a novel bioavailable bh3 mimetic efﬁciently inhibits colon … · the bh3 mimetic mediates its...

Cancer Therapy: Preclinical

A Novel Bioavailable BH3 Mimetic EfficientlyInhibits Colon Cancer via Cascade Effects ofMitochondriaXuefeng Wang1,2, Chen Zhang3, Xiangming Yan1, Bin Lan2,4, Jianyong Wang1,Chongyang Wei1, Xingxin Cao3, Renxiao Wang3, Jianhua Yao3, Tao Zhou5, Mi Zhou3,Qiaoling Liu1, Biao Jiang3, Pengfei Jiang6, Santosh Kesari6, Xinjian Lin6, and Fang Guo1,4

Abstract

Purpose:Gossypol and its analogs, through their ability tobindto and inactivate BH3 domain-containing antiapoptotic proteins,have been shown to inhibit the growth of various human cancercells in culture and xenograft models. Here, we evaluated theantitumor efficacy of a novel gossypol derivative and BH3mimet-ic ch282-5 (2-aminoethanesulfonic acid sodium-gossypolone) incolon cancer models. Several innovative combination strategieswere also explored and elaborated.

Experimental Design: Ch282-5 was synthesized by modifyingthe active aldehyde groups and R groups of gossypol according toa computer-aided drug design program. The stability of ch282-5was examined by high-performance liquid chromatography, andcytotoxic effects of ch282-5 on colon cancer cells were assessed byMTS assay. Activation of mitochondrial apoptotic pathway bych282-5 was evidenced with a series of molecular biology tech-niques. In vivo antitumor activity of ch282-5 and its combination

with chloroquine, rapamycin, oxaliplatin, and ABT-263 was alsoevaluated in colon cancer xenograft models and experimentalliver metastasis models.

Results: Ch282-5 showed antiproliferative and pro-cell deathactivity against colon cancer cells both in vitro and in vivo, and theresponse to the drug correlated with inhibition of antiapoptoticBcl-2 proteins, induction of mitochondria-dependent apoptoticpathway, and disruption of mitophagy and mTOR pathway.Ch282-5 also suppressed liver metastasis produced by intrasple-nic injection of colon cancer cells. Furthermore, ch282-5 couldpotentiate the effectiveness of oxaliplatin and rescue ABT-263efficacy by downregulation of Mcl-1 and elevation of plateletnumber.

Conclusions: Thesefindings provide a rational basis for clinicalinvestigation of this highly promising BH3 mimetic in coloncancer. Clin Cancer Res; 22(6); 1445–58. �2015 AACR.

IntroductionBcl-2 family prosurvival proteins are abundantly expressed in a

wide variety of human cancers and associated with oncogenesis,tumor proliferation, metastasis, and chemoresistance (1). There-

fore, targeting Bcl-2 draws a great deal of attention in cancer drugdevelopment. In recent years, Bcl-2 small-molecule inhibitorssuch as ABT-737, ABT-263, ABT-199, andGX-15-070have enteredclinical trials. Gossypol, a polyphenol derived from cottonseedoil, was the first natural compound discovered that demonstratedinhibition of Bcl-2 family proteins (2). NMR and fluorescencepolarization competitive binding experiments demonstrated thatgossypol can bind to the BH3 domain of the Bcl-2 family anti-apoptotic proteins in the tumor cells thus initiating caspase-dependent apoptosis events (3). However, the clinical use ofgossypol is limited by its toxicity problemmost likely due to tworeactive aldehyde groups and by its high hydrophobicity (4). As aresult, many derivatives of gossypol have been generated includ-ing AT-101 and ApoG2 that have entered clinical trials (4).

We previously reported a gossypol derivative named "com-pound-7" (6-APA-Na-gossyplone) as a Bcl-2 inhibitor that dem-onstrated competitive binding to the BH3 domain of Bcl-xL (Ki¼1.76 mmol/L) causing release of proapoptotic proteins Bim andBax, and ultimately leading to apoptosis (5). Compound-7 notonly inhibited the growth of several cancer cell lines in vitro butalso suppressed tumor growth in vivo with less toxicity whencompared with natural gossypol. More importantly, com-pound-7 could potentiate the antitumor effects of 5-fluorouracil(5-FU) both in vitro and in vivo in colon cancer models. However,the log P of compound-7 is 8.33 indicating its poor absorptionand permeation (6), which limits a possible clinical application.The stability of compound-7 is also not appreciable.

1Laboratory of Tumor Targeted Therapy, Shanghai AdvancedResearch Institute, Chinese Academy of Sciences, University of Chi-nese Academy of Sciences, Shanghai, China. 2Institute of HealthSciences, Shanghai Institute for Biological Sciences, Chinese Acade-my of Sciences & Shanghai Jiao Tong University School of Medicine,Shanghai, China. 3CAS Key Laboratory of Synthetic Chemistry ofNatural Substance, Shanghai Institute of Organic Chemistry, Univer-sity of Chinese Academy of Sciences, Shanghai, China. 4Key Labora-toryof SystemsBiomedicine (Ministryof Education), Shanghai Centerfor Systems Biomedicine, Shanghai Jiao Tong University, Shanghai,China. 5Academy of Life Science, Shanghai University, Shanghai, Chi-na. 6DepartmentofMedicineandUCSanDiegoMooresCancerCenter,University of California-San Diego, La Jolla, California.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Fang Guo, Laboratory of Tumor Targeted Therapy,Shanghai Advanced Research Institute, Chinese Academy of Sciences, Univer-sity of Chinese Academy of Sciences, 99 Haike Road, Shanghai 201210, China.Phone: 8618-6162-13863; Fax: 8602-1203-50910; E-mail: [email protected] orXinjian Lin, UC San Diego Moores Cancer Center, Department of Medicine,University of California-San Diego, 3855 Health Sciences Drive, La Jolla, CA92093-0819, Tel: (858) 822-1115; Fax: (858) 822-1111; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-0732

�2015 American Association for Cancer Research.

ClinicalCancerResearch

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Mitochondria is an important place of biologic oxidation andenergy conversion for eukaryotic cells, involving cell homeostasis,proliferation, motility, aging, death, and other biologic processes.Bcl-2 family inhibitors could damage mitochondria and changepermeability of mitochondrial membrane, uncoupling oxidativephosphorylation. ATP excessive consumption induces necrosis orswelling mitochondria triggers apoptosis by released cytochromec. Timely removal of damaged mitochondria is essential for thenormal growth and metabolism of cells. Mitochondrial autop-hagy (mitophagy) is a specific phenomenon of autophagy byselectively removing damaged mitochondria (7). Autophagy isgenerally regarded as a prosurvival factor under cellular stress, andautophagy inhibition could enhance killing effects in tumor cellsboth in vivo and in vitro (8). Therefore, the strategy to inhibitautophagy to enhance the effectiveness of chemotherapy is con-stantly attempted.

As of 2015, colorectal cancer has been the third most commoncancer in United States (9). More than half of the patients withcolorectal cancer are expected to develop metastatic disease, aquarter of whom would have distant metastatic lesions at diag-nosis, often in the liver (10). Therefore, antimetastatic propertybecomes a compulsory requirement for a colon cancer drugcandidate. mTOR is a Ser/Thr kinase that plays a key role inregulation of a wide range of biologic processes including cellgrowth, proliferation, survival, autophagy, metabolism, and cyto-skeletal organization. The frequent hyperactivation of mTORsignaling in cancer cells has made it an attractive target fortherapeutic intervention and has stimulated the development ofa number of mTOR inhibitors, some of which have alreadyundergone clinical trials (11). mTOR is also positioned upstreamand downstream of multiple oncogenic pathways such as AKT,ERK, and STAT signaling that may contribute to some of theresistance that can occur with mTOR-targeted therapies (12).

Combination therapy is a common strategy for cancer clinicaltreatment. Combination of two or more chemotherapeutics notonly augments the cytotoxicity but also reduces the drug resis-tance and side effects. The current clinical therapy of colon canceremploys a combination of oxaliplatin and 5-fluorouracil (5-FU),both of which are DNA-damaging agents (13). Thus, discoveryand development of new targeted drugs for colon cancer treat-ment is urgently needed. Accumulating evidence also indicates

that a single BH3 mimetic may not be sufficient as a monother-apeutic to treat cancer patients and the best clinical outcome maybe achieved by appropriate drug combinations. Combination ofABT-737 or ABT-263 with autophagy inhibitors and other tar-geted agents has proved beneficial in preclinical and clinicalcancer treatment (14).

