MOLECULAR CARCINOGENESIS

A Novel DNA Intercalator, 8-MethoxyPyrimido[40,50:4,5]Thieno (2,3-b)Quinoline-4(3H)-One Induces Apoptosis in Cancer Cells, Inhibitsthe Tumor Progression and Enhances Lifespan inMice With Tumor

Sheetal Sharma,1 Kuppusamy Panjamurthy,1 Bibha Choudhary,1,2 Mrinal Srivastava,1

MS Shahabuddin,1 Ranjit Giri,3 Gopal M. Advirao,4 and Sathees C. Raghavan1*1Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka, India2Institute of Bioinformatics and Applied Biotechnology, Biotech Park, Electronics City, Bangalore, Karnataka, India3National Brain Research Center, Manesar, Gurgaon, Haryana, India4Department of Biochemistry, Kuvempu University, Davanagere, Karnataka, India

Polycyclic aromatic molecules such as ellipticine intercalate into double-stranded DNA and interfere with physiologi-

cal functions. In the present study, we evaluate the chemotherapeutic potential of MPTQ on animal models and itsmode of action. In order to test the antitumor activity, monohydrochloride of MPTQ was orally administered in micebearing tumor. Results showed a significant inhibition of tumor growth compared to that of untreated controls. More

importantly, mean lifespan of tumor bearing animals treated with MPTQ was significantly higher as compared to thatof untreated tumor bearing mice suggesting that the treatment affected viability of cancerous cells, but not of normalcells. Consistent with this, we find that administration of MPTQ to normal mice did not cause any major side effects

as observed upon hematological and serum profiling. We also found that MPTQ induces cytotoxicity in cancer celllines, by activating apoptosis both by intrinsic and extrinsic pathways. Thus, MPTQ could be used as a potential cancertherapeutic agent. � 2011 Wiley Periodicals, Inc.

Key words: chemotherapy; DNA intercalator; double-strand breaks; extrinsic pathway of apoptosis; DNA damage;

anticancer drug

INTRODUCTION

DNA, the genetic material of an organism, con-trols cellular functions and has been a target fortreatment of cancer. DNA intercalators are a classof cancer therapeutic agents, which can intercalatebetween specific sequences [1]. Although biophysi-cal studies such as CD, UV-fluorescence, differen-tial scanning calorimetry etc. help in decipheringthe binding of these molecules, cytological techni-ques are important to prove their physiologicalimportance.Polycyclic aromatic molecules such as ellipticine

intercalate into double-stranded DNA and affectmajor physiological functions. Pyrimidothienoqui-nolines, a new class of structural analogues of ellip-ticine, have been described as novel and rationalintercalating ligands with potential antitumor ac-tivity [2]. However, many intercalators currentlyused are highly toxic to normal cells and therefore,design and synthesis of novel molecules that donot affect the normal cellular physiology are ofprime importance.Quinolines are one of the DNA intercalators,

which have been extensively studied for their DNA

binding properties. Sandramycin is a naturally oc-curring quinoline having an anti-tumorigenic ac-tivity [2,3]. DNA binding properties of manyderivatives of quinolines, after addition of sidechains containing various functional groups werestudied [4,5]. A binding affinity of 104–106 M�1 toDNA has been shown for pyrimidothieno/selenoloquinolines with methoxy, morpholino, diethyla-mino, propylamino, oxochloro, anilino, butyla-mino, or piperazino substitutions showing that

Additional Supporting Information may be found in the onlineversion of this article.

Abbreviations: EAC, Ehrlich ascites carcinoma; ROS, reactive oxy-gen species; PI, propidium iodide; DLA, Dalton’s lymphoma; ALP,alkaline phosphatase; IHC, immunohistochemistry.

Sheetal Sharma and Kuppusamy Panjamurthy contributed equal-ly to the work.

Authors declare that there is no competing interest.

*Correspondence to: Department of Biochemistry, Indian Insti-tute of Science, Bangalore 560 012, Karnataka, India.

Received 10 May 2011; Revised 2 December 2011; Accepted 6December 2011

DOI 10.1002/mc.21867

Published online in Wiley Online Library(wileyonlinelibrary.com).

� 2011 WILEY PERIODICALS, INC.

length and position of side chains also play a piv-otal role in determining cytotoxicity [6–11]. Previ-ous studies from our laboratory have shown thecytotoxic properties of 8-methoxy pyrimido[40,50:4,5]thieno(2,3-b)quinoline-4(3H)-one (MPTQ)and 4-morpholinopyrimido[40,50:4,5]thieno(2,3-b)quinoline (morpho-PTQ) in four different cancercell lines [12]. We found that MPTQ is more po-tent than morpho-PTQ in inducing cytotoxicity[12]. However, within the cells these compoundsneed to have good pharmacokinetic and pharma-codynamic properties such as good permeability,transportation, high unbound serum fraction,availability in the active form before binding andinteracting with DNA. In fact, it has been observedthat failure of most of the intercalating drugs isdue to their poor pharmacokinetic abilities [2,13].This can be improved by synthesizing salts of suchcompounds [2,13].

In the present study, we report the efficacy of mono-hydrochloride of 8-methoxy pyrimido[40,50:4,5]-,50:4,5]thieno (2,3-b)quinoline-4(3H)-one, which iswater soluble, as a potential antitumor drug. Usingvarious techniques, we show that MPTQ inducescytotoxicity by activating both extrinsic and in-trinsic pathways of apoptosis. Further, we reportthat MPTQ treatment on mice bearing differenttypes of tumors resulted in substantial reductionin tumor progression and extended the life spansignificantly compared to control animals bearingtumor, suggesting that MPTQ could be used as apotential cancer therapeutic agent without adverseeffects.

MATERIALS AND METHODS

Chemicals and Reagents

All the chemicals and reagents used in the pres-ent study were obtained from Sigma Chemical Co.(St. Louis, MO), Amresco (OH), and SRL (Mumbai,India). Annexin V-FITC and antibodies were pur-chased from Santa Cruz Biotechnology (CA) andCell Signaling Technology (MA).

Synthesis and Characterization of MPTQ

Synthesis and characterization of 8-methoxypyrimido[40,50:4,5]thieno(2,3-b)quinoline-4(3H)-one has been described previously [8,12,14,15]. Forthe present study, a monohydrochloride of MPTQwas synthesized. The identity of the molecule wasconfirmed by mass spectrometry: 283 (M þ , 100);254 (5.32); 253 (5); 241 (7.2); 240 (46); 212 (7); IR([Nujol] max 1670 (CO), 3100 (NH) CM-1); NMR[Delta 4.1 (3H,s, OCH3), 7.5–7.7(1H,d,H-9;J ¼ 10Hz), 7.6 (1H,s,H-7), 8.3–8.5(1H,d,H-10;J ¼ 10 Hz); 8.93(1H,s,H-11); 9.7(1H,s,H-2)]. Thestock solution of MPTQ was prepared by dissolvingit in water.

