autophagy inhibition synergistically enhances …...preclinical development autophagy inhibition...

Preclinical Development

Autophagy Inhibition Synergistically Enhances AnticancerEfficacy of RAMBA, VN/12-1 in SKBR-3 Cells, and TumorXenografts

Abhijit M. Godbole1,4, Puranik Purushottamachar1, Marlena S. Martin1, Constantine Daskalakis3, andVincent C.O. Njar1,2

AbstractVN/12-1 is a novel retinoic acidmetabolism blocking agent discovered in our laboratory. The purpose of the

studywas to elucidate themolecularmechanismof anticancer activity ofVN/12-1 inbreast cancer cell lines and

in tumor xenografts.We investigated the effects of VN/12-1 on induction of autophagy and apoptosis in SKBR-

3 cells. Furthermore, we also examined the impact of pharmacologic and genomic inhibition of autophagy on

anticancer activity of VN/12-1. Finally, the antitumor activity of VN/12-1 was evaluated as a single agent and

in combinationwith autophagy inhibitor chloroquine in an SKBR-3mouse xenograft model. Short exposure of

low dose (<10 mmol/L) of VN/12-1 induced endoplasmic reticulum stress, autophagy, and inhibited G1–S

phase transition and caused a protective response. However, a higher dose of VN/12-1 initiated apoptosis

in vitro. Inhibition of autophagy using either pharmacologic inhibitors or RNA interference of Beclin-1

enhanced anticancer activity induced by VN/12-1 in SKBR-3 cells by triggering apoptosis. Importantly,

VN/12-1 (5 mg/kg twice weekly) and the combination of VN/12-1 (5 mg/kg twice weekly) þ chloroquine

(50 mg/kg twice weekly) significantly suppressed established SKBR-3 tumor growth by 81.4% (P < 0.001 vs.

control) and 96.2% (P < 0.001 vs. control), respectively. Our novel findings suggest that VN/12-1may be useful

as a single agent or in combination with autophagy inhibitors for treating human breast cancers. Our data

provides a strong rationale for clinical evaluation of VN/12-1 as single agent or in combination with

autophagy inhibitors. Mol Cancer Ther; 11(4); 898–908. �2012 AACR.

IntroductionBreast cancer is the most common neoplasia in women.

Despite significant advances in treatment, breast cancerremains incurable because of emergence of alternativepathways adopted by cancer cells to overcome the effectsof anticancer therapy. To overcome this issue, a rationalapproach would be to concomitantly target clinicallyrelevant cellular abnormalities with combination therapy

or to use a potentmultitargeted agent.Mounting evidenceindicates that the antitumor effects of all-trans retinoicacid (ATRA) are attributed to its ability to interfere withmultiple facets of oncogenic signaling pathways (1, 2).Moreover, ATRA proved to be an effective anticanceragent to treat hematologic cancers (3). However, severalfactors compromise the widespread clinical use of ATRA.They include low in vitro anticancer potency, limitedbioavailability, and unfavorable pharmacokinetic beha-viors due to rapid metabolism by CYP26 enzymes (4, 5).Consequently, the structural modification of ATRA todevelop novel retinoic acid metabolism blocking agents(RAMBA)with improved potency andmetabolic stabilityhas been the focus of our group for many years. OurRAMBAs are considered to be atypical, because in addi-tion to being potent inhibitors of ATRAmetabolism, theyalso possess potent intrinsicmultiple anticancer activities.

4-(�)-(1H-imidazol-1-yl)-(E)-retinoic acid (VN/14-1)and its corresponding methyl ester, VN/12-1 (Fig. 1Aand Supplementary Fig. S1) are among our lead RAMBAs(6). Previously, we had reported that flow cytometricanalysis of breast cancer cells treated with our RAMBAsrevealed significant growth arrest with only weak apo-ptosis (7), a phenomenon also reported for a variety ofretinoids (8). In an effort to search for strategies that couldenhance cancer apoptosis mediated by RAMBAs, we

Authors' Affiliations: 1Department of Pharmaceutical Sciences, JeffersonSchool of Pharmacy, 2Kimmel Cancer Center, 3Department of Pharma-cology & Experimental Therapeutics, Thomas Jefferson University, Phila-delphia, Pennsylvania; and 4Department of Pharmacology & ExperimentalTherapeutics, University of Maryland School of Medicine, Baltimore,Maryland

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

Current address for A.M. Godbole, P. Purushottamachar, M.S. Martin, andV.C.O. Njar: Department of Pharmacology and Experimental Therapeutics,Center for Biomolecular Therapeutics (CBT), University ofMaryland Schoolof Medicine, Baltimore, MD.

