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Research Article

Paricalcitol Enhances the ChemopreventiveEfficacy of 5-Fluorouracil on an Intermediate-TermModel ofAzoxymethane-InducedColorectalTumors in RatsAdel Galal El-Shemi1,2, Bassem Refaat1, Osama Adnan Kensara3,Amr Mohamed Mohamed1,4, Shakir Idris1, and Jawwad Ahmad1

Abstract

Colorectal cancer is a common cancer with high mortality rate.Despite being the standard anti–colorectal cancer drug, 5-fluoro-uracil (5-FU) exhibits only limited therapeutic benefits. Herein,we investigated whether paricalcitol, a synthetic vitamin D ana-logue with potential antitumor properties, would enhance thechemopreventive efficacy of 5-FU on an intermediate-term (15weeks) model of colorectal tumors induced by azoxymethane(AOM) in rats. After AOM injection, 5-FU was administeredduring the 9th and 10th weeks (12 mg/kg/day for 4 days, then6mg/kg every other day for another 4 doses), whereas paricalcitol(2.5 mg/kg/day; 3 days/week) was given from the 7th to the 15thweek. At week 15, the animals were euthanized and their resectedcolons were examined macroscopically and microscopically.Quantitative RT-PCR was used to measure the transcriptionactivities of Wnt, b-catenin, DKK-1, CDNK-1A, NF-kB, and

COX-2 genes, and ELISA was used to quantify the protein levelsof b-catenin, COX-2, HSP90, and VEGF. IHC was additionallyused to measure b-catenin, HSP90, and inducible nitric oxidesynthase (iNOS). Compared with their individual therapy, com-bination of 5-FU and paricalcitol showed more significant reduc-ing effect on numbers of grown tumors and large aberrant cryptsfoci. Mechanistically, paricalcitol and 5-FU had cooperatedtogether to repress the expression of procancerousWnt, b-catenin,NF-kB, COX-2, iNOS, VEGF, andHSP-90more, and to upregulatethe expression of antitumorigenesis DKK-1 and CDNK-1A, com-pared with their monotherapies. Our findings suggest that com-bined use of paricalcitol with 5-FU exhibits an augmentingchemopreventive effect against colorectal tumors, and mightpotentially be useful for chemoprevention in colorectal cancerpatients. Cancer Prev Res; 9(6); 491–501. �2016 AACR.

IntroductionAccording to the World Health Organization's International

Agency for Research on Cancer, colorectal cancer represents thethirdmost frequent cancer inmen and second in women, rankingas the fourth leading cause of cancer-related deaths worldwide(1). The limited therapeutic efficacy of its current chemotherapyrepresents the most important challenge in colorectal cancermanagement. With this aspect, 5-fluorouracil (5-FU)-basedtherapy, either alone or in combination with other cytotoxicagents (e.g., irinotecan, leucovorin, or oxaliplatin), remains thestandard approach (2–4). Overall, 5-FU still exhibits limitedefficacy with low tumor response rate ranging between 7% and

17 % with its monotherapy and 35%–39% when given withother chemotherapeutic agents (3, 4). More importantly,although the recent addition of new targeted agents, such asbevacizumab and cetuximab, to the standard chemotherapyprovides hope for more effective therapy in advanced colorectalcancer, they have shown only modest benefit and are subject toboth primary and secondary resistance, like traditional chemo-therapy, which ultimately leads to treatment failure (4). Thus,development of potential alternative or combinational colo-rectal cancer chemopreventive and therapeutic strategies is aparamount medical demand. To that end, vitamin D and itsanalogues are the most attractive agents in this setting (5, 6).

There is a compelling body of evidence that calcitriol [1,25(OH)2D3], the active form of vitamin D, may not only reduce therisk of colorectal cancer and other human cancers but also repressthe tumor cell resistance toward the cytotoxic effects of 5-FU andother anticancer chemotherapeutic agents, and regulate the activ-ities of various genes that impact cancer cell proliferation, differ-entiation, apoptosis, angiogenesis, invasion, and drug resistance(5–7). However, such potential antitumor activity of calcitriol isachieved only when it is given in supraphysiologic doses thatwill lead to significant hypercalcemia and hypercalciuria sideeffects that hinder and impede its clinical usefulness for thispurpose (7, 8). As a clinically relevant task, synthesis of non- orless calcemic vitamin D analogues has therefore been initiated toachieve or even potentiate the antiproliferative/tumoricidal prop-erties of calcitriol but precludes its calcemic side effects (7, 8). In

1Department of Laboratory Medicine, Faculty of Applied MedicalSciences, Umm Al-Qura University, Holy Makkah, Saudi Arabia.2Department of Pharmacology, Faculty of Medicine, Assiut University,Assiut, Egypt. 3Department of Clinical Nutrition, Faculty of AppliedMedical Sciences, Umm Al-Qura University, Holy Makkah, Saudi Ara-bia. 4Department of Animal Medicine, Faculty of Veterinary Medicine,Assiut University, Assiut, Egypt.

