Derivative with potent in vivo anticancer activity.pdf

Diphenyl Difluoroketone:A Curcumin Derivative with Potent In vivo Anticancer Activity

Dharmalingam Subramaniam,1Randal May,1Sripathi M.Sureban,1Katherine B.Lee,4 Robert George,4Periannan Kuppusamy,5Rama P.Ramanujam,3Kalman Hideg,6 Brian K.Dieckgraefe,4Courtney W.Houchen,1and Shrikant Anant1,2,4

1Section of Digestive Diseases and Nutrition,Department of Medicine and2Department of Cell Biology,University of Oklahoma Health Sciences Center;3Swaasth,Inc.,Oklahoma City,Oklahoma;4Division of Gastroenterology,Department of Internal Medicine,Washington University School of Medicine,Saint Louis,Missouri;5Dorothy M.Davis Heart and Lung Research Institute,Ohio State University

Medical Center,Columbus,Ohio;and6Institute of Organic and Medicinal Chemistry,University of Pecs,Pecs,Hungary

Abstract

Diphenyl difluoroketone(EF24),a molecule having structural similarity to curcumin,was reported to inhibit proliferation of a variety of cancer cells in vitro.However,the efficacy and in vivo mechanism of action of EF24in gastrointestinal cancer cells have not been investigated.Here,we assessed the in vivo therapeutic effects of EF24on colon cancer https://www.wendangku.net/doc/615456511.html,ing hexosaminidase assay,we determined that EF24inhibits proliferation of HCT-116and HT-29colon and AGS gastric adenocarcinoma cells but not of mouse embryo fibroblasts. Furthermore,the cancer cells showed increased levels of activated caspase-3and increased Bax to Bcl-2and Bax to Bcl-xL ratios,suggesting that the cells were undergoing apoptosis.At the same time,cell cycle analysis showed that there was an increased number of cells in the G2-M phase.To determine the effects of EF24in vivo,HCT-116colon cancer xenografts were established in nude mice and EF24was given i.p.EF24significantly suppressed the growth of colon cancer tumor xenografts.Immunostaining for CD31showed that there was a lower number of microvessels in the EF24-treated animals coupled with decreased cyclooxygenase-2,interleukin-8,and vascular endothelial growth factor mRNA and protein expression.Western blot analyses also showed decreased AKT and extracellular signal-regulated kinase activation in the tumors.Taken together,these data suggest that the novel curcumin-related compound EF24is a potent antitumor agent that induces caspase-mediated apoptosis during mitosis and has significant therapeutic potential for gastrointestinal cancers.[Cancer Res2008;68(6):1962–9]

Introduction

Colorectal carcinoma is the second leading cause of cancer mortality in the United States with f55,000deaths in2006(1–3). Because conventional therapies,including surgical rejection, chemotherapy,and radiation,are often inadequate at treating the disease,the need for new treatment options has grown more critical.Epidemiologic studies suggest that diet plays a major role in the prevention of many cancers,and curcumin(a common flavoring agent in the spice turmeric)may be a dietary component responsible for lower rates of colorectal cancer in certain part of India(4).Curcumin has also been used in Indian folk medicine to treat several ailments,and recent preclinical and clinical studies show that curcumin has several anticancer and antiangiogenic properties(5,6).Its antitumor properties include cancer growth inhibition and apoptosis induction in a variety of cultured cancer cell lines in vitro.Additionally,curcumin has shown the ability to inhibit tumorigenesis in vivo(7–13).The use of curcumin in antiangiogenesis includes the inhibition of vascular endothelial cell proliferation in vitro and capillary tube formation and growth in vivo(14,15).The published report and our previous studies showed that curcumin inhibits epidermal growth factor(EGF)-mediated signaling and growth of intestinal adenomas in APC min/+ mice(16).However,due to the low cancer-killing potency and poor intestinal absorption characteristics of curcumin(17,18),bioavail-ability of the compound has limited its use.Consequently, analogues of curcumin with similar safety profiles but increased anticancer activity and solubility have recently been developed (13,14,19).One compound in particular,diphenyl difluoroketone (EF24),is active in anticancer screens and has considerably less toxic effect than the commonly used chemotherapeutic drug cisplatin(17,20).In this article,we show results of our in vitro experiments showing that EF24inhibits cell proliferation,prevents colony formation,and promotes G2-M arrest of colon and gastric cancer cells.Our in vivo studies with HCT-116colon cancer cell tumor xenografts in nude mice revealed that EF24suppresses cancer cell proliferation and angiogenesis and induces cell cycle arrest and apoptosis,coupled with the reduction of the expression of colon cancer–promoting genes,including cyclooxygenase-2 (COX-2),interleukin-8(IL-8),and vascular endothelial growth factor (VEGF).