Corresponding Author: Vincent C. O. Njar, Department of Pharmacologyand Experimental Therapeutics, Center for Biomolecular Therapeutics(CBT), University of Maryland School of Medicine, Baltimore, MD. Phone:410-706-6364; Fax: 410-706-0032; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0860

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(4) April 2012898

on July 24, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst February 14, 2012; DOI: 10.1158/1535-7163.MCT-11-0860

http://mct.aacrjournals.org/

looked for possible prosurvival pathways that may beactivated in response to RAMBAs. VN/12-1 does notbind to or transactivate retinoic acid receptors (7), andits exact molecular target (other than CYP26 inhibition)is yet to be identified. Herein, we report the induction ofpronounced autophagy by VN/12-1–treated SKBR-3cells and tumors.Autophagy is an intracellular process in which pro-

teins and cytoplasmic organelles are degraded (9). It hasbeen implicated in various physiologic processes such assurvival in stress, response to starvation and pathogen-esis (10, 11), and a mechanism of cell protection againstdrug-induced apoptosis (12–14). It is now generallyaccepted that the accumulation of misfolded proteinsin the lumen of the endoplasmic reticulum (ER) results

in cellular stress that initiates a specialized response,designated ER stress response (15). In most cases, induc-tion of cellular ER stress subsequently leads to activationof autophagy (16, 17).

Whereas the estrogen receptor-a-proficient breasttumors respond to several therapies, estrogen receptor-a–deficient tumors are less sensitive partly because ofactivation of growth factor signaling pathways such ashumanepidermalgrowth factor receptor-2 (Her-2; ref. 18).Therefore, there is an urgent need for development ofeffective therapy against this type of breast cancer. In thisstudy, we primarily focused on an estrogen receptor-a–deficient cell line that overexpresses Her-2 (SKBR-3cells). We note that SKBR-3 xenograft model is one of theless commonly investigated model because of reported

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Figure 1. Effect of VN/12-1 on the growth of human breast cancer cells and ER stress markers in vitro. A, chemical structures of VN/12-1 and chloroquine. B,VN/12-1 inhibits the growth of MCF-7 (square), SKBR-3 (filled circle), MDA-MB-231 (unfilled circle), and MCF-10A cells (triangle). Curves generated from anMTT assay after 96 hours of exposure to VN/12-1. Points, mean of replicates from 3 independent experiments; bars, SE. Solid line, best-fit sigmoidaldose response (variable slope). C, cellswere exposed to10mmol/LVN/12-1 for timepoints shown.After each timepoint, the compoundwaswashedawayandcells were maintained in normal growth medium for 96 hours, and cell viability wasmeasured with anMTT assay.�, P < 0.05; ��,P < 0.01. D(a) and (b), effect ofVN/12-1 on ER stress markers. Cells were treated with indicated concentrations of VN/12-1 for 6 hours [D(a)] or 24 hours [D(b)]; whole-cell lysates weretested for BiP and p-eIF2a. T-Thapsigargin (20 mmol/L) was used as a positive control. Total eIF2a was used as loading control.

Efficacy Modulation of RAMBAs by Autophagy Inhibition

www.aacrjournals.org Mol Cancer Ther; 11(4) April 2012 899




difficulty of SKBR-3 cells to form tumors innudemice (19).However, a few research groups have successfully usedthis xenograft model in severe-combined immunode-ficient (SCID) mice previously (20, 21).

This study evaluated the potency and antitumor activ-ity of VN/12-1, as a single agent and in combination withchloroquine (Fig. 1A; ref. 15), an autophagy inhibitor thatblocks lysosome acidification and autophagosome degra-dation. We show that VN/12-1 has dual effects on thecancer cells based on the dose. Low dose (<10 mmol/L) ofVN/12-1 induces autophagy, ER stress, and cell-cyclearrest as an immediate protective response in SKBR-3cells and xenograft tumors. Importantly, we show thatinhibition of autophagy using either pharmacologic inhi-bitors (e.g., chloroquine) or RNA interference of essentialautophagy gene Beclin-1 potentiates apoptotic cell deathinduced by VN/12-1. Notably, VN/12-1 producedimpressive tumor inhibitory effects when used alone orin combination with chloroquine (P < 0.001).

Materials and MethodsFor cell culture, cell growth inhibition, Western blot,

Annexin fluorescein isothiocyanate (FITC), electron andimmunofluorescence microscopy, short interfering RNA(siRNA), statistical analysis, and in vivo studies, see Sup-plementary Data Materials and Methods.