In this study, we identified a new gossypol derivative ch282-5by changing the active aldehyde groups of gossypol into ketogroups to reduce side effects, and replacing the R group ofgossypol with sodium taurate to enhance hydrophilicity. Wefound that ch282-5 selectively induced colon cancer cell deathby specific and competitive binding to the BH3 domain of Bcl-2family antiapoptotic proteins. Inhibition of mitophagy and dis-ruption of mTOR signaling enhanced the antitumor effects ofch282-5. Ch282-5 significantly inhibited experimental livermetastasis and rescued ABT-263 efficacy by downregulation ofMcl-1 and increasing the platelet count. Taken together, thesefindings provide new understanding of themechanisms bywhichthe BH3 mimetic mediates its antitumor effects and facilitates itsclinical development.

Materials and MethodsCh282-5 synthesis

Sodium2,20-(1E,10E)-(6,60,7,70-tetrahydroxy-5,50-diisopropyl-3,30-dimethyl-1,10,4,40-tetraoxo-1,10,4,40-tetrahydro-2,20-binaph-thyl-8,80-diyl) bis (methan-1-yl-1-ylidene) bis (azan-1-yl-1-yli-dene) diethanesulfonate. Taurine (250 mg, 2 mmol) was addeddrop-wise to a solution of NaOH (88 mg, 2.2 mmol) in dryethanol (20mL) at 40�C under Ar to obtain taurate. Gossypolone(546 mg, 1 mmol) was then added drop-wise to the reactionmixture at room temperature. The solution turned red wineimmediately and the reaction mixture was stirred at room tem-perature for 1.5 hours then warmed to 40�C for another 0.5 hour.The resulting reactionmixturewas filtered under reduced pressureto give a red solid powder, which was then washed with dryethanol (2�10mL) anddry ether (3�10mL) to give a Schiff baseproduct (604 mg) with a yield of 75%. 1H NMR (500 MHz,DMSO) d 14.19 (s, 2H), 9.25(s, 4H), 3.97(s, 4H), 3.79–3.86 (m,2H), 2.82–2.84 (m, 4H), 1.90 (s, 6H), 1.36 (t, J ¼ 6.2 Hz, 12H),13C NMR(126 MHz, DMSO) d 186.70, 184.92, 168.65, 165.21,153.25, 144.89, 137.38, 130.96, 126.22, 123.70, 106.70, 106.06,56.00, 49.96, 48.48, 27.52, 20.10, 19.94, 18.53, 14.02, LR-ESI:[M-2H]þ 802.2, [M-2NaþH]þ 759.3; [M-Na]þ 781.1, [M-2Naþ H] þ 759.1(Supplementary Figs. S1 and S2).

Cell cultureHuman colon cancer cell linesHCT116 andHT29 (ATCC)were

cultured in McCoy 5A medium, SW620 in L-15 medium, andmouse colon cell line CT26 in 1640 medium in a 5% CO2

humidified atmosphere at 37�C. All media were supplementedwith 10% FBS (Gibco), 1% penicillin and streptomycin (Invitro-gen). Other cell lines were cultured in growth medium as perATCC recommendations.

Cell viability assayThree thousand or 5,000 cells were treated with ch282-5 or

other combinations for different time in 96-well plates in afinal volume of 100 mL and viability was assessed by CellTiter96 AQueous One Solution Cell Proliferation Assay (MTSassay).

Translational Relevance

Antiapoptotic Bcl-2 family proteins are well recognized astargets for the development of novel anticancer therapeutics.Gossypol and its analogs are known to have potent proapop-totic effects through inhibition of antiapoptotic Bcl-2 familymembers while toxicity remains a major problem. In thisstudy, we investigated the efficacy of a novel bioavailablegossypol derivative and BH3 mimetic, ch282-5, in coloncancer models. We found that while achieving reduction inside effects ch282-5 inhibited the growth of xenografted orexperimentally metastatic colon cancer, potentiated antican-cer effects of oxaliplatin, and rescued ABT-263 efficacy bydownregulation of Mcl-1 and increasing platelets. Theseresults demonstrate that ch282-5 is a potent Bcl-2 inhibitorwith less adverse effects andwould provide rationale for futureclinical studies in colon cancer.

Wang et al.

Clin Cancer Res; 22(6) March 15, 2016 Clinical Cancer Research1446




Flow cytometryFor apoptosis, ch282-5–treated cells were incubated with

Annexin V-FITC and propidium iodide (PI) for 15 minutes atroom temperature in the dark. For cell cycle, ch282-5–treated cellswere fixed in 70% ethanol at 4�C for 30minutes, and labeledwithPI (5mg/mL) in thepresence of RNaseAat 37�C indarkness for 30minutes. Then, the samples were analyzed by FACScan flowcytometer (Becton Dickinson). Analysis of the results was carriedout using FlowJo 7.6.1 software.

Immunoblot assayCoimmunoprecipitation (Co-IP) experiments were completed

using a Pierce Direct IP Kit. Cell lysates were equally loaded to10%–12% SDS-polyacrylamide gels, electrophoresed, and trans-ferred to an Immobilon-P PVDF Transfer Membrane (0.45 mm,Millipore).Membraneswere blocked for 1 hour in TBS–Tween-20containing 5% nonfat milk and then incubated with primaryantibodies at 4�C overnight. After being washed 3 times, themembranes were incubated with horseradish peroxidase(HRP)-linked secondary antibodies at room temperature for 1hour. Themembranes were visualized by a SuperSignal West PicoChemiluminescent Substrate according to the manufacturer'sinstructions. All experiments were repeated three times.

Electron microscopyCh282-5–treated HCT116 cells were fixed in ice-cold 2% glu-

taraldehyde, rinsed with PBS, postifixed in 1% osmium tetroxidewith 0.1% potassium ferricyanide, dehydrated through a gradedseries of ethanol (30%–100%), and embedded in Epon.Ultrathinsections (65 nm)were stainedwith 2%uranyl acetate andReynodlead citrate and examined at 80 KV using a PHILIP CM-120transmission electron microscope.

Fluorescence microscopeFor TUNEL assay, ch282-5–treated cells were fixed with 4%

paraformaldehyde in PBS for 15 minutes at room temperature.After washing with PBS, cells were processed in permeabilizationsolution for 2minutes at 4�C, washed with PBS, and incubated inTUNEL reaction mixture for 1 hour at 37�C in the dark. Nucleiwere stained with Hocest33342. For JC-1 assay and Mito-tracker/Lyso-tracker staining, the treated cells were incubated with JC-1,Mito-tracker, or Lyso-tracker for 20 to 30 minutes at 37�C, andthen washed twice with PBS. Samples were observed under afluorescence microscope. Three random fields were recorded forstatistical analysis.

Invasion and migration assayFor invasion assay, the transwell system (24 wells, 8 mm pore

size with polycarbonate membrane; Corning Costar) was coatedwith 3 mg/mL Matrigel (BD Biosciences). Three hours later, 5 �105 CT26 cells in 100 mL serum-free 1640 medium were seededinto the top chambers and 1,000 mL 1640 medium containing20% FBS and 10 mmol/L ch282-5 was added into the bottomchamber. Twenty-four hours later, cells that did not invadethrough the pores of the transwell inserts were removed with acotton swab and the inserts were fixed in cold methanol for 10minutes and then stained with 0.1% crystal violet. For migrationassay, the experiments were performed as above except that cellswere plated on top of uncoated (Matrigel-free) inserts. Themigrated or invaded cells were photographed under a lightmicroscope.

In vitro scratch assayCT26-MSCV and CT26-MSCV-AEP cells were seeded in 24-well

plates. When the cells grew to 90% confluence, a scratch wasintroduced through the cell monolayer using a 200 mL pipette tip.The cells were washed with PBS and immediately incubated with10 mmol/L ch282-5. The scratch area was photographed at 0 and24 hours after the drug exposure.