Materials and Methods

Cells and reagents.HCT-116,HT-29colon,and AGS gastric adenocar-cinoma cells(all from American Type Culture Collection)were grown in DMEM and RPMI1640containing10%heat-inactivated fetal bovine serum (FBS;Sigma Chemical Co.)and1%antibiotic-antimycotic solution (Mediatech,Inc.)at37j C in a humidified atmosphere of5%CO2.Curcumin was purchased from LKT Laboratories.EF24was synthesized at the Institute of Organic and Medicinal Chemistry,University of Pecs(Pecs, Hungary).

Proliferation and apoptosis assays.To assess proliferation,cells were seeded onto96-well plates at a density of1?103per well and allowed to adhere and grow overnight in10%heat-inactivated FBS containing DMEM or RPMI1640.The cells were then treated with increasing doses of EF24in 10%FBS containing DMEM or RPMI1640.Analysis of cell proliferation was performed by enzymatic assay as described above(21).For apoptosis,

Note:Supplementary data for this article are available at Cancer Research Online (https://www.wendangku.net/doc/615456511.html,/).

Requests for reprints:Shrikant Anant,Section of Digestive Diseases and Nutrition,Department of Medicine,University of Oklahoma Health Sciences Center, Oklahoma City,OK73126.Fax:405-271-5450;E-mail:shrikant-anant@https://www.wendangku.net/doc/615456511.html,.

I2008American Association for Cancer Research.

doi:10.1158/0008-5472.CAN-07-6011

Research Article

caspase-3/7activity was measured using the Apo-ONE Homogeneous Caspase-3/7Assay kit(Promega).Both floating and attached cells were included in these analyses.

Cell cycle analysis.Cells were plated at a density of5?105per well on six-well plates.After treatment with EF24for the indicated period,both floating and attached cells were collected into flow cytometry tubes and centrifuged at1,000rpm for5min to obtain cell pellets.The supernatant was discarded,and the cells were washed with PBS and then recentrifuged. The cells were resuspended in100A L PBS,3mL ofà20j C ice-cold70% ethanol were added,and the cells were then incubated for1h at4j C.The cells were washed twice with PBS and10mg/mL RNase A was added. Propidium iodide was added to the tubes at a final concentration of 0.05mg/mL and incubated at4j C for30min in the dark.Cell cycle analysis was performed with a Becton Dickinson FACScan using an FL2detector with a bandpass filter at specifications of585F21nm.In each analysis, 10,000events were recorded.Results were analyzed with ModFit LT software (Verity Software House).

Colony formation assay.Briefly,six-well dishes were seeded with 500viable cells in complete medium and allowed to grow for24h.The cells were then incubated in the presence or absence of various concentrations of EF24for up to48h.The EF24-containing medium was then removed, and the cells were washed in PBS and incubated for an additional10days in complete medium.Each treatment was done in triplicate.The colonies obtained were washed with PBS and fixed in10%formalin for10min at room temperature and then washed with PBS followed by staining with hematoxylin.The colonies were counted and compared with untreated cells.

HCT-116cell tumor xenograft in mice.Five-week-old male athymic nude mice purchased from The Jackson Laboratory were used for in vivo experiments;they were maintained with water and standard mouse chow ad libitum and used in protocols approved by the University’s Animal Studies Committee.Animals were injected with1?106HCT-116cells in the left and right flank and allowed to form xenograft.EF24(200A g/kg body weight)in5%Na2HCO3buffer alone was given i.p.daily for23days.Tumor size was measured weekly.At the end of treatment,the animals were sacrificed,and the tumors were removed and weighed for use in histology (H&E,COX-2,VEGF,and CD31)and gene expression studies.