Corresponding Author: Adel Galal El-Shemi, Associate Professor, Departmentof Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-QuraUniversity, PO Box 7607, Holy Makkah, Saudi Arabia; and Department ofPharmacology, Faculty of Medicine, Assiut University, Egypt. Phone: 9665-0965-5135; Fax: 9661-2 527-0000, ext. 4242; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-15-0439

�2016 American Association for Cancer Research.

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spite of their well-known vitamin D receptor (VDR)-mediatedactions, their precise mechanisms and why specific analogueshave superagonistic effects on specific tissues or diseases still haveto be deciphered (8). Among these analogues, paricalcitol (19-nor-1a-25-dihydroxyvitamin D2), a direct acting VDR activatorthat is clinically approved by the FDA for the treatment ofsecondary hyperparathyroidism, has recently gained attention ona variety of disease modalities, including cancer, due to its lesscalcemic effects, wider therapeutic window, and an equipotentialactivity as calcitriol in several in vivo and in vitro systems (8–11). Incancer patients, paricalcitol may have potential safety and feasi-bility in women with metastatic breast cancer (12), and in menwith advanced prostate cancer (13). Paricalcitol has also demon-strated potential suppressive effects on a variety of human cancercells and preclinical tumormodels such as pancreatic cancer (14),gastric cancer and peritoneal metastasis (10), uterine fibroids(15), androgen-dependent prostate cancer cell model (16), andhuman leukemic cells (17).

Taken together, paricalcitol may be a potential chemopreven-tive agent in cancer therapy, especially for patients with lowresponse or who fail in conventional therapies (10, 11, 15).However, there remains insufficient information concerning itsbenefits on colorectal cancer. Therefore, the current study wasaimed to investigate whether paricalcitol would improve andsynergize the anti–colorectal cancer effect of 5-FU, and toidentify the possible mechanisms underlying such synergy onan intermediate-term model of colorectal neoplasia induced byazoxymethane (AOM) in rats. AOM-induced colorectal tumorsand carcinogenesis in rats and mice have been proven as anoutstanding rodent model that closely mirror the phases andfeatures of human colorectal cancer [aberrant crypts foci (ACF)–adenoma–adenocarcinoma and carcinoma sequence] in a time-dependent manner post-AOM injection, and commonly used toassess new chemopreventive and therapeutic strategies and toprovide new insights into the pathophysiologic mechanismsand risk factors of human colorectal cancer (18–20). Ourfindings showed that monotherapy with either 5-FU or pari-calcitol resulted in a significant chemopreventive effect on thismodel; however, their combination exhibited amore significantefficacy to repress the morphologic, histopathologic, andmolecular changes that were observed in this model. Furtherstudies are still essential to realize the potential clinical value ofthis combination in human patients with colorectal cancer.

Materials and MethodsDrugs and chemicals

Paricalcitol (Zemplar 5 mg/mL vials) was obtained from Abb-Vie Ltd., while AOM and 5-FU were purchased from Sigma-Aldrich. All other chemicals used were of the highest commercialgrade.

Animals, induction of colorectal tumorigenesis, and treatmentapproach

All experimental protocols and procedures of the current studywere approved by the Institutional Animal Care and Use Com-mittee at the University of Umm Al-Qura (Holy Makkah, SaudiArabia), and in accordance with the US Public Health ServicePolicy onHumane Care andUse of Laboratory Animals. A total of75 adult male Wistar rats, weighing 220–250 g, were housed inclean, sterile, and polyvinyl cages (5 rats/cage), maintained on

standard laboratory pellet diet and water ad libitum, and kept in atemperature-controlled air conditioned environment at 22–24�Cwith a 12-hour dark/light cycle.

For induction of colorectal tumorigenesis, AOM was dis-solved in normal saline and injected subcutaneously at a doseof 15 mg/kg, once weekly for 2 weeks as described previously(18). According to the subsequent treatment schedules, the ratswere randomly categorized into the following 5 groups (15 rats/group): group 1 (normal controls): received only normal saline;group 2 (AOM group): AOM-injected rats and left withouttreatment; group 3 (5-FU group): AOM-injected rats and thentreated with 5-FU; group 4 (paricalcitol group): AOM-injectedrats and then treated with paricalcitol; and group 5 (5-FUþparicalcitol group): AOM-injected rats and then treated with 5-FU plus paricalcitol combination therapy. In its designatedgroups, 5-FU was freshly prepared in normal saline and injectedintraperitoneally (i.p.) during the 9th and 10th weeks post-AOM injection in a dosage regime similar to that used in thetreatment of human colorectal cancer (12 mg/kg/day for suc-cessive 4 days, then 6 mg/kg every other day for 4 doses), whileparicalcitol was administered in a dosage regimen of 2.5 mg/kg/day i.p., 3 days/week; starting from the 7th week post-AOMinjection and continued till the end of the study (week 15 post-AOM injection). The dose of paricalcitol was chosen on thebasis of our tested pilot experiments and previously publishedreports (14).

