Small Molecule Therapeutics

Phyllanthusmin Derivatives Induce Apoptosis andReduce Tumor Burden in High-Grade SerousOvarian Cancer by Late-Stage AutophagyInhibitionAlexandria N. Young1, Denisse Herrera1, Andrew C. Huntsman2, Melissa A. Korkmaz3,Daniel D. Lantvit1, Sarmistha Mazumder4, Shamalatha Kolli4, Christopher C. Coss4,Salane King4, HongyanWang4, StevenM. Swanson5, A. Douglas Kinghorn2, Xiaoli Zhang6,Mitch A. Phelps4, Leslie N. Aldrich3, James R. Fuchs2, and Joanna E. Burdette1

Abstract

High-grade serous ovarian cancer (HGSOC) is a lethalgynecological malignancy with a need for new therapeutics.Many of the most widely used chemotherapeutic drugs arederived from natural products or their semi-synthetic deri-vatives. We have developed potent synthetic analogues of aclass of compounds known as phyllanthusmins, inspiredby natural products isolated from Phyllanthus poilanei Beille.The most potent analogue, PHY34, had the highest potencyin HGSOC cell lines in vitro and displayed cytotoxic activitythrough activation of apoptosis. PHY34 exerts its cytotoxiceffects by inhibiting autophagy at a late stage in thepathway, involving the disruption of lysosomal function.The autophagy activator, rapamycin, combined with

PHY34 eliminated apoptosis, suggesting that autophagyinhibition may be required for apoptosis. PHY34 wasreadily bioavailable through intraperitoneal administrationin vivo where it significantly inhibited the growth ofcancer cell lines in hollow fibers, as well as reduced tumorburden in a xenograft model. We demonstrate that PHY34acts as a late-stage autophagy inhibitor with nanomolarpotency and significant antitumor efficacy as a single agentagainst HGSOC in vivo. This class of compounds holdspromise as a potential, novel chemotherapeutic anddemonstrates the effectiveness of targeting the autophagicpathway as a viable strategy for combating ovarian cancer.Mol Cancer Ther; 17(10); 2123–35. �2018 AACR.

IntroductionThe most lethal gynecologic malignancy is ovarian cancer, of

which high-grade serous ovarian cancer (HGSOC) is the mostcommon and deadly subtype (1, 2). The overall 5-year survivalrate (46%) of HGSOC has not noticeably changed in the last 30years (1, 3). Treatment for HGSOC generally involves cytoreduc-tive surgery and a combination of platinum-based (e.g., cisplatin)and taxane (e.g., paclitaxel) chemotherapy. Although first-linetreatment is initially effective, relapse with chemoresistanceoccurs rapidly in up to 80% of cases (4). Though targeted

therapies, such as PARP inhibitors and antiangiogenesis agents,have recently been approved for recurrent disease, gains in overallsurvival have been lagging and new drug development for thisdisease is warranted (4–7).

Natural products, like paclitaxel, and their derivatives pro-vide a rich source of new chemotherapeutic agents. Phyl-lanthusmins (PHYs), which were isolated from various Phyl-lanthus species, are an example of a promising class of anti-proliferative natural product compounds (8, 9). In 2014, bio-assay-guided fractionation of Phyllanthus poilanei extractsafforded PHYD, a novel and particularly potent member ofthis class, which exhibited nanomolar activity against a humancolon cancer cell line (HT-29). Preliminary in vivo assays indi-cated that PHYD was able to inhibit the growth of HT-29embedded intraperitoneally in fibers in athymic nude micewithout displaying gross toxicity. The arylnaphthalene lignanlactone substructure of PHYs makes them similar to the arylte-tralin, etoposide, a known DNA topoisomerase II inhibitor.However, PHYD was not able to inhibit this enzyme, indicatinga novel mechanism of action (9).

Compounds with similar structures to PHYs, especially thosewith a diphyllin core, have been described as V (vacuolar)-ATPase inhibitors that affect lysosomal acidification in variouscell types (10–12). Acidification of the lysosome plays a vitalrole in autophagy, the process by which cells recycle theirintracellular components, organelles, and macromolecules(13). During times of cell stress, autophagy may act as a survivalmechanism, whereas too much intracellular recycling can

1Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,University of Illinois at Chicago, Chicago, Illinois. 2Division ofMedicinal Chemistryand Pharmacognosy, College of Pharmacy, The Ohio State University, Colum-bus, Ohio. 3Department of Chemistry, University of Illinois at Chicago, Chicago,Illinois. 4Department of Pharmaceutics and Pharmaceutical Chemistry, Collegeof Pharmacy, The Ohio State University, Columbus, Ohio. 5PharmaceuticalSciences Division, School of Pharmacy, University of Wisconsin, Madison,Wisconsin. 6Department of Biomedical Informatics, College of Medicine, TheOhio State University, Columbus, Ohio.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

CorrespondingAuthor: Joanna E. Burdette, University of Illinois at Chicago, 900S. Ashland Rm 3206, Chicago, IL 60607. Phone: 312-996-6153; Fax: 312-996-7107; E-mail: joannab@uic.edu

doi: 10.1158/1535-7163.MCT-17-1195

�2018 American Association for Cancer Research.

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induce cell death (14). Cancer cells may become dependent onautophagy to make intracellular building blocks available fortheir own energetic and proliferative needs or to eliminatedysfunctional or damaged organelles over time (15, 16).Inhibiting or amplifying autophagy to lethal levels in thesecells may be a viable option for treating disease (14, 15, 17,18). Cancer, and ovarian cancer in particular, appears to bereliant on the process of autophagy as evidenced by a recentanalysis of haploinsufficiency networks, which found autop-hagy and compensatory proteostasis pathways to be the mostdisrupted pathways in ovarian cancer as compared with 20other cancer types (19). A number of recent studies are begin-ning to uncover a role for autophagy in combating HGSOCchemoresistance (20–27) and are investigating potentialautophagy-targeting compounds preclinically for efficacyagainst ovarian cancer (28–32), including V-ATPase inhibitors(33–35).

Building on previous results (9), we synthesized a number ofanalogues, selecting for potency against ovarian cancer cell lines.In this study, we report on the in vitro and in vivo activity of thethree most potent analogues, PHY25, 30, and 34. These com-pounds inhibit late-stage autophagy, followed by apoptotic celldeath in HGSOC. Our data exemplify how iterative medicinalchemistry optimization of a natural product can generate potentlate-stage autophagy inhibitors that trigger apoptosis with nano-molar cytotoxicity in vitro and induce significant reduction oftumor burden in vivo.

Materials and MethodsPHY synthesis, characterization, and purity

PHY analogues were synthesized with functionalized carbo-hydrates to the aglycone core and characterized with 1H- and13C-nuclear magnetic resonance (NMR) (SupplementaryFig. S1). Prior to biological testing, purity was determined byhigh-performance liquid chromatography (SupplementaryFig. S2A for PHY25, S2B for PHY30, and S2C for PHY34).Briefly, diphyllin glycoside starting materials (b-D-glycosyldiphyllin, a-L-arabinosyl diphyllin, and b-D-galactosyl diphyl-lin) were prepared according to a recent publication (36).Following the selective tert-butyldimethylsilyl protection ofb-D-glycosyl diphyllin, per-methylation was cleanly accom-plished (37). Fluoride-mediated desilylation then affordedPHY25 (Supplementary Fig. S1A). PHY30 and PHY34 (Supple-mentary Fig. S1B and S1C, respectively) were prepared from theirrespective glycosyl starting materials via an acid-catalyzed acetalformation (38). The experimental data for PHY30 are in agree-ment with the same compound that was recently disclosed as inintermediate in the first total synthesis of phyllanthusminD (39).