In vivo studiesFive- to 6-week-old male Nu/Nu mice and BALB/c mice were

purchased from Shanghai SLAC Laboratory Animal Center andcare of the mice was consistent with the guidelines of ShanghaiModel Organisms Research Center. In toxicity study, Nu/Numicewere given 10% ethanol as vehicle control, 120 mmol/kg/day ofch282-5 or gossypol for 12 consecutive days. After treatment,small intestine and colon were fixed in 4% paraformaldehyde(PFA) and stained with hematoxylin and eosin (H&E). In efficacystudies, Nu/Nu mice or BALB/c mice received subcutaneousinjection of 5 � 105 CT26 cells, 1 � 106 HCT116 or HT29 cellsin the rightflank area.Once tumors grewup to 60mm3,micewererandomly divided into various groups with six mice per group atleast. The mice were treated with 120 mmol/kg ch282-5 (intragas-trically; vehicle was 10% ethanol, 60% Phosal 50 PG/30% PEG400/10% ethanol or 82% PBS/5% Tween-80/5% PEG 400/8%ethanol), 120 mmol/kg CQ (intraperitoneally; vehicle asdescribed above), 20mg/kg rapamycin (intraperitoneally; vehicleas described above), 3 mg/kg oxaliplatin (intraperitoneally; vehi-cle was 5% glucose solution), 50mg/kg ABT-263 (intragastrically;vehicle was 60% Phosal 50 PG/30% PEG 400/10% ethanol) ortheir given combination every other day. The tumors were mea-sured by length and width every 3 days, and tumor volume wascalculated by the formula: (length�width2)�0.52. In experimen-tal liver metastasis study, 1 � 105 CT26 cells were injected intospleen of Balbc mice. Mice were treated with 120 mmol/kg ch282-5 every other day, until any one of mice died first. The livers ofdead mice were fixed by 4% PFA, and the survival dates wererecorded.

Statistical analysisAll experiments were performed at least thrice, and the

results were expressed as mean � SD wherever applicable.GraphPad Prism 5 software was used for statistical analysis.Student t-test was used to compare the differences betweenvariables. P value < 0.05 was considered statistically significant(�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

ResultsCh282-5 is an efficacious Bcl-2 family inhibitor withoutobvious side effects

To augment the bioavailability of the gossypol derivativecompound-7 that we have previously reported (5), we introduced2-aminoethanesulfonic acid sodium into R groups to develop anovel gossypol derivative ch282-5 (MW 804) based on theformation of a gossypol Schiff base (Fig. 1A–C and Supplemen-tary Fig. S3). In light of Lipinski Rule of Five, ideal drugs shouldexhibit log P values in the range from�0.4 toþ5.6. We applied aforecasting system that we established to calculate the hydropho-bic constant of ch282-5 (log P¼ 4.63) which is much lower thanthat of compound-7 (log P ¼ 8.33), indicating an increase ofhydrophilicity (Supplementary Table S1). As expected, over 12

A Novel Bioavailable BH3 Mimetic

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mmol ch282-5 can be dissolved in 100 mL H2O at 37�C,compared with 9.3813 mmol compound-7 and 0.0019 mmolgossypol. For a stability test, ch282-5 was dissolved in PBSbuffer (pH 7.4) for 180 minutes and above 70% identifiablech282-5 could be detected by high-performance liquid chro-matography, which indicated a better stability of ch282-5 at asimilar condition of physiology (Supplementary Fig. S4). Inaddition, the predicted acute toxicity of ch282-5 was belowcompound-7 (Supplementary Table S1). Therefore, ch282-5might be more amenable to absorption, distribution, andmetabolism than compound-7.

Molecular models of ch282-5 binding with Bcl-2, Bcl-xL, andMcl-1 (PDB code: 4AQ3, 1YSI, and 4HW2) were simulated bythe FlexX software (Fig. 1D–F), which disclosed a high affinityof ch282-5 to the target proteins. A fluorescence polarization–based assay further confirmed that His-Bcl-2, His-Bcl-xL, and His-Mcl-1 bound to the FAM-Bim-BH3 peptide with a Kd value of400 nmol/L, 100 nmol/L, and 250 nmol/L, respectively (Supple-mentary Fig. S5A–S5C), and ch282-5 competitively disrupted theinteraction of Bcl-2 (Ki 3.0 mmol/L), Bcl-xL (Ki 1.6 mmol/L), Mcl-1(Ki 0.254 mmol/L), and proapoptotic proteins (SupplementaryFig. S5D–S5F).

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Figure 1.Ch282-5 is an efficacious Bcl-2 family proteins inhibitor without obvious side effects. Chemical structure of gossypol (A), 6-APA-Na-gossypolone:6-aminopenicillanic acid sodium- gossypolone (compound 7; B), and ch282-5: 2-aminoethanesulfonic acid sodium-gossypolone (C). Molecular model studiesby the FlexX software, molecular model of Bcl-2 (D), Bcl-xL (E), and Mcl-1 (F) binding with ch282-5. Toxicity test in colon cancer xenograft model. G, thebody weight of Nu/Nu mice was measured every day. H, tumor volume at day 15 after commence of gossypol or ch282-5 treatment every other day. I, percentagesurvival curves of Nu/Nu mice with different treatments; Results of biochemical indexes (J) and routine blood test (K). L, ch282-5–treated mice haveno obvious damage in H&E–stained small intestine (b) and colon (e) comparedwith the control (a and d), while gossypol lead to small intestine basementmembranehyperplasia (c) and colon expansion (f).

Wang et al.





To assess the antitumor activity and possible side effects ofch282-5 in vivo, we measured the mice body weight, tumorvolume, survival rate, biochemical indexes, and routine bloodtest aswell as performedpathologic examinations onmiceorgans.Body weight and survival of ch282-5–treated mice had no sig-nificant reduction compared with the control, whereas the loss ofbody weight and a decrease of survival in gossypol-treated micewere significant (Fig. 1G and I). In contrast, treatmentwith ch282-5 at the same dose significantly inhibited the tumor growth ascompared with either gossypol- or vehicle-treated group (Fig.1H). We compared biochemical indexes (Fig. 1J) and routineblood tests (Fig. 1K) between vehicle control, 120 mmol/kggossypol, and 120 mmol/kg ch282-5 treated mice. An increase inhigh-density lipoprotein (LDL-C) indicating liver damage wasseen in gossypol-treated mice not in ch282-5–treated group. Adecrease in alkaline phosphatase (ALP), creatinine (CRE), glucose(GLU), and inorganic phosphate may indicate gossypol break-down and absorption. Ch282-5 raised inorganic phosphate levelin contrast to the control and gossypol group, so we had toconfirm or exclude the nephrotoxicity by other indexes. Asch282-5 decreased uric acid (UA) level and caused no significantchanges in urea nitrogen (BUN) and potassium (Kþ) levels, wemay exclude the possibility of renal toxicity. Ch282-5 doubled thelevel of white blood cell (WBC). Both gossypol and ch282-5treatment markedly increased blood platelet count with a largereffect for gossypol, which implicated a possible risk of thrombo-sis. In addition, ch282-5 caused a most noticeable increase ofneutrophilic granulocyte. A decrease in lymphocyte, monocyte,and eosinophil granulocyte was observed in both groups. Histo-logic examinations revealed that Ch282-5–treated mice had nodamage in the small intestine and colon (Fig. 1L, b and e)compared with the controls (Fig. 1L, a and d), whereas gossypolcaused small intestine basement membrane hyperplasia (Fig. 1L,c) and colon expansion (Fig. 1L, f).

Ch282-5 inhibits cell proliferation by cell-cycle arrest and themitochondria-dependent apoptosis

To assess the growth inhibitory effect of ch282-5 on coloncancer cells, 40 different cancer cell lines were treated with ch282-5 and tested for cell viability byMTS assay. The results showed thatch282-5 significantly suppressed the growth of multiple cancercell lines including colon, breast, prostate, and gastric cancer(Supplementary Table S2). Human colon cancer cells were sen-sitive to ch282-5 with the EC50 between 5 and 20 mmol/L (72-hour continuous exposure). Treatment of HCT116, HT29,SW620, or CT26 cells with ch282-5 reduced cell viability in adose- and time-dependent manner (Fig. 2A and SupplementaryFig. S6A). Ch282-5–induced growth inhibition was further con-firmed by a colony formation assay (Supplementary Fig. S6B).

