A Novel Arylurea Fatty Acid That Targets the ... - ACS Publications

18 Sep 2017 - and high-risk neuroblastoma.7,8 Polyunsaturated fatty acids ... Brief Article .... serum-free medium (37 °C, 20 min) and subjected to f...
0 downloads 0 Views 2MB Size
Download PDF
Brief Article Cite This: J. Med. Chem. 2017, 60, 8661-8666

pubs.acs.org/jmc

A Novel Arylurea Fatty Acid That Targets the Mitochondrion and Depletes Cardiolipin To Promote Killing of Breast Cancer Cells Tristan Rawling,*,† Hassan Choucair,‡ Nooshin Koolaji,‡ Kirsi Bourget,‡ Sarah E. Allison,‡ Yong-Juan Chen,§ Colin R. Dunstan,§ and Michael Murray‡ †

School of Mathematical and Physical Sciences, Faculty of Science, University of Technology Sydney, Ultimo, New South Wales 2007, Australia ‡ Discipline of Pharmacology, School of Medical Sciences, Sydney Medical School, University of Sydney, Sydney, New South Wales 2006, Australia § School of Aerospace, Mechanical and Mechatronic Engineering, University of Sydney, Sydney, New South Wales 2006, Australia S Supporting Information *

ABSTRACT: Cancer cell mitochondria are promising anticancer drug targets because they control cell death and are structurally and functionally different from normal cell mitochondria. We synthesized arylurea fatty acids and found that the analogue 16-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid (13b) decreased proliferation and activated apoptosis in MDA-MB-231 breast cancer cells in vitro and in vivo. In mechanistic studies 13b emerged as the prototype of a novel class of mitochondrion-targeted agents that deplete cardiolipin and promote cancer cell death.

■

INTRODUCTION Oncology drugs target pathways in tumor cells that promote proliferation or activate cell death.1 New classes of anticancer agents with novel mechanisms of action are required to provide additional therapeutic alternatives in cancer chemotherapy as sole agents or in combination with other drugs. The cancer cell mitochondrion is a promising anticancer drug target because of its role in apoptotic cell death.2 Cytochrome c is stored within the mitochondrial intermembrane space and, in response to proapoptotic stimuli, is released into the cytoplasm where it activates executioner caspases such as caspase-3/7.3 Furthermore, the tumor cell mitochondrion is functionally and structurally distinct from those in noncancerous cells, which provides an opportunity for selective cytotoxicity.2 The atypical phospholipid cardiolipin (CL) regulates apoptosis by modulating the release of cytochrome c from the mitochondrial membrane.4 Interest in the capacity of lipids and lipid metabolites to modulate cellular homeostasis is increasing.5,6 The alkylphospholipid analogues, typified by miltefosine and perifosine, have found clinical application in the treatment of amebal infections and high-risk neuroblastoma.7,8 Polyunsaturated fatty acids undergo biotransformation to metabolites that modulate intracellular signaling and the viability of cancer cells. Thus, the ω-6 arachidonic acid is converted by cyclooxygenases and cytochromes P450 to prostaglandin E2 and the epoxyeicosatrienoic acids (EETs), respectively, that drive tumor proliferation and promote metastasis.9 In contrast, certain ω-3 polyunsaturated fatty acid metabolites are antitumorigenic. Zhang et al. © 2017 American Chemical Society

found that epoxides of docosahexaenoic acid inhibited primary tumor growth and metastasis in tumor-bearing mice.10 Similarly, the ω-3-17,18-epoxide metabolite of eicosapentaenoic acid (ω-317,18-epoxy-EPA) inhibited cell viability by arresting cell cycle progression and activating apoptosis.11 Development of fatty acid epoxides as therapeutics is complicated by their in vivo instability, including degradation by soluble epoxide hydrolase (sEH).12 Indeed, coadministration of the sEH inhibitor trans-4[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid promotes in vivo antitumorigenic activity of ω-3-epoxydocosahexaenoic acids,10 and adamantylurea sEH inhibitors mimic the action of epoxides independent of sEH inhibition, possibly by interacting directly with a putative EET receptor.13 An alternative approach is replacement of the unstable epoxide moiety with bioisosteric functionalities such as the corresponding ureas. Indeed, this technique has been used to develop more metabolically stable mimetics of ω-314 and ω-615−19 epoxides, and one such compound is a potential antiarrhythmic agent now in phase 1 clinical trials.20 In addition to mimicking the parent epoxides, these ureas may also inhibit sEH and amplify the in vivo effects of endogenous epoxides.12 Using ω-3-17,18-epoxy-EPA as a lead compound, we recently reported the synthesis of the saturated ω-3-17,18-epoxyeicosanoic acid (1, Figure 1). 1 retained the antiproliferative and proapoptotic activities of ω-3-17,18-epoxy-EPA in breast cancer cells.21 Interestingly, Falck et al. found that functional epoxide Received: May 21, 2017 Published: September 18, 2017 8661

