oligomannurarate sulfate sensitizes cancer cells to doxorubicin by inhibiting atypical activation of...

Oligomannurarate sulfate sensitizes cancer cells to doxorubicin byinhibiting atypical activation of NF-jB via targeting of Mre11

Jing Zhang, Xianliang Xin*, Qin Chen*, Zuoquan Xie, Min Gui, Yi Chen, Liping Lin, Jianming Feng, Qiuning Li, Jian Ding

and Meiyu Geng

Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, Peoples Republic of China

Aberrant regulation of nuclear factor kappa B (NF-jB) transcription factor is involved in cancer development, progression

and resistance to chemotherapy. JG3, a marine-derived oligomannurarate sulfate, was reported as a heparanase and NF-jB

inhibitor to significantly block tumor growth and metastasis in various animal models. However, the detailed functional

mechanism remains unclear. Here, we report that JG3 inhibits NF-jB activation by specifically antagonizing the doxorubicin-

triggered Ataxia-telangiectasia-mutated kinase (ATM) and the sequential MEK/ERK/p90Rsk/IKK signaling pathway but does not

interfere with TNF-a-mediated NF-jB activation. This selective inactivation of the specific NF-jB cascade is attributed to the

binding capacity of JG3 for Mre11, a major sensor of DNA double-strand breaks (DSB). Based on this selective mechanism,

JG3 showed synergistic effect with doxorubicin in a panel of tumor cells and did not affect immune system function as shown

in the in vivo delayed-type hypersensitivity (DTH) and hemolysis assays. All these highlight the clinical potential of JG3 as a

favorable sensitizer in cancer therapy. In addition, identification of Mre11 as a potential target in the development of NF-jB

inhibitors provides a platform for the further development of effective anticancer agents.

Nuclear factor kappa B (NF-jB) is a ubiquitously expressedfamily of transcription factors, which participates in a widespectrum of cellular functions, such as the cell cycle,apoptosis, migration as well as immune and inflammatoryresponses.1–3 It is clear that aberrant activation of NF-jBand the signaling pathways regulating NF-jB activity areinvolved in cancer development and progression as well asresistance to chemotherapy.4–6

NF-jB activation is subject to complex regulation. Underbasal conditions, NF-jB is sequestrated in the cytoplasm bythe inhibitor of jB (IjB). Restricted NF-jB is released via

three distinct signaling pathways, specifically, the canonical,alternative and atypical modes, which are induced by stimulisuch as tumor necrosis factor a (TNF-a), CD40 ligand andgenotoxic agents, respectively.7–9 The canonical and alterna-tive ways play an important role in innate and adaptiveimmunity.10 Different from the classical or alternative pathwayinitiated by cytokines, the ‘‘atypical’’ pathway is triggered byDNA damage in the nucleus, which is a common feature ofgenotoxic agents, such as doxorubicin, VP16 and CPT.11–14

Several chemotherapeutic agents induce NF-jB activation,which, in turn, is used by tumor cells to achieve resistance to

Key words: oligosaccharide sulfate, NF-jB inhibitor, doxorubicin, Mre11, DNA double-strand breaks

Abbreviations: ATM: ataxia-telangiectasia-mutated kinase; CPT: camptothecin; DOX: doxorubicin; DSB: double-strand breaks; DTH: delayed-type

hypersensitivity; EMSA: electrophoretic mobility shift assay; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IjB: inhibitor of kappaB; IKK:

IkappaB kinase; i.p.: intraperitoneal; NF-jB: nuclear factor kappa B; NEMO: NF-jB essential modulator; RIP: receptor-interacting protein; s.c.:

subcutaneous; SRBC: sheep red blood cells; siRNA: small interfering RNA; TNF-a: tumor necrosis factor a; VP16: etoposide

Additional Supporting Information may be found in the online version of this article

Grant sponsor: Natural Science Foundation of China for Distinguished Young Scholars; Grant number: 30725046; Grant sponsor: National

Basic Research Program Grant of China; Grant number: 2003CB716400; Grant sponsor: Natural Science Foundation of China for

Innovation Research Group; Grant number: 30721005; Grant sponsor: Knowledge Innovation Program of Chinese Academy of Sciences;

Grant number: KSCX2-YWR-25; Grant sponsor: Key New Drug Creation and Manufacturing Program; Grant number: 2009ZX09103-073;

Grant sponsor: 863 Hi-Tech Program of China; Grant number: 2006AA020602

*X.X. and Q.C. contributed equally to this work

DOI: 10.1002/ijc.26021

History: Received 12 Sep 2010; Accepted 2 Feb 2011; Online 8 Mar 2011

Correspondence to: Meiyu Geng or Jian Ding, Shanghai Institute of Materia Medica, 555 Zuchongzhi Road, Shanghai 201203, Peoples