Blood sampling and isolation of whole colonAt the end of the study, rats of the different groups were

weighed, fasted overnight, and then euthanized under diethylgeneral anesthesia (Fisher Scientific UK Ltd). After euthanasia,blood sample (4mL) was collected from the rat's vena cava into aplain tube, and used to measure the serum levels of calcium, 25-OH vitamin D, kidney function tests (creatinine, BUN, and urea),and liver function enzymes [alkaline phosphatase (ALP), alaninetransaminase (ALT), and aspartate aminotransferase (AST)] byusing Cobas e411 (Roche Diagnostics International Ltd), accord-ing to the manufacturer's instructions. Subsequently, the wholecolon fromrectum to cecumwas gently resected,flushedwithPBS,and slit opened longitudinally. The surface area of each isolatedcolon (length�width in cm2) wasmeasured, and then the wholecolon was immersed in 10% (v/v) neutralized formalin forovernight between layers of filter papers with the mucosa on theupper side.

Quantification of grown tumors and large ACF in the colorectaltissues

The grown tumors on the mucosae of the isolated colons wereblindly counted by the naked eye by two observers. Next, thecolon was cut into 3 portions: proximal, middle, and distalsegments; and each segment was stained with 0.2% methyleneblue solution for 1.5–2 minutes, placed on a microscope slidewith the mucosal side upward, and examined under a dissectingmicroscope to count the small tumors, that were not detected bythe naked eye, and large ACF (containing 4 or more aberrantcrypts) according to previously published criteria (21). Aftercounting process, a micro-feather scalpel blade was used underthe dissecting microscope to excise the colorectal specimens thathad tumors and ACF from the surrounding normal tissues to beused for the subsequent histopathologic, molecular, ELISA, andimmunohistochemical examinations.

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Histopathologic examinationThe colorectal tissue specimens were first destained frommeth-

ylene blue by 80% ethanol, and then processed for paraffinembedding and sectioning (4–5 mm tissue sections) and hema-toxylin and eosin (H&E) staining by using the conventionalmethods. The stained tissue sections were microscopically exam-ined for the presence of ACF, colonic adenomas, and adenocarci-nomas. On the basis of the crypt architecture and nuclear features,ACF were microscopically classified into hyperplastic ACF (nodysplasia) or dysplastic ACF (elongated, crowded, and pseudos-tratified nuclei, increased nucleus-to-cytoplasm ratio; reducednumber of goblet cells; back-to-back glands; and markedlydecreased interglandular stroma) as previously identified byestablished criteria (22). A colonic adenoma consists of prolifer-ative and hyperplastic glands, whereas colonic adenocarcinoma iscomposed of dysplastic glands invading the submucosa andmuscular layers (20).

RNA extraction, cDNA synthesis, and qRT-PCR analysisColorectal specimens were homogenized in RNAlater solution

(Ambion), and then total RNA was isolated by using the PurelinkRNAMini Kit (Applied Biosystems), following themanufacturer'sinstructions. The quality and the quantity of the extracted RNAwere measured by the Nanodrop equipment (BioSpec-nano,Shimadzu Corporation). Up to 200 ng of the extracted totalRNA was employed in the reverse transcription step for cDNAsynthesis and by using a high capacity RNA-to-cDNA ReverseTranscription Kit (Thermo Fisher Scientific). qRT-PCR was con-ducted using the 2�DDCt method on the following target rat genes:Wnt (NM_001105714.1), b-Catenin (AF397179.1), Dickkopf-1[DKK-1; (NM_001106350.1)], Cyclin-dependent kinase inhibi-tor 1A (CDNK-1A; NM_080782.3), NF-kB (NM_001008349.1),and COX-2 (AF233596.1). In addition, b-actin (NM_031144.3)was used as an internal reference (housekeeping gene) to stan-dardize the data of the target genes. The nucleotide primersequences of these seven rat origin genes were summarizedin Table 1. All reactions were performed in triplicate and usingPower SYBR Green Master Mix (Applied Biosystems, ThermoFisher Scientific) and the StepOnePlus Real-Time PCR System(Applied Biosystems). Briefly, 10 mL SYBR Green, 7 mL DNase/RNase–free water, 1 mL of each primer (5 pmol), and 1 mL cDNA(25 ng) were mixed in each well of the PCR plate, and theamplification was conducted under the following conditions:40 cycles (15 seconds at 95�C and 1 minute at 65�C). Data wereanalyzed using a comparative threshold cycle (Ct) technique,normalized against the Ct values of b-actin and expressed asfold-change compared with the normal control group.

ELISAThe levels of b-catenin, COX-2, HSP-90, and VEGF were quan-

titatively measured in the colorectal tissue specimens by ELISA

technique. Briefly, the tissue specimens were homogenized inRIPA lysis buffer containing protease inhibitors (Santa CruzBiotechnology Inc), centrifuged, and then their harvested super-natants were stored in �20�C until use. During the assays, thetotal protein concentration in each samplewas adjusted tomake afinal concentration of 500 mg/mL, and the concentrations of thecandidate proteinsweremeasured byusing commercial ELISA kits(Cusabio) and a fully automated ELISA system (Human Diag-nostics), according to themanufacturers' instructions. All sampleswere processed in duplicate and the results are presented as ng/mLfor HSP-90, pg/mL for b-catenin and VEGF, and pmol/mL forCOX-2.