Chemicals and reagentsATRA and 4-hydroxytamoxifen (Supplementary Fig.

S1) were purchased from LKT Laboratories Inc., whereasVN/12-1, other RAMBAs, letrozole (Supplementary Fig.S1; ref. 22), and 3-methyl adenine (3-MA; ref. 23) weresynthesized in our laboratory. Chloroquine (15) was pur-chased from Sigma Aldrich.

Cell growth inhibition (MTT colorimetric assay)MTT (Sigma Aldrich) assay was carried out as previ-

ously described (24).

Cell-cycle analysisCell-cycle analysis was done using method as previ-

ously described (24).

In vivo tumor growthAll animal studies were done according to the guide-

lines approved by the Institutional Animal Care and UseCommittee of Thomas JeffersonUniversity (Philadelphia,PA). Female SCID mice were obtained from the NationalCancer Institute (Frederick,MD). The compounds used inthis study and their doses are as shown in Fig. 5A. SeeSupplementary section for details.

ResultsVN/12-1 inhibits the growth of breast cancer celllines

To determine whether VN/12-1 exerts a growth inhib-itory effect, MCF10A, MCF-7, SKBR-3, andMDA-MB-231

cells were treated with VN/12-1 and cell viability wasanalyzed usingMTT assay (Fig. 1B; Supplementary TableS1). VN/12-1 did not induce any significant growth inhib-itory effects in immortalized noncancerous MCF10A cellline. However, it was quite potent against the cancer celllines with IC50 in the low mmol/L range. Clinically usedanticancer drugs ATRA, 4-hydroxytamoxifen, and letro-zole also effectively inhibited the growth of MCF-7 cells(Supplementary Table S1). However, their potencies weresignificantly less than that ofVN/12-1 against SKBR-3 andMDA-MB-231 cells.

Short exposure ofVN/12-1 to breast cancer cells doesnot reduce cell viability

Cancer cells have fundamental differences in theirmetabolism and sensitivity to preconditioning, autophagy,and apoptosis, so the dosage and duration of treatmentwith the therapeutic agents is important. Typically, shortincubation time of treatment induces a protective mecha-nism byway of autophagy (25). To test this, MCF-7, SKBR-3, and MDA-MB-231 cells were treated with 10 mmol/LVN/12-1, and the compoundwas washed away at varioustime points (Fig. 1C). The cells were then maintained inregular growthmedia until the endpoint of theMTT assay.AsshowninFig. 1C, continuousexposure toVN/12-1 foratleast 24 to 36 hours was needed to reduce the cell viabilityby approximately 20% for all the cells tested (P < 0.05). Thisindicated that the induction of growth inhibitory effect ofVN/12-1 is time dependent. Collectively, these data alsosuggested that the cells treated with VN/12-1 for a shortertime (<36 hours) manage to counter the growth inhibitoryeffect of VN/12-1, possibly, by a protective mechanism.

VN/12-1 induces endoplasmic reticulum stressManycancer therapies including retinoids are known to

induce ER stress in various cancers (26–28). The trans-duction of ER stress is mediated by the binding of thechaperone BiP (binding immunoglobulin protein) to themisfolded proteins and its concomitant dissociation fromPERK (protein kinase RNA-like ER kinase; ref. 29). Thisdissociation results in its activation and subsequent ini-tiation of a cascade of downstream signals (e.g., attenu-ation of protein translation by inhibition phosphorylationof eIF2) that ultimately aim to preserve cellular homeo-stasis facilitating cell survival. Prolonged ER stress, how-ever, beyond levels at which cellular homeostasis can bemaintained becomes proapoptotic by upregulatingCHOP (CCAAT-enhancer-binding protein homologousprotein) and IRE1-a (17, 30). To test the role of ER stress,Western blot analysis was carried out on SKBR-3 lysatestreated with VN/12-1 for 6 and 24 hours. As shownin Figs. 1D (a) and 1D (b), there was an upregulation ofeIF2a phosphorylation (Ser51) and BiP in response toVN/12-1 within 6 hours, which was further enhanced in24 hours. PERK phosphorylation remained unchanged(data not shown). These data showed that induction of ERstress is a concentration-dependent early event (i.e., after 6hours) following VN/12-1 treatment.

Godbole et al.