Animals

Mice were maintained as per the principles andguidelines of the ethical committee for animal careof Indian Institute of Science (IISc) in accordancewith Indian National Law on animal care and use.Swiss albino and BALB/c mice, 8–10 wk old, weigh-ing 18–22 g were purchased from central animalfacility, IISc, India and used for the study. The ani-mals were housed in polypropylene cages and pro-vided standard pellet diet (Agro Corporation PvtLtd, Bangalore, India) and water ad libitum. Thestandard pellet diet is composed of 21% protein,5% lipids, 4% crude fiber, 8% ash, 1% calcium,0.6% phosphorus, 3.4% glucose, 2% vitamin, and55% nitrogen-free extract (carbohydrates). Themice were maintained under controlled conditionsof temperature and humidity with a 12 h light/dark cycle. The experimental design of the presentstudy was approved by Institutional Animal EthicsCommittee (Ref. CAF/Ethics/125/2007/560), IISc,Bangalore, India.

Cell Lines and Culture Conditions

The Philadelphia chromosome positive, chronicmyeloid leukemia cell line, K562 and mouse breastcancer cell line, Ehrlich ascites carcinoma (EAC)were purchased from National Centre for Cell Sci-ence, Pune, India. K562 cell line was cultured inRPMI1640 (Sera Lab, West Sussex, UK) containing10% FBS (GIBCO BRL, NY), 100 U of PenicillinG/mL, and 100 mg of streptomycin/mL at 378C ina humidified atmosphere containing 5% CO2. EACcell line was grown in DMEM containing 10% FBS(GIBCO BRL), 100 U of Penicillin G/mL and100 mg of Streptomycin/mL as specified above.

Trypan Blue Exclusion and MTT Assays

The effect of MPTQ (5, 10, 20, and 50 mM) onviability of K562 cells was determined by trypanblue exclusion assay as described [16,17]. Datafrom three independent experiments with two rep-licates in each case is presented as a graph witherror bars. MTT assay was performed to determinethe effect of MPTQ (5, 10, 20, and 50 mM) on pro-liferation of K562 cells [16,18]. The absorbance ofexperimental samples was divided by absorbanceof untreated control, which is presented as per-centage of inhibition and shown in a bar diagram.In both cases, untreated cells were used as control.Error bars were calculated based on a minimum ofthree independent experiments. IC50 values wereestimated after 72 h treatment.

Detection of Intracellular ROS by Flow Cytometry

The level of total intracellular reactive oxygenspecies (ROS) production was measured by usingcell permeable fluorescent probe, 2,7-dichloro-dohydro fluorescein diacetate (H2DCFDA). The

2 SHARMA ET AL.

Molecular Carcinogenesis

esterified form of H2DCFDA can enter the cellmembrane and is deacteylated by intracellularesterases. The resulting compound, dichlorofluoro-scein reacts with H2O2 to give fluorescent dichloro-fluoroscein [19]. K562 cells were treated withMPTQ (10 mM) for different time points (30 min,1, 2, 4, 6, 18, and 24 h), stained with H2DCFDA(5 mM) and analyzed by flow cytometry. Cellstreated with H2O2 were used as positive control.

Live and Dead Cell Assay

Live and dead cell assay is a sensitive assay tomonitor changes in the ratio of live and dead cellsin the total cell population. MPTQ treated K562cells (1, 2, 5, and 10 mM) were collected after 48 h,washed and resuspended in PBS solution contain-ing RNase A (50 mg/mL), EDTA (100 mM), and ethi-dium bromide (0.05%). A minimum of 10,000 cellswere acquired using flow cytometry wherein loga-rithmic scale was used on the X-axis to separatelive and dead cell population, instead of conven-tional linear scale commonly used for cell cycleanalysis. The data were analyzed using WinMDI2.8 software and presented as histogram.

Annexin V-FITC/Propidium Iodide Cytometric Analysis andConfocal Microscopy

Annexin V-FITC apoptosis detection kit (SantaCruz Biotechnology, CA) was used to detect earlyand late apoptotic cell death as described earlier[17,19]. In brief, after 72 h of MPTQ treatment (1, 5,and 10 mM), the cells were harvested and washed inPBS (48C), resuspended in a buffer containing10 mM HEPES–NaOH (pH 7.4), 1.4 M NaCl, 25 mMCaCl2 and incubated with annexin V-FITC (0.2 mg/ml) and propidium iodide (PI) (0.05 mg/ml). Cellswere then subjected to FACS analysis and the resultsobtained were shown as histogram.Confocal fluorescence microscopy was per-

formed to visualize the apoptotic cells generatedupon treatment with MPTQ based on morphologi-cal changes. Annexin V-FITC/PI stained cells wereviewed under an inverted Zeiss confocal laser-scan-ning microscope (Ziess Meta 510 LSM; Carl Zeiss,Jena, Germany) as described [20].

Western Blot Analysis

Cell extract was prepared from K562 (1, 2, 5, and10 mM) and EAC (0.5, 1, 2, and 5 mM) cells treatedwith MPTQ for 48 h as described earlier [17,21]. Inbrief, cells were harvested, washed with PBS, resus-pended in lysis buffer (RIPA, 25 mM Tris (pH 7.6),150 mM NaCl, 1% NP-40, 1% sodium deoxycho-late and 0.1% SDS; 1 mL of RIPA buffer for5 � 106 cells) containing protease inhibitors andincubated on ice for 1 h. The pellet was sonicatedand supernatant containing proteins was col-lected by centrifugation (14,000 rpm for 15 min).

The protein concentration was determined byBradford’s assay.For western blot analysis, �40 mg protein was

resolved over 8–10% SDS–polyacrylamide gel. Fol-lowing gel electrophoresis, proteins were trans-ferred to PVDF membrane (Millipore, MA).Membranes were blocked with 0.1% Tween-20in PBS containing 5% nonfat dry milk for 2 hat room temperature. Appropriate primary anti-bodies (human reactive anti-PCNA, BAD, BCL2,CASPASE3, CASPASE8, CASPASE9, p73, FAS,KU70, KU80, and TUBULIN from Santa CruzBiotechnology; BCL-XL, PUMA, GAPDH, SMACfrom Cell Signaling Technology, PARP1 and p53from (Calbiochem, Merck Chemicals, Dermstadt,Germany) and CDC25a from EMD Biosciences(Merck, Dermstadt, Germany) were added andincubated overnight at 48C. Following washing(0.1% Tween-20 in PBS), the membrane was incu-bated with appropriate HRP-conjugated secondaryantibody (2 h at 48C). The blot was developedusing chemiluminescent solution (ImmobilonTM

western, Millipore) and scanned by gel documenta-tion system (LAS 3000, FUJI, Tokyo, Japan). Blotswere stripped subsequently as per standard proto-cols and reprobed with anti-GAPDH or TUBULIN.