Immunohistochemical analysisImmunohistochemical staining for b-catenin, HSP-90, and

inducible nitric oxide synthase (iNOS) was performed onparaffin sections following the conventional protocol. Theprimary antibodies used were as follows: polyclonal goat IgGantibodies (1: 100) against rat HSP-90-a/b (N-17), iNOS (N-20), and b-catenin (C-18; Santa Cruz Biotechnology Inc).Biotinylated anti-goat secondary antibodies (1:200) conjugatedwith horseradish peroxidase complex (Santa-Cruz Biotechnol-ogy Inc) were used, and staining process was developed by DABchromogen substrate and counterstained with Gill hematoxy-lin. The intensity of staining was assessed using H-score for-mula as follows: H-score ¼ P

Pí (í þ1), where í represents theintensity of positively stained cells (0 ¼ negative; 1 ¼weak; 2 ¼moderate; and 3 ¼ strong) and Pí is the percentage (0%–100%)of positively stained cells (23).

Statistical analysisResults were expressed as mean � SD. Comparisons of data

between groups were made using one-way ANOVA, with post hoccomparisons using Dunnett multiple comparison test. The dif-ference between datawere considered to be statistically significantwhen P < 0.05, and to be very significant when P < 0.01.

ResultsInhibitory effects of paricalcitol and/or 5-FU on colorectaltumor growth and large ACF formation

In the current study, the chemopreventive effects of mono- andcombination therapy with paricalcitol (Pcal) and 5-FU in inhibit-ing the early colorectal tumorigenesis stages and tumor growthwere examined in an intermediate-term model (15 weeks) ofAOM-induced colorectal carcinogenesis in rats. As shown in Figs.1 and 2, in comparison with normal controls (Figs. 1 and 2; panel1A), rats received AOM and left with treatment developed asignificant number of gross tumors and distortions on theircolorectal mucosae (Figs. 1 and 2; panel 2A). However, treatmentof these AOM-injected rats with either 5-FU (Figs. 1 and 2; panel3A) or paricalcitol (Figs. 1 and 2; panel 4A) had significantly

Table 1. Primer sequences used in the qRT-PCR for detection of the transcription activities of Wnt, b-catenin, DKK-1, CDNK-1A, NF-kB, COX-2, and b-actin genesincluding the corresponding genes accession numbers

Gene Forward Reverse

Wnt (NCBI: NM_001105714.1) 50-AGC TGG GTT TCT GCT ACG TT-30 50-AAT CTG TCA GCA GGT TCG TG-30

b-Catenin (NCBI: AF397179.1) 50-TTC CTG AGC TGA CCA AAC TG-30 50-GCA CTA TGG CAG ACA CCA TC-30

DKK-1 (NCBI: NM_001106350.1) 50-ATT CCA GCG CTG TTA CTG TG-30 50-GAA TTG CTG GTT TGA TGG TG-30

CDNK-1A (NCBI: NM_080782.3) 50-AGA AGG GAA CGG GTA CAC AG-30 50-ACC CAT AAG AAG GGC AGT TG-30

NF-kB (NCBI: NM_001008349.1) 50-CAG AGC TGG CAG AGA GAC TG-30 50-TAC GAA GGA GAC TGC CAC TG-30

COX-2 (NCBI: AF233596.1) 50-AAT CGC TGT ACA AGC AGT GG-30 50-GCA GCC ATT TCT TTC TCT CC-30

b-actin (NCBI: NM_031144.3) 50-CGG TCA GGT CAT CAC TAT CG-30 50-TTC CAT ACC CAG GAA GGA AG-30

Paricalcitol Potentiates the Efficacy of 5-FU Against CRC

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decreased the number of the grown tumors and the topographicmucosal alterations. More importantly, the lowest number ofgrown tumors was observed in rats treated with 5-FU/paricalcitolcombination therapy (Figs. 1 and 2; panel 5A). Furthermore,examination of the resected colons of the different studied groupsby dissecting microscope following methylene blue stainingshowed normal mucosal and crypt appearance in the normalcontrol group (Fig. 2; panel 1B), but colons of rats injected withAOM and not treated developed several micro-tumors and ACF(Fig. 2; panel 2B). On the other hand, the number of these micro-tumors and ACF were significantly diminished when these dis-eased animals were treated with either 5-FU (Fig. 2; panel 3B) orparicalcitol (Fig. 2; panel 4B), and dual therapy with paricalcitoland 5-FUpreserved/restored the normalmucosal architecture andwas associated with the highest reducing effect on the develop-ment of such micro-tumors and ACF (Fig. 2; panel 5B).