Mol Cancer Ther; 11(4) April 2012 Molecular Cancer Therapeutics900




VN/12-1 induces autophagy and inhibits G1–S phasetransitionAutophagy represents a failed attempt to adapt to stress

and survival (31). ER stress can also trigger autophagy (16,17). The observation that VN/12-1 mediated induction ofER stress andmodest anticancer activity following shortertreatment of VN/12-1 led us to hypothesize that SKBR-3cells undergo a protective response, possibly autophagy,at lower mmol/L concentrations of VN/12-1 or upon shortexposure.To better understand themorphologic changes induced

by VN/12-1, transmission electron microscopy was car-ried out on SKBR-3 cells treated with vehicle control (Fig.2A) or VN/12-1 (10 mmol/L) for 24 hours [Fig. 2B (a), (b),and (c)]. There was an abundance of autophagosomes inthe images from the treatment group. VN/12-1 treatmentcaused nucleolar disruption, cytoplasmic vacuolization[Fig. 2B (a)], distortion ofmitochondrial shape, and dilatedER [an evidence of ER stress; Fig. 2B(b)]. Numerous autop-hagosomes in various stages of maturation containing

cellular organelles were also noted [Fig. 2B(c)]. Anotherefficient way to confirm autophagy is by measuring lapi-dated form of LC3 (LC3B), which is formed from the LC3 amicrotubule-associated protein (32). On the fluorescencemicroscopy, the presence of autophagosomes is indicatedby punctate pattern of LC3B staining unlike uniform stain-ing in the absence of autophagy (32). On fluorescencemicroscopy, 20 mmol/L treatment of VN/12-1 for 24 hoursincreased thenumber and size of punctateLC3Baggregates[Fig. 2C(b)] compared with control [Fig. 2C(a)]. Collective-ly, these studies showed that VN/12-1 can induce mor-phologic features of autophagy in SKBR-3 cells.

To confirm the upregulation of biochemical markers,immunoblots were carried out to probe various autop-hagy markers. Chloroquine inhibits the fusion of autop-hagosomes with lysosomes and thus leads to accumula-tion of lapidated formof LCB (LC3B; ref. 33). Following 24hours incubation, the cells treated with VN/12-1 showeda dose-dependent upregulation of LC3B [Fig. 3A(a)].Nevertheless, there is a need to further discriminate

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Figure 2. VN/12-1 induces the formation of autophagosomes. A andB, transmission electronmicroscopy images of VN/12-1–treatedSKBR-3 cells. Cells weretreatedwith ethanol (A) or 10mmol/LVN/12-1 [B (a), (b), (c)] for 24hours. A andB(a), represent comparisonof nuclear andnucleolarmorphology (arrowheads); Aand B(b), represent normal versus VN/12-1–treated mitochondria [solid arrows in A vs. arrowhead in B(b)]; B(a), VN/12-1–treated cell with vacuolatedcytoplasm (solid arrow); B(b), VN/12-1–treated cell with dilated ER showing ER stress (solid arrow); and B(c), early (solid arrow) and late (arrowhead)autophagosomes containing cellular organelles. C, SKBR-3 cells were treated with ethanol (a) or 10 mmol/L VN/12-1 (b) for 24 hours. VN/12-1 induces LC3aggregation [punctate staining–peculiar of autophagy formation as shown in figure 2C(b)].






between 2 physiologically distinct scenarios—increasedautophagic flux without impairment in autophagic turn-over versus impaired clearance of autophagosomes (31).For these studies, we next exposed the cells to VN/12-1 inthe presence of chloroquine (a weak lysosome stabilizingbase). Cotreatment with chloroquine resulted in enhance-ment of LC3B band [Fig. 3A(b)]. Beclin-1 is an importantautophagy-relatedprotein, and itsupregulation correlateswith induction of autophagy (34). Both, dose-dependent[Fig. 3A(a)] and time-dependent (Supplementary Fig.S2A) Beclin-1 upregulation in Western blots further sup-ported our premise of autophagy induction by VN/12-1.

Under various conditions, an inverse relationship isfound between the percentage autophagy and the degreeof phosphorylation of p70S6 kinase and Akt, key proteinsin Akt-mTOR pathway (35–37). In our studies, a dose-dependent downregulation of phosphorylation of p70S6K(Thr389) andAkt (S473) followingVN/12-1 treatmentwasevident, which correlates well with induction of autop-hagy [Fig. 3A(a)]. Following the additionof chloroquine toVN/12-1, therewas an abolishment of reversible ER stressmarker, BiP, and induction of the irreversible ER stress

markers–IRE1-a and CHOP (Fig. 3B). This indicated thatthe addition of chloroquine to VN/12-1 pushes the cellsfrom reversible ER stress to irreversible ER stress,which isa well-known trigger for apoptosis (29). Thus, our datasuggested that VN/12-1–induced autophagy due toenhanced induction rather than decreased clearance.Upregulation of LC3Bandabolishment ofAKTphosphor-ylation and p70S6K phosphorylation were also evident inMCF-7 cells (Supplementary Fig. S2B), suggesting thatVN/12-1–mediated autophagy is not cell line dependent.Basal level of autophagy, as indicated by LC3B formation,was evident in the untreated MCF-7 cells.