To determine the factors responsible for the reduction in cellviability, we considered three aspects: apoptosis, necrosis, andcell-cycle suppression. Necroptosis is characterized by cell swell-ing, plasmamembrane permeabilization, mitochondria dysfunc-tion, and release of mitochondrial content to cytoplasm (15). Allcharacteristics of necroptosis are similar to apoptosis except noDNA fragmentation. We found that ch282-5–treated cells died ofapoptosis with DNA fragmentation (Fig. 2B and C). Flow cyto-metric analysis showed that 35% to 40% of HCT116 and HT29cells treated with 20 mmol/L ch282-5 were at the early and latestages of apoptosis (Fig. 2D). Moreover, ch282-5 induced apo-ptosis in a time- and dose-dependent fashion. Immunoblots

showed that PARP were activated by ch282-5 (Fig. 2E), whichconfirmed apoptotic cell death induced by ch282-5. Meanwhile,in both HCT116 and HT29 cells, ch282-5 induced Sub-G1 phasearrest in a dose- and time-dependent manner (Fig. 2F). Thisphenomenon was confirmed by decreased Cyclin D1 andincreased CHOP expression (Fig. 2G). CHOP was identified asa C/EBP-homologous protein, which suppresses cell-cycle pro-gression from G1–S phase.

Mitochondrial damage is a principal incentive of apoptosis. Tofurther interrogate themechanism of ch282-5–induced apoptosisin colon cancer cells, transmission electron microscopy, andfluorescence microscopy were employed. We observed pyknosisnucleus, apoptotic vesicles, damaged spinal, mitochondriavacuoles in ch282-5–treated cells, whereas normal nuclei andmitochondriawere observed in control cells (Fig. 2H). Changes ofmitochondrial membrane potential (Dcm) were assessed in afluorescence microscope with the mitochondrial-specific dualfluorescence probe JC-1. We observed green fluorescence in thech282-5–treated HCT116 cells and HT29 cells but red fluores-cence in the untreated control (Fig. 2I), suggesting that ch282-5significantly damaged mitochondria.

Ch282-5 regulates Bcl-2 and IAP family proteins and inducescaspase-dependent apoptosis pathway

Coimmunoprecipitation was used to detect ch282-5 inducedcompetitive disruption of the connectionbetweenMcl-1 andBim,Noxa in ch282-5–treated HCT116 cells (Fig. 3A) as dissociatedproapoptotic proteinswouldoligomerize tomitochondrialmem-brane and formed pores. As a result,mitochondria were damaged,and mitochondria contents were released to cytoplasm in ch282-5–treated cells (Fig. 3B). The data further demonstrated thatch282-5 induced a cytochrome c–initiated apoptosis pathway.Meanwhile, an increase in phosphorylated-JNK (Fig. 3C) mightpromote Bak and Bax dissociating from the 14-3-3 protein, andencourages the progression of Bak and Bax oligomer into andpermeabilizes themitochondriamembrane (16).Hence, a certaindegree of Bak and Bax reduction occurred in the cytoplasm(Supplementary Fig. S7A). Dissociated Bcl-2 family antiapoptosisproteins were significantly decreased (Fig. 3D). Real-time quan-titative PCR excludedmRNA regulation andproteasome inhibitorMG132 could restrain this reduction (3D and Supplementary Fig.S7B and S7C), that is to say, partial dissociated Bcl-2, Bcl-xL, andMcl-1 were ubiquitinated and degraded (17). IAP family proteinsinhibit caspase function to impede apoptosis. Ch282-5 inducedSmac/Diablo and AIF release from the mitochondria, which candisrupt connection of IAP proteins and caspase proteins. It isknown that dissociated IAP family proteins could be degraded byautoubiquitination (18). Therefore, decreases of c-IAP1, c-IAP2,XIAP, and survivinwere observed andMG132 could restrain thesedecreases to a certain extent (Fig. 3E).

Damaged mitochondria would inevitably cause a drop in ATPlevel. The AMPK is a sensor of cellular energy status, which isactivated by an elevated AMP/ATP ratio (19). We detected anincreased phosphorylation of AMPKa at the site of Thr172 (Fig.3F), which would inhibit mTOR signaling. Recent studies showthat survivin is regulatedbyPI3K/Akt/p70s6K1pathway (20), andsome Raf kinase inhibitors could induce mTOR-dependent sur-vivin downregulation (21). Therefore, we went further to inves-tigate the effects of ch282-5 on mTOR, Akt, and p70s6k pathway.As expected, ch282-5 significantly reduced phosphorylation ofmTOR at Ser2448 and Ser2481site (Fig. 3F), mTOR was






Control 12 h-10 μmol/L 12 h-20 μmol/L 24 h-10 μmol/L 24 h-20 μmol/L

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G1: 47.9%S: 20.8%G2: 6.06%Sub-G1 15.4%Super-G2 9.80%

G1: 50.8%S: 17.4%G2: 5.24%Sub-G1 22.2%Super-G2 4.36%

G1: 23.9%S: 34.8%G2: 3.64%Sub-G1 32.4%Super-G2 5.24%

G1: 5.13%S: 23.4%G2: 3.74%Sub-G1 61.1%Super-G2 6.57%

G1: 43.0%S: 16.2%G2: 21.9%Sub-G1 3.49%Super-G2 15.4%

G1: 53.0%S: 9.33%G2: 19.9%Sub-G1 6.53%Super-G2 11.3%

G1: 61.5%S: 8.84%G2: 12.8%Sub-G1 11.0%Super-G2 5.78%

G1: 50.6%S: 11.0%G2: 15.4%Sub-G1 16.1%Super-G2 6.88%

G1: 48.4%S: 11.9%G2: 13.23%Sub-G1 21.68%Super-G2 4.85%

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Figure 2.Ch282-5 inhibits cell proliferation by cell-cycle arrest and themitochondria-dependent apoptosis. A, cells were treatedwith 0, 5, 10, 20 mmol/L ch282-5 for 48 hoursand subjected to the MTS assay. B, cells were treated with ch282-5 for 24 hours and then stained with Hoehst33342 and TUNEL. C, record and analysis ofpositive tunel puncta by GraphPad Prism 5 software based on three random fields. Cells were treated with 10 mmol/L or 20 mmol/L ch282-5 for 12 or 24 hours,respectively, and apoptosis (D) and cell cycle (F) were determined by Annexin V–FITC/PI labeling using flow cytometry. Cells were treated with ch282-5 for24 hours, and PARP, PARP-1 (E), Cyclin D1 and CHOP (G) were detected by Western blot analysis. H, representative electron micrographs of HCT116 cells (a–f) andSW620 cells (g–i) treated with or without 20 mmol/L ch282-5 for 24 hours. Contrasted normal nucleus (a), pyknosis nucleus (b), apoptotic bodies (c), normalmitochondria (d andg), damagedmitochondria ridge (e and h),mitochondria vacuolization (f and i). I, HCT116 andHT29 cellswere treatedwithout 10mmol/L ch282-5for 6 hours, and then imaging technique was used to assess the change of mitochondrial membrane potential of cells treated with ch282-5 by the JC-1 method.

Wang et al.





phosphorylated at Ser2448 via the PI3 kinase/Akt signaling path-way and autophosphorylated at Ser2481 (22, 23). Akt and Erkpromote mTORC1 signaling through phosphorylation of aGTPase activator protein (GAP), referred to as tuberous sclerosiscomplex 2 (TSC2; ref. 24), and the tuberin/hamartin (TSC2/TSC1) complex inhibits mTOR activity indirectly by inhibitingRheb (25). In accord with these previous observations, we foundthat the level of P-Akt (Ser473), P-Erk, and Rheb proteins was allreduced (Fig. 3F). The decrease of phosphorylated mTOR led toinhibition of p70s6k (an activator of translation) and activationof 4E-BP1 (an inhibitor of translational initiation), and then

activated 4E-BP1 further resulted in inhibition of p65 (transcrip-tion factors of the NF-kB; Fig. 3G). Inhibition of p70s6k and p65could directly cause a reduction in the synthesis of some proteinssuch as Bcl-2 and IAP family antiapoptotic proteins.

Decrease in antiapoptotic proteins and activation of proapop-totic proteins (Fig. 3D, E, and H and Supplementary Fig. S7A)induced the caspase-dependent apoptosis pathway. Caspase-9combines with cytochrome c forming apoptosome. Activatedforms of caspase-3, 6, 7, 9, and Lamin-A/C (a key protein of cellshrinkage and membrane blebbing) were all presented in thech282-5–treated cells (Fig. 3I).

