Paricalcitol Enhances the Chemopreventive Efficacy of 5-Fluorouracil on an Intermediate-Term Model of Azoxymethane-Induced Colorectal Tumors in Rats

Published OnlineFirst March 28, 2016; DOI: 10.1158/1940-6207.CAPR-15-0439

Research Article Cancer Prevention Research Paricalcitol Enhances the Chemopreventive Efﬁcacy of 5-Fluorouracil on an Intermediate- Term Model of Azoxymethane-Induced Colorectal Tumors in Rats Adel 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. COX-2 genes, and ELISA was used to quantify the protein levels Despite being the standard anti–colorectal cancer drug, 5-fluoro- of b-catenin, COX-2, HSP90, and VEGF. IHC was additionally uracil (5-FU) exhibits only limited therapeutic benefits. Herein, used to measure b-catenin, HSP90, and inducible nitric oxide we investigated whether paricalcitol, a synthetic vitamin D ana- synthase (iNOS). Compared with their individual therapy, com- logue with potential antitumor properties, would enhance the bination of 5-FU and paricalcitol showed more significant reduc- chemopreventive efficacy of 5-FU on an intermediate-term (15 ing effect on numbers of grown tumors and large aberrant crypts weeks) model of colorectal tumors induced by azoxymethane foci. Mechanistically, paricalcitol and 5-FU had cooperated (AOM) in rats. After AOM injection, 5-FU was administered together to repress the expression of procancerous Wnt, b-catenin, during the 9th and 10th weeks (12 mg/kg/day for 4 days, then NF-kB, COX-2, iNOS, VEGF, and HSP-90 more, and to upregulate 6 mg/kg every other day for another 4 doses), whereas paricalcitol the expression of antitumorigenesis DKK-1 and CDNK-1A, com- (2.5 mg/kg/day; 3 days/week) was given from the 7th to the 15th pared with their monotherapies. Our findings suggest that com- week. At week 15, the animals were euthanized and their resected bined use of paricalcitol with 5-FU exhibits an augmenting colons were examined macroscopically and microscopically. chemopreventive effect against colorectal tumors, and might Quantitative RT-PCR was used to measure the transcription potentially be useful for chemoprevention in colorectal cancer activities of Wnt, b-catenin, DKK-1, CDNK-1A, NF-kB, and patients. Cancer Prev Res; 9(6); 1–11. 2016 AACR.

Introduction 17 % with its monotherapy and 35%–39% when given with other chemotherapeutic agents (3, 4). More importantly, According to the World Health Organization's International although the recent addition of new targeted agents, such as Agency for Research on Cancer, colorectal cancer represents the bevacizumab and cetuximab, to the standard chemotherapy third most frequent cancer in men and second in women, ranking provides hope for more effective therapy in advanced colorectal as the fourth leading cause of cancer-related deaths worldwide cancer, they have shown only modest benefitandaresubjectto (1). The limited therapeutic efficacy of its current chemotherapy both primary and secondary resistance, like traditional chemo- represents the most important challenge in colorectal cancer therapy, which ultimately leads to treatment failure (4). Thus, management. With this aspect, 5-fluorouracil (5-FU)-based development of potential alternative or combinational colo- therapy, either alone or in combination with other cytotoxic rectal cancer chemopreventive and therapeutic strategies is a agents (e.g., irinotecan, leucovorin, or oxaliplatin), remains the paramount medical demand. To that end, vitamin D and its standard approach (2–4). Overall, 5-FU still exhibits limited analogues are the most attractive agents in this setting (5, 6). efficacy with low tumor response rate ranging between 7% and There is a compelling body of evidence that calcitriol [1,25 (OH)2D3], the active form of vitamin D, may not only reduce the 1Department of Laboratory Medicine, Faculty of Applied Medical risk of colorectal cancer and other human cancers but also repress Sciences, Umm Al-Qura University, Holy Makkah, Saudi Arabia. the tumor cell resistance toward the cytotoxic effects of 5-FU and 2Department of Pharmacology, Faculty of Medicine, Assiut University, other anticancer chemotherapeutic agents, and regulate the activ- Assiut, Egypt. 3Department of Clinical Nutrition, Faculty of Applied Medical Sciences, Umm Al-Qura University, Holy Makkah, Saudi Ara- ities of various genes that impact cancer cell proliferation, differ- bia. 4Department of Animal Medicine, Faculty of Veterinary Medicine, entiation, apoptosis, angiogenesis, invasion, and drug resistance Assiut University, Assiut, Egypt. (5–7). However, such potential antitumor activity of calcitriol is Corresponding Author: Adel Galal El-Shemi, Associate Professor, Department achieved only when it is given in supraphysiologic doses that of Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura will lead to significant hypercalcemia and hypercalciuria side University, PO Box 7607, Holy Makkah, Saudi Arabia; and Department of effects that hinder and impede its clinical usefulness for this Pharmacology, Faculty of Medicine, Assiut University, Egypt. Phone: 9665- purpose (7, 8). As a clinically relevant task, synthesis of non- or 0965-5135; Fax: 9661-2 527-0000, ext. 4242; E-mail: [email protected] less calcemic vitamin D analogues has therefore been initiated to doi: 10.1158/1940-6207.CAPR-15-0439 achieve or even potentiate the antiproliferative/tumoricidal prop- 2016 American Association for Cancer Research. erties of calcitriol but precludes its calcemic side effects (7, 8). In