Because autophagy represents a phase of dormancy orarrest, we expected VN/12-1 to have negative effect oncell-cycle proteins such as cyclin D1. Although chloro-quine alone did not have any major effect on cyclin D1expression, VN/12-1 (10 mmol/L) alone or in combinationwith chloroquine completely abolished the expression ofcyclin D1 (Fig. 3B). Analysis of cell-cycle distribution byflow cytometry revealed that VN/12-1 treatment resultedin a significantly higher % increase in G0–G1 populationafter 24 hours (Supplementary Fig. S2C).

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Figure 3. VN/12-1 induces autophagy markers and cell-cycle arrest in SKBR-3 cells. A(a) and (b), effect of VN/12-1 on autophagy markers. Cells were treatedwith indicated concentrations of VN/12-1 and chloroquine for 24 hours; whole-cell lysateswere tested for LC3B, Beclin-1, p-p70S6K, and p-Akt. b-Actin, totalAkt, and total p70S6K were used as control. B, cells were treated with indicated concentrations of VN/12-1 and/or chloroquine for 24 hours. Whole-celllysates were tested for ER stress markers and cyclin D1. V-VN/12-1, C-chloroquine. The number following V or C indicates the concentration in mmol/L.C, SKBR-3 cells were treated with indicated concentrations of VN/12-1 and IC20 concentrations of chloroquine [C(a)] and 3-methyladenine [3-MA; C(b)]for 96hours in anMTTassay.Columnsare themeanof viable cells in 3 experiments; bars, SE. ��,P<0.D, cellwere transfectedwith si-Beclin-1or si-Scrambledas described above. After 72 hours, they were treated with indicated concentrations of VN/12-1 and cell viability was assessed by MTT assay asdescribed above. �, P < 0.05; ��, P < 0.01. CHL, chloroquine.

Godbole et al.





Pharmacologic inhibition of autophagy enhances thegrowth inhibitory activity of VN/12-1To assess whether VN/12-1–induced autophagy con-

tributed to survival response of the cells, we measuredVN/12-1–induced reduction in cell viability in the pres-ence or absence of IC20 concentrations of known inhibitorsof autophagy: chloroquine (15) and 3-methyladenine (3-MA; refs. 33, 38). When combined with VN/12-1 both theautophagy inhibitors enhanced the anticancer potency ofVN/12-1 [P< 0.01; Fig. 3C (a) andC (b)].Wealso found thatVN/12-1, combined with either chloroquine (Supplemen-tary Fig. 2D) or 3-MA (Supplementary Fig. S2E), resultedin synergistic effects, as assessed by isobologram analysisusing themedian effect principle of ChouandTalalay (39).These data suggested that VN/12-1 and autophagy inhi-bitors elaborate unique individual effects that are comple-mentary when these compounds are used together.To quantitatively evaluate the effect of the combination

of VN/12-1 with chloroquine, the dose reduction index(DRI; ref. 40) and combination index (CI) were calculatedfrom the data (Supplementary Table S2) using Calcusynsoftware program at 50% to 95% inhibition levels of cellviability. DRI values indicate that the synergic combina-tion can result in 2- to 8-fold reduction of the VN/12-1dose and 3- to 9-fold reduction of chloroquine dose toachieve 50% to 95% cell growth inhibition, respectively,compared with the dose if they were used as single agent.The values of the CI suggest that the combination ofVN/12-1 and chloroquine was synergistic at 50% to95% of growth inhibition. Collectively, these results con-firmed that autophagy is a protective cellular responsethat lowers anticancer potency of VN/12-1, and its inhi-bition by pharmacologic means enhances the growthinhibition by VN/12-1.