Republic of China, Tel.: 86-21-50806072, Fax: 86-21-50806072, E-mail: [email protected] or [email protected]

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Int. J. Cancer: 130, 467–477 (2012) VC 2011 UICC

International Journal of Cancer

IJC

anticancer drugs. So, inhibiting NF-jB activation appears tobe a promising option to improve the efficacy of conven-tional anticancer therapies.15–17 Accordingly, considerableefforts have been made to develop novel NF-jB inhibitors.To date, over 780 inhibitors targeting the NF-jB pathwayhave been identified, but few are clinically available.18 Themajor reason is that most inhibitors are general inhibitorstargeting classical pathway, which leads to inevitable side-effects.18–20 The theory of selective inhibition NF-jB signal-ing pathway has attracted more and more attention, but stilllack of successful examples.4,21 The highlighted requirementfor selectively targeting NF-jB signaling components is thedriving force for developing novel NF-jB inhibitors that aredistinct from conventional compounds.

JG3, a novel marine-derived semisynthetic oligosaccharide,was first reported to significantly inhibit angiogenesis andtumor metastasis in animal models by targeting heparanase.22

Previous experiments by our group further demonstrated thatJG3 significantly inhibited in vivo tumor growth throughblocking NF-jB activation, but the functional mechanismwas not clear.23 In our study, we try to figure out the under-lying mechanism of NF-jB inhibition and extend the poten-tial clinical usage of JG3.

Material and MethodsMaterials

JG3 was obtained by semisynthesis after sulfate modificationby reacting oligomannurarate with CLSO3H in formamide.The pH of products was adjusted to 7.0 with 4M NaOH anddesalted using Sephadex G-10. The product peak was pooledand freeze-dried. The molecular weights of JG3 were ana-lyzed with high performance gel permeation chromatography(HPGPC) using a G3000PWxl column (300 mm � 7.8 mm)(TOSOH, Japan). The structure of JG3 is depicted in Supple-mental Figure 1a.

JG3 mAb was generated by hybridoma fusions of BALB/Cmouse spleen cells and NS-1 myeloma cells. This mAb dis-plays high affinity for JG3 with a KD value of 2.33 � 10�9

M, as determined using SPR. Analysis of the rate of cross-reactivity in an ELISA assay showed that the mAb did notreact with the carrier proteins, BSA or OVA.

Electrophoretic mobility shift assay

BEL-7402 cells were pretreated with 50 lg/ml JG3 for 24 hror left untreated and exposed to 1 lM doxorubicin for 12 hror 10 ng/ml TNF-a for 30 min. Nuclear extracts were pre-pared as described previously.24 The supernatant was quanti-fied using the BCA protein assay kit. Electrophoretic mobilityshift assay (EMSA) was performed using the LightShiftVR

Chemiluminescent EMSA kit (Pierce, Rockford, IL) with NF-jB biotin-labeled oligonucleotide.

NF-jB luciferase reporter assays

The NF-jB luciferase reporter assay was performed asdescribed earlier.25 Briefly, cells were cotransfected with 3 �jBL and renilla luciferase expressing plasmid (internal con-trol to normalize transfection efficiency) with Lipofectamine2000, according to the manufacturer’s instructions (Invitro-gen, CA). After drug treatment for different time periods,firefly and renilla luciferase activities were assessed using adual luciferase reporter gene assay kit (Beyotime, China). Thefollowing equation was applied: NF-jB transcriptional activ-ity ¼ (relative light units of firefly luciferase/relative lightunits of renilla luciferase) � 100.

Western blot analysis

Whole-cell protein lysates and cytoplasmic or nuclear extractswere electroblotted onto nitrocellulose membranes andprobed with p-IKK (S176/180), p-IjB (S32), IjB, p-p65(S536), p-ATM (S1981), p-MEK1/2 (S217/221), p-P44/42MAPK (T202/Y204), p-P90Rsk (S380), p-P38MAPK(T180/Y182) antibodies (1:1000 dilution, Cell signaling, Bev-erly, MA) as well as p65(RelA) and GAPDH antibodies(1:1000, Santa Cruz, CA).

Affinity chromatography

Whole cell lysates of BEL-7402 were collected and subjectedto affinity chromatography using beads bound to JG3. Col-umns were washed with 0.15 M NaCl solution to wash awayunbound protein. A graded concentration of elution bufferwas used to elute proteins bound to JG3.

Molecular modeling

DOCK 4.0 was used for conformational screening based onthe X-ray crystal structure of the Mre11 protein reported inthe Brookhaven protein database. Residues within 5 Å of theactive center, Domains I and II of Mre11 were extracted sep-arately as the binding pocket for docking. During dockingsimulation, different conformational isomers of trimannura-rate were used to present JG3.