Real-time reverse transcription-PCR analysis.Total RNA isolated from HCT-116cells and tumor xenograft using Trizol reagent was reverse transcribed with SuperScript II reverse transcriptase in the presence of random hexanucleotide primers(all from Invitrogen).Complementary DNAs were then used for real-time PCR using JumpStart Taq DNA polymerase(Sigma Chemical)and SYBR Green nucleic acid stain(Molecular Probes).Crossing threshold values for individual genes were normalized to h2microglobulin.Changes in mRNA expression were expressed as fold change relative to control.Primers used in this study were as follows: h2microglobulin,5?-GAGTGCTGTCTCCATGTTTGATG-3?and5?-CTCTA-AGTTGCCAGCCCTCCT-3?;IL-8,5?-CTCTTGGCAGCCTTCCTGATT-3?and 5?-TATGCACTGACATCTAAGTTCCTTTAGCA-3?;COX-2,5?-GAATCATTCA-CCAGGCAAATTG-3?and5?-TCTGTACTGCGGGTGGAACA-3?;and VEGF, 5?-AGCGCAAGAAATCCCGGTA-3?and5?-TGCTTTCTCCGCTCTGAGC-3?.

Western blot analysis.Cell lysates were subjected to PAGE and blotted onto Immobilon polyvinylidene difluoride membranes(Millipore).Anti-bodies were purchased from Cell Signaling Technology and Santa Cruz Biotechnology,Inc.,and specific proteins were detected by the enhanced chemiluminescence system(Amersham Pharmacia Biotech).

IL-8ELISA.The HCT-116cells were treated with EF24for24h.The cell-free supernatants and tumor xenograft proteins were measured by IL-8using an enzyme immunoassay kit(Pierce Biotechnology)using the manufacturer’s suggested protocol.

Immunohistochemistry.Tissues embedded in paraffin were cut to a section of4A m,deparaffinized,and treated with citrate buffer.Then,they were blocked with avidin/biotin for20min.The slides were incubated with anti–COX-2or VEGF or CD31for overnight at4j C.Subsequently,the slides were treated with secondary antibody with horseradish peroxidase goat anti-rabbit for COX-2or VEGF and goat anti-rat for CD31staining for 1h and developed with3,3?-diaminobenzidine(Sigma-Aldrich).Finally,the slides were counterstained with hematoxylin.

Statistical analysis.All values are expressed as the mean F SE.Data were analyzed using a paired two-tailed t test.A P value of<0.05was considered statistically significant.

Results

EF24inhibits intestinal cancer cell proliferation.We first determined the effect of EF24and curcumin on cell proliferation in a variety of cultured cancer cell lines(Fig.1A).EF24significantly suppresses proliferation of colon cancer cell lines HCT-116and HT-29and a gastric cancer cell line(AGS)within a24-h period, which continues to72h(Fig.1B).More importantly,the effects were observed at a dose of1A mol/L,a dose at which curcumin had no significant effect on HCT-116cell proliferation.Similar results were observed with esophageal,lung,breast,pancreatic,and cervical cancer cells(data not shown).In contrast,EF24did not affect the proliferation of normal mouse embryonic fibroblasts cells treated with5A mol/L concentration,whereas hydrogen peroxide significantly affected it(Fig.1C),suggesting that EF24is not toxic to normal cells.To determine the long-term effect of EF24treat-ment,cells were treated with1A mol/L EF24for24h,following which the cells were allowed to grow in normal medium.EF24 treatment suppresses colony formation in a dose-dependent man-ner in all three cell lines(Fig.1D),suggesting that EF24effects on the cells are irreversible.Again,at this1A mol/L dose,curcumin did not affect cell growth and colony formation(Fig.1D).