Genomic silencing of Beclin-1 enhances the growthinhibitory activity of VN/12-1To determine whether autophagy inhibition by way of

silencing an important autophagy gene, Beclin-1, enhancesthe anticancer activity of VN/12-1, siRNA technologywasused. For Beclin-1, the siRNAsequence that showed90% to100%knockdownof theproteinwas selected for the studies(Supplementary Fig. S2F). As expected, SKBR-3 cells trea-ted with VN/12-1 alone showed a decrease in viability bytheMTTassaywhencomparedwith thevehicle treated (nosiRNA) and scrambled sequence siRNA (scrambledsiRNA) controls (P < 0.05; Fig. 3D). Cells treated withscrambled siRNA plus VN/12-1 showed no change inactivity compared with VN/12-1 alone (P > 0.05). In theabsence ofVN/12-1, siRNAtargetingBeclin-1 exerted littleeffects on SKBR-3 cell viability (P > 0.05), but the combi-nation of VN/12-1 with siRNA for Beclin-1 resulted in adramatic reduction in cell viability (P < 0.0005; Fig. 3D).

VN/12-1 induces caspase-dependent apoptosisLiterature evidence suggests that the compounds that

are autophagy inducers at smaller dose can induce apo-ptosis at higher dose (9). In many situations, autophagy

accompanies rather than causes apoptosis. Immunoblotswere done to determine whether VN/12-1 has apoptoticactivity.Thehallmarksofapoptosis,PARPcleavage,andanupregulation of proapoptotic protein Bad were evidentwhen VN/12-1 was combined with chloroquine (Fig.4A). Twenty micromol/L VN/12-1 as a single agent initi-ated apoptosis in cells as shown by Annexin/PI staining.However, additionof chloroquine toVN/12-1 significantlyenhanced the percentage of apoptotic cells (P < 0.001; Fig.4B).Pancaspase inhibitorz-vad-fmkwasused todeterminewhetherVN/12-1–mediatedapoptosiswas caspasedepen-dent. Pretreatmentwith 30mmol/L z-vad-fmk reversed thegrowth inhibitory effects of VN/12-1 and its combinationwith chloroquine in MTT assay (P < 0.01; Fig. 4C).

Preliminary toxicology and pharmacokinetics of VN/12-1 in SCID mice

Before assessment of the in vivo antitumor efficacy ofVN/12-1, we conducted preliminary toxicity and pharma-cokineticsstudies.Preliminary toxicity studies inSCIDmiceshowed that VN/12-1 was not toxic as a single agent or incombination with chloroquine in the subcutaneous doses2.5 and 5 mg/kg twice a week. In addition, preliminarypharmacokinetics following subcutaneous administrationof 20mg/kg ofVN/12-1 showed that the peakplasma levelafter was 41.38 mg/mL and the mean t1/2 was 6 hours.Details of these studies will be reported in a future article.

VN/12-1 inhibits the growth of SKBR-3 xenograftsWe evaluated the ability of VN/12-1 to inhibit the

growth of SKBR-3 xenografts when given via subcutane-ous administration. Mice were divided into 8 groups asdetailed in the Methods section. As shown in [Fig. 5A(a)and 5A(b)]. Geometric means of starting tumor sizeswerecomparable across the 8 groups and ranged from 208 to266mm3 (P¼ 0.224). SupplementaryTable S3 summarizesthe estimated tumor growth parameters (daily tumorgrowth rate and tumor doubling time), whereas Supple-mentary Table S4 shows the P values for all the pairwisecomparisons among the 8 treatment groups. Daily tumorgrowth was not significantly different between ATRAalone and VN/12-1 2.5 mg/kg alone (3.9% vs. 3.6%, P¼ 0.551) but was significantly slower with VN/12-1 5.0mg/kg (2.2%, P ¼ 0.001 vs. both ATRA and VN/12-1 2.5mg/kg; Supplementary Table S3).

The combination of VN/12-1 (either dose) with chlo-roquine slowed tumor growth compared with treatmentwith VN/12-1 alone (1.9% vs. 3.6% for the low dose, 1.2%vs. 2.2% for the high dose, P ¼ 0.001 for both). Thecombination of VN/12-1 at 5 mg/kg with chloroquinewas better than the combination of VN/12-1 (2.5 mg/kg)with chloroquine (1.2% vs. 1.9%, P ¼ 0.086). The combi-nation of VN/12-1 (at either dose) with chloroquine wasbetter than the combination of ATRA with chloroquine(P ¼ 0.001 for both comparisons).

Representative pictures of tumors from each group areshown in Fig. 5B. Figure 5C(a) and (b) show the averagetumor weights and the body weights of the mice in






different groups, respectively. As shown in Fig. 5C(a),tumors in treatment groups involving the combination ofVN/12-1 (either dose, 2.5 mg/kg and 5 mg/kg twice aweek) and chloroquine had statistically significant reduc-tion of tumor weights (P < 0.05 and P < 0.01, respectively).However, therewasno significant change inbodyweightsof mice in any group.