Surface plasmon resonance assay

The JG3 binding sequence on Mre11 was determined with theBIAcore X surface plasmon resonance apparatus. Briefly, basedon the results of molecular modeling, we synthesized two wild-type binding and two mutant sequences (1, E227RWDFGDYEVRYE239WDGIKFK246ER248YG250; 2, ERWDFGDYEVRYAWDGIKFAEA YG; 3, I274DVK277IKGS281 and 4, IDVAIKGS).JG3 was immobilized on CM5 sensor chips, and unreacted sur-face moieties were blocked with ethanolamine. Changes inmass due to the binding response were recorded as resonanceunits. All binding experiments were performed at 25�C with aconstant flow rate of 10 AL/min HBS-EP.

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468 JG3, a novel selective NF-kB inhibitor


siRNA interference

The Mre11 siRNA sequence used was 50-GCC UCG AGUUAU UAA GAA ATT-30 (225-245). Cells were transfectedwith siRNA using Oligofectamine 2000 reagent, according tothe manufacturer’s instructions (Invitrogen, CA).

Terminal transferase dUTP nick end labeling (TUNEL) assay

BEL-7402 cells (5 � 105 cells/ml were pretreated with 50 lg/mlJG3 for 12 hr or left untreated, followed by exposure to 1 lMdoxorubicin for 48 hr. After treatment, the TUNEL assay wasperformed according to the manufacturer’s instructions (Roche,Basel, Switzerland) and images were obtained using the Olym-pus BX51 UV fluorescence microscope.

Flow cytometric analysis

Cells were treated in a similar manner as above, fixed in 70%ice-cold ethanol, double labeled with annexin V FITC andpropidium iodide and subjected to flow cytometry (FACSCa-libur, Becton Dickinson, USA). FITCþ/PI-, representing earlystage apoptotic cells, was analyzed.

Delayed-type hypersensitivity (DTH) assay

Six- to eight-week old BALB/C mice were randomly dividedinto negative control (normal saline), positive control (cyclo-phosphamide) and treatment (JG3) groups, with 10 mice ineach group. On the first day, the mice in positive control groupwere treated with 200 mg/kg cyclophosphamide once via i.p.and mice in JG3 group started treatment with 20 mg/kg/dayJG3 via s.c. The mice in negative control group were treatedwith normal saline via s.c. On the third day, all the micereceived an i.p. injection of 5% sheep red blood cells (SRBC).The mice in JG3 group continued treating with 20 mg/kg JG3for 1 week. One week later, animals were challenged with 5%SRBC (0.05 ml) inoculated into the right footpad. A compara-ble volume of normal saline was injected into the left footpadas the control. The DTH reaction was recorded after 24 hr andfootpad swelling was measured through comparing the thick-ness of right and left footpads with a digital caliper. Aftermeasurement, all the mice were euthanized. The protocol wasapproved by the IACUC of the institute.

Hemolysis assay

Animal grouping and drug administration were similar tothose described for the DTH assay. On the third day, micereceived an i.p. injection of 5% SRBC(0.2 ml/mouse). Oneweek later, serum samples were collected and react withSRBC. The hemolysis amount will be measured through platereader with 413 nm as test wavelength. The OD valuereflected the hemolysin level. After blood collection, all themice were euthanized. The protocol was approved by theIACUC of the institute.

Statistical analysis

Data are presented as means 6 SE, and differences consid-ered significant at p < 0.05 were determined using the Stu-dent’s t test.

Details of cell lines information and a number of assaysare described in Supplementary Methods.

ResultsJG3 selectively inhibits doxorubicin-, but not TNF-a-,triggered NF-jB activation

Our previous study demonstrated that JG3 can inhibit NF-jBactivation both in vitro and in vivo, but the underlying mech-anism remains unclear. Given JG3 represses p65, a key mem-ber of NF-jB, nuclear translocation without affecting itsexpression, it is thus likely that JG3 blocks p65 translocationthrough direct interaction. Using Co-IP assay, we found thatthe monoclonal antibody raised against JG3 could not pulldown the p65 protein in the cell lysate of BEL-7402 cells thatwere exposed to 50 lg/ml of JG3 for 24 hr, suggesting nodirect interaction between p65 and JG3(Supplemental Fig. 1b).