DOI: 10.1021/acs.jmedchem.7b00701 J. Med. Chem. 2017, 60, 8661−8666

Journal of Medicinal Chemistry

Brief Article

4b−13b to decrease ATP production was assessed in the highly aggressive MDA-MB-231 breast cancer cell line. Consistent with previous findings, the alkylurea 221 and arylureas 4b−12b were inactive after 24 or 48 h of treatment (Figure 2A, Figure 1SA in Supporting Information), but analogue 13b, which carries a 4chloro, 3-trifluoromethyl substituted aromatic ring (Scheme 1), effectively decreased ATP production. Thus, after 24 h, 13b (10 μM) decreased ATP production to 72 ± 14% (P < 0.05) of control (Figure 2A) and longer-term treatment for 48 h produced a more pronounced decrease to 30 ± 5% (P < 0.001; Figure 1SA in Supporting Information). To substantiate the decrease in ATP viability in 13b-treated cells, we assessed the impact of the arylureas on cell cycle kinetics using flow cytometry. Treatment of MDA-MB-231 cells with 13b (10 μM, 24 h) produced a marked increase in the proportion of cells in sub-G1 phase and decreases in cells in G0G1, S, and G2M phases (Figure 1SB in Supporting Information). In contrast, the inactive analogues 2, 4b, and 11b minimally altered cell cycle distribution (Figure 1SC in Supporting Information). Using crystal violet staining 13b, but not 4b or the disubstituted analogues 11b and 12b, decreased cell adherence and viability (Figure 1SD in Supporting Information). This indicates that 13b selectively decreases the viability of MDA-MB-231 cells by impairing energy production and cell cycle progression. Activation of Apoptosis by Arylurea 13b. Because 13b increased the proportion of cells in sub-G1 phase (20 ± 3% versus 1.4 ± 0.03% in control), we tested its capacity to activate apoptosis in MDA-MB-231 cells. 13b (10 μM) strongly increased the activity of the executioner caspases-3/7 after 24 h to 203 ± 2% of control (P < 0.001; Figure 2B), but the other analogues were inactive even after 48 h of treatment (Figure 2B, Figure 2SA in Supporting Information). To further substantiate the capacity of 13b to produce cell death, MDA-MB-231 cells were subjected to annexin V-FITC/7aminoactinomycin D (annexin V/7AAD) staining. Treatment of MDA-MB-231 cells with 13b for 48 h increased the proportion of cells that exhibited early apoptosis (annexin V staining alone, quadrant E4, 11 ± 2% of cells versus 6.6 ± 1.4% in control, P < 0.001; Figure 2C) and late apoptosis (dual staining, quadrant E2, 20 ± 4% versus 5.9 ± 0.9% in control, P < 0.01; Figure 2C). The increase in 7AAD-stained cells (quadrant E1; 5.9 ± 2.1% versus 1.5 ± 0.2% in control, P < 0.001) is consistent with DNA staining following cell membrane disruption. As expected, the proportion of unstained viable cells (quadrant E3) was lower after 13b treatment (62.4 ± 5.9% versus 86.2 ± 2.3% in control, P < 0.001; Figure 2C). In contrast, annexin V/7AAD staining of MDA-MB231 cells was unaltered after treatment with the inactive analogues 2, 4b, and 11b (Figure 2SB in Supporting Information). We tested the capacity of 13b to impair the viability of other cell lines. As shown in Figure 2SC in Supporting Information, 13b decreased ATP production in T47D, MDA-MB-468, and MCF-7 breast cancer cells, especially after longer-term treatments. 13b also increased caspase-3/7 activity in T-47D cells but not MDA-MB-468 or MCF-7 cells (Figure 2SD in Supporting Information). Importantly, 13b did not impair the viability of well-differentiated MCF-10A breast cells (Figure 2SE in Supporting Information). 13b Targets the Mitochondrial Membrane in MDA-MB231 Cells. The capacity of the active agent 13b to impair ATP production and to activate caspase-3/7 suggested that the mechanism of action involves mitochondrial disruption in MDAMB-231 cells. This possibility was tested using the membrane-