Other compoundsPurchased compounds included two chemotherapeutics

[paclitaxel (Sigma; #T7402) and etoposide (Sigma; #E1383)],the autophagy inducer [rapamycin (Sigma; #R8781)], an early-stage autophagy inhibitor [PIKIII (Cayman Chem; #17002)],and two late-stage autophagy inhibitors [bafilomycin A1 (Sig-ma #SML1661 and LC Labs #B-1080 in Fig. 4) and chloroquine(Sigma; #C6628)] (40). Structures of non–FDA-approved smallmolecules may be found here: PIKIII (41) and bafilomycin A1(42). All compounds were suspended in dimethyl sulfoxide(DMSO), and final vehicle concentrations in all experimentsdid not exceed 0.1% except where noted.

Cell cultureThe ovarian cancer (OVCAR3 andOVCAR8), colon cancer (HT-

29), melanoma (MDA-MB-435), and breast cancer (MDA-MB-231) cell lines were purchased from the American Type CultureCollection. OVCAR8-RFP cells expressing red fluorescent proteinwere a gift from Sharon Stack at the University of Notre Dame,immortalized human ovarian surface epithelial cells (IOSE80)were a gift from Nelly Auersperg at the University of Vancouver,and eGFP-LC3HeLa andmCherry-eGFP-LC3HeLa cell lines werea gift from Ramnik Xavier at Massachusetts General Hospital.OVCAR3 was grown in RPMI 1640 supplemented with 20% FBS,1% penicillin/streptomycin (P/S), and 10 mg/mL insulin.OVCAR8 and OVCAR8-RFP were grown in DMEMwith 10% FBSand 1% P/S. HT-29 and MDA-MB-435 cells were grown in RPMI1640 with 10% FBS and 1% P/S. MDA-MB-231 cells were grownin DMEMwith 5% FBS, 1% L-glutamine, 1% P/S, and 0.02 mg/mLinsulin. IOSE80 wasmaintained in v/v 50%Medium 199 and v/v50% MCBD with 15% FBS, 1% P/S, 1% L-glutamine, and 11 ng/mL epithelial growth factor. HeLa cells were cultured in DMEMwith 8.8%FBS, 1.8% L-glutamine, and1%P/S. Cultured cellsweremaintained in a humidified incubator at 37�C in 5% CO2. Cellswere passaged a maximum of 20 times. Cell lines were validatedby short tandem repeat analysis and tested mycoplasma-free in2017.

Cell viability assayCells were seeded in 96-well, clear, flat-bottomed plates at

2,500 to 5,000 cells per well, depending on the cell line, andallowed to attach overnight. Compounds suspended in DMSOwere diluted to final concentrations and added to the cells. Thefinal vehicle concentration was 0.25% to achieve a wide doserange. Cells were incubated for 24, 48, or 72 hours. Cellularprotein contentwasmeasuredusing a sulforhodamineB assay as ameasure of cell survival (43). Treatment measurements werenormalized to vehicle, and dose–response curves with corre-sponding IC50 values were generated using Graphpad PrismSoftware.

Two-dimensional foci assayOVCAR3 andOVCAR8were plated at a concentration of 400 or

200 cells per 60 mm dish, respectively, allowed to attach over-night, and treated as indicated for 72 hours. Foci were allowed togrow in nontreated medium with media changes every 3 days.When two-dimensional (2D) foci became visible (day 20 forOVCAR3 and day 14 for OVCAR8), they were fixed with 4%paraformaldehyde, stained with 0.05% crystal violet, and imagedwith the FluorChem E system (ProteinSimple). Foci counts werequantified with ImageJ software (NIH) and normalized to thevehicle control. Significance was determined using one-wayANOVA with Dunnett multiple comparisons, compared with thevehicle control.

Immunoblot analysisAfter culture and treatment, cells were lysed in RIPA lysis

buffer (50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 1% TritonX-100, 0.1% SDS) with protease (Roche Applied Science) andphosphatase (Sigma) inhibitors, incubated at –80�C, and cen-trifuged. Protein concentration was obtained using the bicinch-oninic acid assay (Pierce), and 15 mg protein per sample wasseparated by SDS-PAGE and transferred to nitrocellulose oractivated 0.45 mm PVDF membranes (Thermo Fisher). After 5%

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milk block, the following primary antibodies were applied toblots at a 1:1,000 concentration: GAPDH as a loading control(Cell Signaling Technology; #2118), PARP (Cell SignalingTechnology; #9542), or LC3B (Cell Signaling Technology;#2775). Membranes were incubated with 1:1,000 anti-rabbitsecondary antibody (Cell Signaling Technology; #7074) priorto visualization of signal with SuperSignal West Femto sub-strate (Thermo Fisher) and imaging on a FluorChem E system(ProteinSimple).

Annexin V/propidium iodine stainingCells were seeded into T25 flasks, allowed to attach

overnight, and treated as indicated for 3 days. Cells weresubjected to an Annexin V–FITC/Propidium Iodide Apopto-sis Assay (Nexcelom Biosciences) according to instructionsprovided by the manufacturer, except with 10 mL Annexin V–FITC reagent per sample. Fluorescence was detected using aK2 Cellometer (Nexcelom Biosciences). Data were analyzedwith the FCS Express program (De Novo Software). Eachreplicate contained at least 2,000 cells for quantification offluorescent signal. Statistics were generated with one-wayANOVA with Dunnett's multiple comparisons to vehiclecontrol within each group (Fig. 2B) or Tukey's multiplecomparisons (Fig. 6C).

LC3B puncta and autophagic flux assayHeLa cells stably expressing eGFP-LC3 (LC3 puncta assay) or

stably expressing mCherry-eGFP LC3 (autophagic flux assay)were seeded in 384-well plates at 3,000 cells per well (44). Afterattachment overnight, test compounds and controls were movedinto assay plates using a 96-well pin tool (V&P Scientific) and aliquid handler, BiomekNXP Lab AutomationWorkstation (Beck-man Coulter). Assay plates were incubated at 37�C for 4 (LC3puncta assay) or 24 (autophagic flux assay) hours. Cells were thenfixedwithw/v 4% formaldehyde (ThermoScientific) in PBS for 12minutes, washed with PBS, DNA-stained with 2 mg/mL Hoechst33342 (Molecular Probes) in PBS for 12 minutes, and dilutedwithPBS. Plateswere sealed using PlateMax Semi-Automatic PlateSealer (Axygen). Plates were imaged at 10X by an automatedfluorescence microscope, ImageXpress Micro XLS (MolecularDevices), using DAPI and FITC filters (both assays) and TexasRed filter for the autophagic flux assay. Four sites were imaged perwell. The number of puncta per cell was quantified using theMetaXpress High-Content Image Analysis Software with Trans-fluor ApplicationModule (Molecular Devices). The average num-ber of autophagosomes (eGFPþ/mCherryþ; yellow puncta) andautolysosomes (eGFP–/mCherryþ; red puncta) per cell was quan-tified using CellProfiler Software (Broad Institute). Treatmentmeasurements were normalized to vehicle, and dose–responsecurves with corresponding EC50 values were generated usingGraphPad Prism Software.

Lysotracker and acridine orange stainingCells were plated according to the manufacturer's instruc-

tions on 8-well, culture-treated m-slides (Ibidi USA Inc.) for livecell confocal imaging. Cells were treated as indicated andincubated in either 1 mg/mL acridine orange (Sigma) in PBSfor 15 minutes or 50 nmol/L Lysotracker Red DND-99 (Invi-trogen) in PBS for 30 minutes before imaging on a Zeiss LaserScanning 710 confocal microscope. Images were obtained withZen software (Zeiss).

