothiazines (PTZs) have been used for the antipsychotic drugs for centuries. However, some of these PTZs have been reported to exhibit antitumor effects by targeting various signaling pathways in vitro and in vivo. Thus, this study was aimed at exploiting trifluoperazine, one of PTZs, to develop potent antitumor agents. This effort culminated in A4 [10-(3-(piperazin-1-yl)propyl)-2-(trifluoromethyl)-10H-phenothiazine] which exhibited multi-fold higher apoptosis-inducing activity than the parent compound in oral cancer cells. Compared to trifluoperazine, A4 demonstrated similar regulation on the phosphorylation or expression of multiple molecular targets including Akt, p38, and ERK. In addition, A4 induced autophagy, as evidenced by increased expression of the autophagy biomarkers LC3B-II and Atg5, and autophagosomes formation. The antitumor activity of A4 also related to production of reactive oxygen species and adenosine monophosphate-activated protein kinase. Importantly, the antitumor utility of A4 was extended in vivo as it, administrated at 10 and 20 mg/kg intraperitoneally, suppressed the growth of Ca922 xenograft tumors. In conclusion, the ability of A4 to target diverse aspects of cancer cell growth suggests its value in oral cancer therapy.

Pharmacological exploitation of the phenothiazine antipsychotics to develop novel antitumor agentsA drug repurposing strategy

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Chia-HsienWu, Li-Yuan Bai,, Ming-HsuiTsai, Po-Chen Chu,, Chang-Fang Chiu,, MichaelYuanchien Chen,, Shih-Jiuan Chiu, Jo-Hua Chiang & Jing-RuWeng

Phenothiazines (PTZs) have been used for the antipsychotic drugs for centuries. However, some of in vitro and in vivo H-phenothiazine] which exhibited multi-fold higher apoptosis-inducing activity than the parent in vivo aspects of cancer cell growth suggests its value in oral cancer therapy.

Oral cancer is the sixth leading cause of cancer death worldwide, accounting for over 350,000 deaths per year1.

The combined use of tobacco, betel nut, and alcohol has been shown to a major risk factor for the development of oral cancer due to their carcinogenic eect in the oral muscosa2. Although many therapeutic options are available for the treatment of oral cancer, many patients eventually develop therapeutic resistance. Thus, there is an urgency to develop new chemotherapeutic agents for patients who have developed advanced oral cancer.

Phenothiazines (PTZs) represent a major class of antipsychotic drugs, widely used for the treatment of schizophrenia, and bipolar disorder3,4. PTZs were originally developed as anti-psychotropic agents due to their inhibitory activity against the dopamine D2 receptor4,5. Recently, some of these PTZs have been reported to exhibit antitumor eects by targeting various signaling pathways, including those mediated by protein kinase C, calmodulin-dependent enzymes, P-glycoprotein, and protein phosphatase 2A57. Consequently, recent years have wintnessed the modications of PTZs to develop dierent targeted agents, including the triazole derivatives as farnesyltransferase inhibitors8 and the N-benzoylated derivatives as tubulin inhibitors911 (Fig.1A).

In this study, we report the use of triuoperazine as a lead compound to develop antitumor agents with improved antitumor efficacy and reduced toxicity. This structure optimization eort resulted in a proof-of-concept compound, A4, which exhibited multi-fold higher potency as compared to the parent compound without appreciable cytotoxicity to normal oral human keartinocytes (NHOKs). Mechanistically, A4 retained the activity of

College of Medicine, China Medical Division of Hematology and Oncology, Department of Internal Medicine, China Graduate Institute of Clinical Medical Science, China Medical Institute of Basic Medical Sciences, College of Medicine, National Cheng