Preparation of Ehrlich Ascites Carcinoma (EAC) andDalton’s Lymphoma (DLA) Cells Followed by Induction

of Tumor

EAC cells collected from donor mice (Swiss albino)were resuspended in sterile saline. A fixed numberof viable cells were injected into the peritonealcavity of each recipient mouse and were allowedto multiply. The cells were withdrawn, diluted insaline and injected (1 � 106 cells/animal) to rightthigh tissue of experimental animals for develop-ing the solid tumor. Similarly, DLA cells collectedfrom donor mice were diluted in saline andinjected (0.25 � 106 cells/animal) to the peritone-um of experimental mice (BALB/c) for developingthe liquid tumor.

Determination of the Anticancer Effect of MPTQ onTumor Development in Mice

A total number of 30 Swiss albino mice wereused per batch, in which 20 were injected withEAC cells (1 � 106 cells/animal) into right thighfor the development of solid tumor. Ten animalswithout tumor or MPTQ treatment served as no tu-mor control (Group I). EAC injected animals weredivided into 2 groups of 10 each. Group II wasconsidered as tumor control and received no treat-ment. Group III was treated with MPTQ (1 mg/kg,b. wt; determined based on pilot studies) by oraladministration using oral gavage starting after12 d of tumor development (6 doses over a periodof 2 wk). In the case of group II and III, diameter

METHOXY PTQ INHIBITS TUMOR PROGRESSION IN MICE 3

Molecular Carcinogenesis

of developing tumor was measured using verniercalipers at alternative days for entire life span oftumor animal and tumor volume was calculatedusing the formula V ¼ 0.5 � a � b2, where ‘‘a’’and ‘‘b’’ indicates the major and minor diameter,respectively [22]. At the end of 25th and 45th dayof experimental period, one animal from eachgroup was sacrificed and tissues of interest werecollected from untreated control, tumor controland MPTQ treated animals and processed for histo-logical evaluation. Each experiment was repeatedthree independent times.

In order to determine the effect of MPTQ on thesurvival of mice injected with DLA cells, a total of17 BALB/c mice were used per batch, out of which12 were injected with DLA cells (0.25 � 106 cells/animal) into the peritoneum for the developmentof tumor. DLA injected animals were dividedinto 2 groups. Group I comprising of five mice wasconsidered as tumor control and received no treat-ment. Group II (7 animals) was treated with MPTQ(10 mg/kg, body wt; determined based on pilotstudies) by oral administration (15 continuousdoses over a period of 2 wk). In both the groups,progression of tumor was monitored by checkingtheir weight. Weight changes during tumor devel-opment were then plotted as a function of time.

Determination of Effect ofMPTQ on the Survival of TumorBearing Animals

The percentage of increased lifespan was calcu-lated and compared to that of control animals.The death pattern of control and MPTQ treatedanimals due to EAC or DLA induced tumor devel-opment was recorded and fold increase in the life-span was calculated by using the formula [(T � C)/C],where ‘‘T’’ indicates the mean life span of treatedanimals and ‘‘C’’ indicates the mean life span ofcontrol mice [22–24].

Evaluation of Toxicity of MPTQ in Mice

A total number of 15 mice were used per batch,in which 10 were fed with MPTQ (1 mg/kg; 6 dosesover a period of 2 wk) and other 5 served as thecontrol. Side effects were monitored by measuringtheir weight changes during administration ofMPTQ. The mean weight in untreated control wasthen compared to that of treated mice. Mice wereanesthetized on 21st day and blood was collectedas per standard protocol. The blood plasma was an-alyzed using Neubauer’s chamber, and mean val-ues of total red blood cells (RBCs) and white bloodcells (WBCs) are presented. To further check fortoxicity induced by MPTQ treatment, liver andkidney function tests, such as alkaline phosphatase(ALP), alkaline transferase (ALT), creatinine andurea were performed. Values are presented asmean � SEM for both the controls and MPTQ ad-ministered mice.

Histopathological Evaluation

The tissues of interest (tumor and liver) collectedfrom the animals were processed as per standardprotocol and embedded in paraffin. Microtomesectioning was done at 3–5 mm in a rotary micro-tome (Leica Biosystems, Wetzlar, Germany) andstained with hematoxylin and eosin [25,26]. Eachsection was evaluated by light microscopy andimages were captured (Carl Zeiss).

Immunohistochemistry (IHC) Analysis

IHC staining was performed on formalin fixed,paraffin embedded tissues that were sectioned at5 mm thickness. Slides were de-paraffinized, rehy-drated and treated with 3% H2O2 in PBS. Antigenretrieval was done using 0.01 M sodium-citratebuffer followed by blocking in PBST containing 1%BSA and 10% FBS. Primary antibody incubation(Ki67, BID, or 53BP1) was carried out for overnightat 48C. Slides were washed and incubated with ap-propriate biotinylated secondary antibody for 1 hat room temperature. Following washing, slideswere incubated with streptavidin–HRP (1:1000).Slides were washed (PBS containing 0.1% Tween-20) and color was developed by DAB þ H2O2.Slides were counterstained with hematoxylin,washed and mounted in DPX (Sigma-Aldrich,USA). Images were taken using light microscope(Carl Zeiss, Jena, Germany).

Statistical Analysis

Values are expressed as mean � SEM for controland experimental samples and statistical analysiswas performed by one-way ANOVA followed byStudent’s t-test using GraphPad software prism 5.1.The values were considered statistically significant,if the P-value was <0.05.

RESULTS

Cytotoxic Effect of MPTQ on Human Leukemic Cells

In the present study, we investigate the mecha-nism of action of monohydrochloride of MPTQ,which is water soluble (Suppl. Figure 1) on humanchronic myelogenous leukemia cell line, K562. Pre-viously, we showed that MPTQ (soluble in DMSO)can affect cell viability in four different cancer cellsand had a comparable IC50 value (�10 mM) [12].However, mechanism by which MPTQ induces celldeath was not understood. To compare the effectof water soluble MPTQ on cell viability, K562 cellswere treated with 5, 10, 20, and 50 mM of the com-pound and subjected to both trypan blue exclusionand MTT assays. Cells grown without MPTQ servedas control. Results showed that addition of MPTQsignificantly affected cell viability in a dose-depen-dent manner (Figure 1A). These results suggestthat water soluble MPTQ is more potent than

4 SHARMA ET AL.

Molecular Carcinogenesis

DMSO soluble form (IC50 value �5 mM for watersoluble form) (Figure 1A). Results of MTT assay fur-ther supported such a dose dependent effect ofMPTQ on cell proliferation (Figure 1B). Theseresults further confirm MPTQ induced cytotoxicityin K562 cells.

MPTQ Induces Intracellular Reactive Oxygen Species (ROS)

Excessive generation of ROS is an indication ofcellular events leading to cell death. We wonderedwhether MPTQ treatment could induce ROS pro-duction. To test this, K562 cells treated with MPTQ(10 mM) for different time points were incubatedwith H2DCFDA (5 mM) and subjected to FACSanalysis. Results showed that MPTQ treatmentresulted in an increase in ROS in a time-dependent

manner (Figure 1C). Excessive ROS production wasevident as early as 30 min, which was more thanthe positive control, H2O2 (Figure 1C). Interesting-ly, further increase in the incubation did not evokethe similar ROS production till 24 h. Hence, theresults suggest that MPTQ induces ROS productionat early time points indicating cellular damage andthis could help in inducing the cell death.