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ch282-5 (μmol/L)

Figure 3.Ch282-5 regulates Bcl-2 and IAP family proteins and induces caspase-dependent apoptosis pathway. A, coimmunoprecipitation was used to detect ch282-5–induced competitive disrupting the connection of Mcl-1 and Bim, Noxa in HCT116 cells treated with 20 mmol/L ch282-5 for 6 hours. B, cells were treated withch282-5 for 30minutes, and then thedistributionsof Cyto-c, Smac, andAIF inboth cytoplasmandmitochondrionwere analyzedbyWesternblot analysis. HCT116 andHT29 cells were treated with 0, 10, 20 mmol/L ch282-5 with or without 1 mmol/L MG132 for 24 hours, and then Bcl-2 family antiapoptotic proteins: Bcl-2,Bcl-xL, Mcl-1 (D) and IAP family proteins: c-IAP1, c-IAP2, XIAP, Survivinwere detected byWestern blot analysis (E). HCT116 and HT29 cells were treatedwith 0, 10, 20mmol/L ch282-5 for 24 hours, and then JNK, P-JNK (C), mTOR, P-mTOR, AMPKa, P-AMPKa, AKT, P-AKT, Erk1/2, P-Erk1/2, Rheb (F), p70s6k, P-p70s6k,4E-BP1, P-4E-BP1, p65, P-p65 (G), Bak and Bim (H), caspase family proteins, Lamin A/C were detected by Western blot analysis (I).






Ch282-5 antitumor effect is augmented by disturbingmitophagy and mTOR pathway

Given that Bcl-2 inhibitors simultaneously induce mitochon-drial damage in tumor cells and activate mitochondrial qualitycontrol, and mitophagy has emerged as a key mechanism in thisquality control for the elimination of superfluous or damagedmitochondria (26), we sought to determine whether ch282-5triggers mitophagy in colon cancer cells. Electron microscopyultrastructural analyses revealed a series of characteristic phenom-ena of mitophagy in ch282-5–treated HCT116 and SW620 cellsthat included an increase of primary lysosome (Fig. 4A, a, whitearrow), secondary lysosome (Fig. 4A, a, black arrow), damagedmitochondria wrapped in the autophagosome (Fig. 4A, b, d, ande), secondary lysosome with a monolayer membrane structurefused with the autophagosome to become autolysosome (Fig. 4A,c, blackarrow), anddigested autolysosome (Fig. 4A, c,white arrow,and 4A, f). Colocalization stainingofMito-tracker and Lyso-trackercan be used to track mitophagy (27). We observed an increase ofcolocalization (yellow) staining in ch282-5–treated CT26 cells inan early phase (Fig. 4B), and the similar phenomenawere all foundin HCT116, HT29, and SW620 cells (Supplementary Fig. S8A–S8C), clearly indicating that ch282-5 induced a typical mitophagy.Ch282-5–induced mitophagy was also confirmed in HCT116-GFP-LC3b and HT29-GFP-LC3b cell lines as evidenced by GFP-LC3b punctuate aggregation (Fig. 4C and D). Moreover, decreasedBeclin-1, Lamp-2, andp62, and increasedLC3I/IIwere all unfoldedin a late phase of mitophagy (Fig. 4E). These results suggest thatch282-5 induced dose-dependentmitophagy in colon cancer cells.Coimmunoprecipitation study revealed that ch282-5 competitive-ly dissociated Beclin-1 from Bcl-2, Bcl-xL, and Mcl-1 service tomitophagy (Supplementary Fig. S8D–S8F).

It is conceivable that combining ch282-5 with a mitophagyinhibitor may augment inhibitory effects of ch282-5 on coloncancer cells. Indeed, 5 mmol/L or 10 mmol/L CQ can significantlypotentiate the growth inhibitory effect of ch282-5 on HCT116and HT29 cells (Fig. 4F and G). Mitophagy inhibitors 3-MA andWortmannin could also sensitize these cells to ch282-5 (Supple-mentary Fig. S8G and S8H). Colocalization of lyso-tracker–labeled lysosome and GFP-labeled autophagosome suggestedthat CQ plays a role in blocking autophagosome fusion withlysosome in ch282-5–treated cells (Supplementary Fig. S8I). TheJC-1 method, DNA ladder assay, and Western blot analysis allshowed that CQ could enhance ch282-5–induced mitochondria-dependent apoptotic cell death (Supplementary Fig. S8J–S8L).The assumption that disruption of mitophagy could augmentch282-5–induced inhibition on colon cancer cells were validatedin HCT116 and HT29 xenograft models. We obtained the resultsthat the antitumor effect of ch282-5 or CQ alone at the same doseof 120 mmol/kg/2 days was similar, whereas combined treatmentsignificantly enhanced the effect (Fig. 4I and J).

A triple combination of CQ, rapamycin, and ch282-5 wasfurther investigated in the colon cancer models. To our surprise,this triple combination therapy remarkably inhibited CT26 cellsboth in vitro (Fig. 4H) and in vivo (Fig. 4K) compared with singleuse or double combination. A similar result was also obtainedwith HCT116 cells (Supplementary Fig. S8M). We next investi-gated the effects of triple combination on IAP family proteins byWestern blot analysis. The results showed that phosphorylation ofmTORwas reduced to themaximumextent by triple combination(Fig. 4L), and P-p70s6k was completely disappeared under thistreatment (Fig. 4M). Accordingly, downstreamproteins of 4E-BP1

and p65were also suppressed and the greatest degree of reductionwas seen in IAP family proteins (Fig. 4M andN). In the same time,we observed the autophagy flux by detecting p62, LC3I/II, Lamp1,and Lamp2 (Supplementary Fig. S8N).

Ch282-5 suppresses liver colonization of colon cancer cellsThe intrasplenic injection of human cancer cells into the nude

mice produces experimental metastases in the liver, and cellsinjected into the spleen reach the liver parenchyma within min-utes (28). Thus, the intrasplenic implantation of cells measurestheir ability to grow in the liver parenchyma and the effect of drugtreatment represents its efficacy in suppression of establishedmetastasis. As shown in Fig. 5A, treatment with 120 mmol/kgch282-5 every other day substantially increased survival of thetreated mice. We observed that the liver volume of ch282-5–treatedmice was slightly smaller than that of vehicle-treatedmice,and ch282-5–treated mice had less metastasis (SupplementaryFig. S9A). To further clarify that antimetastatic effect of ch282-5was mediated through suppression of established metastasis, weset up two groups of experimental liver metastasis models, andthen treated one group at day 1 and the other at day 8 with thesamedose of ch282-5 after the intrasplenic injectionofCT26 cells.There was no significant difference in the inhibition of livermetastasis between the two groups (Fig. 5B and SupplementaryFig. S9B) implying that ch282-5 did not inhibit the process ofCT26 cells migrated from the spleen to the liver but rathersuppressed the proliferation of metastasized cells in the liver.

In vitro experiment demonstrated that ch282-5 could signifi-cantly reduce migration and invasion of CT26 cells (Fig. 5C)although moderate reduction in cell viability by 10 mmol/Lch282-5 treatment (data not shown) may partially contribute tothe less invasive capabilities of the cells. Tumormetastasis may beassociatedwith STAT, AKT, ERK, andmTORpathway (12, 29).Wefound STAT3, STAT5b, STAT6, and their phosphorylation formswere reduced by ch282-5 (Fig. 5D), and phosphorylated AKT, Erk,andmTORwere all decreased (Fig. 5E). Ch282-5 also reduced theexpression of metastasis-related proteins MMP-2, MMP-9, AEP,Cathepsin-B, and Cathepsin-L (Fig. 5F and I). We validated thatAEP also increasedmetastatic potential of colon cancer by scratchassay. AEP-overexpressing CT26 cells migrated faster than AEP-WT CT26 cells, and ch282-5 slowed down the cell migration inboth cell types (Fig. 5G and H).