The histopathologic findings (Fig. 2) were also consistent withthe macroscopic/microscopic observations and showed the pres-ence of many large ACF (>4 crypt/focus) with hyperplastic anddysplastic features, and multiple tubular adenomas in the colo-rectal tissues of rats injected with AOM and left without anytreatment (AOM group; Fig. 2; panel 2C). However, treatment ofthese AOM-injected rats with either 5-FU (Fig. 2; panel 3C),paricalcitol (Fig. 2; panel 4C), or 5-FU plus paricalcitol (Fig. 2;

panel 5C) had attenuated the development of such large ACF andtubular adenomas; and the highest attenuating effect wasobserved with 5-FU/paricalcitol dual therapy. Taken together,these results indicate that therapy with paricalcitol not onlyattenuates colorectal cancer initiation, but is also able to enhancethe chemopreventive efficacy of 5-FU in this disease modality.

qRT-PCR findingsTo provide mechanistic insights into the observed chemo-

preventive effects of paricalcitol and 5-FU therapy on thisrodent model of the early stages of colorectal cancer, geneexpression study was conducted by qRT-PCR to determine therelative mRNA expression patterns of Wnt, b-catenin, NF-kB,and COX-2 pro-oncogenes that have important role in colo-rectal cancer development and progression, as well as of DKK-1and CDNK-1A as examples of well-established colorectal can-cer–suppressive genes. As illustrated in Fig. 3, there was asignificant upregulation in the mRNA expression ofWnt, b-cate-nin, NF-kB, and COX-2 genes, and a significant decrease in thein the mRNA expression of DKK-1 and CDNK-1A in AOMgroup, in comparison with normal control group. In contrary,treatment of the diseased animals with paricalcitol and 5-FUhad synergistically cooperated to modify the altered mRNAexpression patterns of these target genes (Fig. 3).

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Figure 1.Effects of paricalcitol (Pcal) and/or 5-FU on the numbers of grown tumors (A–C) and large aberrant crypts foci (ACF; D) in the colorectal tissues of AOM-induced ratcolorectal tumors. The grown tumors on colorectal tissues were counted by the naked eye (A), and under a dissectingmicroscope after methylene blue staining (B).Large ACF containing 4 or more aberrant crypts (D) were also counted under a dissecting microscope following methylene blue staining. Data are representedas mean � SD. a, P < 0.05 versus AOM group; b, P < 0.05 versus AOM/5-FU group; c, P < 0.05 versus AOM/paricalcitol group; and d, P < 0.01 versus.AOM group.

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ELISA and IHC findingsTo further explore the possible underlying mechanisms that

could mediate the observed chemopreventive effects of paricalci-tol and 5-FU therapy, we quantitatively measured the proteinconcentrations of b-catenin, COX-2, HSP-90, and VEGF by ELISAin the colorectal tissue homogenates of the different groups.Compared with the normal control group, the concentrations ofb-catenin (Fig. 4A), HSP-90a (Fig. 4B), COX-2 (Fig. 4C), andVEGF (Fig. 4D) were significantly elevated in the colorectal tissuehomogenates of AOM group. However, the concentrations ofthese candidate molecules were significantly reduced when theseAOM-injected rats were treated with either paricalcitol or 5-FU.

Interestingly, cotherapywith paricalcitol and 5-FUhad resulted inmore reduction in the colorectal levels of b-catenin, COX-2, HSP-90, and VEGF proteins compared with paricalcitol or 5-FUmono-therapy (Fig. 4).

Data of immunohistochemical assays (Fig. 5) were also insymmetry with ELISA results and demonstrated low expressionof b-catenin (Fig. 5; panels 1A and 1F), iNOS (Fig. 5; panels 2Aand 2F), and HSP-90 (Fig. 5; panels 3A and 3F) in the colorectaltissues of normal controls. In contrast, a marked expression ofeither b-catenin (Fig. 5; panels 1B and 1F), iNOS (Fig. 5; panels2B and 2F), or HSP-90 (Fig. 5; panels 3B and 3F) was observedin the colorectal tissues of rats injected with AOM and left

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Figure 2.Representative photos of macroscopicand microscopic appearance ofcolorectal mucosa of (1) normal controlgroup, (2) AOM group, (3) AOM/5-FUgroup, (4) AOM/paricalcitol group, and(5) AOM/5-FU/paricalcitol group. Thecolorectal mucosae of the differentgroups were examined by gross (panelA), dissecting microscopy followingstaining with 0.2% methylene blue(panel B), and light microscopy atmagnifications �100 and �200following staining with H&E (panel C).Black arrowhead, gross tumorsobserved by naked eye; yellowarrow, large ACF (>4 crypts/focus).Pcal, paricalcitol.






without treatment, particularly in the glandular epithelium,and the combination treatment with 5-FU and paricalcitol wassynergistically interacted to diminish the expression of theseprocancerous proteins in the colorectal tissues of AOM-injectedrats (Fig. 5; panels 1E and 1F, 2E and 2F, and 3E and 3F,respectively).

The biochemical findingsThe synthesis of vitamin D analogues, including paricalcitol,

was initiated to achieve the therapeutic properties of calcitriolbut precluded its hypercalcemic side effects (8, 9). In agree-ment, the biochemical analyses of the current study (Table 2)did not show any significant differences in the serum levels ofcalcium, liver function enzymes, and renal function parametersamong the different animal groups, confirming the noncalce-mic property of the applied paricalcitol dosage regimen, alsosuggesting the hepato-renal safety of paricalcitol/5-FU combi-nation therapy.