MPTQ Induces Cell Death by Apoptosis

We have used live dead cell assay, a sensitiveflow cytometry based assay, to monitor changes inthe ratio of live to dead cell population followingMPTQ treatment (1, 2, 5, and 10 mM) on K562cells. Results showed a significant increase in deadcell population from 5 mM onwards (Figure 2A and

Figure 1. 8-Methoxypyrimido[40,50:4,5]thieno(2,3-b) quinoline-4(3H)-one (MPTQ) induces cytotoxicity in K562 cells. (A) Trypanblue dye exclusion assay was used to determine the cell viability onchronic myelogenous leukemia (K562) cells after MPTQ (5, 10, 20,and 50 mM) treatment. Following addition of MPTQ, viable cellswere counted every 24 h, until they attained stationary phase,which is presented as a graph. (B) MTT assay showing the cell pro-liferation on K562 cells at 24, 48, and 72 h of MPTQ treatment. In

both panels, error bars shown are based on three independentexperiments. P values are indicated by asterisk and calculated com-paring the mean of control cells with mean of MPTQ treated cells(�P < 0.05, ��P < 0.01, ���P < 0.001). (C) Detection of intracellu-lar ROS production by flow cytometry. K562 cells treated withMPTQ at 10 mM for different time points were harvested and test-ed for ROS production using flow cytometry. H2O2 treated cellswere used as positive control.

METHOXY PTQ INHIBITS TUMOR PROGRESSION IN MICE 5

Molecular Carcinogenesis

B). Based on this and above results, it is evidentthat MPTQ induces cytotoxicity in a concentra-tion-dependent manner.

Next, we were interested in exploring the mech-anism of MPTQ induced cell death. Translocationof phosphatidyl serine from the inner to the outermembrane (which can be detected by annexin Vstaining) is one of the major events in apoptosisand is widely used as a measure to distinguish be-tween apoptosis and necrosis. Therefore, we testedwhether MPTQ induces apoptosis. MPTQ treatedK562 cells (1, 5, and 10 mM, 48 h) were harvested,stained with annexin V-FITC and PI and subjectedto flow cytometric analysis. Results showed a con-centration-dependent increase in population ofearly and late apoptotic cells upon MPTQ treat-ment (Figure 2C). However, we could not find anyenhancement in the population of necrotic cellsupon addition of increasing concentrations of

MPTQ (Figure 2C). These results suggest thatMPTQ activates apoptosis to induce cytotoxicity.Further, confocal microscopy was used to visualizethe early and late apoptotic cells (Figure 2D)Results showed cells stained with annexin V-FITCalone (green fluorescence), which are early apopto-tic cells (Figure 2D, b) and annexin V-FITC and PI(green and red fluorescence), which are late apo-ptotic cells (Figure 2D, c–e). Besides, we couldobserve distinct changes in morphology both atcellular and nuclear level. As expected, controlcells did not show any significant intake ofannexin V or PI (Figure 2D, a).

MPTQ Induces Apoptosis by Extrinsic and Intrinsic

Pathways

In order to study the mechanism by whichMPTQ induces apoptosis, expression level of differ-ent apoptotic proteins was studied. For this, cell

Figure 2. Detection of mode of cell death induced by MPTQ. (A)Analysis of live and dead cell population following MPTQ treat-ment. K562 cells were treated with MPTQ (1, 2, 5, and 10 mM),cells were harvested after 48 h, and analyzed by flow cytometry.The live and dead cell populations are represented as a histogram.K562 cells without addition of MPTQ were used as control. (B) Bardiagram showing dead and live cell population following MPTQtreatment. Results shown are based on the data presented in panelA. (C) Detection of early and late apoptotic cells by annexin V andPI staining. K562 cells were cultured with increasing concentrationsof MPTQ (1, 5, 10 mM each) for 48 h and processed for annexin

V-FITC/PI double staining. The cells were then subjected to flowcytometric analysis and data obtained is shown as histogram. (D)Confocal imaging of apoptotic cells using Zeiss laser scanning mi-croscopy. MPTQ treated cells were stained with annexin V-FITCand PI and subjected to microscopic evaluation. Control cells (a)were negative for both annexin V and PI indicating live cells withcellular integrity, cells in the early apoptotic stage stained only withannexin V appeared green in color (b), cells in the late stage ofapoptosis, stained with both annexin V and PI appeared green andred in color (c–e).

6 SHARMA ET AL.

Molecular Carcinogenesis

lysates were prepared from both untreated controlcells and MPTQ treated K562 cells (0.5, 1, 2, and5 mM, for 48 h). MPTQ being a DNA intercalator,may lead to DNA damages, which could upregulatep53, thereby resulting in apoptosis.Therefore, we analyzed the levels of p53 and p73

proteins and they showed a dose-dependentincrease in expression (Figure 3A and B). Previousstudies have also shown such an upregulation ofp53, in a concentration dependent manner upontreatment with methyl methane sulfonate and5-fluorouracil [27,28]. However, its downstreamtarget PUMA did not show any significant changein expression although a drastic reduction wasobtained at 5 mM (Figure 3A and B). p53 is knownto regulate both the extrinsic and intrinsic path-ways by inducing FAS expression. Consistent tothis, an increase in the expression of FAS wasobserved in a concentration-dependent manner(Figure 3C and D). Immunoblotting showed adose-dependent increase in the levels of cleavedCASPASE8 suggesting an induction of apoptosisthrough extrinsic pathway (Figure 3C and D).CASPASE3 is known as an effector caspase and isinvolved in the final step of apoptosis. Westernblot analysis showed an increase in the activationof CASPASE3 (Figure 3C and D).Further, we observed an increase in the levels of

BAD, which is a pro-apoptotic protein (Figure 3Aand B). As expected, MPTQ treatment led to thedownregulation of anti-apoptotic proteins, BCL2and BCL-xL (Figure 3A and B). We also observed adose-dependent increase in the cleaved CASPASE9,which is an indicator of intrinsic pathway of apo-ptosis. SMAC/DIABLO is known to bind to IAPs(inhibitors of apoptotic proteins) which are boundto caspases and thus allow caspases to be accessibleto apoptotic machinery. Based on multiple ex-periments, we found an increase in the levelsof SMAC/DIABLO upon treatment with MPTQ(Figure 3C and D). One of the downstream effectormolecules of CASPASE3, PARP1, was also analyzed.However, no significant difference in its expressionwas observed following MPTQ treatment (Figure 3Aand B). Every blot described herein was reprobedusing GAPDH antibody to ensure equal loading ofproteins. Taken together, above results indicatethat MPTQ may induce apoptosis through both in-trinsic and extrinsic pathways.