Because JG3 does not bind to P65, we assumed that JG3inhibits NF-jB activation by targeting upstream molecule(s).There are three distinct pathways, including classical, alterna-tive and atypical pathways, which can stimulate NF-jB activa-tion. Of them, the p65 activation mainly responds to the clas-sical and atypical pathways. We, thus, selected TNF-a anddoxorubicin,9,13,21,26,27 two typical stimuli to trigger the classi-cal and atypical pathways, respectively. At first, we examinedthe influence of JG3 on classical pathway stimulated by TNF-a. We found that 10 ng/ml of TNF-a rapidly activated IKKand increased phosphorylation of p65 in 5 min and thenfollowed by the IjB degradation in 10 min, indicative of theNF-jB activation. However, pretreatment with JG3 at 50 lg/mlfor 24 hr did not reverse the activation of TNF-a-triggeredupstream molecules (Fig. 1a), indicating that that JG3 does notaffect classical pathway. For further confirmation, we examinedthe effect of JG3 on the interaction of NF-jB with DNA andtranscriptional activity triggered by TNF-a. Pretreatment withJG3 at 50 lg/ml for 24 hr failed to interfere with TNF-a-trig-gered DNA-NF-jB binding event (Fig. 1c). Similar results wereobtained in reporter assay, as evidenced by the findings thatJG3 took little influence on the TNF-a-driven NF-jB transcrip-tional activity (Fig. 1d).

Having demonstrated that JG3 selectively inhibited NF-jBactivation independent of the TNF-a-stimulated classicalpathway, we focused on the effect of JG3 on atypical pathwaydriven by doxorubicin. Different from the classical onestimulated by TNF-a, we found that 1 lM of doxorubicinslowly led to the phosphorylation of IKK and p65 in 4 hr,followed by IjB degradation in 8 hr. Notably, pretreatmentwith 50 lg/ml of JG3 for 24 hr caused a marked reduction inphosphorylation of IKK and p65 and blocked IjB degrada-tion accordingly (Fig. 1b). To fully substantiate this issue, wealso measured the NF-jB-DNA binding and transcriptionalactivity under the same condition. Similarly, using EMSA,

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Figure 1. Effect of JG3 on NF-jB activation induced by TNF-a or doxorubicin. BEL-7402 cells were pretreated with JG3 for 24 hr and then

stimulated with TNF-a (a) or doxorubicin (b) for the indicated times. The whole-cell lysates were tested by Western blot with indicated

antibodies. c, EMSA assay for NF-jB DNA binding. The nuclear extracts of intact BEL-7402 cells acted as negative control (lane 1). The

nuclear extracts of BEL-7402 cells stimulated by TNF-a 10 ng/ml for 30 min were loaded in lane 2. 100� unlabeled NF-jB consensus

oligonucleotide were added in binding action for competition action (lane 3). P65 antibody was added in binding action to specifically

super-shift p65 protein (lane 4). Samples pretreated with JG3 50 lg/ml for 24 hr before adding TNF-a 10 ng/ml were loaded in lane 5;

stimulated by doxorubicin 1 lM for 12 hr were loaded in lane 6; pretreated with JG3 50 lg/ml for 24 hr before adding doxorubicin were

loaded in lane 7. Densitometric analysis was used to quantify the binding band, and the number is labeled below the band. d, The effect

of JG3 on NF-jB transcriptional activity triggered by TNF-a or doxorubicin was assessed by reporter assay. The treatment is similar with

EMSA assay. Comparisons between two groups were made by Student’s t-test, n ¼ 9, ##p < 0.01 compared to negative control group;

*p < 0.05 compared to doxorubicin stimulation group.

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based on the densitometric analysis, we found that JG3(50 lg/ml, 24 hr) attenuated approximately 35% DNA-NF-jB binding induced by doxorubicin, as compared to that indoxorubicin-stimulated group (Fig. 1c). Similarly, in reporterassay, JG3 significantly antagonized doxorubicin-triggeredNF-jB transcriptional activity about one third as comparedto that in doxorubicin-stimulated group (Fig. 1d).

Together these, we demonstrated that JG3 selectivelyinhibited doxorubicin-driven atypical NF-jB activation butnot the concanical TNF-a-mediated NF-jB activation.

JG3 arrests doxorubicin-triggered ATM activation and

sequential MEK/ERK/P90Rsk signaling pathway

Doxorubicin-driven DNA double strand breaks can be rap-idly responded by ATM,28 which in one cascade initiates theactivation of a MEK/ERK/IKK signaling pathway26 and inthe other cascade recruits NEMO together with RIP into nu-cleus, in a more complicated manner, to active NF-jB.29 Wefirst focused on the effects of JG3 on the ATM/MEK/ERKsignaling pathway. Using western blotting, we found that1 lM of doxorubicin triggered phosphorylation of ATM andactivation of the MEK/ERK/P90Rsk signaling pathway. Intri-guingly, pretreatment with 50 lg/ml of JG3 for 24 hr effec-tively inhibited doxorubicin-induced ATM phosphorylationas well as the MEK1/2/ERK1/2/P90Rsk signaling pathway(Fig. 2a). To further confirm the selective inhibition of JG3,we also examined the effect of JG3 on the same pathwaystimulated by TNF-a. Consistent with our previous findings,JG3 had no effect on the activation of ERK1/2/P90Rskinduced by TNF-a (Fig. 2b).