Discussion5-FU, either alone or in combination with other chemothera-

peutic agents, remains the standard drug in the treatment ofhuman colorectal cancer; however, low response rates and devel-opment of resistance to 5-FU therapy still represent a majorchallenge (2–4). On the other hand, the potential antitumorproperties of paricalcitol, a synthetic less calcemic vitamin Danalogue directly activates VDR, have recently attracted a specificdeal of attention (9, 10, 13–16).Herein, an intermediate-term (15weeks) model of colorectal tumorigenesis was induced in rats byAOM, a commonly used in vivomodel for the experimental studyof human colorectal cancer at several aspects (18–20); our studywas designed to investigate the chemopreventive efficacy ofparicalcitol and 5-FU alone and in combination, and whethertheir cotherapy resulted in an enhanced chemopreventive effectthan individual therapy on this model. Interestingly, in compar-ison with their monotherapy, cotherapy with paricalcitol and 5-FU had resulted in augmenting effects in inhibiting the formation

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Figure 3.qRT-PCR findings show themodulatory effects of paricalcitol (Pcal), 5-FU, and their combinations on the relativemRNA expression ofWnt (A), b-catenin (B),NF-kB(C), COX-2 (D), DKK-1 (E), and CDKN1A (F) genes in AOM-induced rat colorectal tumors. a, P < 0.01 versus normal controls; b, P < 0.05 versus normalcontrols; c, P < 0.05 versus AOM group; d, P < 0.05 versus AOM/5-FU group; e, P < 0.05 versus AOM/paricalcitol group; and f, P < 0.01 versus AOM group.

El-Shemi et al.





of preneoplastic large ACF and the grade of cellular dysplasia, andin reducing the number of grown colorectal tumors, whichcollectively are the main macroscopic and microscopic featuresduring the short- to intermediate term of AOM model (18–20).Moreover, paricalcitol and 5-FU had also synergized to modulatea number of molecular pathways and candidate molecules thattheir dysregulations play crucial roles in the development andprogression of human and experimental colorectal cancer diseasesuch as Wnt/b-catenin pathway (24, 25), DKK-1 (26–29),CDKN1A (30, 31), NF-kB (32–34), COX-2 (35), iNOS (33),VEGF (35), and HSP-90 (2, 36). In turn, our findings not onlysupport the importance of paricalcitol as an adjuvant agent incancer therapy (10, 12–16), but also are in harmony with thehypothesis that vitamin D or its analogues improve tumor cellsensitivity and tumoricidal efficacy of 5-FU (6, 7).

Development and progression of colorectal cancer is multigen-ic andheterogeneous inorigin, and also has clinical importance aspredictors of disease prognosis and treatment response (37). Inthis view, aberrant activation ofWnt/b-catenin signaling pathwayis highly implicated in the induction and dissemination of mosthuman cancers, including colorectal cancer (24), and significantallocation in thedevelopment of 5-FUandmultidrug resistance inhuman cancer therapy (38, 39). Mechanistically, upon binding

with their target plasma membrane receptors/coreceptors, Wntproteins induce a series of downstream signaling events resultingin b-catenin dephosphorylation and stabilization. This allowsb-catenin, the main effector protein of this signaling, to translo-cate into the nuclei wherein it stimulates the transcription ofseveral oncogenes, particularly in cells derived from the intestinalcrypts (25, 40). At this point, approximately 80% of all humancolorectal cancers have aberrant overactivations inWnt/b-cateninsignal and its downstream components in the colorectal cells(25). On the other hand, the expression pattern of Dkk-1, aninhibitor of Wnt/b-catenin pathway, by blocking Wnt signalingreceptor complexes and contributing to colon cancer suppression,is remarkably downregulated in the colonic biopsies of colorectalcancer patients, and its downregulation is disclosed as a biomark-er of chemoresistance and poor clinical outcome (25–29).Although the overall available data still have discrepancies,Dkk-1 has been found to not only act as an inhibitor of Wnt/b-catenin signaling but also has additional b-catenin–indepen-dent tumor suppressor, antiangiogenesis, and antimetastasisactions in colorectal cancer disease (27, 41). Likewise, CDNK-1A, a tumor suppressor gene encoding a potent cell-cycle inhib-itory factor (CDKN1A, p21, or CIP1), is downregulated or evenlost in most colorectal cancer cases (30), and some colorectal

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Figure 4.ELISA findings of the modulatory effects of paricalcitol (Pcal), 5-FU, and their combinations on the protein concentrations of b-catenin (A), HSP90 (B), COX-2 (C),and VEGF (D) in the colorectal tissues of AOM-induced rat colorectal tumors. Data are represented as mean � SD. a, P < 0.01 versus normal controls; b,P < 0.05 versus normal controls; c, P < 0.05 versus AOM group; d, P < 0.05 versus AOM/5-FU group, e, P < 0.05 versus AOM/paricalcitol group; and f, P < 0.01 versusAOM group.