Effect of MPTQ on Cell Cycle Regulatory and DNADamage Response Proteins

To check the effect of MPTQ on cell cycle regula-tory proteins, PCNA was used as a marker for cellu-lar proliferation [29]. We did not find any changein the levels of PCNA upon treatment with MPTQ(Figure 3E and F). We also did not observe any sig-nificant difference in the levels of CDC25a exceptat 5 mM, which is a marker of G1/S transition

(Figure 3E and F). We were also interested in theeffect of MPTQ on DNA damage proteins whichare early indicators of any DNA damage. We foundan upregulation in the levels of KU70 and KU80upon increasing concentration of MPTQ whichindicates DNA damage induced by MPTQ (Figure 3Aand B).Since we are using EAC to develop a syngeneic

tumor model, we checked for the effect of MPTQon EACs ex vivo. Results showed an increase in thelevels of activated CASPASE3, 8, and 9 followingMPTQ treatment of 0, 1, 2, 5, and 10 mM (Suppl.Figure 3). We observed downregulation of BCL2upon treatment with MPTQ from 1 mM onwards.In these experiments, tubulin was used as the load-ing control.

MPTQ Treatment Led to the Reduction of Tumor in Mice

Tumor was induced in Swiss albino mice usingEAC cells, which was used for investigating the an-ticancer activity of MPTQ in an animal model. Atotal of 30 mice were taken and divided into threebatches, containing 10 mice each. Each batch in-cluded tumor bearing mice (control), treated withMPTQ (experimental) and mice with no tumors(negative control). The experiment was repeatedthree independent times. To determine the opti-mum dose required for administration of MPTQ,we performed pilot studies using dosage from 1 to30 mg/kg. Results showed that 1 mg/kg was suffi-cient to reduce the tumor load on treated animals(Suppl. Figure 2). After 11th day of EAC injection(small tumor was visible), the animals were treatedwith six doses in a duration of 2 wk. Resultsshowed significant reduction in tumor volumeupon treatment with MPTQ as compared to un-treated tumor bearing animals (Figure 4A). Besides,we found that 67% (20/30) of the mice with tumorwere dead by 50th day of tumor development,when not administered with MPTQ, while 80%(24/30) of the mice survived upon treatment withMPTQ (Figure 4A and B). The gross appearance ofthigh tissue containing tumor, liver and spleen ofnegative control, untreated tumor control and MPTQtreated mice showed a proportional morphologicaldifference (Figure 4C and data not shown).Besides we have also tested the effect of MPTQ

on tumors developed following injection of DLAcells. A total of 34 mice were taken and dividedinto two batches, each containing 17 mice. Eachbatch included tumor bearing mice (control, 5)and treated with MPTQ (experimental, 7), while 5animals served as no tumor control. Based on pilotstudies, concentration of MPTQ for animal studieswas determined to be 10 mg/kg body weight (datanot shown). The animals were treated with 15doses of MPTQ immediately after injection of DLAcells. Results showed a significant reduction in thebody weight of the animal upon treatment with

METHOXY PTQ INHIBITS TUMOR PROGRESSION IN MICE 7

Molecular Carcinogenesis

MPTQ as compared to untreated tumor bearinganimals (Figure 4E). Besides, we found that alluntreated mice were dead by 25th day of tumordevelopment, while 60% of the tumor bearingmice survived upon treatment with MPTQ(Figure 4D–F and data not shown). The gross ap-pearance of mice showed a proportional morpho-logical difference between untreated tumor controland MPTQ treated mice (Figure 4D and data notshown).

MPTQ Treatment Improves the Survival of Tumor BearingMice Significantly

We found that lifespan of MPTQ treated EAC tu-mor bearing mice increased significantly, com-pared to that of control animals with tumor(Figure 4B). While the mean lifespan of controlanimals was 44.1 d after tumor development, themean lifespan of tumor bearing mice treated withMPTQ was 255.7 d indicating �4.8-fold increase inlife span of tumor bearing mice (Figure 4B). There-fore, our results demonstrate that MPTQ treatmentsignificantly improved the survival of tumor bear-ing animals. However, the effect of MPTQ on life-span of mice treated with DLA cells induced tumorwas less compared to EAC (Figure 4F).

Histopathological studies were performed on tu-mor and liver tissues of control and MPTQ treatedexperimental animals at two different time points(25 and 45 d). Sections through tumor tissue of a25-day-old mouse showed hematoxylin stainednuclei indicating cell proliferation in the thigh tis-sue where EAC was injected, while in the case ofcontrols, no other cells other than the nuclei ofskeletal muscle were stained (Suppl. Figure 4A andC). Interestingly, tumor tissue from MPTQ treatedmouse tissue showed a significant reduction inproliferating cells (Suppl. Figure 4B). Tissue sec-tions from thigh region after 45th day of MPTQtreatment showed negligible number of proliferat-ing cells which was comparable to the normal tis-sue, while proliferating cells were abundant inmice bearing tumor, where no treatment was given(Suppl. Figure 4C and D). To analyze whetherMPTQ had any adverse effect on other tissues, sec-tions through liver of control and experimentalanimals, stained with hematoxylin and eosin wereobserved (Suppl. Figure 4E(a) and F(a)). Sectionsthrough liver tissue from MPTQ treated animals af-ter 25th day exhibited normal morphology of

hepatocytes, while liver from a tumor bearingmouse showed irregular shaped hepatocytes(Suppl. Figure 4E(b) and F(b)). In contrast, liverfrom mouse after 45 d of tumor induction showedincrease in number of basophilic cells as comparedto normal controls and MPTQ treated animals(Suppl. Figure 4E(c) and F(c)). There was no changein morphology of liver upon treatment withMPTQ indicating that it does not exhibit any sideeffects. Thus, our results show that MPTQ can beutilized as a potent anti-carcinogenic agent withno evident adverse effects.In order to study the possible side effects due to

MPTQ treatment, mice were administered withMPTQ (1 mg/kg). Results showed that there wasno significant effect on total weight of the ani-mals, due to the treatment (Figure 5A). To furthercorroborate our findings, we performed hemato-logical and serum profile on mice administeredwith MPTQ at day 21. Results showed no signifi-cant difference in the number of RBCs and WBCsupon treatment with MPTQ (Figure 5B). Further,we found no significant difference in the levels ofALP and alanine aminotransferase (ALT), themarkers of normal liver function, upon treatmentwith MPTQ (Figure 5B). We also could not findany significant difference in the levels of creatinineand urea in the serum which indicates efficientkidney function (Figure 5B). Thus, our data indi-cate that MPTQ treatment does not result in anymajor side effects.Ki67 is a marker for cellular proliferation [30,31].

The tumor cell proliferation was investigated byimmunohistochemical staining for Ki67 on tissuesections derived from untreated tumors and MPTQtreated animals bearing tumor. Results showed effi-cient Ki67 and nuclear staining in tumor sectionsupon immunostaining, while number of Ki67 posi-tive tumor cells was substantially less when micewere treated with MPTQ (Figure 6A). As expected,thigh sections from wild type animals were nega-tive for Ki67 staining (Figure 6A). Further, weobserved that the expression of p53 binding pro-tein 1 (53BP1), and proapoptotic protein BID wassignificantly high in MPTQ treated tissue (25thday of treatment) as compared to tumor tissue(Figure 6B and C), further suggesting the activationof apoptosis in tumor cells in mice. Therefore, ourresults suggest that MPTQ treatment significantlyinhibited tumor progression in mice.