We then examined the effect of JG3 on NEMO/RIP cas-cade. Stimulation with 1 lM of doxorubicin did not lead to amarked nuclear translocation of NEMO/RIP. Similarly, JG3pretreatment also did not affect their nuclear translocation(Fig. 2c). Our results collectively indicated that JG3 failed toinhibit NEMO/RIP axis but selectively suppressed doxorubi-cin-driven NF-jB activation by specifically interfering withATM/MEK/ERK/IKK pathway.

JG3 suppresses doxorubicin-induced NF-jB activation via

interaction with Mre11

To determine the molecular basis of how JG3 inhibits doxor-ubicin-induced NF-jB activation, we focused on ATM first,because JG3 did not cause obvious cell growth inhibition(Fig. 4a) nor DNA damage, but effectively inhibited ATMphosphorylation. For this, we assumed that JG3 might physi-cally interact with ATM. Unexpectedly, coimmunoprecipita-tion assay revealed that no directly interaction between JG3and ATM (data not shown).

To find out the exact target of JG3, JG3-based affinity chro-matography was used. Whole-cell lysates of BEL-7402 werecollected and subjected to affinity chromatography using beadsbound to JG3. The potential target proteins that bind to JG3were eluted and identified by mass spectrum. Through screen-ing, we found that JG3 can bind to Mre11. Mre11 is a memberof MRN complex that plays an important role in both recruit-ment of ATM to the sites of DNA damage and efficient activa-tion of ATM.30 Using JG3-based affinity chromatographycombined with western blotting analysis, we found that JG3exhibited high binding affinity for Mre11 (Fig. 3a, left) but

Figure 2. Effect of JG3 on the signaling pathway activation induced by doxorubicin or TNF-a. a and b, Effect of JG3 on the ATM/MEK1/2/

Erk1/2 /P90Rsk signaling pathway stimulated by doxorubicin or TNF-a. c, Effect of JG3 on NEMO/RIP pathway stimulated by doxorubicin.

BEL-7402 cells were pretreated with JG3 for 24 hr and then stimulated with doxorubicin or TNF-a for the indicated times. Western blot was

performed with indicated antibodies. GAPDH and Rad50 act as quality and loading control for cytoplasm and nuclear samples, respectively.

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Figure 3. Interactions between JG3 and Mre11. a, JG3 binding to Mre11. All samples eluted with different concentrations of NaCl buffer in

affinity chromatography were examined with Mre11, Rad50 and NBS1 antibodies, respectively, as shown in the left panel. In the right

panel, the co-immunoprecipitation assay was applied to examine binding between Mre11 and JG3 in cells. BEL-7402 cells were incubated

with 50 lg/ml JG3 for 24 hr, and whole-cell lysates were subjected to precipitation with a monoclonal antibody against JG3 (4) or Nbs1

antibody as a positive control (1). Intact BEL-7402 was exposed to JG3 antibody as a negative control (3). Protein samples pulled down

with these antibodies and whole cell lysates (2) were examined with the Mre11 antibody in Western blot analysis. b, The binding pattern

between JG3 with Mre11. Computer molecular simulation was applied with a JG3 trimer. The colored stick structure reflects the

oligosaccharide and the gray block or blue line and band around it indicate the conformation and secondary structure of Mre11. c, Binding

between JG3 and small peptides of Mre11. The binding curves of JG3 with I274DVK277IKGS281 (left), E227RWDFGDYEVRYE239WDGIKFK246E

R248YG250 (middle) and E227RWDFGDYEVRYA239 WDGIKFA246EA248YG250 (right) were determined using surface plasmon resonance. d, Role

of Mre11 in NF-jB activation. BEL-7402 cells were co-transfected with pNF-luc and Renilla as well as Mre11 siRNA or negative control,

mock sequence, followed by treatment with JG3 and doxorubicin. NF-jB transcriptional activity in normal untreated cells was designated

‘1’, and values of other groups derived by comparison with the controls. The upper right panel depicts the siRNA blocking effect on Mre11

expression. Comparisons between the two groups were made with the student’s t-test, n ¼ 9, ##p < 0.01 compared to the negative group,

**p < 0.01, compared to the DOX only treatment group. [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

Figure 4. JG3 sensitizes tumor cells to doxorubicin. a, JG3 potentiates the anti-proliferation effect of doxorubicin. In left panel, the cells were

only treated with 50 lg/ml JG3 at 37�C for 84 hr. The inhibitory rate is presented as means 6 SE of three independent experiments. In the

right panel, the cells seeded in 96-well plates were pretreated with 50 lg/ml JG3 for 12 hr or left untreated, followed by treatment with

gradient concentrations of doxorubicin at 37�C for an additional 72 hr. Cell viability was determined using the MTT or Sulforhodamine B

assay. IC50 values were determined as means 6 SE of three independent experiments. The broken line indicates the average IC50 of

doxorubicin single group and the line represents average IC50 of the doxorubicin and JG3 combination group. The student’s t-test was

applied to compare significant differences between single treatment and combination groups, *p < 0.05, **p < 0.01. B, JG3 enhances

apoptosis induced by doxorubicin. BEL-7402 cells were pretreated with 50 lg/ml JG3 for 12 h, followed 1 lM doxorubicin at 37�C for 48 hr.