cancer patients have anti-CDKN1A autoantibodies in their colo-rectal tissues (31). On the basis of these facts, it is conceivable thattargeting these crucial colorectal tumorigenesis pathways,through repression of Wnt/b-catenin activity and/or stimulationof Dkk-1 and CDNK-1A, may hold tremendous therapeuticpotential in treating colorectal cancer and other cancers, andin enhancing the cytotoxic effects of chemotherapeutic agents(25, 41). Interestingly, data of the current study are in agreementand showed that cotherapy with paricalcitol and 5-FU signifi-cantly interacted to repress the overexpressed Wnt and b-catenin,and upregulated the decreased Dkk-1 and CDNK-1A in the

colorectal tissues of the chemically induced colorectal cancermodel, suggesting a cooperative mechanism between the twodrugs that, in part, might be behind their observed tumoricidaleffect. In support of our observations, paricalcitol therapy haspreviously shown tomediate blockade ofWnt/b-catenin signaling(40) and upregulate CDNK-1A (p21; ref. 14), and 1,25(OH)2D3(the most active form of vitamin D) is a multilevel repressor ofWnt/b-catenin signaling pathway and its downstream targetproinflammatory and oncogenes (42). Furthermore, Aguileraand colleagues (28) reported that in a dose- and VDR-dependentmanner, 1a,25-dihydroxyvitamin D3 activates the humanDKK-1

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Figure 5.Findings of immunohistochemical assaysdemonstrate the modulatory effects ofparicalcitol (Pcal), 5-FU, and theircombinations on the proteinconcentrations of b-catenin (A), HSP90(B), and iNOS in the colorectal tissues ofazoxymethane (AOM)-induced ratcolorectal tumors (C). Data arerepresented as mean � SD. a, P < 0.01versus normal controls; b,P<0.05 versusnormal controls; c, P < 0.05 versus AOMgroup; d, P < 0.05 versus AOM/5-FUgroup; e, P < 0.05 versus AOM/Pcalgroup; and f, P < 0.01 versus AOM group.

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gene promoter with subsequent high expression levels of DKK-1RNA and protein in different phenotypes of human colon cancercells. Furthermore, in vivo therapy with a less calcemic vitamin Danalogue in a xenografted model of colorectal cancer in immu-nodeficient mice has resulted in antitumor action associated withsignificant induction of DKK-1 gene transcription and inhibitionof Wnt/b-catenin signaling (28).

Indeed, human colorectal cancer is a life-threatening compli-cation of inflammatory bowel diseases with a complex patho-genesis, in which NF-kB, COX-2, and iNOSmay provide a crucialmechanistic link between inflammation and carcinogenesis(32, 33). With this concept, aberrant activation of NF-kB hasbeen shown to regulate the expression of many tumorigenesisgenes involved in cellular transformation, proliferation, inflam-mation, angiogenesis, invasion, metastasis, and numerous otherpotentially carcinogenic processes (43). NF-kB induces COX-2gene expression and drug resistance genes (10), and COX-2upregulation directly correlates with colorectal cancer progressionand poor prognosis in human patients (35). iNOS levels andactivities are also increased in human adenocarcinomas and incolon tumors chemically induced in rats, to induce chronicinflammation and to create amicroenvironment that favors coloncarcinogenesis (33). Moreover, angiogenesis mediated by VEGFand other pivotal angiogenic facilitators plays a crucial role indevelopment, neovascularization, progression and metastasis ofcolorectal cancer and other human cancers, andCOX-2 appears tobe importantly involved with multiple aspects in this phenom-enon, particularly by overexpressing VEGF (35). Coherently,suppression of NF-kB, iNOS, COX-2, and VEGF could representtherapeutic potential in colorectal cancer therapy (35, 43). Inharmony, data of the current study showed that paricalcitol and 5-FU alone or in combination significantly decreased the expressionof NF-kB, iNOS, COX-2, and VEGF in the colorectal tissues ofdiseased rats, and their combination was cooperatively acted tofurther downregulate these tumorigenesis molecules. In agree-ment with our findings, several lines of evidence have suggestedthe robust capacity of either vitamin D or its analogues, includingparicalcitol, to disruptNF-kB, COX-2, VEGF, and/or iNOS-depen-dent inflammation, tumor promotion, and carcinogenesis. Forinstance, paricalcitol treatment had suppressedCOX-2 andNF-kBexpression in colon, leukemic, and gastric cancer cells (10, 44),and attenuated VEGF in renal diseases (45). In a constant line,vitaminDhas intrinsically shown to inhibitNF-kB activity, reduceNF-kB protein levels, and downregulate a variety of NF-kB targetgenes in a variety of inflammatory and cancer cell types (46, 47).Mechanistically, activation of VDR by vitamin D or its analoguesresults in VDR/IkB kinase beta protein (IKKb) physical interaction

and stimulation/stabilization of the NF-kB inhibitory protein a(IkBa), all ofwhich are responsible for blockingNF-kBbinding toDNA and inhibition of its canonical activation pathway (47).Similarly, earlier in vitro studies on cancer cells revealed that 5-FUexerts direct inhibitory effect on NF-kB and on nitric oxideproduction mediated by NF-kB (48, 49). Administration ofselective inhibitors of NF-kB (34), or COX-2 (50) had previouslyseen to augment the antitumor effects of 5-FU on colon cancer,and was also associated with suppression of VEGF and the tumorvessel density (50). In short, we may speculate that there was adirect and indirect crosstalk between paricalcitol and 5-FU ininhibiting NF-kB and concomitantly in repressing the colorectaltumorigenesis effects of iNOS, COX-2, and VEGF.