Figure 1. Chemical structures of the saturated fatty acid epoxide 1 and alkylurea 2.

mimetics retained at least one double bond.14,15 Whether 1 exerts its anticancer action by the same mechanism as ω-3-17,18epoxy-EPA or functions by a different mechanism is the focus of ongoing investigation. The epoxide ring in 1 presents a metabolic liability. We previously evaluated simple alkylurea isosteres, including 2 (Figure 1), but these were relatively nonpotent and only minimally altered breast cancer cell viability in vitro.21 In the present study we report the development of arylurea analogues of 2 that also possess a ω-terminal phenyl ring, a structural moiety used in stabilized lipid analogues to prevent ω and ω-1 hydroxylation.5 We found that analogue 13b markedly decreased the viability of MDA-MB-231 breast cancer cells in vitro and in vivo when administered to nude mice carrying MDA-MB-231 xenografts. In mechanistic studies, we found that 13b targeted the mitochondrion and decreased CL content in tumor cells, which promoted apoptotic cell death. Considered together, 13b has emerged as the prototype of a new class of lipid-derived agents with potential anticancer activity.

■

RESULTS Synthesis of Arylurea Fatty Acids. A series of arylureas was prepared according to our previously reported synthesis of the alkylurea 2 (Scheme 1).21 Briefly, the ethyl ester protected amino Scheme 1. Synthesis of Arylureas 4b−13ba

a

Reagents and conditions: (i) aryl isocyanate, rt, 2 h; (ii) 1.5 M NaOH, ethanol, rt, 3 h.

fatty acid 3 was reacted with aryl isocyanates under anhydrous conditions to afford the arylureas 4a−13a, which were readily purified on silica gel in good yields. The ethyl ester protecting groups were subsequently removed by base-catalyzed hydrolysis, and acidification of the reaction mixture resulted in precipitation of carboxylic acids 4b−13b that were isolated by filtration in excellent yield and purity. Impaired ATP Production by an Arylurea in Breast Cancer Cells. The capacity of the urea-based analogues 2 and 8662

DOI: 10.1021/acs.jmedchem.7b00701 J. Med. Chem. 2017, 60, 8661−8666

Journal of Medicinal Chemistry

Brief Article

Figure 2. (A) Effects of 2, 4b−11b, and 13b (10 μM, 24 h) on ATP formation by MDA-MB-231 cells. Basal ATP formation in MDA-MB-231 cells at 24 h was (0.34 ± 0.02 nmol)/(7.5 × 104 cells). (B) Effects of 2, 4b−11b, and 13b on caspase-3 activity in MDA-MB-231 cells (10 μM, 24 h). Basal caspase3 activity at 24 h was (1.5 ± 0.2 units)/(7.5 × 104 cells). (C) Annexin V/7AAD staining in MDA-MB-231 cells after treatment with 13b (10 μM, 48 h): black, 13b-treated cells; blue, control cells (CTL). (D) JC-1 red:green fluorescence ratio in MDA-MB-231 cells (10 μM, 24 h). (E) MDA-MB-231 cells were treated with DMSO (panels i−iii) or 13b (10 μM, 6 h, panels iv−vi), washed twice with serum-free medium, and then incubated with JC-1 in serum-free medium (37 °C, 20 min) and subjected to fluorescence microscopy; cells were counter-stained with Hoechst 33342. All data are the mean ± SEM from three separate experiments. Different from DMSO-treated control: (∗) P < 0.001, (**) P < 0.01, (***) P < 0.05.