Representative images of TUNEL analysis from three independent experiments yielding similar results are shown in the left panel. The scale

represents 100 lm. In the right panel, flow cytometric analysis was also performed to determine the cell apoptosis level. The student’s t-test

was applied to compare differences between the two groups. c, NF-jB activation status of cells in which JG3 exerts different sensitizing

effects were examined with the immunofluorescence assay. Cells were pretreated with 50 lg/ml JG3 for 12 hr or left untreated, followed by

incubation with 1 lM doxorubicin for 12 hr. Representative images from three independent experiments yielding similar results are shown.

The scale represents 30 lm. d, Intracellular drug concentration. Untreated BEL-7402 cells were used as blank control (red). Cells were

treated solely with 5 lM doxorubicin for 2 hr (presented in green). Combination group cells were pretreated with 50 lg/ml JG3 for 12 hr and

followed by 5 lM doxorubicin for 2 hr (pink). Flow cytometric analysis was performed to determine the intracellular doxorubicin level. [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

failed to bind to the other two members of the MRN complex,Rad50 and NBS. To examine whether this setting can be repli-cated within cells, we performed a coimmunoprecipitationassay. The whole-cell lysates of BEL-7402 cells that incubatedwith 50 lg/ml JG3 for 24 hr were subjected to precipitationusing monoclonal antibody raised against JG3. Consistent withJG3-based affinity chromatography data, Mre11 protein waspulled down by the JG3 antibody, further supporting that JG3binds to MRE11. Consistently, JG3 failed to pull down Rad50or NBS1 protein (supplementary Fig. 3), suggesting a specificinteraction of JG3 with Mre11.

To further clarify the binding mode between Mre11 andJG3, computer molecular simulations were applied. A JG3trimer was selected as the docking probe. Among more than20 distinct docking runs using conformations of interest, thetrimer docked strongly to amino acids on the b-sheet struc-ture of domain II of Mre11, including R237, E239, K246,R248, K277 and R303 with hydrogen bond as a driving force(Fig. 3b). Based on the simulation prediction, two separatepeptides, EG-24(07) (E227RWDFGDYEVRYE239WDGIKF-K246ER248YG250) and IS-8 (I274DVK277IKGS281), were thensynthesized. In parallel, two mutated peptides, EG-24(08)(E227RWDFGDYEVRYA239WDGIKFA246EA248YG250) andIS-8-2 (I274DVA277 IKGS281), were synthesized. The SPRassay revealed that both IS-8 and EG-24(07) bound stronglyto JG3, yielding KD values of 2.99E-05 and 1.65E-06, respec-tively. By contrast, mutation at K277 (IS-8-2) led to a nearlycomplete loss of JG3 binding, whereas mutations at E239,K246 and R248 in EG-24(08) resulted in a 100-fold decreasein binding affinity (Fig. 3c). All these substantiate that E239,K246, R248 and K277 are critical for JG3-Mre11 binding.

To examine whether interactions between JG3 and Mre11are functionally involved in doxorubicin-stimulated NF-jBactivation, Mre11 expression was knocked down via siRNAmanipulation. The status of NF-jB activation was measuredusing a reporter gene assay. On doxorubicin stimulation, NF-jB, in particular, was activated. Treatment with Mre11siRNA potently inhibited doxorubicin-induced NF-jB activa-tion compared to the mock control, indicating a criticalinvolvement of Mre11 in doxorubicin-stimulated NF-jB acti-vation. Similarly, JG3 also abrogated doxorubicin-triggeredNF-jB activation as compared to the mock control, but failedto inhibit NF-jB activation on knock down of Mre11 (Fig.3d). All these strongly suggested that Mre11 is involved inJG3-mediated NF-jB inactivation.

JG3 selectively sensitizes tumor cells to doxorubicin-driven

events

NF-jB inhibitors were reported as favorable agents to sensi-tize chemotherapy.31,32 The above results demonstrated thatJG3 selectively inhibit doxorubicin-induced NF-jB activation.So, we extended to evaluate the sensitizing effects of JG3 oncell proliferation inhibition induced by doxorubicin. JG3 at50 lg/ml didnot cause obvious cell growth arrest in a panelof human tumor cell lines, with an average cell growth inhib-

itory rate of 9.31% (Fig. 4a left). However, the pretreatmentof JG3 at 50 lg/ml for 12 hr led to a marked increase in dox-orubicin-triggered growth inhibition, with an average IC50

value of 0.202 lM, about 44% of the doxorubicin-alonegroup (with an average IC50 of 0.455 lM) (Fig. 4a right).