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spite of their well-known vitamin D receptor (VDR)-mediated standard laboratory pellet diet and water ad libitum, and kept in a actions, their precise mechanisms and why specific analogues temperature-controlled air conditioned environment at 22–24C have superagonistic effects on specific tissues or diseases still have with a 12-hour dark/light cycle. to be deciphered (8). Among these analogues, paricalcitol (19- For induction of colorectal tumorigenesis, AOM was dis- nor-1a-25-dihydroxyvitamin D2), a direct acting VDR activator solved in normal saline and injected subcutaneously at a dose that is clinically approved by the FDA for the treatment of of 15 mg/kg, once weekly for 2 weeks as described previously secondary hyperparathyroidism, has recently gained attention on (18). According to the subsequent treatment schedules, the rats a variety of disease modalities, including cancer, due to its less were randomly categorized into the following 5 groups (15 rats/ calcemic effects, wider therapeutic window, and an equipotential group): group 1 (normal controls): received only normal saline; activity as calcitriol in several in vivo and in vitro systems (8–11). In group 2 (AOM group): AOM-injected rats and left without cancer patients, paricalcitol may have potential safety and feasi- treatment; group 3 (5-FU group): AOM-injected rats and then bility in women with metastatic breast cancer (12), and in men treated with 5-FU; group 4 (paricalcitol group): AOM-injected with advanced prostate cancer (13). Paricalcitol has also demon- rats and then treated with paricalcitol; and group 5 (5-FUþ strated potential suppressive effects on a variety of human cancer paricalcitol group): AOM-injected rats and then treated with 5- cells and preclinical tumor models such as pancreatic cancer (14), FU plus paricalcitol combination therapy. In its designated gastric cancer and peritoneal metastasis (10), uterine fibroids groups, 5-FU was freshly prepared in normal saline and injected (15), androgen-dependent prostate cancer cell model (16), and intraperitoneally (i.p.) during the 9th and 10th weeks post- human leukemic cells (17). AOM injection in a dosage regime similar to that used in the Taken together, paricalcitol may be a potential chemopreven- treatment of human colorectal cancer (12 mg/kg/day for suc- tive agent in cancer therapy, especially for patients with low cessive 4 days, then 6 mg/kg every other day for 4 doses), while response or who fail in conventional therapies (10, 11, 15). paricalcitol was administered in a dosage regimen of 2.5 mg/kg/ However, there remains insufficient information concerning its day i.p., 3 days/week; starting from the 7th week post-AOM benefits on colorectal cancer. Therefore, the current study was injection and continued till the end of the study (week 15 post- aimedtoinvestigatewhetherparicalcitolwouldimproveand AOM injection). The dose of paricalcitol was chosen on the synergize the anti–colorectal cancer effect of 5-FU, and to basis of our tested pilot experiments and previously published identify the possible mechanisms underlying such synergy on reports (14). an intermediate-term model of colorectal neoplasia induced by azoxymethane (AOM) in rats. AOM-induced colorectal tumors Blood sampling and isolation of whole colon and carcinogenesis in rats and mice have been proven as an At the end of the study, rats of the different groups were outstanding rodent model that closely mirror the phases and weighed, fasted overnight, and then euthanized under diethyl features of human colorectal cancer [aberrant crypts foci (ACF)– general anesthesia (Fisher Scientific UK Ltd). After euthanasia, adenoma–adenocarcinoma and carcinoma sequence] in a time- blood sample (4 mL) was collected from the rat's vena cava into a dependent manner post-AOM injection, and commonly used to plain tube, and used to measure the serum levels of calcium, 25- assess new chemopreventive and therapeutic strategies and to OH vitamin D, kidney function tests (creatinine, BUN, and urea), provide new insights into the pathophysiologic mechanisms and liver function enzymes [alkaline phosphatase (ALP), alanine and risk factors of human colorectal cancer (18–20). Our transaminase (ALT), and aspartate aminotransferase (AST)] by findings showed that monotherapy with either 5-FU or pari- using Cobas e411 (Roche Diagnostics International Ltd), accord- calcitol resulted in a significant chemopreventive effect on this ing to the manufacturer's instructions. Subsequently, the whole model; however, their combination exhibited a more significant colon from rectum to cecum was gently resected, flushed with PBS, efficacy to repress the morphologic, histopathologic, and and slit opened longitudinally. The surface area of each isolated molecular changes that were observed in this model. Further colon (length width in cm2) was measured, and then the whole studies are still essential to realize the potential clinical value of colon was immersed in 10% (v/v) neutralized formalin for this combination in human patients with colorectal cancer. overnight between layers of filter papers with the mucosa on the upper side.