Figure 3. Effect of MPTQ on the expression of apoptotic pro-teins. Cell lysate was prepared after 48 h of addition of MPTQ(0.5, 1, 2, and 5 mM) in K562 cells. The untreated cells grown for48 h were used as control. Proteins (�40 mg) were resolved bySDS–PAGE and used for Western blot analysis using appropriateprimary and secondary antibodies. (A) and (B) Western blot analysisand its quantification showing expression of p53, p73, PUMA,

BAD, BCL2, BCL-XL, PARP1, KU70, and KU80. ‘‘0’’ is untreatedcontrol. (C) and (D) Western blot analysis and its quantificationshowing expression of CASPASE8, FAS, CASPASE3, SMAC/DIABLO,and CASPASE9. (E) and (F) Western blot analysis and its quantifica-tion of PCNA and CDC25a following treatment with MPTQ. In allpanels, GAPDH was used as an internal loading control.

8 SHARMA ET AL.

Molecular Carcinogenesis

Figure 3.

METHOXY PTQ INHIBITS TUMOR PROGRESSION IN MICE 9

Molecular Carcinogenesis

DISCUSSION

Development of potential chemotherapeuticagents with fewer side effects is the major chal-lenge in cancer therapy [32]. DNA intercalatingagents are a common class of compounds for thetreatment of cancer. In a previous study, synthesisof ellipticine structural analogues, pyrimidothieno-quinolines possessing DNA intercalating activityand potential anticancer properties was reported[2]. It has been shown that such DNA intercalatingagents interfere with the physiological functions ofthe cells such as transcription and replication bybinding to the DNA and resulting in cell death[33,34]. We have previously reported the synthesisand anticancer activity of a new class of DNA

intercalating and cytotoxic molecules, pyrimido[40,50:4,5]thieno(2,3-b)quinolines, having a tetra-cyclic condensed quinoline system [6,35] andpyrimido[40,50:4,5]selenolo(2,3-b)quinolines [18,36],having a seleno atom in the aromatic ring. Howev-er, modifications in the hydrophilic alkyl sidechain (butylamino) result in potent cytotoxic ac-tivity in leukemic cells. By using various biophysi-cal studies, it has been shown that these smallmolecules were able to interact with DNA. Basedon preliminary cytotoxic studies using differentcancer cell lines, we have identified MPTQ as apotential anticancer agent [12]. Inspired by thepreliminary study, we have synthesized the mono-hydrochloride of MPTQ (water soluble form) toimprove the pharmacokinetics of the compound

Figure 4. Effect of MPTQ on tumor progression in mice. Tumorswere induced in mice by injecting EAC cells or DLA cells. (A) Sixdoses of MPTQ were orally administered every alternate day from12th day of EAC cell injection. Data shows volume of tumor atdifferent time intervals, with and without treatment of MPTQ.Data shown is described from three independent batches of experi-ments containing 10 animals each. In all the cases, error bars indi-cate standard error from three independent experiments. P valuesindicated by asterisk are calculated by comparing the mean of EACalone groups with mean of MPTQ treated groups (�P < 0.05,��P < 0.01). (B) Kaplan–Meier survival curves of MPTQ treatedSwiss albino mice. (C) Gross appearance of control and experimen-tal animals and their selected organs on 25th day of MPTQ treat-ment, (a) control mouse neither treated with tumor cells norMPTQ, (b) mouse bearing tumor, (c) tumor bearing mouse after

treatment with MPTQ, (d) thigh tissue of normal mouse, (e) thightissue of a tumor bearing mice, (f) thigh tissue of a MPTQ treatedmouse, (g) liver of a normal mouse, (h) liver of a mouse containingtumor, (i) liver of a MPTQ treated mouse, (j) spleen of a normalmouse, (k) spleen of a mouse containing tumor, (l) spleen of aMPTQ treated mouse. (D) Gross appearance of control and MPTQtreated animals following injection into BALB/c mice with Dalton’slymphoma cells. (a) control mouse neither treated with tumor cellsnor MPTQ, (b) mouse bearing tumor, (c) tumor bearing mouse af-ter treatment with MPTQ. (E) Graphical representation of weightchanges in mice bearing tumor (DLA) followed by oral administra-tion of MPTQ. (F) Kaplan–Meier survival curves of mice with Dal-ton’s lymphoma following MPTQ treatment.

10 SHARMA ET AL.

Molecular Carcinogenesis

and here we show that this compound inhibits tu-mor progression and improves the lifespan of tu-mor bearing mice.The presence of the small alkyl methoxy group

and the substituted oxygen in the aromatic ring ofMPTQ may facilitate its entry into the cells. Thiscould lead to generation of excessive reactive oxy-gen free radicals, thereby resulting in cell death. Itcan also intercalate with DNA and interfere withcellular processes.EAC cells possessing malignant feature of cancer

is used commonly for inducing tumors in mice,and used for evaluating anti-cancer activity ofsmall molecules in vivo [22–24,37,38]. Our resultsshow that MPTQ treatment showed a significantreduction in tumor size, without affecting otherorgans. It is also important to note that concentra-tion of compound used was remarkably low. Morethan fourfold increase in lifespan of tumor bearinganimals was observed following MPTQ treatment,when compared to untreated control animals withtumor. This suggests a significant increase in the

survival and inhibition of tumor growth upon ad-ministration of MPTQ in tumor bearing animals.Histological evaluation of tumor and normal tis-sues in conjunction with hematological and enzy-matic assays on animals administered with MPTQfurther suggest that its effect was mostly restrictedto tumor cells.Although MPTQ treatment led to the regression

of tumor in mice injected with DLA cells, its effectwas less as compared to EAC generated tumor.However, even in this case, a significant increasein the life span of tumor animals was observed.Thus, taken together our data suggest that the wa-ter-soluble nature of MPTQ makes it a potent can-cer therapeutic agent.

MPTQ Activates Apoptotic Pathways to InduceCytotoxicity in Cancer Cells

We have used one of the cancer cell lines (K562)to study the mechanism by which MPTQ inducescell death. Initial results showed a dose- and time-dependent activation of apoptosis. Induction of

Figure 5. Evaluation of side effects caused due to administration of MPTQ. Mice were orally administeredwith six doses of MPTQ (1 mg/kg) every alternate day. (A) Data showing average weight changes in the boththe controls (n ¼ 5) and MPTQ treated mice (n ¼ 10). In all the cases, error bars indicate SEM. P-value wascalculated by comparing the mean of no treatment animals with the mean of MPTQ treated groups(�P < 0.05). (B) Hematological and serum profile on mice administered with MPTQ at day 21. Values indicatedare mean � SEM (n ¼ 10).