μg)/(1.05 × 107 cells), compared with (91 ± 9 μg)/(1.05 × 107 cells) in control). Addition of the monounsaturated fatty acid oleic acid has been reported to prevent cellular CL depletion by the saturated palmitic acid (0.1 mM) in MDA-MB-231 breast cancer cells.22 In the present study, oleic acid protected MDAMB-231 cells against CL/phosphatidylglycerol depletion elicited by 13b (Figure 3B). In parallel, oleic acid treatment also prevented the 13b-mediated increase in caspase-3/7 activity and apoptotic cell death (Figure 3C). As part of the present study we also assessed other potential mechanisms that could operate in 13b-mediated MDA-MB-231 cell death. However, neither the Ca2+ chelator BAPTA-AM (5, 25 μM; Figure S3B in Supporting Information) or the mitochondrial permeability transition pore inhibitor cyclosporin A (10, 20 μM; Figure S3C in Supporting Information) altered the 13b-mediated decrease in ATP formation. We also found no evidence that 13b increased the production of reactive oxygen species in MDA-MB-231 cells. Flow cytometry with 2′,7′-dichlorofluorescin diacetate revealed no increase in pro-oxidant stress after 2−6 h of treatment with 13b, and co-treatment with α-tocopherol or other antioxidants did not prevent the 13b-mediated loss of cell viability (not shown). Together, these findings strongly implicate the loss of

permeable redox-active cationic dye JC-1. JC-1 monomers fluoresce green in the cell cytoplasm but form aggregates that fluoresce red in the electronegative environment of the intact inner mitochondrial membrane. 13b treatment (10 μM, 24 h) decreased the red:green fluorescence ratio in JC-1 stained cells (to 32 ± 3% of control; P < 0.001; Figure 2D), consistent with mitochondrial impairment and disruption. By comparison, inactive analogues, which minimally altered other end points of cell viability, did not affect the JC-1 ratio. In accord with these findings, fluoresence microscopy in cells that were treated with 13b (10 μM, 6 h) showed a striking increase in JC-1 monomers (green) and a decrease in JC-1 aggregates (red; Figure 2E). The mitochondrial actions of 13b were assessed further. CL is an atypical phospholipid that maintains the integrity of the mitochondrial membrane; mitochondrial disruption releases CL and initiates apoptosis.4 Addition of 13b to MDA-MB-231 cells decreased the mitochondrial content of CL and its precursor phosphatidylglycerol to 61 ± 7% of control (Figure 3A); in contrast, CL was not depleted by the inactive analogue 2. The decrease in mitochondrial CL/phosphatidylglycerol was selective because no change in the plasma membrane phospholipid phosphatidylcholine was produced in 13b-treated cells ((88 ± 7 8663

DOI: 10.1021/acs.jmedchem.7b00701 J. Med. Chem. 2017, 60, 8661−8666

Journal of Medicinal Chemistry

Brief Article

Figure 3. (A) 13b, but not the inactive analogue 2, decreased the mitochondrial content of CL and its precursor phosphatidylglycerol in MDA-MB-231 cells. (B) Oleic acid protected MDA-MB-231 cells against CL/phosphatidylglycerol depletion elicited by 13b. (C) Oleic acid prevented the 13bmediated increase in caspase-3/7 activity and apoptotic cell death. (D) Dose-dependent effects of 13b on the in vivo growth of MDA-MB-231 cell xenografts after intraperitoneal administration. Female nu/nu mice (7 weeks of age; n = 5−8 per group) received 4 × 105 cells/100 μL by subcutaneous injection into the 4th mammary fat pad. Body weights and tumor sizes were measured. (E) Histological analysis showing the increase in TUNEL (apoptosis) and decrease in Ki67 (proliferation) staining in sections from 13b-treated and control mice. DAPI (4′,6-diamidino-2-phenylindole) was used to stain nuclei. All data from cell experiments are the mean ± SEM from three separate experiments. Different from control: (∗) P < 0.001, (**) P < 0.01, (***) P < 0.05. Scale bar: 50 μm.