Notably, the potential carcinogenic role ofHSP-90 in activationof various oncogenic proteins and growth factors is currently ofintense interest, particularly in colorectal cancer, which is whyspecific HSP90 inhibitors are currently being investigated aspotential anticancer drugs (36).More importantly, HSP90 depen-dence acquired resistance to 5-FU therapy, as well as its potentialrole as a proangiogenic factor for the induction of VEGF and iNOSfor de novo angiogenesis has been reported recently (2). Withthis concept, treatment of 5-FU–resistant colon cancer cellswith selective HSP90 inhibitors had repressed primary colorectaltumor growth, circulation in the blood, and metastatic tumordevelopment (2). Remarkably, our data are consistent andshowed a significant increase in HSP90 expression in the AOMgroup compared with normal controls, and cotreatment withparicalcitol and 5-FU resulted in a more significant repressionon HSP90 expression compared with their monotherapy. On thebasis of our findings and on the given potential carcinogenic roleof HSP-90 in colorectal cancer, we therefore reasoned to hypoth-esize that paricalcitol could promote the efficacy of 5-FU and theregression of the chemically induced colon cancer partly bydownregulating the expression of HSP90, although the currentavailable data on the possible effects of vitaminDor its analogueson this protein in colorectal cancer or other cancers are limited.

In conclusion, this study suggests that paricalcitol augmentsthe therapeutic efficacy of 5-FU on colorectal cancer disease bymodulating several pro- and anticancerous molecules thathave important roles in the regulation of colorectal tumorcells' DNA damage, growth, differentiation, inflammation,angiogenesis, and metastasis. These molecular pathwaysinclude Wnt/b-catenin pathway, NF-kB, COX-2, iNOS, VEGF,and HSP90. In this context, our findings have therapeuticimpact considering ongoing clinical development of paricali-tol/5-FU combination therapy for the treatment of colorectalcancer patients; however, further studies are warranted to

Table 2. Effects of paricalcitol and/or 5-FU on the serum levels of calcium, liver function enzymes, and renal function parameters, and on body weight in AOM-induced rat colorectal tumors

Parameters Controls AOM AOMþ5-FU AOMþPcal AOMþPcalþ5-FU

Calcium (mg/dL) 9.23 � 0.71 9.05 � 0.83 9.12 � 0.33 9.92 � 0.36 9.33 � 0.45ALP (IU/L) 122.6 � 11.2 125.7 � 9.7 120.8 � 12.4 117.3 � 13.5 119.3 � 12.4ALT (U/L) 67 � 2.4 71.2 � 6.7 68.7 � 4.1 67.3 � 4.1 65.8 � 3.4AST (U/L) 92.4 � 24.2 105.8 � 26.7 109 � 21.6 101 � 18.6 97 � 12.1Creatinine (mg/dL) 0.22 � 0.03 0.2 � 0.06 0.2 � 0.03 0.19 � 0.03 0.21 � 0.05Urea (mg/dL) 48.6 � 5.1 50.3 � 4.3 50.6 � 9.5 48.3 � 5.8 49.0 � 3.7BUN (mg/dL) 22.2 � 2.4 24.4 � 1.9 26.3 � 4.4 22 � 2.7 21.4 � 2.1Body weight (g) 231.5 � 20 221.9 � 23.01 238.4 � 13.6 228.3 � 19.2 230.7 � 19.4

NOTE: Data are represented as mean � SD.Abbreviations: AOM, Azoxymethane; 5-FU, 5-Fluorouracil; Pcal, Paricalcitol.






evaluate this therapeutic combination and also to explore itsprecise antitumor mechanisms.

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

Authors' ContributionsConception and design: A.G. El-Shemi, O.A. Kensara, A.M. MohamedDevelopment of methodology: A.G. El-Shemi, B. Refaat, A.M. Mohamed,S. Idris, J. AhmadAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.G. El-Shemi, B. Refaat, O.A. Kensara,A.M. Mohamed, S. Idris, J. AhmadAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.G. El-Shemi, B. RefaatWriting, review, and/or revision of the manuscript: A.G. El-Shemi,A.M. Mohamed

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Refaat, O.A. Kensara, S. Idris, J. AhmadStudy supervision: A.G. El-Shemi, B. Refaat, O.A. Kensara

AcknowledgmentsThe authors thank Mr. M. Al-Malki and Mr. H. Alshareef (Faculty of Applied

Medical Sciences, Umm Al-Qura University) for processing the samples.

Grant SupportThis project was funded by theNational Science, Technology and Innovation

Plan (MARRIFAH)-King Abdul Aziz City for Science and Technology (KACST),the Kingdomof Saudi Arabia (award number 12-MED2965-10). A.G. El-Shemi,B. Refaat, O.A. Kensara, and A.M. Mohamed are the recipients of the grant.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 31, 2015; revised February 2, 2016; accepted March 17,2016; published OnlineFirst March 28, 2016.

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2016;9:491-501. Published OnlineFirst March 28, 2016.Cancer Prev Res Adel Galal El-Shemi, Bassem Refaat, Osama Adnan Kensara, et al. Azoxymethane-Induced Colorectal Tumors in Rats5-Fluorouracil on an Intermediate-Term Model of Paricalcitol Enhances the Chemopreventive Efficacy of

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