Ch282-5 potentiates effectiveness of oxaliplatin and rescuesABT-263 efficacy

A more rational approach to utilize Bcl-2 inhibitors in clinicalapplications is the combination with other targeted agents. In thisregard, the anticancer property of ch282-5 in combination withclinical and preclinical anticancer drugs against colon cancer wasexamined in vitro and in vivo. Oxaliplatin, widely used for coloncancer chemotherapy, and another BH3 mimic compound ABT-263 were selected for our drug combination study. As shownin Fig. 6A and D, the combination of ch282-5 and oxaliplatin orABT-263 had a better growth inhibitory effect than used alone. InCT26 xenograft models, the combination of ch282-5 at 120mmol/kg/2 days with oxaliplatin at 3 mg/kg/2 days or ABT-263at 50mg/kg/2 dayswas considerablymore effective than ch282-5,oxaliplatin, or ABT-263 alone (P < 0.05) without enhancedtoxicity (Fig. 6B, C and E). ABT-263 is a potent Bcl-2 familyinhibitor as it can bind to Bcl-1, Bcl-xL, Bcl-w, but not Mcl-1(30), while ch282-5 is sensitive to Mcl-1. We assumed that

Wang et al.





combination of these two may be beneficial in terms of targetingdifferent Bcl-2 family antiapoptotic proteins and possible reduc-tion of ABT-263 side effects. ABT-263 had entered phase II clinicaltrial but its use is limited by the adverse effect of platelet reduction(31). We found that ch282-5 alone increased the platelet countand could rescuemore than 50%of platelet drop induced by ABT-263 (Fig. 6F). These observations indicate that ch282-5 not onlyaugments ABT-263–curative effect, but also possibly reduces a riskof thrombocytopenia.

DiscussionThe American Cancer Society provides an estimate that

45,890 men and 47,200 women will be diagnosed with colo-rectal cancer and 26,100 men and 23,600 women will die ofthis disease in 2015 (9). Although some styles of colorectalcancer can be effectively treated with combination chemother-apy, many cancer patients die of metastasis and multidrugresistance. In recent years, a series of excellent Bcl-2 inhibitorshave been developed to solve these problems. A natural Bcl-2inhibitor gossypol has drawn a great deal of attention fromcancer researchers in the field, but is limited in clinical applica-tions for its low hydrophilicity and negative side effects (4, 32).We previously reported a series of gossypol derivatives, one ofwhich is referred as "compound-7" that demonstrated a rela-tively positive effect on colon cancer treatment (5). Com-pound-7 competitively binds to Bcl-xL inducing apoptosis incells and inhibiting tumor growth in the CT26 xenograft model.However, stability and hydrophobicity still limit its value inclinical application.

Ch282-5 was conceived on the basis of the compound-7synthesis process by changing the active aldehyde group ofgossypol into keto group in an attempt to reduce side effects. Asexpected, ch282-5 caused less severe liver injury than gossypolbut almost no damage to the small intestine or colon and didnot affect the digestion and absorption. Ch282-5 significantlyimproved the immune system in tumor bearing mice comparedwith gossypol. Furthermore, the additional step of replacing theR group of gossypol with sodium taurate to enhance hydro-philicity was also taken. Ch282-5 may adapt to kill the cancercells that overexpress Mcl-1 due to its high affinity to Mcl-1.Possibly, ch282-5 is complementary to the small-molecule Bcl-2 inhibitors that have a low affinity binding to Mcl-1, such thatthe combination of ch282-5 and ABT-263 obtained a favorableresult. In addition, ch282-5 has appropriate hydrophobicityconstant and stability for medicinal applications.

Multiple cancer cell lines were sensitive to ch282-5, such asbreast cancer, colon cancer, gastric cancer, glioma, lung cancer,melanoma, pancreatic cancer, prostate cancer, etc. (Supplemen-tary Table S2), which indicates that ch282-5 has a broad-spectrumantitumor property. Just like any other Bcl-2 inhibitors, ch282-5inhibits tumor cells in both time- and dose-dependent mannerand induces typical mitochondrion-dependent apoptotic celldeath. As proposed in Supplementary Fig. S10B for ch282-5induced mitochondria cascade effects, ch282-5 competitivelybinds to the BH3 domain of Mcl-1 and releases proapoptoticproteins Bim and Noxa with decreased Bcl-2 prosurvival proteinsin HCT116 cells (Fig. 3E and I). Ch282-5 phosphorylates SAPK/JNK then promotes Bak/Bax dissociation from the 14-3-3 proteinand oligomerizes into the mitochondrial membrane, resulting inrelease of mitochondrial contents though membrane channels

(16). Cytoplasmic cytochrome c combinedwith caspase-9 formedactivated apoptosome that activated caspase-3, 6, and 7. ReleasedAIF from mitochondria directly enters the nucleus and partici-pates in DNA damage event. IAPs regulate ubiquitin-dependentsignaling events that participate in activation of NF-kB andMAPKpathways that control expression of critical genes related toinflammation, immunity, cell migration, and cell survival (33).AIF and Smac/Diablo can relieve the inhibition of IAP familyproteins to caspase-3, 7, and 9. Furthermore, dissociated IAPfamily proteins were degraded by autoubiquitination (18). Wehave excluded the possibility that a decrease in antiapoptotic Bcl-2family protein level results from change in RNA level. In addition,both lysosomal-dependent autophagy degradation and lysosom-al-independent ubiquitin degradation can reduce proteins level.Ubiquitin degradation can be inhibited byMG132, a proteasomeinhibitor (17). We confirmed that dissociated Bcl-2, Bcl-xL, andMcl-1 underwent ubiquitination degradation by MG132. Disso-ciated Bim is alternatively spliced into threemajor informs BimEL,BimL, and BimS. BimS is the most cytotoxic and is transientlyexpressed during apoptosis, BimEL and BimL may be sequesteredto the dynamic motor complex and released from this complexduring apoptosis (34). Apoptosis is closely related to aG0–G1 cell-cycle arrest (35). Under ch282-5 stress, the cell cycles of HCT116and HT29 were blocked at the Sub-G1 phase. Correspondingly,changes in key protein markers of the G1 phase transition to S-phase, decreased Cyclin D1, and accumulated CHOP wereobserved (36, 37).

Autophagy and apoptosis often occur in the same cell,mostly in a sequence in which autophagy precedes apoptosis(38). Nonetheless, if the cell commences apoptosis, autophagycan be inactivated, in part owing to the caspase-8–mediatedcleavage of Beclin-1 which is essential to autophagy (8). Inac-tivating mutation of caspase-8 has been identified in variouskinds of colorectal cancers, which prevents the recruitment ofthe wild-type form of caspase-8 to active death receptors,thereby inhibiting apoptosis (39, 40). SEM, fluorescencemicroscopy, coimmunoprecipitation, and Western blot analy-sis confirmed ch282-5–induced autophagy. Hence, we hadbetter prevent autophagy at occurrence stage by inhibitors.Ch282-5 competitively binds to the BH3 domain of Bcl-2,Bcl-xL, and Mcl-1 and then releases Beclin-1. Combination ofreleased Beclin-1 with PI3K, p150, and hVPS34 was involved inthe formation of autophagosome membranes (41). CQ inhi-bits autophagy by disrupting the fusion of autophagosome andlysosome (42). Both wortmannin and 3-MA inhibit autophagyby inactivating PI3K (43). As expected, CQ, wortmannin, and3-MA enhanced ch282-5–induced apoptotic cell death to someextent in HCT116 and HT29 cells. In combination treatment,CQ also plays a role in inhibiting autophagy and enhancesch282-5–induced mitochondria-dependent apoptosis, result-ing in increased DNA fragmentation and the release of mito-chondrial contents. Numerous preclinical studies havefound that inhibition of autophagy by CQ restores chemosen-sitivity and promotes tumor cell death by diverse anticancertherapies (42).

Although there is a dispute between autophagy inhibition andinduction as a cancer therapeutic strategy, preclinical and clinicaltrials of these modalities were underway (44). Autophagy inhi-bition is a common combined strategy in Bcl-2–targeting therapy(45), while autophagy induction is also occasionally attempted inthis targeting therapy (46). This may seem contradictory, because






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Wang et al.





we often fix the protein in one kind of signal pathway, thinkingthat regulationof this proteinwouldonly affect "its pathway" thusignoring functions of the protein beyond "its pathway." Autop-hagy inhibitor CQ is currently under clinical stage I/II/III inves-

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Figure 5.Ch282-5 suppresses liver colonization of colon cancer cells. Experimental liver metastasis models of CT26 were treated with ch282-5 until the first mouse dead, thesurvival curve (A). B, group 1 and group 2 were treated with 120 mmol/kg ch282-5 at first and eighth day after the establishment of the experimental livermetastasismodel and then follow the same treatment as figure descript. C, Twenty-four hour treatmentwith 0 and 10 mmol/L of ch282-5 showed inhibitedmigrationand invasion of CT26 cells. HCT116 cells and CT26 cells were treated with 0, 10, and 20 mmol/L ch282-5 for 24 hours, and then STAT3, P-STAT3, STAT5b,STAT6 and P-STAT6 (D), AKT, P-AKT, Erk1/2, P-Erk1/2, mTOR, P-mTOR (E), MMP-2, MMP-9, Pro-AEP, Active-AEP (F), Cathepsin-B, Cathepsin-L were detected byWestern blot analysis (I). G, scratch assay of CT26-MSCV cells and CT26-MSCV-AEP cells treated with 0 and 10 mmol/L ch282-5 for 24 hours. H, themigration inhibition was transformed to the percentage of the initial distance between the two edges.