In vivo assaysAll animals were treated in accordance with theNIH Guidelines

for the Care and Use of Laboratory Animals and the establishedAnimal Care and Use Committees at the Ohio State University(OSU, protocol #2009A0196-R2) of the University of Illinois atChicago (UIC, protocol #16-035). In vivo studies conducted atOSU (MTD, bioavailability) utilized ICRmalemice 6weeks in ageandaround20grams inweight (Harlan Laboratories), and studiesat UIC (hollow fiber assay, intraperitoneal xenografts) utilizedNCr nu/nu athymic female mice 6 to 8 weeks in age (Taconic).Mice used in all in vivo assays were housed in a temperature- andlight-controlled environment under 12:12 hour light:dark cyclesand provided food and water ad libitum.

Solubility testingPHY34 was prepared at a starting concentration of 120 mg/mL

inDMSO, 50mg/mL in PEG300 and 30%HPbCD, or 1mg/mL inPBS and centrifuged at 13,500 rpm for 10 minutes. The solublefraction was serially diluted, and 100 mL was analyzed by absorp-tion spectrometry using a BioTek Synergy HT Multi-Mode Micro-plate Reader. The dilution of the original formulation within thelinear range of the calibration curve was used to assess theconcentration of PHY compounds in the soluble fraction. Datawere acquired by using Software Gen5 2.05 (BioTek).

MTD testingMice were administered PHY34 in the formulation of 50%

PEG300:50% saline intravenously at dose levels of 3, 1, 0.6, and0.3 mg/kg until the maximum tolerated dose (MTD) was obtain-ed. MTD is defined as the highest dose that can be administeredto a mouse without causing unacceptable toxicity. MTDs wereobtained starting at 10x the intravenous MTD by the intraperito-neal route or 50x the intravenous MTD by the oral route and de-creasing doses down to 3x or 1x the intravenous MTD. Five micewere used at each dosing level for each route of administration.

Bioavailability studiesA sterile dosing solution of PHY34 was prepared in 50%

PEG300:50% saline. Mice were dosed at the MTD via intrave-nous (0.6 mg/kg) or intraperitoneal (1.8 mg/kg) injections or75 mg/kg for oral administration. The injection volumes were100 mL for intravenous and 200 mL for intraperitoneal and oralroute per mouse. Around 30 mice for each dosing route wereused to perform plasma pharmacokinetic (PK) study. At eachtime point after dosing, 3 mice were euthanized by CO2

asphyxiation, and blood was immediately collected via cardiacpuncture then transferred to heparinized tubes. PHY34 wasquantified using LC-MS/MS (LC, liquid chromatography; MS,mass spectrometry) with a Dionex RSLC nano LC system and aTSQ Quantiva mass spectrometer. Briefly, 200 mL internalstandard solution (200 ng/mL PHY1, analogue of PHY34, inDMSO/isopropal alcohol/acetonitrile, 5/10/10 v/v/v) wasadded to 100 mL mouse plasma followed by centrifugation at4�C and 13,500 rpm for 5 minutes. After nitrogen drying, 100mL water was added to reconstitute the analytes, which wereloaded in an autosampler vial for LC-MS/MS analysis. Analyteswere separated on an Extend C-18 column (50 � 2.1 mm, 3.5mm) using a gradient of water and isopropanol, each with 0.1%formic acid. Mass transitions monitored were 583.3 > 381.2(PHY34) and 711.3 > 109.1 [MþH]þ. The calibration curve waslinear from 1 to 2,000 nmol/L in mouse plasma. Plasma

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concentration–time data were analyzed by noncompartmentalmethods using default settings and the NCA analysis object inPhoenix WinNonlin v6.3 (Certara).

Hollow fiber assayOVCAR3, OVCAR8, and HT-29 cells were grown and embed-

ded in biocompatible hollow fibers for xenograft mice intraper-itoneally on day 0 as previously described (45, 46). Paclitaxel (5mg/kg in 20% DMSO:40% PEG300:40% H2O), PHY analogues(0.75 mg/kg in 10% DMSO:40% PEG300:50% H2O), or vehicle(2% DMSO:48% PEG300:50% H2O) were dosed intraperitone-ally onceper day for 4days (days 3–6). Treatment group sizeswereas follows: 3 mice received paclitaxel, 7 received each PHYanalogue, and 6 received vehicle. Mice were sacrificed on day 7,and fibers retrieved. The viable cells were evaluated with a mod-ified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide assay (Promega). Statistics were generated with one-wayANOVA with Holm's multiple comparisons to vehicle control foreach cell line.

Intraperitoneal xenograftOVCAR8-RFP cells were xenografted intraperitoneally (5� 106

cells per mouse), and tumor growth was monitored with theXenogen IVIS Spectrum In Vivo Imaging System (PerkinElmer) aspreviously described (47). When tumors became detectable viaIVIS imaging, mice were separated into treatment groups of 5 to6 mice and dosed once daily 3 times per week with paclitaxel(5 mg/kg), PHY34 (0.75 mg/kg), or vehicle (10% DMSO:40%PEG300:50% H2O) for 3 weeks. Mice were weighed 3 times aweek and imaged once weekly with an exposure of 2 seconds, anFstop of 2, and excitation and emission wavelengths of 535 and620 nm, respectively. Average abdominal radiant efficiency wasquantified with the system's Living Image 4.0 software andnormalized to day 0. After week 3, mice were sacrificed andtumors were collected for immunohistochemistry (IHC). Statis-tics were generated with two-way ANOVA with Dunnett's multi-ple comparisons to vehicle control.

Tumor IHCTumors obtained from the i.p. xenograft experiment were

fixed with 4% paraformaldehyde before dehydration and par-affin sectioning. Sectioning and immunohistochemistry (IHC)were performed using standard histologic procedures. Sectionswere incubated in anti-Ki67 (Abcam; #ab15580), followed byanti-rabbit IgG (Leica Biosystems; #DS9800). Immunoreactiv-ity was visualized with 3,3-diaminobenzidine chromogen andcounterstained with Mayer's hematoxylin. Tissues without pri-mary antibody treatment were used as a negative control.Images were acquired on a Nikon Eclipse E600 microscopeusing a DS-Ri1 digital camera and NIS Elements software(Nikon Instruments).

Statistical analysisData presented are themean� SEM and represent at least three

independent experiments. Statistical analysis was carried outusing GraphPad Prism software and SAS 9.4 for the hollow fiberassay. Statistical significance was determined by ANOVA withDunnett's, Holm's, or Tukey's adjustment for multiple compar-isons, as noted in methods/legends. Adjusted P < 0.05 is consid-ered significant. Symbols used to signify significance include: �,P<0.05; †, P < 0.01; z, P < 0.001; x, P < 0.0001.