Materials and Methods Quantiﬁcation of grown tumors and large ACF in the colorectal Drugs and chemicals tissues Paricalcitol (Zemplar 5 mg/mL vials) was obtained from Abb- The grown tumors on the mucosae of the isolated colons were Vie Ltd., while AOM and 5-FU were purchased from Sigma- blindly counted by the naked eye by two observers. Next, the Aldrich. All other chemicals used were of the highest commercial colon was cut into 3 portions: proximal, middle, and distal grade. segments; and each segment was stained with 0.2% methylene blue solution for 1.5–2 minutes, placed on a microscope slide Animals, induction of colorectal tumorigenesis, and treatment with the mucosal side upward, and examined under a dissecting approach microscope to count the small tumors, that were not detected by All experimental protocols and procedures of the current study the naked eye, and large ACF (containing 4 or more aberrant were approved by the Institutional Animal Care and Use Com- crypts) according to previously published criteria (21). After mittee at the University of Umm Al-Qura (Holy Makkah, Saudi counting process, a micro-feather scalpel blade was used under Arabia), and in accordance with the US Public Health Service the dissecting microscope to excise the colorectal specimens that Policy on Humane Care and Use of Laboratory Animals. A total of had tumors and ACF from the surrounding normal tissues to be 75 adult male Wistar rats, weighing 220–250 g, were housed in used for the subsequent histopathologic, molecular, ELISA, and clean, sterile, and polyvinyl cages (5 rats/cage), maintained on immunohistochemical examinations.