EF24induces cell cycle arrest and apoptosis.At a sufficiently high dose,curcumin is known to induce apoptosis of cancer cells. Cell cycle analysis was performed to determine the mechanism by which EF24affects cancer cells.EF24(1A mol/L)induced growth arrest of the HT-29and AGS cells within24h at the G2-M stage and subsequently at the G0hypodiploid/fragmented DNA stage at 48hours(Fig.2A;Supplementary Fig.S1).Similar results were observed in HCT-116cells(data not shown).These data suggest that the cells were undergoing apoptosis.Caspase-3is a key effector molecule in the apoptosis pathway involved in amplifying the signal from initiator caspases,such as caspase-8(22,23). Increased activation of caspase-3and caspase-7was observed within24h in HCT-116and AGS cells treated with either1or 5A mol/L of EF24(Fig.2B).However,whereas1A mol/L EF24did not induced caspase-3and caspase-7activity in HT-29cells at 24hours,5A mol/L EF24did(Fig.2B).These data further show that EF24was inducing apoptosis(Fig.2B).Western blot analyses of HCT-116cell lysates showed a significant increase in the activated caspase-3in cells treated with5A mol/L EF24(Fig.2C).In addi-tion,5A mol/L EF24inhibited the expression of antiapoptotic genes Bcl-2and Bcl-xL protein but it had no detectable effect on the expression levels of apoptosis-promoting total Bax protein (Fig.2D).This resulted in decreased Bcl-2to Bax and Bcl-xL to Bax protein ratios(Supplementary Fig.S2).These data suggest that EF24is a potent inducer of apoptosis even at low doses where no such effect is observed with curcumin.

EF24inhibits tumor growth.To evaluate the role of EF24in tumor proliferation in vivo,we examined the ability of EF24to suppress the growth of human cancer cell xenografts in nude mice. Colon cancer cell–induced xenograft tumors were allowed to develop and grow to a size of500mm3,following which EF24was given i.p.for3weeks daily.EF24significantly inhibits the growth of the tumor xenografts(Fig.3A).The excised tumors from the EF24-treated animals ranged from200to300mg,whereas those from the control group weighed<120mg(Fig.3B and C).In addition, tumor volumes were significantly decreased(Fig.3D).Whereas the

EF24and Anticancer Cancer Activity

control tumors continue to grow during the treatment period reaching a size of 2,000mm 3,tumors in the EF24-treated animals did not grow.No apparent toxicity effects were observed in the EF24-treated animals (data not shown).Furthermore,tumors began to regress in the EF24-treated animals that were allowed to continue living past the 21-day period (data not shown).In our tests,no control animals were allowed to remain alive after 21days.To evaluate whether EF24administration affects normal physiol-ogy,we treated non–tumor-bearing mice weekly at exactly the same 200A g/kg body weight dose for 3weeks.There was no apparent change in liver weight,spleen weight,or body weight in the animals (data not shown).These data imply that EF24is a potential therapeutic for treatment of colon cancers and that it is relatively nontoxic to mice.

EF24inhibits the signaling of tumor cell survival.Survival signals that originate from extracellular cues result in the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT and/or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)pathways.The PI3K pathway plays a crucial role in the survival of various cell types (24).Activated AKT phosphorylates numerous proteins that have been implicated in the control of the cell cycle to ultimately stimulate cell growth and inhibit apoptosis (25).Western blot analyses of HCT-116cell lysates showed a significant dose-and time-dependent decrease in the EGF-mediated AKT phosphorylation in cells treated with EF24(Fig.4A ;Supplementary Fig.S3).MAPKs are also activated by a range of extracellular signals via protein phosphorylation cascades that relay mitogenic signals to the nucleus,thereby modulating the activity of transcription factors (26–29).The two best-characterized isoforms [p42mapk (ERK-2)and p44mapk (ERK-1)]are directly activated by phosphorylation on specific tyrosine and threonine residues by the dual-specificity ERK kinase.Western blot analyses of HCT-116cell lysates showed a significant dose-dependent decrease in the EGF-mediated ERK phosphorylation in cells treated with EF24(Fig.4B ).In addition,EF24significantly inhibited the tumor xenograft AKT and ERK phosphorylation (Fig.4C and D ).These data suggest that EF24is a potent inhibitor of tumor cell survival through suppression of PI3K and ERK-MAPK

pathways.