Figure 6. Immunostaining for apoptotic markers following treat-ment with MPTQ. IHC staining was performed on formalin fixed,paraffin embedded tissues that were sectioned, deparaffinized,rehydrated, and followed by antigen retrieval. (A) Ki67 immuno-staining of tissue isolated from 25th day tumor (a, g), 25th daytumor tissue following treatment with MPTQ (b, h). (B) BID

immunostaining of tissue isolated from 25th day tumor (c, i), 25thday tumor tissue following treatment with MPTQ (d, j). (C) 53BP1immunostaining of tissue isolated from 25th day tumor tissue (e, k)and 25th day tumor tissue treated with MPTQ (f, l). Magnificationof images shown in panels a–f are 10�, while g–l are 20�.

METHOXY PTQ INHIBITS TUMOR PROGRESSION IN MICE 11

Molecular Carcinogenesis

apoptosis rather than necrosis was confirmed bothquantitatively (FACS) and qualitatively (confocalmicroscopy) following staining with annexinV-FITC/PI. Further, we found an induction of ROSproduction upon treatment with MPTQ, whichmight activate the intrinsic pathway of apoptosis.Generally, apoptosis is regulated through BCL2family proteins comprising of antiapoptotic (BCL2,BCL-XL) and proapoptotic members (BAX, BAD)[39,40]. Western blot analysis showed an alterationin the levels of BCL2/BCL-XL and BAD leading toan increase in the ratio of proapoptotic to antia-poptotic proteins, further suggesting the activationof apoptosis. Besides, we have also noted the acti-vation of CASPASE9, which is specific for intrinsicpathway of apoptosis (Figure 7). Expression analy-sis of proteins involved in apoptosis show thatMPTQ can induce apoptosis by extrinsic pathwaysas well (Figure 7). Supporting evidence for such ahypothesis include MPTQ mediated activation ofFAS and CASPASE8.

We also observed a dose-dependent activation ofCASPASE3, which is an effector caspase. The acti-vation of CASPASE3 leads to a cascade of down-stream events including the activation of nucleaseswhich can cause the degradation of the nuclearDNA leading to apoptosis [41,42]. Thus, our results

suggest that MPTQ triggers the activation of bothextrinsic and intrinsic pathways. Similar resultswere also observed in case of an independentmouse cancer cell line further supporting theconclusion.

CONCLUSION

In summary, binding of MPTQ activate FAS lead-ing to cleavage of CASPASE8 followed by CAS-PASE3 (Figure 7). We also observed evidence ofactivation of intrinsic pathway of apoptosis depen-dent on CASPASE9. Thus, extrinsic as well as in-trinsic pathway of apoptosis is induced by MPTQleading to cell death. In vivo studies on multipletumor models further suggest that MPTQ could beused as a potential cancer therapeutic agent.

ACKNOWLEDGMENTS

We thank Mridula Nambiar, M. Nishana, S.R.Ranganatha, and members of the SCR laboratoryfor discussions and help. This work was supportedby grant from Lady Tata Memorial Trust, UK, andIISc start up grant for SCR. SS is supported by SRFfrom DBT, India and KPM is supported by IIScpostdoctoral fellowship program, India. Authorcontributions: SCR, SS, and KPM designed experi-ments; GMA synthesized and characterized MPTQ;SS, KPM, BC, MS, and SMS performed experiments;SS and SCR interpreted the data and wrote themanuscript.

REFERENCES

1. Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L.Anthracyclines: Molecular advances and pharmacologicdevelopments in antitumor activity and cardiotoxicity.Pharmacol Rev 2004;56:185–229.

2. Martinez R, Chacon-Garcia L. The search of DNA-interca-lators as antitumoral drugs: What it worked and what didnot work. Curr Med Chem 2005;12:127–151.

3. Matson JA, Bush JA. Sandramycin, a novel antitumor anti-biotic produced by a Nocardioides sp. Production, isola-tion, characterization and biological properties. J Antibiot(Tokyo) 1989;42:1763–1767.

4. Boger DL, Chen JH, Saionz KW, Jin Q. Synthesis of keysandramycin analogs: Systematic examination of the inter-calation chromophore. Bioorg Med Chem 1998;6:85–102.

5. Gopal M, Shahabuddin MS. Biological properties of 8-methoxypyrimido[4(1),5(1):4,5]thieno(2,3-b)quinoline-4(3H)-one, a new class of DNA intercalating drugs. Indian J MedRes 2004;119:198–205.

6. Nandeeshaiah SK, Ambekar SY. Synthesis, Dimroth rear-rangment and blood platelet disaggregation property ofpyrimido[40,50:4,5]selenolo(2,3-b)quinolines: A new classof condensed quinoline. Indian J Chem 1998;37:995–1000.

7. Gopal M, Shenoy S, Doddamani LS. Antitumor activity of4-amino and 8-methly-4-(3-diethyl aminopropylamino)pyr-imido[40,50:4,5]thieno(2,3-b)quinoline. J Photochem Pho-tobiol B 2003;72:69–78.

8. Gopal M, Shahabuddin MS, Inamdar SR. Interaction be-tween 8-methoxypyrimido [40,50:4,5]thieno(2,3-b)quino-line-4(3H)-one antitumour drug and deoxyribo nucleicacid. Proc Indian Acad Sci (Chem Sci) 2002;114:687–696.

Figure 7. Proposed mechanism of MPTQ induced apoptosis incancer cells. Treatment of cells with MPTQ induced apoptosis pri-marily through extrinsic pathway wherein upregulation of FASleads to the activation of CASPASE8. Cleaved CASPASE8 activatesthe effector CASPASE3 leading to cell death. MPTQ treatment alsoresulted in the activation of intrinsic pathway of apoptosis. Evi-dence for it include upregulation of proapoptotic protein BAD andconcomitant upregulation of p53, which results in the downregula-tion of BCL2, the antiapoptotic protein. Downregulation of BCL2leads to loss of mitochondrial membrane permeability, which inturn may release cytochrome c, leading to the activation of theinitiator CASPASE9. This results in the activation of the apoptoticprotein cascade wherein CASPASE3 gets cleaved.

12 SHARMA ET AL.

Molecular Carcinogenesis

9. Shahabuddin MS, Gopal M, Raghavan SC. Intercalating,cytotoxic, antitumour activity of 8-chloro and 4-morpholi-nopyrimido [40,50:4,5]thieno(2,3-b)quinolines. J Photo-chem Photobiol B 2009;94:13–19.

10. Shahabuddin MS, Gopal M, Raghavan SC. Intercalatingand antitumour activity of 4-oxopyrimido[40,50:4,5]thieno(2,3-b)quinoline-4(3H)-one. J Cancer Mol 2007;3: 139–146.

11. Gopal M, Veeranna S. 4-Anilinopyrimido[40,50:4,5]selenolo-(2,3-b)quinoline and 4-piperazino pyrimido[40,50:4,5]-selenolo(2,3-b)quinoline: New DNA intercalating chromo-phores with antiproliferative activity. J Photochem Photo-biol B 2005;81:181–189.