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Histopathologic examination technique. Briefly, the tissue specimens were homogenized in The colorectal tissue specimens were first destained from meth- RIPA lysis buffer containing protease inhibitors (Santa Cruz ylene blue by 80% ethanol, and then processed for paraffin Biotechnology Inc), centrifuged, and then their harvested super- embedding and sectioning (4–5 mm tissue sections) and hema- natants were stored in 20C until use. During the assays, the toxylin and eosin (H&E) staining by using the conventional total protein concentration in each sample was adjusted to make a methods. The stained tissue sections were microscopically exam- final concentration of 500 mg/mL, and the concentrations of the ined for the presence of ACF, colonic adenomas, and adenocarci- candidate proteins were measured by using commercial ELISA kits nomas. On the basis of the crypt architecture and nuclear features, (Cusabio) and a fully automated ELISA system (Human Diag- ACF were microscopically classified into hyperplastic ACF (no nostics), according to the manufacturers' instructions. All samples dysplasia) or dysplastic ACF (elongated, crowded, and pseudos- were processed in duplicate and the results are presented as ng/mL tratified nuclei, increased nucleus-to-cytoplasm ratio; reduced for HSP-90, pg/mL for b-catenin and VEGF, and pmol/mL for number of goblet cells; back-to-back glands; and markedly COX-2. decreased interglandular stroma) as previously identified by established criteria (22). A colonic adenoma consists of prolifer- Immunohistochemical analysis b ative and hyperplastic glands, whereas colonic adenocarcinoma is Immunohistochemical staining for -catenin, HSP-90, and composed of dysplastic glands invading the submucosa and inducible nitric oxide synthase (iNOS) was performed on fi muscular layers (20). paraf n sections following the conventional protocol. The primary antibodies used were as follows: polyclonal goat IgG RNA extraction, cDNA synthesis, and qRT-PCR analysis antibodies (1: 100) against rat HSP-90-a/b (N-17), iNOS (N- Colorectal specimens were homogenized in RNAlater solution 20), and b-catenin (C-18; Santa Cruz Biotechnology Inc). (Ambion), and then total RNA was isolated by using the Purelink Biotinylated anti-goat secondary antibodies (1:200) conjugated RNA Mini Kit (Applied Biosystems), following the manufacturer's with horseradish peroxidase complex (Santa-Cruz Biotechnol- instructions. The quality and the quantity of the extracted RNA ogy Inc) were used, and staining process was developed by DAB were measured by the Nanodrop equipment (BioSpec-nano, chromogen substrate and counterstained with Gill hematoxy- Shimadzu Corporation). Up to 200 ng of the extracted total lin. The intensity of stainingP was assessed using H-score for- RNA was employed in the reverse transcription step for cDNA mula as follows: H-score ¼ Pí (í þ1), where í represents the synthesis and by using a high capacity RNA-to-cDNA Reverse intensity of positively stained cells (0 ¼ negative; 1 ¼ weak; 2 ¼ Transcription Kit (Thermo Fisher Scientific). qRT-PCR was con- moderate; and 3 ¼ strong) and Pí is the percentage (0%–100%) DDC ducted using the 2 t method on the following target rat genes: of positively stained cells (23). Wnt (NM_001105714.1), b-Catenin (AF397179.1), Dickkopf-1 [DKK-1; (NM_001106350.1)], Cyclin-dependent kinase inhibi- Statistical analysis tor 1A (CDNK-1A; NM_080782.3), NF-kB (NM_001008349.1), Results were expressed as mean SD. Comparisons of data post hoc and COX-2 (AF233596.1). In addition, b-actin (NM_031144.3) between groups were made using one-way ANOVA, with was used as an internal reference (housekeeping gene) to stan- comparisons using Dunnett multiple comparison test. The dif- fi dardize the data of the target genes. The nucleotide primer ference between data were considered to be statistically signi cant P < fi P < sequences of these seven rat origin genes were summarized when 0.05, and to be very signi cant when 0.01. in Table 1. All reactions were performed in triplicate and using Power SYBR Green Master Mix (Applied Biosystems, Thermo Results fi Fisher Scienti c) and the StepOnePlus Real-Time PCR System Inhibitory effects of paricalcitol and/or 5-FU on colorectal fl m m (Applied Biosystems). Brie y, 10 L SYBR Green, 7 L DNase/ tumor growth and large ACF formation – m m RNase free water, 1 L of each primer (5 pmol), and 1 L cDNA In the current study, the chemopreventive effects of mono- and (25 ng) were mixed in each well of the PCR plate, and the combination therapy with paricalcitol (Pcal) and 5-FU in inhibit- amplification was conducted under the following conditions: ing the early colorectal tumorigenesis stages and tumor growth 40 cycles (15 seconds at 95 C and 1 minute at 65 C). Data were were examined in an intermediate-term model (15 weeks) of C analyzed using a comparative threshold cycle ( t) technique, AOM-induced colorectal carcinogenesis in rats. As shown in Figs. C b normalized against the t values of -actin and expressed as 1 and 2, in comparison with normal controls (Figs. 1 and 2; panel fold-change compared with the normal control group. 1A), rats received AOM and left with treatment developed a significant number of gross tumors and distortions on their ELISA colorectal mucosae (Figs. 1 and 2; panel 2A). However, treatment The levels of b-catenin, COX-2, HSP-90, and VEGF were quan- of these AOM-injected rats with either 5-FU (Figs. 1 and 2; panel titatively measured in the colorectal tissue specimens by ELISA 3A) 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,andb-actin genes including 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