Figure 1.EF24inhibits intestinal cancer cell proliferation.A,the topological structures of curcumin (diferuloylmethane)and EF24(diphenyl difluoroketone).B,EF24inhibits proliferation of HCT-116,HT-29,and AGS cells.These cells were incubated with increasing doses of EF24(0.01–10A mol/L)for 24-,48-,and 72-h periods and analyzed for cell proliferation using hexosaminidase enzyme activity.EF24treatment resulted in a significant dose-dependent decrease in cell number in all three cells when compared with untreated controls and curcumin.C,EF24does not affect the proliferation of normal mouse embryonic fibroblast cells treated with 5A mol/L EF24for 48h using hexosaminidase enzyme activity.D,EF24inhibits cell growth as indicated by colony formation assays using HCT-116,HT-29,and AGS cells.These cells were incubated with 1A mol/L EF24or curcumin for 24h and subsequently allowed to grow into colonies.As indicated by counts performed 10d after incubation,EF24,but not curcumin,inhibits colony-forming units.Results are representative of three independent experiments.

Cancer Research

EF24inhibits expression of cancer-promoting genes and angiogenesis.COX-2,a key rate-limiting enzyme in prostaglandin production,is overexpressed at multiple stages of colon carcinogen-esis (30–32).Knockdown of COX-2activity by either nonsteroidal anti-inflammatory drugs or by deletion of the COX-2gene results in the suppression of intestinal polyposis in APC min/+mice (30–32).Pre-vious studies have shown increased levels of COX-2mRNA in colo-rectal adenomas,adenocarcinomas,and colon cancer cell lines (33,34).Therefore,we next determined the effects of EF24treat-ment on COX-2expression.EF24treatment reduced COX-2mRNA and protein levels in HCT-116cells (Fig.5A and B ).Interestingly,protein levels of both COX-2and VEGF were rapidly reduced within 2hour of treatment with EF24,suggesting the presence of posttrans-lational control mechanisms in regulating expression of these genes.COX-2expression was significantly lower in EF24-treated nude mice tumor xenografts when compared with the control

tumors

Figure 2.EF24induces cancer cell apoptosis.A,DNA content (propidium iodide)and cell cycle analysis of EF24-treated cells.HT-29cells were treated with

EF24for 24and 48h.M1,sub-G 1-G 0peak;M2,G 1phase;M3,S phase;M4,G 2-M phase.Apoptosis was measured as the percentage of cells containing hypodiploid amounts of DNA (sub-G 1-G 0peak).EF24treatment leads to increased number of cells in the G 2-M phase after 24h as indicated by the increase in M4peaks compared with control.Increase in M1peaks at 48h indicates that EF24induces apoptosis after 48h.Graphs are representative of data collected from at least three experiments.B,EF24treatment induces apoptosis in HCT-116,HT-29,and AGS cells.HCT-116,HT-29,and AGS cells incubated with 1A mol/L (top )and 5A mol/L (bottom )of EF24were analyzed for apoptosis.EF24treatment increased number of apoptotic cells compared with either curcumin-treated or untreated control.*,P <0.001.C,EF24induces caspase-3,an apoptosis mediator.Lysates from HCT-116cells incubated with 5A mol/L EF24were analyzed by Western blotting for caspase-3protein expression levels using rabbit anti–caspase-3antibody.EF24-treated cells show cleaved (activated)caspase-3,whereas untreated cells have no cleaved caspase-3.D,EF24reduces expression of antiapoptotic proteins Bcl-2and Bcl-xL in treated cells when compared with untreated cells.Lysates from HCT-116cells incubated with 5A mol/L EF24were analyzed by Western blotting for Bcl-2,Bcl-xL,and Bax protein expression levels.Whereas both Bcl-2and Bcl-xL were reduced,Bax expression was not affected by EF24treatment.

EF24and Anticancer Cancer Activity

(Fig.6A and C ).Immunohistochemistry showed a diffused cyto-plasmic staining for COX-2in the epithelial cells in the control tumors,and the expression was higher subepithelial myofibro-blasts (Fig.6D ).However,COX-2staining was significantly reduced in both the epithelial cells and myofibroblasts in EF24-treated tumors.