■

DISCUSSION The development of novel well-tolerated anticancer agents could provide valuable options in the treatment of cancer patients. This study has identified arylurea 13b as the first in a new class of potential anticancer agents with activity against MDA-MB-231 and other tumor cells. 13b did not produce observable toxicity in mice or impair the viability of well-differentiated breast-derived MCF10A cells. In previous studies, we reported that the saturated epoxy fatty acid 1 was antiproliferative and proapoptotic in MDA-MB-231 breast cancer cells.21 Replacement of the epoxide group in 1 with an alkylurea group produced analogues like 2 that exhibited very low anticancer activity in cells.21 In the present study, we show that incorporation of a 3-trifluoromethyl, 4-chloro-substituted terminal aromatic system into 2 produced the novel arylurea 13b that had significant anticancer activity in vitro and in vivo. In contrast, several other 4-mono- and 3,4-disubstituted analogues were inactive. Arrest of MDA-MB-231 cells in S and G2M phases by 13b and the failure of cells to complete mitosis are consistent with observed impairments in cellular ATP production. In addition to antiproliferative actions 13b also activated cell killing, as reflected by the increases in sub-G1 phase, caspase-3/7 activity, and annexin V/7AAD staining. Apoptosis appears to be the major initial death mechanism that is activated in 13b-treated cells,

mitochondrial CL in the mechanism of 13b-mediated cell killing. It is possible that alternative mechanisms may operate, but we found no evidence for effects of 13b on Ca2+ homeostasis, production of reactive oxygen species, or the mitochondrial permeability transition pore. In Vivo Activity of 13b in Mice Carrying MDA-MB-231 Breast Cancer Xenografts. 13b emerged from in vitro testing as a novel molecule with potentially promising anticancer properties. Further evaluation was undertaken in animal models. In initial experiments 13b was found to be well tolerated in Balb/ c mice (2.5−40 mg/kg ip, 5 days treatment). Weight gain was normal in 13b-treated animals, and there were no other indications of toxicity. The capacity of 13b to kill MDA-MB-231 cells in vivo was assessed in xenografted nu/nu mice. As shown in Figure 3D, a dose-dependent decrease in tumor volume was noted from 32 days of treatment. In addition, increased TUNEL staining, consistent with activation of apoptosis, and decreased staining for the proliferative marker Ki67 were noted in isolated tumors (Figure 3E). Taken together, 13b is active in vivo and decreases tumorigenesis from MDA-MB-231 xenografts by inhibiting proliferation and increasing apoptosis. 8664

DOI: 10.1021/acs.jmedchem.7b00701 J. Med. Chem. 2017, 60, 8661−8666

Journal of Medicinal Chemistry

Brief Article

ities are unlikely because the inactive analogues 4b−12b are structurally similar to 13b. Taken together, 13b has emerged as the first in a new class of agents with activity against cancer cells produced by targeting of the tumor cell mitochondrion and CL depletion. Further studies are now warranted to define the structural requirements that underlie the anticancer activity of 13b analogues so that the therapeutic potential of this novel class of agents may be optimized.

although plasma membrane disruption following prolonged treatment enables DNA staining by 7AAD and is consistent with extensive tumor cell destruction. 13b was assessed for in vivo efficacy in female nu/nu mice that had been inoculated with MDA-MB-231 breast cancer cells in mammary fat pads. Tumor volume was decreased in a dosedependent fashion during treatment with 13b. In untreated tumors Ki67 staining was extensive but was decreased in tumor sections from 13b-treated mice, consistent with the antiproliferative actions of 13b seen in vitro. In addition, the increase in positive TUNEL staining in 13b-treated tumor sections compared with control tumors is consistent with the evidence for increased apoptosis in cells in vitro. It has been proposed recently that the cancer cell mitochondrion could be a novel therapeutic target because the organelle is structurally and functionally different from those in normal cells.2 There are fewer mitochondria in cancer cells, and whereas oxidative phosphorylation is the major pathway of ATP generation in normal cells, in highly aggressive tumors cells ATP generation occurs primarily by aerobic glycolysis (the Warburg effect).23 Instead, the cancer cell mitochondrion is adapted to increased production of macromolecules required for rapid cell replication.23 The mitochondrion is also a critical regulator of apoptotic cell death, and in cancer cells these pathways are frequently less responsive to cytotoxic agents. Thus, the development of novel agents that selectively target the mitochondria in tumor cells could lead to the development of new therapeutic strategies.2,23 The redox active dye JC-1 fluoresces red in cells whose mitochondria have intact membranes and fluoresces green when in the cytoplasm or when the mitochondrial membrane potential is disrupted. Studies with JC-1 showed that the active analogue 13b, but not the inactive analogues 2 and 4b−11b, targeted MDA-MB-231 cell mitochondrion and rapidly depolarized the inner membrane. There is some evidence that other oncology drugs may interact with mitochondria in breast cancer cells, but the kinetics and selectivity of mitochondrial impairment appear to be somewhat different from that produced in MDA-MB-231 cells by 13b. Thus, the estrogen receptor antagonist tamoxifen (∼1 μM) impaired the mitochondrial membrane potential in MCF-7 tumor cells after ∼1 h but also in normal mammary epithelial cells.24,25 Similar findings were made with doxorubicin; however the duration of treatment required for mitochondrial disruption was relatively long at ∼24−72 h.26,27 In comparison, the present findings indicate that 13b disrupted the MDA-MB231 cell mitochondrion within 6 h (Figure 3E). We also found that the multikinase inhibitor sorafenib disrupted the mitochondrion in these cells (not shown). Because 13b and sorafenib contain the same aryl substitution pattern, the role of that feature in mitochondrial impairment should now be evaluated in greater detail. In the present study 13b decreased the mitochondrial content of CL and its precursor phosphatidylglycerol in MDA-MB-231 cells. This was selective for CL because no change in plasma membrane phosphatidylcholine content was noted. Cellular CL depletion was mechanistically important because supplementation by oleic acid protected MDA-MB-231 cells against 13bmediated CL depletion and apoptotic cell death. Alternative hypotheses for the actions of 13b are possible. 13b is structurally similar to arylurea-based sEH inhibitors that recapitulate the cellular effects of fatty acid epoxides by slowing their degradation.12 13b may also possess surface activity that could disrupt the mitochondrial membrane. However, these possibil-