We then extended to examine the effects of JG3 on doxor-ubicin-induced apoptosis using BEL-7402 cells as a represen-tative cell model. Data from the TUNEL assay showed thatpretreatment with JG3 at 50 lg/ml enhanced doxorubicin-induced apoptosis, as compared to doxorubicin-treated alone(Fig. 4b left). Similar results were obtained in the FACSassay. As shown in Figure 4b right, a single treatment withJG3 did not induce apoptosis, whereas the application of 1lM of doxorubicin for 12 hr resulted in 9.7% apoptosis.Strikingly, the combination treatment induced a significantapoptosis, with the apoptotic rate increasing to 17.6% (p <

0.05), nearly 2-fold increase than doxorubicin alone.Next, we evaluated the correlation between NF-jB activa-

tion status and JG3-triggered sensitizing effects. We selectedthree kinds of cell lines: drug-sensitive liver carcinoma BEL-7402 cells, a less sensitive ovarian carcinoma SK-OV-3 cellline and an insensitive oral epidermoid carcinoma KB-3-1cell line, as representative cell lines. We used p65 as a probeto reflect NF-jB activation status. Under basal conditions,p65 was localized mainly in the nucleus, suggesting the con-stitutive activation of NF-jB in BEL-7402 cells. However, inboth SK-OV-3 and KB-3-1 cell lines, the majority of p65 waslocalized in the cytoplasm, indicating the inactive status ofNF-jB. After exposure to 1 lM of doxorubicin for 12 hr,p65 was significantly activated and translocated to the nu-cleus in all three cell lines examined. However, the concomi-tant presence of JG3 induced a dramatic blockage on doxoru-bicin-induced NF-jB activation in BEL-7402 cells, a partialsuppression in SK-OV-3 cells and limited inhibition in KB-3-1 cells (Fig. 4c). These data suggested that the sensitization todoxorubicin was closely correlated with NF-jB-inactivatingpotency by JG3.

To exclude out that JG3 enhances doxorubicin activity viaincreasing the intracellular concentration of doxorubicin, theintracellular concentrations of doxorubicin were examined inthe presence or absence of JG3. Because doxorubicin exhibitsself-fluorescence, the intensity of intracellular fluorescencewas adopted to reflect its intracellular concentration throughthe FACS assay. We found that the fluorescence intensity inthe doxorubicin group was significantly higher than that thatof the blank group. However, the fluorescence intensity inthe JG3 treatment group was identical to that of the doxoru-bicin group, indicating that JG3 does not influence intracellu-lar concentration of doxorubicin (Fig. 4d).

JG3 does not interfere with immune function

Classical NF-jB activation plays an important role in innateand adaptive immune responses.10 Given JG3 can selectivelyinhibit doxorubicin-induced NF-jB activation bypassing theclassical one mediated by TNF-a, we thus proposed that JG3

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might not interfere with immune function. To test this hy-pothesis, we examined the effects of JG3 on immune system,using standard cell-mediated immunity model, delayed-typehypersensitivity (DTH) assay and humoral immunity model,hemolysis assay. From Figure 5a, we found that the increasein footpad thickness reach to 0.34 mm in negative controlgroup, whereas reference drug cyclophosphamide significantlyinhibited the paw swelling, which means the DTH model wassuccessfully established. But the paw thickness in JG3-treatedgroup (20 mg/kg/day) is almost the same with that of thenegative control, indicating JG3 did not interfere with T cellfunction. Similarly, in hemolysis assay, dosing with 20 mg/kg/day JG3 for 9 days induced no decrease in hemolysin con-centration compared to negative control group, indicatingthat JG3 does not result in B-cell function suppression inBALB/c mice (Fig. 5b). All these data supported that theselective inhibition of JG3 on NF-jB activation does notinterfere with immune function.