VEGF and IL-8are potent inducers of capillary growth into the tumor,and without angiogenesis,tumor growth normally stops at a diameter of about 1to 2mm.Prostaglandins and the other tumor-promoting mediators are known to induce the expression of VEGF and IL-8in epithelial cells.Hence,we also determined the effect of EF24on expression of these two genes in colon cancer cells.Both VEGF and IL-8expression were significantly reduced in HCT-116cells (Fig.5A–C ).Similarly,VEGF and IL-8levels were significantly reduced in the EF24-treated tumor xenografts (Fig.6A–C ).We also determined the effect of EF24on tumor vascularization by staining for the endothelial-specific antigen CD31.As shown in Fig.6D ,EF24treatment leads to a significant reduction in CD31staining and to the obliteration of the normal vasculature that is associated with tumor angiogenesis.We also calculated the microvessel density and found it to be significantly decreased following EF24treatment (Fig.6D ).

Discussion

Our results indicate that the novel curcumin analogue EF24possesses great potential as a promising anti–colon therapeutic agent.Colon cancer is a common malignancy in economically

developed countries,particularly in North America,Europe,and Australia,and has emerged as one of the leading causes of cancer-related deaths in the Western world.The significant morbidity of surgery,radiation,and chemotherapy for colon cancer has led searches for less toxic alternative therapies.Many studies have shown that curcumin suppresses the proliferation of a variety of tumor cells,including breast,colon,oral,lung,melanoma,mye-loma,leukemia,and prostate carcinoma (10,11,35–43).The data presented in the article show that EF24selectively inhibits the proliferation of colon cancer cells,suppresses the formation of colon cancer cell colonies,inhibits cell survival signaling,promotes cell cycle arrest and apoptosis,and inhibits cancer-promoting genes.In vivo ,EF24decreases tumor size,volume,and microvessel density and suppresses mRNA expression and protein levels of the colon cancer–promoting genes COX-2,IL-8,and VEGF .

Because curcumin is poorly absorbed through the intestine,more potent and soluble curcumin analogues have been developed (17).Furthermore,as shown in our studies,at low doses,curcumin did not have a therapeutic effect.Similar results were observed in studies on squamous cell carcinomas,where a dose of 3.75A mol/L curcumin did not affect growth of the cells (44).Consequently,higher doses are required.Synthetic chemical analogues to mole-cularly targeted chemotherapeutic drugs and chemopreventative photochemical confound a myriad of molecular events in host and tumor tissues.These events include the acquisition of self-sufficient growth signals,insensitivity to signals that usually inhibit proliferation,use of survival pathways to evade apoptosis,initia-tion of angiogenesis to ensure sufficient oxygen and

nutrient

Figure 3.EF24inhibits colon cancer tumor xenograft growth in vivo.A and B,HCT-116cells were injected to the flanks of nude mice and palpable tumors were allowed to develop for 7d.Subsequently,EF24was injected daily i.p.for up to 21d.On day 22,tumors were excised and subjected to further analyses.Tumor volumes in EF24given mice were smaller than that of control mice.C,EF24treatment resulted in significantly lower tumor weight when compared with controls.D,tumor size was measured every week.There was a significant reduction in tumor size from EF24-treated animals when compared with untreated controls.

Cancer Research

supply,and attainment of the ability to invade and metastasize (45).EF24molecule interferes with the progression of cancer by disrupting many of the characteristic cancer-promoting events more effectively than curcumin.EF24inhibits proliferation of HCT-116colon cancer cells,promotes apoptosis,prevents angio-genesis of tumors in mice xenografts,and decreases tumor growth.In cell cycle analyses,EF24was observed to increase the number of cells in the G 2-M phase at 24hours.At the same time,there

are

Figure 4.EF24inhibits AKT and ERK activation

required for cell survival.A and B,HCT-116cells were pretreated with 10A mol/L EF24for 2h followed by treatment with EGF (100ng/mL)for the indicated time (0–15min).Lysates from EF24treatment showed significant reduction in the EGF-mediated AKT and ERK phosphorylation at all time points.C and D,AKT and ERK phosphorylation was significantly lower in tumor xenografts from EF24-treated animals when compared with controls.

Figure 5.EF24inhibits the expression of cancer-promoting genes.A,real-time reverse transcription-PCR analysis of total RNA from HCT-116cells following 1A mol/L EF24treatment for 24h showed reduction in the expression of COX-2,VEGF,and IL-8mRNA.B,lysates from EF24treatment caused significant reduction of COX-2and VEGF protein levels in HCT-116cells.C,medium from EF24-treated cells showed significantly lower levels of IL-8protein.