Figure 4.Ch282-5 antitumor effect is augmented by disturbing mitophagy and mTOR pathway. A, HCT116 cells (a–c) and SW620 cells (d–f) were treated with 20 mmol/Lch282-5 for 6 hours. Representative electron micrographs showed: primary lysosome (a, white arrow), and secondary lysosome (a, black arrow),the damaged mitochondria are wrapped in the autophagosome (b, d, and e), secondary lysosome with a monolayer membrane structure is fused with theautophagosome to become autolysosome (c, black arrow), digested autolysosome (c, white arrow, and f). B, 10 mmol/L ch282-5–treated CT26 cells (d–f) werestained with mito-tracker, lyso-tracker and Hoechst33342, and then detected by fluorescence microscopy, a, b, and c as control. C, cells were treated withch282-5 for 6 hours, and then the punctate pattern of GFP-LC3b was detected by fluorescence microscopy. D, record and analysis of GFP-LC3b punctate byGraphPad Prism 5 software based on three random fields. E, cells were treated with ch282-5 for 24 hours, then autophagy-related proteins Beclin-1,Lamp-2, p62, and LC3 I/II were detected byWestern blot analysis. HCT116 cells (F) and HT29 cells (G) were treated with ch282-5, CQ, or combination of ch282-5 andCQ for 72 hours, and then assessed the cell ability by MTS assay. H, CT26 cells were treated with ch282-5, CQ, rapamycin, or every kinds of combination for48 hours, then assessed the cell ability by MTS assay. CQ, ch282-5, or their combinations were used in HCT116 (I) and HT29 (J) xenograft models, recorded tumorvolumes each 3 days. K, CT26 xenograft models were treated with ch282-5, CQ, rapamycin or their combinations, and recorded tumor volumes each 3 days.CT26 cells were treated with 10 mmol/L ch282-5, 5 mmol/L CQ, 200 nmol/L rapamycin or their combinations for 24 hours, and then detected AKT, P-AKT, Erk,P-Erk1/2, mTOR, P-mTOR (L), p70s6k, P-p70s6k, 4E-BP1, P-4E-BP1, p65, P-p65 (M), c-IAP1, c-IAP2, XIAP, and Survivin by Western blot analysis (N).






synthesis (21). At this conjecture, CQand rapamycin seem tohavea common goal of inducing apoptotic cell death (SupplementaryFig. S10A).

Ch282-5 not only effectively inhibited the growth of coloncancer cells xenografted in a nudemice but also suppressed coloncancer cellmigration, invasion, and experimental livermetastasis.We have previously reported that overexpression of AEP is sig-nificant for breast cancer invasion/metastasis (48), and AEP hadbeen a new functional target for tumoricidal prodrug develop-ment and therapy. AEP localized in the front of invasive tumorcells, forming a complex with integrins, and the combined AEPcan activate MMP-2 and Cathepsin-L for metastasis (49).

Combination of ch282-5 and oxaliplatin or ABT-263 seems tobe a feasible strategy against colon cancer. Ch282-5 and oxali-platin do not share the same target, so they might synergize eachother at a lower dose. Although ABT-737 and ABT-263 reducedpalates in vivo, they are still potent Bcl-2 family inhibitors usefulfor several cancer treatments. While ABT-199 alleviated the inhi-bition of platelet to a large extent (31), ABT-737 and ABT-263combination studies are always popular and meaningful. Rapa-mycin rescued ABT-737 induced platelet decrease by unknownmechanisms (46), but rapamycin and ch282-5have similar effectson mTOR activation and IAP family protein regulation, whichmight explain ch282-5–induced platelet reversion.

In summary, the novel gossypol derivate ch282-5 overcomesthe undesirable side effects and hydrophobicity of natural gos-sypol, and improves the stability and bioavailability over com-pound-7. Ch282-5 antitumor effectiveness can be amplifiedthrough combination with CQ and rapamycin, thus making it

an excellent candidate as an anticancer therapeutics. Furthermore,anti-metastasis is a crucial feature of ch282-5. Clarification of themolecular mechanisms responsible for the potent antitumoractivity of ch282-5 in colon and other cancer models meritsfurther study. Because ch282-5 is a relatively safe and inexpensiveagent, further clinical studies are warranted to confirm the exper-imental observations obtained from this study.

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

Authors' ContributionsConception and design: X. Wang, C. Zhang, B. Lan, J. Wang, C. Wei, B. Jiang,X. Lin, F. GuoDevelopment of methodology: X. Wang, B. Lan, X. CaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Yan, R. Wang, T. Zhou, M. ZhouAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Wang, B. Lan, J. Wang, R. Wang, J. Yao, Q. Liu,P. Jiang, S. Kesari, X. Lin, F. GuoWriting, review, and/or revision of the manuscript: X. Wang, X. Yan, J. Wang,C. Wei, R. Wang, P. Jiang, S. Kesari, X. LinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Zhang, X. Yan, Q. Liu, S. Kesari, F. GuoStudy supervision: C. Zhang, B. Jiang

AcknowledgmentsThis work was supported by Shanghai Commission for Science and Technol-

ogy (11DZ1910200), The National Basic Research Program (2011CB510106),The National High Technology Research and Development Program of China(2013AA032201), and The National Natural Science Foundation of China

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Figure 6.Ch282-5 could potentiate effectiveness of oxaliplatin and rescue ABT-263 efficacy. A, SW620 cells and CT26 cells were treated with ch282-5, oxaliplatin,and their combination for 72 hours, and then the cell ability was assessed by MTS assay. CT26 xenograft mice were treated with ch282-5, oxaliplatin, or theircombination, and tumor volume (B) and body weight (C) were analyzed. D, HCT116 cells and CT26 cells were treated with ch282-5, ABT-263, and theircombination for 72 hours, and then the cell ability was assessed by MTS assay. CT26 xenograft mice were treated with ch282-5, ABT-263, or their combination, andtumor volume (E) and routine blood were tested (F).


Wang et al.




(31171308, 81172208, and 81472610) as well as a grant from InterdisciplinaryScience & Youth Innovation Program of Shanghai Advanced Research Institute(2015Y52643232).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 26, 2015; revised September 11, 2015; accepted September15, 2015; published OnlineFirst October 29, 2015.

References1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.2. Wei J, Kitada S, Rega MF, Emdadi A, Yuan H, Cellitti J, et al. Apogossypol

derivatives as antagonists of antiapoptotic Bcl-2 family proteins. MolCancer Ther 2009;8:904–13.

3. Kitada S, Leone M, Sareth S, Zhai D, Reed JC, Pellecchia M. Discovery,characterization, and structure-activity relationships studies of proapop-totic polyphenols targeting B-cell lymphocyte/leukemia-2 proteins. J MedChem 2003;46:4259–64.

4. Kitada S, Kress CL, Krajewska M, Jia L, Pellecchia M, Reed JC. Bcl-2antagonist apogossypol (NSC736630) displays single-agent activity inBcl-2-transgenic mice and has superior efficacy with less toxicity comparedwith gossypol (NSC19048). Blood 2008;111:3211–9.

5. Yan F, Cao XX, Jiang HX, Zhao XL, Wang JY, Lin YH, et al. A novel water-soluble gossypol derivative increases chemotherapeutic sensitivity andpromotes growth inhibition in colon cancer. J Med Chem 2010;53:5502–10.

6. Oprea TI. Property distribution of drug-related chemical databases.J Comput Aided Mol Des 2000;14:251–64.

7. Lemasters JJ. Selective mitochondrial autophagy, or mitophagy, as atargeted defense against oxidative stress, mitochondrial dysfunction, andaging. Rejuvenation Res 2005;8:3–5.