School of School of Pharmacy, Taipei Medical University, Taipei

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Figure 1. Use of triuoperazine as scaolds for developing new anticancer agents. Upper panel, Structureof triuoperazine, Lower panel, structures and potencies for inducing apoptotic death in Ca922 cells of the triuoperazine derivatives A1 to A18. Cell viability was assessed by MTT assays with six replicates. The reported IC50 values are concentrations at which Ca922 cell death measures 50% relative to DMSO control aer 48h exposure in 5% FBS-containing MEM in 96-well plates.

triuoperazine to induce caspase-dependent apoptosis in oral squamous cell carcinoma (OSCC) cells by targeting multiple signaling pathways. Moreover, we obtained evidence that A4 induced autophagic cell death, which might be associated with its ability to activate reactive oxygen species (ROS) production and adenosine

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Figure 2. Antiproliferative eects of triuoperazine and A4 in oral cancer cell lines (Ca922, SCC2095), primary OSCC cells, and NHOKs. (A) Ca922, (B) SCC2095 (C) Primary OSCC cells, and (D) NHOKs (5103/200L) were treated with DMSO vehicle or triuoperazine or A4 at the indicated concentrations. Cell viability was assessed by MTT assay as described in the Material section. Points, mean; bars, S.D. (n =6). *P<0.05, **P< 0.005 compared to the control group.

monophosphate-activated protein kinase (AMPK). More importantly, A4 was eective in vivo in suppressing OSCC xenogra tumor growth, while the triuoperazine-treated counterparts died within a few days.

Results

Structure activity relationship (SAR). In order to improve the antiproliferative eect of triuoperazine, we synthesized three series of N-substituted analogues (Fig.1B). The antiproliferative activities of these agents (A1A18) were evaluated in Ca922 oral cancer cells by MTT assays aer 48 h of drug treatment. Among these derivatives, A4 exhibited the highest antiproliferative potency with IC50 of 4.9M (Fig.1B, etoposide as the positive control), relative to 14M for triuoperazine (Fig.2A). SAR analysis indicates that replacement of the piperazine ring with a dierent heterocycle, such as morpholine (i.e., A3) or piperidine (i.e., A10), and/or changes in

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the length/structure of the linker resulted in substantial loss of antiproliferative potency. Together, these ndings suggested that the propyl-piperazine moeity played an integral role in maintaining the antiproliferative activity. This dierential antitumor activity was also noted in another OSCC cell line, SCC2095 (IC50: A4, 4.5M; triuoperazine, 18 M, Fig.2B). We rationalized that this discrepancy was attributable to the N-methyl function of triuoperazine, of which the stereoelectronic eect hindered the ligand interaction with target proteins.

In addition, A4 was eective in suppressing the viability of primary OSCC cells with IC50 of 5.6 M at 48 h (Fig.2C), while normal humna oral keratinocytes (NHOKs) were insensitive to A4 (Fig.2D). These ndings suggest the discriminative antiproliferative activity of A4 against malignant versus non-malignant oral mucosal cells.

-operazine. We obtained evidence that that A4 retained the ability of triuoperazine to facilitate apoptotic cell death via caspase activation. For example, annexin V/PI staining showed that treatment of Ca922 cells with either agent led to dose-dependent increases in the apoptotic cell population (Fig.3A; etoposide as a positive control). This apoptosis induction was conrmed by Western blot and ow cytometric analyses that showed dose-dependent eects of A4 and triuoperazine on caspase-9 and procapase-8 activation (Fig.3B) and activated caspase-3 expression (Fig.3C), respectively.

Trifluoperazine has been reported to target multiple signaling pathways to induce apoptosis in cancer cells12,13. Consequently, we examined the phosporylation status of a series of key signaling kinases, including Akt and its downstream target mTOR, p38, and ERKs. Western blot anaylsis showed that A4 and triuoperazine dose-dependently decreased the phosphorylation of Akt and mTOR, accompanied by parallel increases in p38 phosphorylation in Ca922 cells (Fig.4). However, A4 and triuoperazine diverged on their respective eects on ERK phosphorylation, i.e., only A4 was eective in suppressing p-ERK levels, indicating a subtle dierence in their pharmacological properties.