■

CONCLUSION We have developed the first member of a promising new class of fatty acid based anticancer agents. The active analogue 13b depolarized the mitochondrial membrane in MDA-MB-231 breast cancer cells leading to extensive cell death. After intraperitoneal injection 13b was also effective in breast cancer cell killing in a mouse xenograft model.

■

EXPERIMENTAL SECTION

■

ASSOCIATED CONTENT

The purity of all test compounds was confirmed to be ≥95% by elemental analysis. General Procedure for Synthesis of 4a−13a. To a suspension of the amine 3 (1.33 mmol) in anhydrous THF (15 mL) under a nitrogen atmosphere was added the appropriate aryl isocyanate (1.40 mmol). The mixture was stirred at room temperature for 2 h and then concentrated under reduced pressure. The residue was purified on silica gel by stepwise gradient elution with dichloromethane/ethyl acetate (100:0 to 70:30), yielding 4a−13a as white solids. General Procedure for Synthesis of 4b−13b. To a solution of 4a−13a (0.5 mmol) in EtOH (30 mL) was added 1.5 M NaOH (10 mL). The solution was stirred at 40 °C for 3 h. The ethanol was removed under reduced pressure, and the residue was acidified with 1 M HCl. The resulting suspension was filtered and the solid were washed with H2O (10 mL) and EtOH (5 mL), yielding the carboxylic acids 4b−13b as white solids. S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00701. Information on general chemistry, analytical data for all compounds, and methods for cell culture, cell-based assays, flow cytometry, animal experiments, and statistics (PDF) Molecular formula strings (CSV)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: (61-2)-9514-7956. E-mail: [email protected]. au. ORCID

Tristan Rawling: 0000-0002-6624-6586 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Martin Conolly for his assistance with the synthetic chemistry, Julie Dwyer and Sarah Cui for technical assistance, and Prof. Christine Clarke, The Westmead Institute, Westmead Hospital, NSW, Australia, for the generous gift of MCF10A cells. This study was supported by grants from the Australian National 8665