8. Marino G, Niso-Santano M, Baehrecke EH, Kroemer G. Self-consumption:the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol 2014;15:81–94.

9. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin2015;65:5–29.

10. O'Connell JB, Maggard MA, Ko CY. Colon cancer survival rates with thenew American Joint Committee on Cancer sixth edition staging. J NatlCancer Inst 2004;96:1420–5.

11. Chiarini F, Evangelisti C, McCubrey JA, Martelli AM. Current treatmentstrategies for inhibiting mTOR in cancer. Trends Pharmacol Sci2015;36:124–35.

12. Fang Z, Tang Y, Fang J, Zhou Z, Xing Z, Guo Z, et al. Simvastatin inhibitsrenal cancer cell growth and metastasis via AKT/mTOR, ERK and JAK2/STAT3 pathway. PLoS ONE 2013;8:e62823.

13. Samuel T, Fadlalla K, Gales DN, Putcha BD,ManneU. Variable NF-kappaBpathway responses in colon cancer cells treated with chemotherapeuticdrugs. BMC Cancer 2014;14:599.

14. Zinn RL, Gardner EE, Dobromilskaya I, Murphy S,Marchionni L, HannCL,et al. Combination treatment with ABT-737 and chloroquine in preclinicalmodels of small cell lung cancer. Mol Cancer 2013;12:16.

15. Giampietri C, Starace D, Petrungaro S, Filippini A, Ziparo E. Necroptosis:molecular signalling and translational implications. Int J Cell Biol2014;2014:490275.

16. Tsuruta F, Sunayama J,Mori Y, Hattori S, Shimizu S, Tsujimoto Y, et al. JNKpromotes Bax translocation to mitochondria through phosphorylation of14-3-3 proteins. EMBO J 2004;23:1889–99.

17. Willis SN, Chen L, Dewson G,Wei A, Naik E, Fletcher JI, et al. ProapoptoticBak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced byBH3-only proteins. Genes Dev 2005;19:1294–305.

18. Srinivasula SM, Ashwell JD. IAPs: what's in a name? Mol Cell 2008;30:123–35.

19. Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensorthat maintains energy homeostasis. Nat Rev Mol Cell Biol 2012;13:251–62.

20. Zhao P,MengQ, Liu LZ, You YP, Liu N, Jiang BH. Regulation of survivin byPI3K/Akt/p70S6K1 pathway. Biochem Biophys Res Commun 2010;395:219–24.

21. Kim YS, Jin HO, Seo SK, Woo SH, Choe TB, An S, et al. Sorafenib inducesapoptotic cell death in human non-small cell lung cancer cells by down-regulating mammalian target of rapamycin (mTOR)-dependent survivinexpression. Biochem Pharmacol 2011;82:216–26.

22. Peterson RT, Beal PA, Comb MJ, Schreiber SL. FKBP12-rapamycin-associ-ated protein (FRAP) autophosphorylates at serine 2481 under translation-ally repressive conditions. J Biol Chem 2000;275:7416–23.

23. Huang S, Houghton PJ. Targeting mTOR signaling for cancer therapy. CurrOpin Pharmacol 2003;3:371–7.

24. Winter JN, Jefferson LS, Kimball SR. ERK and Akt signaling pathwaysfunction through parallel mechanisms to promote mTORC1 signaling.Am J Physiol Cell Physiol 2011;300:C1172–80.

25. Li Y, Corradetti MN, Inoki K, Guan KL. TSC2: filling the GAP in the mTORsignaling pathway. Trends Biochem Sci 2004;29:32–8.

26. Kanki T, Klionsky DJ. The molecular mechanism of mitochondria autop-hagy in yeast. Mol Microbiol 2010;75:795–800.

27. Dolman NJ, Chambers KM, Mandavilli B, Batchelor RH, Janes MS. Toolsand techniques to measure mitophagy using fluorescence microscopy.Autophagy 2013;9:1653–62.

28. Fidler IJ. Rationale and methods for the use of nude mice to study thebiology and therapy of human cancer metastasis. Cancer Metastasis Rev1986;5:29–49.

29. Haricharan S, Li Y. STAT signaling in mammary gland differentiation, cellsurvival and tumorigenesis. Mol Cell Endocrinol 2014;382:560–9.

30. Tse C, Shoemaker AR, Adickes J, AndersonMG,Chen J, Jin S, et al. ABT-263:a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res2008;68:3421–8.

31. Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J, et al.ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumoractivity while sparing platelets. Nat Med 2013;19:202–8.

32. Jia L, Coward LC, Kerstner-WoodCD,Cork RL,GormanGS,Noker PE, et al.Comparison of pharmacokinetic andmetabolic profiling among gossypol,apogossypol and apogossypol hexaacetate. Cancer Chemother Pharmacol2008;61:63–73.

33. Silke J, Meier P. Inhibitor of apoptosis (IAP) proteins-modulators of celldeath and inflammation. Cold Spring Harb Perspect Biol 2013;5:1–19.

34. Puthalakath H, Huang DC, O'Reilly LA, King SM, Strasser A. The proa-poptotic activity of the Bcl-2 familymember Bim is regulated by interactionwith the dynein motor complex. Mol Cell 1999;3:287–96.

35. Terui Y, Furukawa Y, Kikuchi J, Saito M. Apoptosis during HL-60 celldifferentiation is closely related to a G0/G1 cell cycle arrest. J Cell Physiol1995;164:74–84.

36. Lukas J, Bartkova J, Bartek J. Convergence of mitogenic signallingcascades from diverse classes of receptors at the cyclin D-cyclin-depen-dent kinase-pRb-controlled G1 checkpoint. Mol Cell Biol 1996;16:6917–25.

37. Zinszner H, Kuroda M, Wang X, Batchvarova N, Lightfoot RT, Remotti H,et al. CHOP is implicated in programmed cell death in response toimpaired function of the endoplasmic reticulum. Genes Dev 1998;12:982–95.

38. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing:crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol2007;8:741–52.

39. Kim HS, Lee JW, Soung YH, Park WS, Kim SY, Lee JH, et al. Inactivatingmutations of caspase-8 gene in colorectal carcinomas. Gastroenterology2003;125:708–15.

40. Fulda S. Tumor resistance to apoptosis. Int J Cancer 2009;124:511–5.41. Yan Y, Flinn RJ, Wu H, Schnur RS, Backer JM. hVps15, but not Ca2þ/CaM,

is required for the activity and regulation of hVps34 in mammalian cells.Biochem J 2009;417:747–55.

42. Lu Y, Dong S, Hao B, Li C, Zhu K, Guo W, et al. Vacuolin-1 potently andreversibly inhibits autophagosome-lysosome fusion by activating RAB5A.Autophagy 2014;10:1895–905.

43. Hou H, Zhang Y, Huang Y, Yi Q, Lv L, Zhang T, et al. Inhibitors ofphosphatidylinositol 30-kinases promote mitotic cell death in HeLa cells.PLoS ONE 2012;7:e35665.






44. Chen N, Karantza V. Autophagy as a therapeutic target in cancer. CancerBiology Ther 2011;11:157–68.

45. Huang S, Sinicrope FA. Celecoxib-induced apoptosis is enhanced by ABT-737 and by inhibition of autophagy in human colorectal cancer cells.Autophagy 2010;6:256–69.

46. Gardner EE, ConnisN, Poirier JT, Cope L, Dobromilskaya I, Gallia GL, et al.Rapamycin rescues ABT-737 efficacy in small cell lung cancer. Cancer Res2014;74:2846–56.

47. Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in cancer:therapeutic implications. Mol Cancer Ther 2011;10:1533–41.

48. Lin Y, Qiu Y, Xu C, Liu Q, Peng B, Kaufmann GF, et al. Functional role ofasparaginyl endopeptidase ubiquitination by TRAF6 in tumor invasionand metastasis. J Natl Cancer Inst 2014;106:dju012.

49. Liu C, Sun C, Huang H, Janda K, Edgington T. Overexpression of legumainin tumors is significant for invasion/metastasis and a candidate enzymatictarget for prodrug therapy. Cancer Res 2003;63:2957–64.


Wang et al.




2016;22:1445-1458. Published OnlineFirst October 29, 2015.Clin Cancer Res Xuefeng Wang, Chen Zhang, Xiangming Yan, et al. via Cascade Effects of MitochondriaA Novel Bioavailable BH3 Mimetic Efficiently Inhibits Colon Cancer

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