It has been reported that triuoperazine inhibited DNA double strand breaks repair in human larynx carcinoma cells and delayed the ability of -H2AX resolution in DNA-damaged lung cancer cells14,15. In light of the mechanistic link between ROS and DNA damage response to many anti-tumor drugs16,17, we rst examined the cellular levels of ROS in A4-treated Ca922 cells (5 M). As shown in Fig.5A, 5M A4 signicantly increased ROS generation in Ca922 cells aer 48h of treatment (37.1% to 61.8%) which, however, could be rescued by N-acetylcysteine (NAC), a reported antioxidant18. It is noteworthy that this A4-induced ROS generation was accompanied by dose-dependent increases in the phosphorylation of H2AX and p53, hallmarks of DNA damage response19,20, in Ca922 cells (Fig.5B).

It has been reported that triuoperazine induced autophagic degradation without causing cellular damages in glioblastoma cells21. Pursuant to this nding, we obtained several lines of evidence to demonstrate the ability of A4 to induce autophagy. First, transmission electron microscopy revealed autophagosome formation in the cytoplasm aer exposing Ca922 cells to 5 M A4 for 24 h (Fig.6A). Second, Ca922 cells were transiently transfected with GFP-tagged LC3 (GFP-LC3) and exposured 5 M A4, or 100 nM rapamycin (positive control). Confocal uorescene imaging demonstrated that the accumulation of LC3-positive puntca in the cytoplasm in a manner similar to that of rapamycin (Fig.6B). Furthermore, Western blotting showed that A4 dose-dependently increased the expression of LC3B-II and autophagy-related protein (Atg)5 (Fig.6C), one of the essential molecures which forms pre-autophagosomes22,23.

To investigate whether this autophagy induction played a protective or cytotoxic role, we examined the eect of the autophagic inhibitor balomycin A1 (BA; a vacuolar-type H+-ATPase inhibitor) on A4-induced apoptotic cell death. PI/annexin-V analysis demonstrated that co-treatment of Ca922 cells with BA recused A4-induced apoptotic death (Fig.6D). As shown, Western blotting showed that co-treatment with BA led to a lesser extent of PARP cleavage and caspase-9 activation as compare to A4 alone (Fig.6E). Together, these data suggested that A4 induces autophagic cell death, which increased the drug eect on apoptosis in Ca922 cells.

In light of the role of AMPK in inducing auto-phagy through the negative regulation of mTORC124, we further examined the eect of A4 on AMPK activation. Consistent with our premise, Western blotting analysis indicated the ability of A4 to activate AMPK, as manifested by increased phosphorylation levels of AMPK and its downstream target acetyl-CoA carboxylase (ACC) in A4-treated Ca922 cells (Fig.7A). To clarify the role of AMPK, we examined the eect of the AMPK inhibitor compound c on A4s antiproliferative activity. Flow cytometric analysis showed that compound c was able to protect Ca922 cells from A4-induced apoptotic cell death (P< 0.05; Fig.7B,C). Furthermore, Western blot analysis showed that co-treatment of compound c abolished the eect of A4 on AMPK activation, which was accompanied by a lesser extent of PARP cleavage relative to A4 alone (Fig.7C).

We examined the in vivo antitumor efficacy of A4 relative to triuoperazine in Ca922 tumor-bearing mice. Daily administration of A4 at 10 or 20mg/ kg by intraperitoneal injection signicantly inhibited Ca922 xenogra tumor growth by 74% and 81% (P<0.001), respectively (Fig.8A). A4 slightly decreased the body weight of tumor-bearing mice during the rst week of treatment, however, the dierences were not statistically signicant and no further decreases were noted in the following weeks (Fig.8B, P= 0.0795). Although mice receiving parent triuoperazine at 30mg/kg/day had smaller tumor size compared with mice in control group at initial days (Fig.8A), all 6 mice in triuoperazine group had signicant body weight loss from 21.20.5mg to 16.5 0.9mg and died aer 5 days of treatment. This suggested a considerable toxicity of triuoperazine for mice.