ResultsPHY analogues induce cytotoxicity in HGSOC cell lines in vitrowith nanomolar potency

PHYs originally isolated from Phyllanthus poilanei Beille withdemonstrated activity against cancer cell lines in vivo were used asinspiration to generate synthetic analogues with greater potencybased on alterations to the sugarmoiety (9). In order to determineif synthetic analogues were more potent than the natural pro-ducts, a series of cell lines was tested with increasing concentra-tions (0.001 nmol/L to 50 mmol/L) of PHY25, 30, and 34 (Fig.1A). HGSOC cell lines, OVCAR8 and OVCAR3, were selectedbased on their designation as reliable models of HGSOC (48).Colon cancer cell line, HT-29, was also included for continuity asit was the cell line used to discover the original natural productPHY derivatives (9). IOSE80 served as the nontumorigenic cellcontrol (49). Paclitaxel and etoposide were included as positivecontrols. Dose–response curves (Fig. 1B)with corresponding IC50

values (Fig. 1C) were generated. PHY34 was the most cytotoxiccompound in both HGSOC cell lines with an IC50 of 4 nmol/L,more potent than that of paclitaxel (8–10 nmol/L). PHY30 wascytotoxic in bothHGSOCcell lines (IC50¼�12nmol/L), whereasPHY25 was selectively potent against OVCAR8 (IC50 ¼ 17 nmol/L) over OVCAR3 (IC50 ¼ 1.9 mmol/L). PHY30 and 34 were notcytotoxic (IC50 > 50 mmol/L) to the nontumorigenic cell linecontrol, IOSE80. PHY34 was subjected to the NIH NCI's 60Tumor Cell Line Screen (50, 51), which showed PHY34's capa-bility to significantly inhibit the growth of 8 of the 9 types ofcancer represented, including ovarian, colon, melanoma, andleukemia, after 48-hour treatment (Supplementary Fig. S3A andS3B). PHY34 showed greater ability to inhibit the growth ofOVCAR8 as compared with OVCAR3 in this screen. We addition-ally tested PHY25, 30, and 34 against two cell lines from the NCIscreen, melanoma (MDA-MB-435) and triple-negative breastcancer (MDA-MB-231), for 72 hours (Supplementary Fig. S3C).PHYs, especially PHY34, had nanomolar IC50s in both these celllines andweremore cytotoxic againstMDA-MB-231 (PHY34 IC50

in MDA-MB-435 ¼ 23 nmol/L, PHY34 IC50 in MDA-MB-231 ¼5.2 nmol/L).

To further characterize if the PHY compounds were effectivedue to cytostatic versus cytotoxic action, 2D foci assays wereperformed (Fig. 1D), where cells were exposed to drug for 72hours and then foci were allowed to grow in drug-free medium.PHYs displayed greater potency inOVCAR8 than inOVCAR3withall analogues being cytotoxic at 10 nmol/L. In OVCAR3, PHY25and 30 were cytotoxic at 1 mmol/L, and PHY34 was cytotoxic at100 nmol/L. The low concentration (100 nmol/L) of PHY25 and30 was cytostatic in OVCAR3, showing decreased 2D foci forma-tion compared with vehicle (DMSO) control.

PHYs induce apoptosis in vitroIn order to determine the mechanism of cell death in HGSOC

cell lines, Western blot analyses were performed for cleaved PARP(cPARP) as a biomarker for apoptosis. cPARP levels wereincreased in all PHY analogue treatments in OVCAR8 and mostprominently with PHY34 in OVCAR3 (Fig. 2A). Apoptosis induc-tion was confirmed with Annexin V–FITC (AV) and propidiumiodide (PI) co-staining (Fig. 2B). PHY analogue treatment at 10nmol/L resulted in a significant increase in the number of cells inearly and late apoptosis (AVþ,PI– and AVþ,PIþ, respectively) inOVCAR8 as compared with the vehicle control, with a

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Figure 1.

PHYs induce cytotoxicity in HGSOC cell lines in vitrowith nanomolar potency.A, Chemical structures andmolecular weights (M.W.) of PHY25, PHY30, and PHY34.B,Cells were plated and treated with vehicle (DMSO), chemotherapeutic controls (TAX or ETOPO), or PHYs for 72 hours. Dose–response curves were generatedfor two ovarian cancer cell lines (OVCAR8 and OVCAR3), colon cancer cell line (HT-29), and a nontumorigenic control cell line (IOSE80). Curves werenormalized to vehicle (DMSO) control. C, IC50 values generated from dose–response curves (B). D, Cells were plated sparsely and treated with vehicle (DMSO),ETOPO, or PHYs for 72 hours. Treatment was removed, and single cells were allowed to develop into 2D foci for 2 (OVCAR8) or 3 (OVCAR3) weekswithout the presence of drug in order to determine if initial treatment was cytostatic or cytotoxic. 2D foci were stained and quantified. Each experiment wasperformed in at least three biological replicates, and data represent mean� SEM. One-way ANOVA with Dunnett's multiple comparisons to vehicle control (D)wasused to generate P values (� ,P < 0.05; †, P < 0.01; z, P < 0.001; and x, P < 0.0001). TAX, paclitaxel; ETOPO, etoposide; and IC50, concentration at which 50%of cells survive.

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corresponding decrease in nonapoptotic cells (AV–,PI–). OnlyPHY34 treatment (100 nmol/L) resulted in significant increasesin early and late apoptotic cells in OVCAR3. Because PHY34 wasthe most potent compound in both HGSOC cell lines andinduced apoptosis to the highest extent, it was selected for usein remaining studies.

PHY34 reduces tumor burden in vivoPrior to conducting in vivo experiments, the solubility and

MTD of PHY34 were assessed. PHY34 was resuspended invarious solvents at increasing concentrations to elucidate themaximum soluble concentration displayed in mg/mL andmmol/L in Supplementary Fig. S4A. PEG300 was selected forin vivo experiments. Observed MTDs of PHY34 by variousroutes of administration are as follows: 0.6 mg/kg by intrave-nous administration, 1.8 mg/kg by intraperitoneal administra-tion, and >30 mg/kg by oral administration. No toxicities wereseen by oral administration. An intraperitoneal dose of 0.75mg/kg, less than half the MTD, was selected for in vivo studies.

To elucidate the bioavailability of PHY34 administered byvarious routes, plasma from mice dosed with 0.6 mg/kg intrave-nously, 1.8 mg/kg intraperitoneally, or 75 mg/kg orally was

subjected to LC-MS/MS analysis to determine the PHY34 plasmaconcentration over time (Fig. 3A). The plasma concentration–time data were analyzed by noncompartmental methods todetermine PK parameters listed in Supplementary Fig. S4B. Theobserved intraperitoneal bioavailability (F) for PHY34was 56.6%and for oral route was 2.5%. Systemic clearance (Cl) followingoral dose was 194.1 L/hr/kg, roughly 40 times the mouse liverblood flow, suggesting significant extrahepatic clearance. The Tmax

of PHY34 (0.25 hour) suggests very fast absorption of thiscompound orally.

To evaluate the ability of PHYs to induce toxicity in an animalmodel, a hollow fiber assay was performed (Fig. 3B). Cancer celllines (OVCAR3, OVCAR8, and HT-29) embedded in biocompat-ible hollow fibers were implanted intraperitoneally into femalenudemice. All analogues significantly decreasedHT-29 tumor cellsurvival in the hollow fiber. PHY34 significantly reduced cellviability of OVCAR8, but not OVCAR3, and was selected forfurther in vivo study.

OVCAR8 cells fluorescently labeled with RFP (OVCAR8-RFP)were xenografted intraperitoneally into female nudemice. Tumorformation wasmonitored by IVIS imaging, and once tumors wereestablished, they were dosed 3 times a week for 3 weeks. PHY34

Figure 2.

PHYs induce apoptosis in vitro.A,Whole-cell lysateswere obtained from cells treatedwith vehicle (DMSO), chemotherapeutic controls (ETOPO or TAX), or PHYs for72 hours. Representative Western blot analysis displays an increase in apoptosis biomarker, cleaved PARP (cPARP). GAPDH serves as a loading control. B,Cells were treated with vehicle (DMSO), ETOPO, TAX, or PHYs for 72 hours and stained with Annexin V–FITC (AV) and PI. The fluorescent signal of eachcell (n¼ <2,000 cells per replicate) was quantified with a Nexcelom Cellometer. Percentages of nonapoptotic cells (AV–,PI–) and early (AVþ,PI–) and late (AVþ,PIþ)apoptotic cells were quantified. Each experiment was performed in at least three biological replicates, and data represent mean � SEM. One-wayANOVA with Dunnett's multiple comparisons to vehicle control within each group (B) was used to generate P values (� ,P < 0.05; †, P < 0.01; z, P < 0.001;and x, P < 0.0001). ETOPO, etoposide and TAX, paclitaxel.