12. Shahabuddin MS, Nambiar M, Advirao GM, Raghavan SC.Intercalative pyrimido[40,50:4,5]thieno(2,3-b)quinolines in-duce apoptosis in leukemic cells: A comparative study ofmethoxy and morpholino substitution. Invest New Drugs2011;29:873–882.

13. Faulds D, Balfour JA, Chrisp P, Langtry HD. Mitoxantrone.A review of its pharmacodynamic and pharmacokineticproperties, and therapeutic potential in the chemotherapyof cancer. Drugs 1991;41:400–449.

14. Gopal M, Veeranna S, Doddamani LS. Intercalation bind-ing of 4-butylamino pyrimido[40,50:4,5]selenolo(2,3-b)quinoline to DNA: Relationship with invitro cytotoxicity.Spectroscopy Lett 2004;37:347–366.

15. Tilak Raj T, Ambeker SY. Synthesis of pyrimido[40,50:4,5]-,50:4,5]thieno(2,3-b)quinoline-4(3H)-ones. J Chem Res1988;50:537–551.

16. Chiruvella KK, Kari V, Choudhary B, Nambiar M, GhantaRG, Raghavan SC. Methyl angolensate, a natural tetranor-triterpenoid induces intrinsic apoptotic pathway in leuke-mic cells. FEBS Lett 2008;582:4066–4076.

17. Kavitha CV, Nambiar M, Ananda Kumar CS, et al. Novelderivatives of spirohydantoin induce growth inhibition fol-lowed by apoptosis in leukemia cells. Biochem Pharmacol2009;77:348–363.

18. Shahabuddin MS, Nambiar M, Choudhary B, Advirao GM,Raghavan SC. A novel DNA intercalator, butylamino-pyri-mido[40,50:4,5]selenolo(2,3-b)quinoline, induces cell cyclearrest and apoptosis in leukemic cells. Invest New Drugs2010;28:35–48.

19. Chiruvella KK, Raghavan SC. A natural compound, methylangolensate, induces mitochondrial pathway of apoptosisin Daudi cells. Invest New Drugs 2011;29:583–592.

20. Kumar TS, Kari V, Choudhary B, Nambiar M, Akila TS,Raghavan SC. Anti-apoptotic protein BCL2 down-regulates DNA end joining in cancer cells. J Biol Chem2010;285:32657–32670.

21. Sharma S, Choudhary B, Raghavan SC. Efficiency of non-homologous DNA end joining varies among somatic tis-sues, despite similarity in mechanism. Cell Mol Life Sci2011;68:661–676.

22. Noaman E, Badr El-Din NK, Bibars MA, Abou MossallamAA, Ghoneum M. Antioxidant potential by arabinoxylanrice bran, MGN-3/biobran, represents a mechanism for itsoncostatic effect against murine solid Ehrlich carcinoma.Cancer Lett 2008;268:348–359.

23. Ghosh M, Bhattacharya S, Sadhu U, Dutta S, Sanyal U.Evaluation of beta-tethymustine, a new anticancer com-pound, in murine tumour models. Cancer Lett 1997;119:7–12.

24. Attia MA, Weiss DW. Immunology of spontaneous mam-mary carcinomas in mice. V. Acquired tumor resistance

and enhancement in strain A mice infected with mamma-ry tumor virus. Cancer Res 1966;26:1787–1800.

25. Hazra B, Kumar B, Biswas S, Pandey BN, Mishra KP.Enhancement of the tumour inhibitory activity, in vivo, ofdiospyrin, a plant-derived quinonoid, through liposomalencapsulation. Toxicol Lett 2005;157:109–117.

26. Kumagai H, Mukaisho K, Sugihara H, Miwa K, YamamotoG, Hattori T. Thioproline inhibits development of esoph-ageal adenocarcinoma induced by gastroduodenal refluxin rats. Carcinogenesis 2004;25:723–727.

27. Zhan Q, Carrier F, Fornace AJ Jr. Induction of cellular p53activity by DNA-damaging agents and growth arrest. MolCell Biol 1993;13:4242–4250.

28. Ju J, Schmitz JC, Song B, Kudo K, Chu E. Regulationof p53 expression in response to 5-fluorouracil inhuman cancer RKO cells. Clin Cancer Res 2007;13:4245–4251.

29. Celis JE, Celis A. Cell cycle-dependent variations inthe distribution of the nuclear protein cyclin proliferatingcell nuclear antigen in cultured cells: Subdivision of Sphase. Proc Natl Acad Sci USA 1985;82:3262–3266.

30. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U,Stein H. Cell cycle analysis of a cell proliferation-associatedhuman nuclear antigen defined by the monoclonal anti-body Ki-67. J Immunol 1984;133:1710–1715.

31. Cheung AN, Ngan HY, Chen WZ, Loke SL, Collins RJ. Thesignificance of proliferating cell nuclear antigen in humantrophoblastic disease: An immunohistochemical study.Histopathology 1993;22:565–568.

32. Gupta M, Mazumder UK, Kumar RS, Sivakumar T, VamsiML. Antitumor activity and antioxidant status of Caesalpi-nia bonducella against Ehrlich ascites carcinoma in Swissalbino mice. J Pharmacol Sci 2004;94:177–184.

33. Hurley LH. DNA and its associated processes as targets forcancer therapy. Nat Rev Cancer 2002;2:188–200.

34. Dervan PB. Molecular recognition of DNA by small mole-cules. Bioorg Med Chem 2001;9:2215–2235.

35. Tilak Raj T, Ambekar SY. Synthesis of 4-amino pyrimido[40,50:4,5]thieno (2,3-b) quinoline-4(3H)-ones. J Chem Res(S) 1988;50:537–551.

36. Shahabuddin MS, Nambiar M, Moorthy BT, et al. A novelstructural derivative of natural alkaloid ellipticine, MDPSQ,induces necrosis in leukemic cells. Invest New Drugs 2011.29:523–533.

37. Kumar A, D’Souza SS, Nagaraj SR, Gaonkar SL, SalimathBP, Rai KM. Antiangiogenic and antiproliferative effects ofsubstituted-1,3,4-oxadiazole derivatives is mediated bydown regulation of VEGF and inhibition of translocationof HIF-1alpha in Ehrlich ascites tumor cells. CancerChemother Pharmacol 2009;64:1221–1233.

38. Gekeler V, Epple J, Kleymann G, Probst H. Selectiveand synchronous activation of early-S-phase repliconsof Ehrlich ascites cells. Mol Cell Biol 1993;13:5020–5033.

39. Hengartner MO. The biochemistry of apoptosis. Nature2000;407:770–776.

40. Reed JC. Bcl-2-family proteins and hematologic malignan-cies: History and future prospects. Blood 2008;111:3322–3330.

41. Thornberry NA, Lazebnik Y. Caspases: Enemies within.Science 1998;281:1312–1316.

42. Nicholson DW, Ali A, Thornberry NA, et al. Identificationand inhibition of the ICE/CED-3 protease necessary formammalian apoptosis. Nature 1995;376:37–43.

METHOXY PTQ INHIBITS TUMOR PROGRESSION IN MICE 13

Molecular Carcinogenesis