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Figure 3. Evidence of apoptosis for triuoperazine and A4-induced cell death. (A) Annexin V-FITC/ propidium iodide staining. Ca922 cells were treated with DMSO vehicle or triuoperazine or A4 at the indicated concentrations in 5% FBS-supplemented MEM medium for 48h. (B) Western blotting of procaspase-8 and caspase-9 aer the treatment of triuoperazine or A4 in Ca922 cells for 48h. (C) Caspase-3 activation of triuoperazine and A4 in Ca922 cells (n= 4). Staurosporine (Stauro.) as the positive control. *P<0.05; **

P< 0.005 compared to the control group.

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Figure 4. Dose-dependent eects of triuoperazine and A4 on the phosphorylation/expression of Akt, mTOR, p38, and ERK in Ca922 cells. Cells are treated with triuoperazine or A4 at the indicated concentrations in 5% FBS-MEM for 48h and cell lysates were immunoblotted as described in Methods section.

As the development of brand-new therapeutic agentes takes an enormous amount of resources, time, and eort, repurposing of existing drugs by exploiting their o-target mechanisms has become a useful strategy for new drug discovery25,26. In this study, we report the pharmacological exploitation of triuoperazine, an antipsychotic drug, as a proof-of-concept to develop new antitumor agents for oral cancer therapy. The structurally optimized agent A4 exhibited not only higher in vitro/in vivo antitumor efficacy, but also lesser toxicity in normal oral human keratinocytes and tumor-bearing mice. Thus, A4 represents a proof-of-concept compound that the phenothiazine class of antipsychotic drugs could be used as starting points for developing novel antitumor agents for clinical development.

The replacement of hydrogen on the piperazine ring endowed A4 with multifold higher apoptosis-inducing potency. Relative to triuoperazine, A4 displayed similar pattern on modulating multiple molecular targets, including Akt and its downstream eectors mTOR and the MAPK kinases p38 (Fig.4). Its noteworthy that these signaling eectors play the important roles in the development of metastsis and invasion in oral cancer cells2729.

In addition, increased oxidative stress including the long-term use of areca nut, dysfunction of antioxidant enzymes, and DNA damage has been implicated in the pathogenesis of oral cancer3032. Interestingly, a recent paper reported that triuoperazine protected hydrogen peroxide-induced apoptosis in rat pheochromocytoma cells33. In contrast, our present study demonstrated that A4 induced ROS generation, which could be rescue by the antioxidant agent NAC. Meanwhile, our nding of A4-induced DNA damage is consistent with the previous report that triuoperazine impaired DNA repair in lung cancer34. Moreover, we found that autophagy is involved in A4-induced cell death, at leat in part, through AMPK activation. Because pharmacological inhibition of AMPK could partially proteced cells from A4-induced apoptosis, we rationalize that this AMPK activation acts in concert with the inhibition of Akt/mTOR and the activation of p38 to facilitate apoptosis in A4-treated oral cancer cells (Fig.7).

Evidence indicates that autophagy could play a pro-survival or pro-apoptosis role in drug-induced cell death35,36. It has been reported that trifluoperazine-induced autophagy could protect human dopaminergic cells from wild-type alpha-synuclein-induced toxicity37. However, our presente study showed that A4 induced-autophagy played a pro-apoptosis role in oral cancer cells. AMPK, a cellular energy sensor, is one of the molecular targets which modulate autophagy and being the potential target for cancer therapy38. Assessment of the in vivo efficacy of A4 in tumor-bearing nude mice indicated that A4 was eective in suppressing the growth of established xenogra tumors. More importantly, unlike, its parent compound triuoperazine, A4 did not incur acute toxicity in tumor-bearing mice.