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Figure 3.

PHY34 reduces tumor burden in vivo. A, PHY34 was administered to mice via various routes of administration, and plasma PHY34 concentration was measuredover time to generate a plasma concentration–time profile. B, Ovarian cancer (OVCAR8 and OVCAR3) and colon cancer (HT-29) cells were embedded inbiocompatible hollow fibers and implanted intraperitoneal inmice. Micewere treatedwith vehicle, chemotherapeutic control (TAX), or PHYs once per day for 4 days.Fibers were retrieved, and the survival of cells within was quantified. Results were normalized to vehicle control. C, OVCAR8-RFP cells were xenograftedintraperitoneally and allowed to form tumors. Mice were dosed with vehicle, TAX, or PHY34 3 times per week for 3 weeks. Quantification of tumor burdenover time (average radiant efficiency as measured with IVIS imaging) was normalized to day 0, and representative images of OVCAR8-RFP tumors in miceare displayed.D, IHC of Ki67 staining as a marker of proliferation is shown from intraperitoneal xenograft tumors (C). Scale bar, 50 mm. Data represent mean� SEM.One-way ANOVA with Holm's multiple comparisons to vehicle control for each cell line (B) or two-way ANOVA with Dunnett's multiple comparisons tovehicle control (C) was used to generate P values (� ,P < 0.05; †, P < 0.01; z, P < 0.001; and x, P < 0.0001). IP, intraperitoneal; IV, intravenous; PO, orally; TAX,paclitaxel; and H&E, hematoxylin and eosin.

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(0.75 mg/kg) and the positive control, paclitaxel (5 mg/kg),significantly decreased tumor burden based on average abdom-inal radiant efficiency as compared with vehicle at week 3 withpaclitaxel also achieving significant tumor burden reduction atweek 2 (Fig. 3C). No gross toxicities were observed, and micemaintained similar body weights to vehicle animals throughoutthe study in contrast to the paclitaxel control group, which saw asignificant reduction in body weight (Supplementary Fig. S4C).Theproliferativemarker, Ki67,was reduced in tumors treatedwithPHY34, as illustrated by immunohistochemical analysis (Fig. 3D)of tumors from 3 mice per group.

PHYs are late-stage autophagy inhibitorsCompounds with similar diphyllin core structures to PHYs

have been shown to modulate autophagy (10–12). PHY25, 30,and 34 were subjected to two screening assays for autophagymodulators (44). HeLa cells expressing eGFP-LC3B were treatedwith PHYs for 4 hours, fixed, and fluorescent LC3B punctawere counted over a range of doses (9.31 fmol/L to 20 mmol/L;Fig. 4A–C). PHYs, especially PHY34, sustained high levels ofLC3B puncta at low doses (EC50 ¼ 2 nmol/L). In comparison,an early natural product analogue, PHYC (9), had a muchhigher EC50 (979 nmol/L).

Figure 4.

PHYs are late-stage autophagy inhibitors.A–C, HeLa cells stably expressing eGFP-LC3B were treated with PHYs for 4 hours, and the number of LC3B puncta per cellwas quantified as a measure of autophagy modulation. Dose–response curves (A), EC50s (B), and representative photos of bolded concentration in A (C) aredisplayed. D–F, HeLa cells stably expressing mCherry-eGFP-LC3B were treated with PHYs for 24 hours, and the ratio of autophagosomes (eGFPþ/mCherryþ) toautolysosomes (eGFP–/mCherryþ) was quantified as a measure of autophagic flux. Dose–response curves (D), EC50s (E), and representative photos of boldedconcentration inD (F) are displayed. Persistence of high autophagosome/autolysosome ratio (D) or colocalization of signal (yellow in F) is consistent with late-stageautophagy inhibition. Scale bar, 10 mm. Each experiment was performed in at least three biological replicates. BAF, bafilomycin A1 and CQ, chloroquine.

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Because an increase in LC3Bpuncta canbe causedby autophagyinducers or late-stage autophagy inhibitors, an autophagic fluxassaywas performed (Fig. 4D–F),wherein an LC3Bprotein taggedwith both mCherry and eGFP was visualized to count auto-phagosomes (eGFPþ/mCherryþ) and autolysosomes (eGFP–/mCherryþ). Flux was inferred by the acidic stability of the tags:the eGFP signal was attenuated in the acidic lysosomal environ-ment, whereas the mCherry signal remained stable (52). Anaccumulation of autophagosomes, or higher ratio of autophago-somes/autolysosomes, is indicative of a late-stage inhibitor. Bafi-lomycin A1 (BAF) and chloroquine (CQ), both late-stage inhi-bitors, had significantly higher autophagosome/autolysosomeratios. All PHY compounds performed similarly to BAF and CQas late-stage inhibitors of autophagy, and PHY34 had the greatestability to inhibit autophagy of all compounds tested (EC50 ¼ 3.9nmol/L). The original natural product, PHYC, required a muchhigher dose to achieve the same effect (EC50 ¼ 1.1 mmol/L). Anincreased ability to modulate autophagy correlated with theincreased cytotoxicity seen in these synthetic analogues over theirnatural product predecessor.

PHY34 reduces the number and acidity of lysosomes in vitroBecause similar compounds have been hypothesized to mod-

ulate autophagy (10–12), we next visualized lysosomes and theiracidity. OVCAR8 and OVCAR3 cells were treated with PHY34 for24 hours at 100 nmol/L or 1 mmol/L, respectively, and stainedwith either Lysotracker Red or acridine orange tomonitor changesin the lysosomes due to autophagy inhibition using live confocalmicroscopy. A reduction in the fluorescence from the lysosomeswas detected after treatment with PHY34 in both ovarian cancercell lines (Fig. 5A). BAF, a late-stage autophagy inhibitor, almostcompletely eliminated lysosome detection at 50 nmol/L. PIKIII(an early-stage autophagy inhibitor), which should block autop-hagy before the changes to lysosomal expression, was visuallysimilar toDMSO(40). Acridine orange, a greendye that emits red/orange light when sequestered in acidic cellular compartmentslike the lysosome, similarly displayed a dramatic reduction influorescence after treatmentwith PHY34 or BAF (Fig. 5B), suggest-ing a reduction in lysosomal acidification.

PHY34 treatment leads to autophagy inhibition followed byapoptosis induction in vitro

In order to determine if autophagy preceded apoptosis, a seriesof time courses was evaluated. PHY34 induced cytotoxicity earlierand with greater potency in OVCAR8 than OVCAR3 (Fig. 6A;Supplementary Fig. S5 for PHY25 and 30). Western blot analyses(Fig. 6B) supported an earlier induction of apoptosis in OVCAR8based on enhanced cPARP levels after 48hours, whereas itwas notapparent until 72 hours in OVCAR3. Next, PHY34 was combinedwith a late-stage autophagy inhibitor, BAF, and demonstrated anadditive effect on apoptosis induction. To determine if autophagyinhibition was necessary to induce apoptosis, the combination ofa late-stage autophagy inhibitor (PHY34 or BAF) was tested withan autophagy inducer, RAP (40). The presence of RAP reversed theconversion of PARP to cPARP. A rescue of apoptosis was alsoobserved with Annexin V–FITC and PI costaining (Fig. 6C) wherethe addition of RAP to PHY34 treatment significantly decreasedthe number of apoptotic cells as compared with PHY34 alone inboth OVCAR8 at 48-hour treatment and OVCAR3 at 72-hourtreatment. In contrast, PIKIII, an early-stage autophagy inhibitor,did not contribute to apoptosis-mediated cell death. To confirm

that autophagy was directly affected by PHYs and the variouscontrols, the conversion of LC3B-I to LC3B-II was monitored.Correspondingly, an increase in LC3B-II level can be seen in allPHY34- and BAF-treated cell lysates. The increase in LC3B-II canbe seen as early as 24 hours inOVCAR8 and 48 hours inOVCAR3,suggesting that the late-stage inhibition of autophagy precedes theinduction of apoptosis.