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A 20 B 25

15 20

a a 15 10 a b,c,d 10 a

Gross tumors Gross b,c,d 5

Micro-tumor under Micro-tumor 5 dissecng microscope

0 0

AOM AOM Control Control AOM/5-FU AOM/Pcal AOM/5-FU AOM/Pcal

AOM/5-FU/Pcal AOM/5-FU/Pcal C D 35 60 30 50 25 a 40 20 a 30 a 15 b,c,d a 20 10 b,c,d Large ACF Large ACF under Total tumor count tumor Total 5 10 dissecng microscope 0 0

AOM AOM Control Control AOM/Pcal AOM/5-FU AOM/5-FU AOM/Pcal

AOM/5-FU/Pcal AOM/5-FU/Pcal

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 rat colorectal tumors. The grown tumors on colorectal tissues were counted by the naked eye (A), and under a dissecting microscope 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 represented as 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.

decreased the number of the grown tumors and the topographic panel 5C) had attenuated the development of such large ACF and mucosal alterations. More importantly, the lowest number of tubular adenomas; and the highest attenuating effect was grown tumors was observed in rats treated with 5-FU/paricalcitol observed with 5-FU/paricalcitol dual therapy. Taken together, combination therapy (Figs. 1 and 2; panel 5A). Furthermore, these results indicate that therapy with paricalcitol not only examination of the resected colons of the different studied groups attenuates colorectal cancer initiation, but is also able to enhance by dissecting microscope following methylene blue staining the chemopreventive efficacy of 5-FU in this disease modality. showed normal mucosal and crypt appearance in the normal control group (Fig. 2; panel 1B), but colons of rats injected with qRT-PCR findings AOM and not treated developed several micro-tumors and ACF To provide mechanistic insights into the observed chemo- (Fig. 2; panel 2B). On the other hand, the number of these micro- preventive effects of paricalcitol and 5-FU therapy on this tumors and ACF were significantly diminished when these dis- rodent model of the early stages of colorectal cancer, gene eased animals were treated with either 5-FU (Fig. 2; panel 3B) or expression study was conducted by qRT-PCR to determine the paricalcitol (Fig. 2; panel 4B), and dual therapy with paricalcitol relative mRNA expression patterns of Wnt, b-catenin, NF-kB, and 5-FU preserved/restored the normal mucosal architecture and and COX-2 pro-oncogenes that have important role in colo- was associated with the highest reducing effect on the develop- rectal cancer development and progression, as well as of DKK-1 ment of such micro-tumors and ACF (Fig. 2; panel 5B). and CDNK-1A as examples of well-established colorectal can- The histopathologic findings (Fig. 2) were also consistent with cer–suppressive genes. As illustrated in Fig. 3, there was a the macroscopic/microscopic observations and showed the pres- significant upregulation in the mRNA expression of Wnt, b-cate- ence of many large ACF (>4 crypt/focus) with hyperplastic and nin, NF-kB,andCOX-2 genes, and a significant decrease in the dysplastic features, and multiple tubular adenomas in the colo- in the mRNA expression of DKK-1 and CDNK-1A in AOM rectal tissues of rats injected with AOM and left without any group, in comparison with normal control group. In contrary, treatment (AOM group; Fig. 2; panel 2C). However, treatment of treatment of the diseased animals with paricalcitol and 5-FU these AOM-injected rats with either 5-FU (Fig. 2; panel 3C), had synergistically cooperated to modify the altered mRNA paricalcitol (Fig. 2; panel 4C), or 5-FU plus paricalcitol (Fig. 2; expression patterns of these target genes (Fig. 3).