Conclusions

Our results show that the triuoperazine derivative, A4, is a potent antitumor agent in oral cancer cells, which mediates apoptosis, at least in part, through the activation of AMPK, ROS generation, and caspases activation. A4 also induced autophagic cell death, which contributed to its antiproliferative activity. Equally important, A4 exhibited in vivo efficacy in suppressing xenogra tumor growth in nude mice without incurring acute toxicity. Taken together, this study provides a proof of concept for the repurposing of PTZ antipsychotic drugs to develop novel therapeutic agents for cancer therapy.

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Figure 5. ROS generation of A4 in Ca922 cells. (A) Le panel, Flow cytometric analysis of the eect of A4 (5M), alone or in combination with the antioxidant N-acetylcysteine (NAC) for 3h on ROS production. Three independent experiments were performed, and the statistical analysis are presented in right panel, Points, mean; bars, S.D. (n=3). *P< 0.05 compared to the control group. (B) Western blotting analysis of the phosphorylation/expression of H2AX and p53 in Ca922 cells.

Methods

Reagents, Antibodies, and Plasmids. 2-(triuoromethyl)-10H-phenothiazine (Sigma-Aldrich, St. Louis, MO, USA) was used as the starting material to synthesize triuoperazine and triuoperazine derivatives (Fig.1A, Figs S3S16). The identity and purity (>95%) of these synthetic derivatives were identied by proton magnetic resonance spectrometry and HR-EIMS (Figs S5S38). All agents were dissolved in DMSO, diluted in culture medium, and added to cells at a nal DMSO concentration of 0.1%. Antibodies against the following biomarkers were obtained from Cell Signaling Technologies (Danvers, MA, USA): Akt, p-473Ser Akt, p-180/182Thr/Tyr 38, p38, p-2448Ser mTOR, ERK, p-202/204Thr/Tyr ERK, LC3B, Atg5, p-172Thr AMPK, AMPK, p-15Ser p53, p53, p-139Ser H2AX, p-79Ser ACC, ACC, PARP, procaspase-8, and caspase-9. -actin was obtained from Sigma-Aldrich. The GFP-LC3 plasmid was purchased from Addgene (Cambridge, MA, USA). The enhanced chemiluminescence system for detection of immunoblotted proteins was from GE Healthcare (Piscataway, NJ, USA). Other chemicals and reagents were obtained from Sigma-Aldrich unless otherwise noted.

Cell Culture. Ca922 and SCC2095 human oral cancer cells were kindly provided by Professor Susan R. Mallery (The Ohio State University). Ca922 cells were cultured in MEM medium and SCC2095 were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 5 mg/ml of penicillin and 5mg/ml of streptomycin. In addition to the above culture condition, 0.4g/mL hydrocortisone was added in SCC2095 cells. Primary OSCC cells were isolated from freshly surgical specimens according to a protocol approved by the China Medical University Hospital internal review board. Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki and all experimental procedures were carried out in accordance with the approved protocol. Normal human oral keratinocytes (NHOKs) were kindly provided from Dr. Tzong-Ming Shieh (China Medical University) and were maintained in the keratinocyte serum-free medium (Gibco). All cells were cultured at 37C in a humidied incubator containing 5% CO2.

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Figure 6. A4 induced autophagy. (A) Electron microscopic analysis of autophagosome formation aer the treatment of A4 (5M) or DMSO in Ca922 cells for 24h as described in Methods section. Magnication, 12000x. Arrow: autophagosomes. (B) Fluorescent confocal microscopic analysis of A4-induced autophagosome formation in Ca922 cells ectopically expressing GFP-LC3. Cells transiently transfected with GFP-LC3 plasmids were treated with DMSO, 5M A4, or 100nM rapamycin for 48h and then xed by 3.7% paraldehyde and examined by confocal microscopy. Scale bar: 10m. Arrow: autophagosomes. (C) Western blotting of LC3B and Atg5 in Ca922 cells treated with A4 for 48h. (D) Ca922 cells were treated with 7.5M A4 alone or in combination with 1nM balomycin A1 (BA) for 48h, and then annexin V-FITC/PI double-staining analysis was performed. (E) Western blot analysis of the expression of PARP and caspase-9 aer A4 alone or the combination of BA in Ca922 cells.