DiscussionNF-jB transcript factor has been well demonstrated to playan important role in innate and adaptive immune responses,tumor development, progression and resistance to therapeu-tics.1,2,33 Currently, expanding literatures have evidenced thatNF-jB inhibitors can act as an adjuvant of conventionalradiotherapy and chemotherapy, to enhance the treatment ofmany different malignancies in preclinical studies.17,18,20,34–37

Although this strategy is very attractive, there are few clin-ical outcomes. One major obstacle is the mechanism-basedimmunosuppression.16,20,38 In view of the diverse structuresof compounds targeting NF-jB, the majority of NF-jB inhib-itors mainly target IKKb that responses to classical and atypi-cal pathways.10,16,18 As both classical and alternative NF-jB

activation pathways play important roles in innate and adapt-ive immune responses,10 it should be considered the risk ofsevere immunodeficiency after general and prolonged NF-jBinhibition.4,21 Based on this condition, many scholars sug-gested that an effective NF-jB inhibitor for cancer therapyshould prevent NF-jB activation but have limited effects onother signaling pathways.16,21 So, it was supposed to mini-mize systemic toxicity and avoid broad suppression of innateimmunity by selectively targeting specific NF-jB signalingcomponents. Because NF-jB activation pathway is very com-plex and lack of effective target, the development of selectiveinhibitor is quite slow.

In our study, we demonstrated that JG3 functions as aselective NF-jB inhibitor. JG3 specifically arrests atypicalNF-jB activation induced by doxorubicin through inhibitingATM activation and sequent MEK/ERK/IKK pathway, whichbypasses NEMO/RIP pathway. This selective mechanismfavors the attractive synergistic effect of JG3 with doxorubicinin a panel of tumor cells. The additional finding that JG3also sensitized tumor cells to VP16 and other chemothera-peutics, substantiating that JG3 is a choice of combinationwith those chemotherapeutics that induce NF-jB activation(supplementary Fig. 2).

Attractively, JG3 had little influence on TNF-a-drivenNF-jB transcriptional activity. This is evidenced by the factthat JG3 failed to reverse TNF-a-triggered either upstreamactivation or downstream DNA-NF-jB binding or eventualNF-jB transcription. Through both delayed-type hypersensi-tivity (DTH) and hemolysis assays, we further demonstratedthat JG3 does not interfere with immune function in theanimal models. All these data supported that JG3 not only sen-sitized the tumor cells to doxorubicin and other chemothera-peutics but also make low influence on the immune system.

Figure 5. Effects of JG3 on immune system function. a, JG3 does not interfere with cellular immunity in the delayed type hypersensitivity

reaction. On the first day, the mice in positive control group were treated with 200 mg/kg cyclophosphamide once via i.p. and mice in JG3

group started treatment with 20 mg/kg/day JG3 via s.c. The mice in negative control group were treated with normal saline via s.c. On the

third day, all the mice received an i.p. injection of 5% SRBC. The mice in JG3 group continued treating with 20 mg/kg JG3 for 1 week.

After 1 week, all the mice were re-challenged with SRBC and footpad swelling thickness measured to reflect DTH. b, JG3 does not interfere

with humoral immunity in the hemolysis assay. Animal grouping and dosages were similar to those with the DTH assay. Hemolysis was

reflected by OD values at 413 nm. ** means p < 0.01 compared to the negative control group, The two groups were compared to the

Student’s t-test, n ¼ 10.

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This selective effect of JG3 was further found out bas-ing on targeting of Mre11, a member of the Mre11-Rad50-Nbs1complex. The MRN complex can quickly sense geno-toxic stress, then migrates quickly to the site of DNAdamage, binds broken DNA ends and subsequently recruitsATM, facilitating ATM activation.28,30 Our data revealedthat JG3 bind to Mre11 through hydrogen bonds via fourdominant residues, E239, K246, R248 and K277, which arespatially oriented away from the DNA-binding motifs. Af-ter eliminating Mre11with siRNA, we further demonstratedthat Mre11 is involved in NF-jB activation induced bydoxorubicin. Based on these findings, we speculate thatinteractions between JG3 and Mre11 disrupt MRN com-plex formation, in turn blocking the MRN complexresponse to DNA damage and leading to subsequent inac-tivation of ATM and downstream NF-jB. Because Mre11locates in nucleus, it is no chance to involve in TNF-astimulated classical pathway, which explains the selective

effect of JG3 and also provide a clue to develop selectiveNF-jB inhibitors.

In summary, we show for the first time that JG3, a novelsulfated oligosaccharide, obviously sensitize tumor cells todoxorubicin’s treatment through selectively blocking atypicalNF-jB activation pathway. Our study highlights the impor-tance of JG3 as a novel potential chemotherapy sensitizer incancer therapy and also substantiates the hypothesis of selec-tive inhibition on NF-jB activation. In addition, identifica-tion of Mre11 as a key mediator in atypical NF-jB activationaids in conceptualizing the potential value of Mre11 as apromising target in developing selective NF-jB inhibitors.

AcknowledgementsWe are grateful to Dr. Spiros Linardopoulos (Institute for Cancer Research,Sutton, UK) for providing NF-jB-dependent firefly luciferase reporterplasmid 3�jBL and Renilla luciferase reporter plasmid.

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oligomannurarate sulfate sensitizes cancer cells to doxorubicin by inhibiting atypical activation of...

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