In summary, the growth of tumors were significantlyinhibited by all treatments [Figs. 5A(a) and 5A(b)], buttreatments with VN/12-1 (5 mg/kg) alone and its com-

bination with chloroquine were the most effective, withimpressive inhibitory values of 81.4% (P < 0.001 vs. con-trol) and 96.2% (P < 0.001 vs. control), respectively, with-out causing any toxicity.

Effect of VN/12-1 treatment on SKBR-3 tumorprotein expressions

Immunoblot analysis of the tumor lysates confirmedthe activation of autophagic pathway, as there was a risein LC3B expression in the VN/12-1 treatment groups

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Figure 4. VN/12-1 (in combinationwith chloroquine) inducescaspase-dependent apoptosis.A, SKBR-3 cells were treated withindicated concentrations of VN/12-1 or chloroquine for 24 hours andprotein expression of cleavedPARP and Bad was determined.V-VN/12-1, C-chloroquine. Thenumber following V or C indicatesthe concentration in mmol/L (B).The percent of late apoptotic/necrotic cells as determined byAnnexin V–FITC and PI-positivestaining by flow cytometry.��, P < 0.01. SKBR-3 cellswere treated with indicatedconcentrations of VN/12-1 orchloroquine for 24 hours. Thepercentage of cells stained withFITC/PI was calculated usingflow cytometry. C, indicatedconcentrations of VN/12-1,chloroquine, and/or 30 mmol/LZ-vad-fmk used in an MTT assay(as described in Materials andMethods) experiment to determinethe cell viability in SKBR-3 cells.Columns are the mean of 3experiments; bars, SE. �, P < 0.05;��, P < 0.01. CHL, chloroquine.

Godbole et al.





but not in the ATRA group. As in the in vitro results,addition of chloroquine enhanced VN/12-1 upregula-tion of LC3B. PARP cleavage and upregulation of proa-poptotic Bad was noted in all the treated groups (exceptchloroquine group), more so in VN/12-1 and chloro-quine combination groups (Fig. 5D). Thus, these dataidentify apoptosis induction as a major mechanismunderlying the ability of chloroquine to potentiate theantitumor effects of VN/12-1. There was a markedupregulation of CHOP in the groups treated withVN/12-1 alone or with chloroquine. This further sup-ports in vitro findings of activation of ER stress by VN/12-1. These data supported our proposed mechanismthat autophagy is the initial response of the cells to VN/12-1 treatment, and inhibition of VN/12-1–inducedautophagy by chloroquine leads the cells to apoptoticpathway. Cyclin D1 was downregulated in all the treat-ment groups except chloroquine (Fig. 5D). Collectively,these in vivo tumor-suppressive responses exhibited byVN/12-1 or VN/12-1 plus chloroquine strongly correlated

with the mechanisms identified in vitro. The overall mech-anism of action of VN/12-1 and its combination withchloroquine in SKBR-3 human breast cancer cells is sum-marized in Fig. 6.

DiscussionBecause of the heterogeneous nature of breast cancer

(41), combination therapies or multitarget agents arerequired for effective therapy. VN/12-1 has dual actionon SKBR-3 cells based on the dose and duration oftreatment. Shorter exposure of smaller dose inducedprotective effect by autophagy. VN/12-1 initiated apo-ptotic activity at doses higher than 20 mmol/L in SKBR-3 cells in vitro. However, such high plasma VN/12-1levels were not achievable in vivo without producingtoxic effects in the mice. For these reasons, alternativeapproach that combines VN/12-1 with a second agentexhibiting a different mechanism of action wasrequired to further improve the potency of VN/12-1

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Figure 5. VN/12-1 (alone or in combination with chloroquine) inhibits the growth of SKBR-3 xenografts. A, the effect of VN/12-1 (2 doses–2.5 and 5 mg/kgtwice a week), ATRA (5 mg/kg twice a week) alone or in combination with chloroquine (50 mg/kg twice a week) were evaluated in a SKBR-3 xenograftmodel in female SCID mice. Mice (n ¼ 8) were injected subcutaneously. Tumors were measured twice a week. Arithmetic tumor means [A(a)] and,geometric means [A(b)] were plotted against time. A(b) has logarithmic scale on y-axis. P values are indicated separately in SupplementaryTable S4. B, representative tumors from the 8 groups. C(a), mean tumor weights taken upon euthanizing all mice and collecting tumors. �, P < 0.05;��, P < 0.01. Data are mean (� SE). C(b), mean body weights. Mice were weighed once a week for the duration of the study. D, Western blotanalysis of protein expression in SKBR-3 tumors taken from mice. Autophagy marker (LC3B), ER stress marker (CHOP), cell-cycle marker (cyclin D1),apoptosis markers (Bad, PARP cleavage) were probed. CHL, chloroquine.