Cell Viability Analysis. The effect of test agents on cell viability was assessed by the 3-(4,5-dime thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay39 in 6 replicates. Briey, cells (5 103) were seeded and incubated in 96-well, flat-bottomed plates in 10% FBS-supplemented MEM or DMEM/F12 or keratinocyte serum-free medium for 24 h, and were exposed to test agents at the indicated concentrations in 5% FBS-supplemented MEM or DMEM/F12 or keratinocyte serum-free medium for dierent time intervals. Medium was removed and replaced by 200 L of 0.5 mg/ml MTT in the same medium. Aer 2 h incubation, the reduced MTT dye was solubilized in 200L/well of DMSO. Absorbance was determined with a Synergy HT (Bio-Tek, Winooski, Vermont, USA) at 570nm.

Flow Cytometric Analysis. Ca922 cells (5104) were plated and treated with test agents at indicated concentrations in 5% FBS-supplemented MEM medium for 48 h40. Cells were harvested, washed twice in ice-cold PBS, xed in 70% cold ethanol at 4 C for 4 h, followed by spinning at 1200 rpm for 5 min and re-suspending in ice-cold PBS containing 2% PBS. Cells were stained with annexin V-FITC and PI according to the vendors

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Figure 7. Restoration of antiproliferative activity of A4 by inactivating AMPK. (A) The phosphorylation/ expression of AMPK and ACC of A4 in Ca922 cells. Cells were treated with A4 in 5% FBS-supplemented MEM medium for 48h, and cell lysates were immunoblotted as described in Methods. (B) Histogram showing 7.5M A4 alone or in combination with 2.5M compound c (CC) for 48h, and then annexin V-FITC/PI double-staining analysis was performed. (C) The percentage of cells in Q2 and Q4 phases aer the treatment was shown. Data are presented as meanS.D. *P<0.05; **P<0.005. (D) Western blotting analysis of the phosphorylation/expression of AMPK and PARP aer the combination of CC or A4 alone in Ca922 cells.

Figure 8. Eects of A4 on Ca922 xenogra tumor size and mice body weight change. (A) Mice bearing Ca922 xenogras were treated with normal saline control, A4 (10mg/kg/day), A4 (20mg/kg/day), or triuoperazine (30mg/kg/day). The tumor size was recorded every 2 days. (B) Body weight change of mice.

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protocols (BD Pharmingen, San Diego, USA) and analyzed by using BD FACSAria ow cytometer (Becton, Dickinson and Co., Franklin Lakes, NY, USA). For caspase-3 activation, drug-treated cells were assessed using a FITC rabbit anti-active caspase-3 kit, according to the vendors protocols (BD Pharmingen).

Western Blotting. Drug-treated cells were collected, washed with ice-cold PBS, and resuspended in lysis buffer [137 mM NaCl, 1 mM CaCl2, 20 mM Tris-HCl (pH 8), 0.1% SDS, 100 M 4-(2-aminoethyl)-be nzenesulfonyl uoride, 0.5% deoxycholate, 10% glycerol, 1% Nonidet P-40, leupeptin at 10g/mL, and aprotinin at 10 g/mL]. Soluble cell lysates were collected aer centrifugation at 1500 g for 5 min. Equivalent amounts of protein (60100g) from each lysate were resolved in 10% SDS-polyacrylamide gels. Bands were transferred to nitrocellulose membranes and blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBST) and incubated overnight with the corresponding primary antibodies at 4 C. Aer washing with PBST three times, the membrane was incubated at room temperature for 1h with the secondary antibody with PBST and visualized by enhanced chemiluminescence.