EF24and Anticancer Cancer Activity

significantly higher levels of apoptosis.These data imply that EF24treatment leads to mitotic catastrophe in which the proliferating cancer cells undergo cell death but not necessarily an arrest in the G 2-M phase of the cell cycle.This was further supported through our observation that the majority of cells were present in the sub-G 0phase at 48h following treatment.

The effects of EF24on in vivo activity are dependent on the bioavailability of drug at the site of the tumor.Dietary curcumin is poorly absorbed through the intestinal tract.This results in good availability of the compound only to the surface epithelial cells of the intestine and colon but significantly poor availability of the compound to those cells that develop at the deeper tissue locations in the intestine and colon or if the tumor is malignant and is localized in other sites.This has led to the development of analogue with better bioavailability.In our studies,we observed marked suppression of tumor growth in mice xenograft with EF24treatment.However,further studies are needed to confirm and extend the present study to use EF24as an effective therapy for colorectal cancer.Absorption and pharmacokinetic properties of EF24in particular need to be identified in future studies;however,preliminary studies indicate that EF24seems to have low toxicity in liver,kidney,and spleen and allows mice treated with EF24to maintain normal weight gain (19).In addition,EF24,like curcumin,seems to mediate its actions through multiple molecular targets,including COX-2,VEGF,and IL-8.Because COX-2overexpression during colon carcinogenesis causes resistance to apoptosis (46),treatment of colon cancer cells with EF24may potentially restore susceptibility to apoptosis.Furthermore,IL-8overexpression has been correlated with the inflammation-related risk of sporadic colorectal cancer (47),so treatment with EF24may also potentially ameliorate the inflammatory responses that are associated with the development of cancer.Finally,VEGF is important in angiogenesis and promotion of tumor growth,and the ability of EF24to inhibit VEGF expression is yet another molecular mechanism by which EF24may function to prevent colorectal cancer.

In this study,we have shown that EF24suppressed EGF-mediated signaling.However,colon cancer cells,including HCT-116cells,are known to express autocrine factors such as

prostaglandins,

Figure 6.EF24suppresses tumor angiogenesis.A,EF24treatment decreases COX-2,VEGF,and IL-8

mRNA expression in the HCT-116tumor xenografts.B,ELISA analysis of tissue lysates from the EF24-treated mice shows significantly lower levels of IL-8.C,Western blot analysis showed that tissue lysates from the EF24-treated animals have significantly lower

levels of COX-2and VEGF proteins.D,immunohistochemistry shows that EF24treatment significantly reduced the expression of COX-2and VEGF in tumor xenografts.Arrows,proteins are stained brown.Inset in the COX-2immunohistochemistry shows high-power field view of a COX-2–positive stromal cell.Tumor sections were stained for

CD31,an endothelial cell–specific surface marker,and the vessel areas were counted.Microvessel density was significantly reduced in the xenografts of EF24-treated animals.

Cancer Research

insulin-like growth factors,and progastrin,which activate many cellular pathways(48–53).It would be interesting to determine whether EF24is equally potent in inhibiting these signal transduction pathways.

In conclusion,our studies show that EF24treatment of intestinal cancer cells results in growth inhibition in vitro and in vivo.Our in vitro and in vivo studies in combination with the observation that EF24does not affect proliferation of normal human fibro-blasts strongly suggest that EF24has promising potential for use as a therapeutic or chemopreventative agent for intestinal cancer. Similar to curcumin,EF24also seems to have multiple molecular targets and its enhanced potency in cancer cell lines and xenograft tumors renders it a strong candidate for therapeutic applications for colon cancer as well as other cancers and inflammatory disease states.

Acknowledgments

Received10/29/2007;revised1/9/2008;accepted1/16/2008.

Grant support:Hungarian Research Fund OTKA T048334(K.Hideg)and NIH grants AT004118(R.P.Ramanujam),DK62265,and CA109269(S.Anant).

The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solely to indicate this fact.

We thank Drs.Chinthalapalli V.Rao and James H.Wyche for their helpful discussions during the course of these studies.

EF24and Anticancer Cancer Activity

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