and reduce its dose and yet achieve apoptosis. Impor-tantly, immortalized breast MCF10A cells weremuch less sensitive to VN/12-1. This selectivity offersVN/12-1 a clear advantage over many other com-pounds that are in development as potential new breastcancer therapies.

Perhaps the most striking aspect of our data was theeffective inhibition of established SKBR-3 xenograftgrowth by VN/12-1 alone at doses as low as 2.5 mg/kgbody weight, twice weekly, and when combined withchloroquine, with no apparent toxicity as there was nochange in body weight of the mice. Recently, Samaddarand colleagues showed that the combination of 4-hydro-xytamoxifen with 3-MA resulted in potent anticanceractivity against MCF-7 breast cancer cells (42). Wu andcolleagues also showed similar combination strategy inprostate cancer PC-3 model using src family inhibitors incombination with autophagy inhibitors (43). Indeed, anumber of clinical trials have been initiated in patientswith solid and hematopoietic tumors to test the overallhypothesis that autophagy is a mechanism of therapeuticresistance (13).

Overall, following treatment of SKBR-3 cells with VN/12-1, our findings clearly show that VN/12-1 has dualeffects (autophagy and apoptosis) based on the dose andduration of treatment. We also show that (i) autophagyacts as a protective mechanism in SKBR-3 cells; (ii) inhi-bition of autophagy can be exploited to potentiate VN/12-1–induced cell death, and (iii) the combinationofVN/12-1with autophagy inhibitor(s) can reduce the dose of VN/12-1 and thus reduce its toxicity (if any). Aswith our otherleadRAMBAs, the exactmolecular targets (except CYP26)of VN/12-1 are yet to be identified. We envision that this

will be achieved with ongoing studies directed at designand synthesis of appropriate biotin–RAMBAs conjugates(molecular probes that retain potency in bioassays;ref. 44).

In summary, this study provides the first evidencethat a RAMBA rapidly activates autophagy in breastcancer cells and that induction of autophagy by VN/12-1 can be exploited as a target to achieve enhancedantitumor efficacy. We suggest that VN/12-1 or thecombination therapy of VN/12-1 and chloroquine isa potential strategy for the treatment of patients withbreast cancer.

Disclosure of Potential Conflicts of InterestV.C.O. Njar holds an ownership interest in the RAMBAs patents and

technologies thereof. No potential conflicts of interest were disclosed byother authors.

AcknowledgmentsThe authors thank Dr. Gediya for help with the synthesis of RAMBAs

and the Electron Microscopy Facility at TJU for help in acquisition of EMimages.

Grant SupportThis research study was supported by grants from the US Depart-

ment of Defense (PRMRP, W81XW-04-1-0101, V.C.O. Njar) and the USNIH (NIH/NCI, 1R01 CA129379-01A2 and 5R01 CA 129370-02, V.C.O.Njar).

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 October 31, 2011; revised January 17, 2012; accepted February8, 2012; published OnlineFirst February 14, 2012.

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elF2Translational

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TORcomplex

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Figure 6. Mechanisms of action ofVN/12-1 in combination withchloroquine. VN/12-1 induces ERstress. This results in inhibition ofprotein translation, downregulationof key cell-cycle proteins such ascyclin D1 and arrest in G1–S phasecell-cycle transition. Addition ofchloroquine enhances the ERstress and switches it fromreversible (protective) pathway toirreversible (apoptotic) pathway byCHOP upregulation. VN/12-1inhibits PI3K–Akt–mTOR pathwayand activates autophagy. Inhibitionof VN/12-1–mediated autophagyby chloroquine potentiatesapoptosis by activation of caspase9 and PARP cleavage. CHL,chloroquine.

Godbole et al.





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2012;11:898-908. Published OnlineFirst February 14, 2012.Mol Cancer Ther Abhijit M. Godbole, Puranik Purushottamachar, Marlena S. Martin, et al. of RAMBA, VN/12-1 in SKBR-3 Cells, and Tumor XenograftsAutophagy Inhibition Synergistically Enhances Anticancer Efficacy

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