DiscussionThis work demonstrates that the synthetic derivatives, PHY25,

30, and 34, act as effective late-stage autophagy inhibitors able tointerfere with tumor growth in vivo. Inhibition of late-stageautophagy by PHY34 may be required for the resulting apoptoticcell death. Importantly, late-stage autophagy inhibition byPHY34 preceded apoptosis induction, and this chain of eventsoccurred at least 24 hours earlier in the more PHY-sensitive cellline, OVCAR8. Combination treatment with an autophagy induc-er, RAP, rescued the PHY-treated cells from apoptosis. Thisimplies that the apoptotic cell death seen after PHY34 treatmentmay rely on its ability to block autophagy. Bafilomycin A1 andRAP co-treatment displayed a similar rescue, further supportingPHY34's role as a late-stage autophagy inhibitor. PHY34 wasshown to modulate autophagy by its ability to increase bothLC3B puncta and the autophagy marker, LC3B-II. An autophagicflux assay revealed its action as a late-stage inhibitor of thepathway, blocking the final breakdown of the autolysosomes.Overall, these PHY compounds, PHY25, 30, and 34, demonstrat-ed nanomolar cytotoxicity in HGSOC cell lines and were able toreduce tumor burden in vivo.

For this study, derivatization of the PHY class of naturalproducts focused on the modification and optimization of theglycone portion of the molecules and involved the introduc-tion of variously functionalized carbohydrate scaffolds todiphyllin. These efforts provided PHY25 and 30, each of whichpossesses a different chemical moiety thought to be an impor-tant pharmacophore. A hybrid of the two compounds, incor-porating the key pharmacophore from each, was designed andsynthesized, providing PHY34. This technique resulted in theaforementioned improvement of PHY34's in vitro and in vivopotencies as compared with the parent compounds (PHY25and 30).

PHYs exhibit qualities that suggest they are good chemother-apeutic drug leads. Of the three compounds, PHY34 was effectivein vivo against both ovarian and colon cancer models withoutgross toxicity, and in vitrowas not toxic to the normal cell control.The dose of PHY34 required to eliminate tumors in vivowas lowerthan that of paclitaxel (0.75 as compared with 5 mg/kg), and theanimals displayed almost no change in body weight with PHY34treatment. In the NCI 60 Tumor Cell Line Screen, PHYs demon-strated promising growth inhibition in 8 of the 9 tumor typesinvestigated at 48-hour treatment and had nanomolar cytotoxic-ity against two cell lines from this screen, amelanoma and a triple-negative breast cancer cell line, after 72-hour treatment. Of theHGSOC cell lines, OVCAR3was less sensitive to PHY34 treatmentthan OVCAR8, and mechanistic effects of PHY treatment, such asautophagy inhibition and apoptosis induction, occurred 24hourslater in OVCAR3. However, both OVCAR3 and OVCAR8's IC50swere in the nanomolar range, and the delay in cytotoxicitymay beexplained by the slower growth of OVCAR3 in cell culture.Originally, the structure of PHYD was compared with etoposide,

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and both previous in vitro assays studying topoisomerase inhibi-tion and DNA coil relaxation confirmed that these PHYs have adifferentmechanism (9). Our data further demonstrate that PHYsact differentially based on their farmore potent IC50 values in vitroand their strong ability to eliminate colonies after cessation oftreatment. These compounds were bioavailable throughmultipleroutes of administration, including oral. Notably, despite the

relatively low oral bioavailability of 2.5%, oral doses achievedplasma concentration greater than in vitro IC50 values, whichsuggests oral dosing is feasible.

The development of autophagy inhibitors for the treatment ofHGSOC is currently an active area of interest and research. Indeed,an increasing number of compounds are being developed totarget autophagy (28–35). Late-stage autophagy inhibitors, like

Figure 5.

PHY34 reduces the number and acidity of lysosomes in vitro. Ovarian cancer cells (OVCAR8 and OVCAR3) were treated with vehicle (DMSO), negative control(PIKIII), positive control (BAF), or PHY34 for 24 hours and stained with (A) Lysotracker Red DND-99 to stain lysosomes or (B) acridine orange to assesslysosomal acidity. Representative confocal images are displayed. Scale bar, 20 mm. Each experiment was performed in at least three biological replicates. BAF,bafilomycin A1.

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bafilomycin A1 and PHYs, are able to induce apoptotic cell death,whereas the early-stage inhibitor used in this study, PIKIII, requiresadditional cellular stress or nutrient deprivation (40). Large-scalestudies have demonstrated the particular importance of the autop-hagic pathway inovarian cancer (19).One analysis revealed greaterV-ATPase immunoreactivity in epithelial ovarian cancer patient

samples (n ¼ 59) and decreased overall survival in patientsexpressing higher levels of V-ATPase mRNA by analysis of theCancer Genome Atlas data (33), suggesting that V-ATPase expres-sion could serve as a viablebiomarker to stratify patient response toits inhibition. Biomarkers for autophagy, such as Beclin1 andLC3A, have also been shown to correlate with poor prognosis in

Figure 6.

PHY34 treatment leads to autophagyinhibition followed by apoptosisinduction in vitro. A. Cells were platedand treated PHY34 for 24, 48, or 72hours. Dose–response curves weregenerated for two ovarian cancer celllines (OVCAR8 and OVCAR3). Curveswere normalized to vehicle (DMSO)control. B, Whole-cell lysates wereobtained from cells treated withvehicle (DMSO), PHY34, autophagyinducer (RAP), early-stage autophagyinhibitor (PIKIII), or late-stageautophagy inhibitor (BAF) for 24, 48,or 72 hours. Representative Westernblot analysis is displayed, probing forbiomarkers of autophagy (LC3B-II)and apoptosis (cPARP). GAPDHserves as a loading control. C, Cellswere treated with vehicle (DMSO),PHY34, and/or RAP for 48 hours inOVCAR8 and 72 hours in OVCAR3 andstained with Annexin V–FITC and PI toconfirm RAP rescues PHY-inducedapoptosis. The fluorescent signal ofeach cell (n ¼ <2,000 cells perreplicate) was quantified with aNexcelom Cellometer. Percentages ofnonapoptotic cells and apoptotic cellswere quantified. Each experiment wasperformed in at least three biologicalreplicates, and data represent mean�SEM. One-way ANOVA with Tukey'smultiple comparisons (C) was used tocompare treatments within eachgroup. Means that do not share a letterare significantly different (P < 0.05).BAF, bafilomycin A1.

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epithelial ovarian (53) and clear-cell ovarian cancers (54), respec-tively. Future studies should continue toassess theprognostic valueof autophagy markers in HGSOC and work to detail the mechan-isms that govern the potency of these preclinical compounds.