Transient Transfection and Confocal Microscopy. The GFP-LC3 plasmid was transiently transfected into Ca922 cells by using the Fugene HD reagent (Roche, Mannheim, Germany) according to the manufactures protocol41. Cells (2 105/3 mL) were seeded in each well of a six-well plate. Aer 24 h incubation, cells were treated with A4 (5 M) or rapamycin (100 nM) as positive control for 15 min, xed in 2% paraformaldehyde (Merck Millipore, Darmstadt, Germany) for 30min at room temperature, permeabilized with 0.1% Triton X-100 for 20 min, and washed with PBS. GFP uorescence was visualized on a Leica TCS SP2 confocal microscope (Leica Biosystems Nussloch GmbH, Heidelberg, Germany).

Samples were prepared according to an established procedure42. Briey, Ca922 cells were xed in a solution containing 0.2 M sodium cacodylate, 2.5% glutaraldehyde, and 2% paraformaldehyde for 1h. The xed cells were suspended in a buered solution containing 1% osmic acid for 1h, followed by dehydration in a graded ethanol series, wash with acetone, and embedding into EPON epoxy resin. Ultrathin sections (6080nm) were prepared on an ultramicrotome and double-stained with uranyl acetate and lead citrate. All sections were examined and photographed with a Hitachi H-600 transmission electron microscope (Hitachi, Tokyo, Japan).

In vivo. Twenty four male nude mice of 5 weeks of age were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Ca922 cells were cultured in MEM supplemented with 10% heat-inactivated FBS. Each mouse was inoculated subcutaneously with 1107 Ca922 cells in 0.1ml phosphate-buered saline. Tumor diameter was measured twice weekly using calipers and the tumor volume was calculated using a standard formula: width2 length 0.52. Body weights of the mice were measured once weekly. When the mean tumor volume reached 60 mm3, mice were randomized into four groups (n = 6) that received the following treatments: (a) A4 at 10mg/kg body weight qd, (b) A4 at 20mg/kg body weight qd, (c) triuoperazine at 30 mg/kg body weight qd, and (d) normal saline control. All mice received treatments by intraperitoneal injection (50 L/mouse) daily till reaching the endpoint. The criteria for endpoint included death, body weight loss more than 30% or tumor size more than 1200mm3. All animal experiments were performed in accordance with the guidelines of the Animal Welfare Act and The Guide for Care and Use of Laboratory Animals from the Council of Agriculture, Executive Yuan. The in vivo experiment protocol was approved by the Institutional Animal Care and Use Committee of China Medical University (Taichung, Taiwan, IACUC Approval no.: 103-105-N, period of protocol valid from August 01, 2014 to July 31, 2017).

Statistical Analysis. All data are presented as mean S.D. obtained from three independent experiments. Dierences in among group means of tumor volume in vivo were analyzed for statistical signicance using one-way analysis of variance followed by the NeumanKeuls test for multiple comparisons. Dierences were considered signicant *P<0.05, **P< 0.005. Statistical analyses were performed using SPSS for Windows (SPSS,

Chicago, IL, USA).

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Acknowledgements

This work was supported by grant from Ministry of Science and Technology grant (MOST 103-2320-B-039-023-MY3). The authors would like to thank Core Facility Center, Office of Research and Development (Taipei Medical University) for the technical support of transmission electron microscopy.

Author Contributions

C.-H.W. performed the experiments. J.-R.W. conducted the study and wrote the main manuscript. L.-Y.B., M.-H.T. and P.-C.C. prepared Figures 14. C.-F.C., M.Y.C. and S.-J.C. provided advice on the experiments. J.-H.C. prepared Figure S4S7. All authors reviewed the manuscript.

Supplementary information accompanies this paper at http://www.nature.com/srep

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Wu, C.-H. et al. Pharmacological exploitation of the phenothiazine antipsychotics to develop novel antitumor agentsA drug repurposing strategy. Sci. Rep. 6, 27540; doi: 10.1038/srep27540 (2016).

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