DOI: 10.1021/acs.jmedchem.7b00701 J. Med. Chem. 2017, 60, 8661−8666

Journal of Medicinal Chemistry

Brief Article

(15) Falck, J. R.; Kodela, R.; Manne, R.; Atcha, K. R.; Puli, N.; Dubasi, N.; Manthati, V. L.; Capdevila, J. H.; Yi, X.-Y.; Goldman, D. H.; Morisseau, C.; Hammock, B. D.; Campbell, W. B. 14,15-Epoxyeicosa5,8,11-trienoic acid (14,15-EET) surrogates containing epoxide bioisosteres: influence upon vascular relaxation and soluble epoxide hydrolase inhibition. J. Med. Chem. 2009, 52, 5069−5075. (16) Khan, M. A. H.; Liu, J.; Kumar, G.; Skapek, S. X.; Falck, J. R.; Imig, J. D. Novel orally active epoxyeicosatrienoic acid (EET) analogs attenuate cisplatin nephrotoxicity. FASEB J. 2013, 27, 2946−2956. (17) Falck, J. R.; Koduru, S. R.; Mohapatra, S.; Manne, R.; Atcha, K. R.; Manthati, V. L.; Capdevila, J. H.; Christian, S.; Imig, J. D.; Campbell, W. B. 14,15-Epoxyeicosa-5,8,11-trienoic acid (14,15-EET) surrogates: carboxylate modifications. J. Med. Chem. 2014, 57, 6965−6972. (18) Khan, M. A. H.; Pavlov, T. S.; Christain, S. V.; Neckar, J.; Staruschenko, A.; Gauthier, K. M.; Capdevila, J. H.; Falck, J. R.; Campbell, W. B.; Imig, J. D. Epoxyeicosatrienoic acid analogue lowers blood pressure through vasodilation and sodium channel inhibition. Clin. Sci. 2014, 127, 463−474. (19) Khan, M. A. H.; Falck, J. R.; Manthati, V. L.; Campbell, W. B.; Imig, J. D. Epoxyeicosatrienoic acid analog attenuates angiotensin II hypertension and kidney injury. Front. Pharmacol. 2014, 5, 1−7. (20) https://clinicaltrials.gov/ct2/show/NCT03078738. (21) Dyari, H. R. E.; Rawling, T.; Bourget, K.; Murray, M. Synthetic ω3 Epoxyfatty acids as antiproliferative and pro-apoptotic agents in human breast cancer cells. J. Med. Chem. 2014, 57, 7459−7464. (22) Hardy, S.; El-Assaad, W.; Przybytkowski, E.; Joly, E.; Prentki, M.; Langelier, Y. Saturated fatty acid-induced apoptosis in MDA-MB-231 breast cancer cells: A role for cardiolipin. J. Biol. Chem. 2003, 278, 31861−31870. (23) Gogvadze, V.; Orrenius, S.; Zhivotovsky, B. Mitochondria in cancer cells: what is so special about them? Trends Cell Biol. 2008, 18, 165−173. (24) Kallio, A.; Zheng, A.; Dahllund, J.; Heiskanen, K. M.; Haerkoenen, P. Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells. Apoptosis 2005, 10, 1395−1410. (25) Yaacob, N. S.; Kamal, N. N. N. M.; Norazmi, M. N. Synergistic anticancer effects of a bioactive subfraction of Strobilanthes crispus and tamoxifen on MCF-7 and MDA-MB-231 human breast cancer cell lines. BMC Complementary Altern. Med. 2014, 14, 252. (26) Huigsloot, M.; Tijdens, I. B.; Mulder, G. J.; van de Water, B. Differential regulation of doxorubicin-induced mitochondrial dysfunction and apoptosis by Bcl-2 in mammary adenocarcinoma (MTLn3) cells. J. Biol. Chem. 2002, 277, 35869−35879. (27) Yu, P.; Yu, H.; Guo, C.; Cui, Z.; Chen, X.; Yin, Q.; Zhang, P.; Yang, X.; Cui, H.; Li, Y. Reversal of doxorubicin resistance in breast cancer by mitochondria-targeted pH-responsive micelles. Acta Biomater. 2015, 14, 115−124.

Health and Medical Research Council (Grants 1031686 and 1087248).

■

ABBREVIATIONS USED 7AAD, 7-aminoactinomycin; CL, cardiolipin; EET, epoxyeicosatrienoic acid; ω-3-EEA, ω-3-17,18-epoxyeicosanoic acid; sEH, soluble epoxide hydrolase; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide; TUNEL, terminal deoxynucleotidyl transferase nick end labeling