Furthermore, combination treatment with autophagy modu-lators may combat disease recurrence and chemoresistance(20–27), an issue plaguing approximately 80% of HGSOCpatients (4). Of over 20 current clinical trials involving autophagymodulators, almost all involve chloroquine or hydroxychloro-quine, and none are being conducted in ovarian cancer patients.Specific inhibition of V-ATPase subunits by shRNA and inhibitorymonoclonal antibodies increases cisplatin sensitivity and reversesplatinum resistance in ovarian cancer in vitro (55). Specific atten-tion should be paid to elucidate the best therapeutic strategy ofautophagy modulation and combination therapies, includingwhich patients would best respond to its induction or inhibition,and the specific means by which perturbation of the autophagicpathway elicits cytotoxicity via apoptosis. Taken together, thisstudy supports further investigation of autophagy inhibitors forthe treatment ofHGSOCdue to the variety of potentially clinicallyuseful biomarkers inherent in their mechanism of action and thepromise of extending patient survival by delaying chemoresis-tance with strategic combination therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A.N. Young, A.D. Kinghorn, J.R. Fuchs, J.E. BurdetteDevelopment of methodology: A.N. Young, J.E. Burdette

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):A.N. Young,D.Herrera, A.C.Huntsman,M.A.Korkmaz,D.D. Lantvit, S. Kolli, S. King, H. Wang, M.A. Phelps, J.E. BurdetteAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.N. Young, M.A. Korkmaz, S. Kolli, C.C. Coss,X. Zhang, M.A. Phelps, L.N. Aldrich, J.E. BurdetteWriting, review, and/or revision of the manuscript: A.N. Young, D. Herrera,A.C. Huntsman, C.C. Coss, S.M. Swanson, A.D. Kinghorn, X. Zhang,M.A. Phelps, L.N. Aldrich, J.R. Fuchs, J.E. BurdetteAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.C. Huntsman, C.C. Coss, S.M. Swanson,J.E. BurdetteStudy supervision: L.N. Aldrich, J.R. Fuchs, J.E. BurdetteOther (design, synthesis, and characterization of chemical constituentsutilized in the study): A.C. HuntsmanOther (performed the animal studies for PHY compounds and compiling/analysis of data for the solubility studies): S. Mazumder

AcknowledgmentsThe authors gratefully acknowledge the support of grants P01CA125066 and

1F30CA217079 from theNCI of theNIH (Bethesda,MD).We are grateful to theUniversity of Illinois at Chicago Research Resource Center's Center for Cardio-vascular Research and Physiology Core for training and use of the Xenogen IVISSpectrum Imager, the Histology Core for their immunohistochemical proces-sing of tissues obtained from in vivo experiments, and the Core Imaging Facilityfor training and use of the Zeiss LSM 710 Confocal Microscope.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 20, 2017; revised April 6, 2018; accepted July 13, 2018;published first July 17, 2018.

References1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin

2017;67:7–30.2. Kurman RJ. Origin and molecular pathogenesis of ovarian high-grade

serous carcinoma. Ann Oncol 2013;24:x16–21.3. Vaughan S, Coward JI, Bast RC Jr., Berchuck A, Berek JS, Brenton JD, et al.

Rethinking ovarian cancer: recommendations for improving outcomes.Nat Rev Cancer 2011;11:719–25.

4. Korkmaz T, Seber S, Basaran G. Review of the current role of targetedtherapies as maintenance therapies in first and second line treatment ofepithelial ovarian cancer; In the light of completed trials. Crit Rev OncolHematol 2016;98:180–8.

5. Herzog TJ, Monk BJ. Bringing new medicines to women with epithelialovarian cancer: what is the unmet medical need? Gynecol Oncol Res Pract2017;4:13.

6. Rossi L, Verrico M, Zaccarelli E, Papa A, Colonna M, Strudel M, et al.Bevacizumab in ovarian cancer: a critical review of phase III studies.Oncotarget 2017;8:12389–405.

7. Bitler BG, Watson ZL, Wheeler LJ, Behbakht K. PARP inhibitors: clinicalutility and possibilities of overcoming resistance. Gynecol Oncol 2017;147:695–704.

8. Wu SJ, Wu TS. Cytotoxic arylnaphthalene lignans from Phyllanthus oli-gospermus. Chem Pharm Bull (Tokyo) 2006;54:1223–5.

9. Ren Y, Lantvit DD, Deng Y, Kanagasabai R, Gallucci JC, Ninh TN, et al.Potent cytotoxic arylnaphthalene lignan lactones from Phyllanthus poi-lanei. J Nat Prod 2014;77:1494–504.

10. SorensenMG, Henriksen K, Neutzsky-Wulff AV, Dziegiel MH, Karsdal MA.Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogatesacidification of the osteoclastic resorption lacunae and bone resorption.J Bone Miner Res 2007;22:1640–8.

11. Zhao Y, Zhang R, Lu Y, Ma J, Zhu L. Synthesis and bioevaluation ofheterocyclic derivatives of Cleistanthin-A. Bioorg Med Chem 2015;23:4884–90.

12. Lu Y, Zhang R, Liu S, Zhao Y, Gao J, Zhu L. ZT-25, a new vacuolar H(þ)-ATPase inhibitor, induces apoptosis and protective autophagythrough ROS generation in HepG2 cells. Eur J Pharmacol 2016;771:130–8.

13. Stransky L, Cotter K, ForgacM. The function of V-ATPases in cancer. PhysiolRev 2016;96:1071–91.

14. Janku F, McConkey DJ, Hong DS, Kurzrock R. Autophagy as a target foranticancer therapy. Nat Rev Clin Oncol 2011;8:528–39.

15. White E. The role for autophagy in cancer. J Clin Invest 2015;125:42–6.16. Levine B, Klionsky DJ. Development by self-digestion: molecular mechan-

isms and biological functions of autophagy. Dev Cell 2004;6:463–77.17. Duffy A, Le J, Sausville E, Emadi A. Autophagy modulation: a target for

cancer treatment development. Cancer Chemother Pharmacol 2015;75:439–47.

18. Gewirtz DA.The four faces of autophagy: implications for cancer therapy.Cancer Res 2014;74:647–51.

19. Delaney JR, Patel CB, Willis KM, Haghighiabyaneh M, Axelrod J,Tancioni I, et al. Haploinsufficiency networks identify targetable pat-terns of allelic deficiency in low mutation ovarian cancer. Nat Commun2017;8:14423.

20. Zhang N, Dai L, Qi Y, Di W, Xia P. Combination of FTY720 with cisplatinexhibits antagonistic effects in ovarian cancer cells: role of autophagy. Int JOncol 2013;42:2053–9.

21. Wang J, Wu GS. Role of autophagy in cisplatin resistance in ovarian cancercells. J Biol Chem 2014;289:17163–73.

22. Feng X, Li L, JiangH, JiangK, Jin Y, Zheng J.Dihydroartemisinin potentiatesthe anticancer effect of cisplatin via mTOR inhibition in cisplatin-resistantovarian cancer cells: involvement of apoptosis and autophagy. BiochemBiophys Res Commun 2014;444:376–81.

23. Bao LJ, Jaramillo MC, Zhang ZB, Zheng YX, Yao M, Zhang DD, et al. Nrf2induces cisplatin resistance through activation of autophagy in ovariancarcinoma. Int J Clin Exp Pathol 2014;7:1502–13.

Mol Cancer Ther; 17(10) October 2018 Molecular Cancer Therapeutics2134

Young et al.

on January 28, 2021. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 17, 2018; DOI: 10.1158/1535-7163.MCT-17-1195

24. Bao L, Jaramillo MC, Zhang Z, Zheng Y, YaoM, Zhang DD, et al. Inductionof autophagy contributes to cisplatin resistance in human ovarian cancercells. Mol Med Rep 2015;11:91–8.

25. Sun Y, Liu JH, Jin L, Sui YX, Lai L, Yang Y. Inhibition of Beclin 1 expressionenhances cisplatin-induced apoptosis through amitochondrial-dependentpathway in human ovarian cancer SKOV3/DDP cells. Oncol Res 2014;21:261–9.

26. Sun Y, Liu JH, Jin L, Sui YX, Han LL, Huang Y. Effect of autophagy-relatedbeclin1 on sensitivity of cisplatin-resistant ovarian cancer cells to chemo-therapeutic agents. Asian Pac J Cancer Prev 2015;16:2785–91.

27. Khurana A, Roy D, Kalogera E, Mondal S, Wen X, He X, et al. Quinacrinepromotes autophagic cell death and chemosensitivity in ovarian cancerand attenuates tumor growth. Oncotarget 2015;6:36354–69.

28. Correa RJ, Valdes YR, Peart TM, Fazio EN, Bertrand M, McGee J, et al.Combination of AKT inhibition with autophagy blockade effectivelyreduces ascites-derived ovarian cancer cell viability. Carcinogenesis 2014;35:1951–61.

29. Ding YH, Zhou ZW, Ha CF, Zhang XY, Pan ST, He ZX, et al. Alisertib, anAurora kinase A inhibitor, induces apoptosis and autophagy but inhibitsepithelial to mesenchymal transition in human epithelial ovarian cancercells. Drug Des Devel Ther 2015;9:425–64.

30. Zhang C, Qiu X. Andrographolide radiosensitizes human ovarian cancerSKOV3 xenografts due to an enhanced apoptosis and autophagy. TumourBiol 2015;36:8359–65.

31. Zhao X, Fang Y, Yang Y, Qin Y, Wu P, Wang T, et al. Elaiophylin, a novelautophagy inhibitor, exerts antitumor activity as a single agent in ovariancancer cells. Autophagy 2015;11:1849–63.

32. Zi D, Zhou ZW, Yang YJ, Huang L, Zhou ZL, He SM, et al. Danusertibinduces apoptosis, cell cycle arrest, and autophagy but inhibits epithelial tomesenchymal transition involving PI3K/Akt/mTOR signaling pathway inhuman ovarian cancer cells. Int J Mol Sci 2015;16:27228–51.

33. Lee YY, Jeon HK, Hong JE, Cho YJ, Ryu JY, Choi JJ, et al. Proton pumpinhibitors enhance the effects of cytotoxic agents in chemoresistant epi-thelial ovarian carcinoma. Oncotarget 2015;6:35040–50.

34. Patil R, Kulshrestha A, Tikoo A, Fleetwood S, Katara G, Kolli B, et al.Identification of novel bisbenzimidazole derivatives as anticancer vacuolar(H(þ))-ATPase inhibitors. Molecules 2017;22.

35. Lu X, Chen L, Chen Y, Shao Q, Qin W. Bafilomycin A1 inhibits the growthand metastatic potential of the BEL-7402 liver cancer and HO-8910ovarian cancer cell lines and induces alterations in their microRNA expres-sion. Exp Ther Med 2015;10:1829–34.

36. Woodard JL, Huntsman AC, Patel PA, Chai H-B, Kanagasabai R, Karma-hapatra S, et al. Synthesis and antiproliferative activity of derivatives of thephyllanthusmin class of arylnaphthalene lignan lactones. Bioorg MedChem 2018;26:2354–64.

37. Ciucanu I, Kerek F. A simple and rapid method for the permethylation ofcarbohydrates. Carbohydrate Res 1984;131:209–17.

38. Rashid A, Mackie W, Colquhoun IJ, Lamba D. Novel synthesis of mono-sulphated methyl a-D-galactopyranosides. Canadian J Chem 1990;68:1122–7.

39. Liu L, Hu Y, Liu H, Liu DY, Xia JH, Sun JS. First total synthesisof the bioactive arylnaphthyl lignan 4OGlycosides phyllanthusmin

D and 40 0OAcetylmananthoside B. Eur J Org Chem 2017;2017:3674–80.

40. Vakifahmetoglu-Norberg H, Xia HG, Yuan J. Pharmacologic agents target-ing autophagy. J Clin Invest 2015;125:5–13.

41. Dowdle WE, Nyfeler B, Nagel J, Elling RA, Liu S, Triantafellow E, et al.Selective VPS34 inhibitor blocks autophagy and uncovers a role forNCOA4 in ferritin degradation and iron homeostasis in vivo. Nat CellBiol 2014;16:1069–79.

42. Werner G, Hagenmaier H, Drautz H, Baumgartner A, Zahner H. Metabolicproducts of microorganisms. 224. Bafilomycins, a new group of macrolideantibiotics. Production, isolation, chemical structure and biological activ-ity. J Antibiotics 1984;37:110–7.

43. Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicityscreening. Nat Protoc 2006;1:1112–6.

44. Aldrich LN, Kuo SY, Castoreno AB, Goel G, Kuballa P, Rees MG, et al.Discovery of a small-molecule probe for V-ATPase function. J Am ChemSoc 2015;137:5563–8.

45. HollingsheadMG, AlleyMC, Camalier RF, Abbott BJ, Mayo JG,Malspeis L,et al. In vivo cultivation of tumor cells in hollow fibers. Life Sci 1995;57:131–41.

46. Mi Q, Pezzuto JM, Farnsworth NR, Wani MC, Kinghorn AD, Swanson SM.Use of the in vivo hollow fiber assay in natural products anticancer drugdiscovery. J Nat Prod 2009;72:573–80.

47. Lewellen KA, Metzinger MN, Liu Y, Stack MS. Quantitation of intra-peritoneal ovarian cancer metastasis. J Vis Exp 2016(113).

48. Domcke S, Sinha R, LevineDA, Sander C, Schultz N. Evaluating cell lines astumour models by comparison of genomic profiles. Nat Commun 2013;4:2126.

49. Auersperg N, Maines-Bandiera SL, Dyck HG, Kruk PA. Characterization ofcultured human ovarian surface epithelial cells: phenotypic plasticity andpremalignant changes. Lab Invest 1994;71:510–8.

50. Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL,et al. Feasibility of drug screening with panels of human tumor celllines using a microculture tetrazolium assay. Cancer Res 1988;48:589–601.

51. Shoemaker RH. TheNCI60 human tumour cell line anticancer drug screen.Nat Rev Cancer 2006;6:813–23.

52. Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome mat-uration process by a novel reporter protein, tandem fluorescent-taggedLC3. Autophagy 2007;3:452–60.

53. Ju LL, Zhao CY, Ye KF, Yang H, Zhang J. Expression and clinicalimplication of Beclin1, HMGB1, p62, survivin, BRCA1 and ERCC1 inepithelial ovarian tumor tissues. Eur Rev Med Pharmacol Sci 2016;20:1993–2003.

54. Spowart JE, Townsend KN, Huwait H, Eshragh S, West NR, Ries JN, et al.The autophagy protein LC3A correlates with hypoxia and is a prognosticmarker of patient survival in clear cell ovarian cancer. J Pathol 2012;228:437–47.

55. Kulshrestha A, Katara GK, Ginter J, Pamarthy S, Ibrahim SA, Jaiswal MK,et al. Selective inhibition of tumor cell associated Vacuolar-ATPase 'a2'isoform overcomes cisplatin resistance in ovarian cancer cells. Mol Oncol2016;10:789–805.

www.aacrjournals.org Mol Cancer Ther; 17(10) October 2018 2135

Phyllanthusmins Inhibit Autophagy and Induce Apoptosis in Ovarian Cancer

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Published OnlineFirst July 17, 2018; DOI: 10.1158/1535-7163.MCT-17-1195

2018;17:2123-2135. Published OnlineFirst July 17, 2018.Mol Cancer Ther   Alexandria N. Young, Denisse Herrera, Andrew C. Huntsman, et al.   Autophagy InhibitionBurden in High-Grade Serous Ovarian Cancer by Late-Stage Phyllanthusmin Derivatives Induce Apoptosis and Reduce Tumor

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on January 28, 2021. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 17, 2018; DOI: 10.1158/1535-7163.MCT-17-1195