■

REFERENCES

(1) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer 2013, 13, 714−726. (2) Fulda, S.; Galluzzi, L.; Kroemer, G. Targeting mitochondria for cancer therapy. Nat. Rev. Drug Discovery 2010, 9, 447−464. (3) Li, P.; Nijhawan, D.; Budihardjo, I.; Srinivasula, S. M.; Ahmad, M.; Alnemri, E. S.; Wang, X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997, 91, 479−489. (4) Lutter, M.; Fang, M.; Luo, X.; Nishijima, M.; Xie, X.-S.; Wang, X. Cardiolipin provides specificity for targeting of tBid to mitochondria. Nat. Cell Biol. 2000, 2, 754−761. (5) Murray, M.; Hraiki, A.; Bebawy, M.; Pazderka, C.; Rawling, T. Antitumor activities of lipids and lipid analogues and their development as potential anticancer drugs. Pharmacol. Ther. 2015, 150, 109−128. (6) Currie, E.; Schulze, A.; Zechner, R.; Walther, T. C.; Farese, R. V. Cellular fatty acid metabolism and cancer. Cell Metab. 2013, 18, 153− 161. (7) Sundar, S.; Jha, T. K.; Thakur, C. P.; Engel, J.; Sindermann, H.; Fischer, C.; Junge, K.; Bryceson, A.; Berman, J. Oral miltefosine for Indian visceral leishmaniasis. N. Engl. J. Med. 2002, 347, 1739−1746. (8) Kushner, B. H.; Cheung, N.-K. V.; Modak, S.; Becher, O. J.; Basu, E. M.; Roberts, S. S.; Kramer, K.; Dunkel, I. J. A phase I/Ib trial targeting the Pi3k/Akt pathway using perifosine: Long-term progression-free survival of patients with resistant neuroblastoma. Int. J. Cancer 2017, 140, 480−484. (9) Panigrahy, D.; Edin, M. L.; Lee, C. R.; Huang, S.; Bielenberg, D. R.; Butterfield, C. E.; Barnes, C. M.; Mammoto, A.; Mammoto, T.; Luria, A.; Benny, O.; Chaponis, D. M.; Dudley, A. C.; Greene, E. R.; Vergilio, J.-A.; Pietramaggiori, G.; Scherer-Pietramaggiori, S. S.; Short, S. M.; Seth, M.; Lih, F. B.; Tomer, K. B.; Yang, J.; Schwendener, R. A.; Hammock, B. D.; Falck, J. R.; Manthati, V. L.; Ingber, D. E.; Kaipainen, A.; D’Amore, P. A.; Kieran, M. W.; Zeldin, D. C. Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice. J. Clin. Invest. 2012, 122, 178−191. (10) Zhang, G.; Panigrahy, D.; Mahakian, L. M.; Yang, J.; Liu, J.-Y.; Lee, K. S. S.; Wettersten, H. I.; Ulu, A.; Hu, X.; Tam, S.; Hwang, S. H.; Ingham, E. S.; Kieran, M. W.; Weiss, R. H.; Ferrara, K. W.; Hammock, B. D. Epoxy metabolites of docosahexaenoic acid (DHA) inhibit angiogenesis, tumor growth, and metastasis. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 6530−6535. (11) Cui, P. H.; Petrovic, N.; Murray, M. The ω-3 epoxide of eicosapentaenoic acid inhibits endothelial cell proliferation by p38 MAP kinase activation and cyclin D1/CDK4 down-regulation. Br. J. Pharmacol. 2011, 162, 1143−1155. (12) Morisseau, C.; Hammock, B. D. Impact of soluble epoxide hydrolase and epoxyeicosanoids on human health. Annu. Rev. Pharmacol. Toxicol. 2013, 53, 37−58. (13) Olearczyk, J. J.; Field, M. B.; Kim, I.-H.; Morisseau, C.; Hammock, B. D.; Imig, J. D. Substituted adamantyl-urea inhibitors of the soluble epoxide hydrolase dilate mesenteric resistance vessels. J. Pharmacol. Exp. Ther. 2006, 318, 1307−1314. (14) Falck, J. R.; Wallukat, G.; Puli, N.; Goli, M.; Arnold, C.; Konkel, A.; Rothe, M.; Fischer, R.; Muller, D. N.; Schunck, W.-H. 17(R),18(S)Epoxyeicosatetraenoic acid, a potent eicosapentaenoic acid (EPA) derived regulator of cardiomyocyte contraction: structure-activity relationships and stable analogues. J. Med. Chem. 2011, 54, 4109−4118. 8666

DOI: 10.1021/acs.jmedchem.7b00701 J. Med. Chem. 2017, 60, 8661−8666