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1B 1C Normal controls

2B 2C AOM

Figure 2. group Representative photos of macroscopic and microscopic appearance of colorectal mucosa of (1) normal control group, (2) AOM group, (3) AOM/5-FU 3A group, (4) AOM/paricalcitol group, and (5) AOM/5-FU/paricalcitol group. The colorectal mucosae of the different groups were examined by gross (panel 3B 3C A), dissecting microscopy following staining with 0.2% methylene blue 5-Fu (panel B), and light microscopy at group magniﬁcations 100 and 200 following staining with H&E (panel C). Black arrowhead, gross tumors observed by naked eye; yellow 4A arrow, large ACF (>4 crypts/focus). Pcal, paricalcitol. 4B 4C Pcal group

5B 5C

Pcal/5-FU group

ELISA and IHC findings Interestingly, cotherapy with paricalcitol and 5-FU had resulted in To further explore the possible underlying mechanisms that more reduction in the colorectal levels of b-catenin, COX-2, HSP- could mediate the observed chemopreventive effects of paricalci- 90, and VEGF proteins compared with paricalcitol or 5-FU mono- tol and 5-FU therapy, we quantitatively measured the protein therapy (Fig. 4). concentrations of b-catenin, COX-2, HSP-90, and VEGF by ELISA Data of immunohistochemical assays (Fig. 5) were also in in the colorectal tissue homogenates of the different groups. symmetry with ELISA results and demonstrated low expression Compared with the normal control group, the concentrations of of b-catenin (Fig. 5; panels 1A and 1F), iNOS (Fig. 5; panels 2A b-catenin (Fig. 4A), HSP-90a (Fig. 4B), COX-2 (Fig. 4C), and and2F),andHSP-90(Fig.5;panels3Aand3F)inthecolorectal VEGF (Fig. 4D) were significantly elevated in the colorectal tissue tissues of normal controls. In contrast, a marked expression of homogenates of AOM group. However, the concentrations of either b-catenin (Fig. 5; panels 1B and 1F), iNOS (Fig. 5; panels these candidate molecules were significantly reduced when these 2B and 2F), or HSP-90 (Fig. 5; panels 3B and 3F) was observed AOM-injected rats were treated with either paricalcitol or 5-FU. in the colorectal tissues of rats injected with AOM and left

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A 8 B 6 a 7 5 a 6 4 5 4 3 3 2 Wnt mRNA 2 c c

β-catenin mRNA c

Relave expression c 1 1 b,d,e,f Relave expression b,d,e,f 0 0 Control AOM 5-FU Pcal 5FU/Pcal Control AOM 5-FU Pcal 5FU/Pcal

8 a C 8 a D 7 7 6 6 5 5 b,c b,c b,c b,c 4 4 3 3 b,d,e,f COX-2 mRNA NF-κB mRNA 2 d,e,f 2 Relave expression Relave expression Relave expression Relave expression 1 1 0 0 Control AOM 5-FU Pcal 5FU/Pcal Control AOM 5-FU Pcal 5FU/Pcal

a,d,e,f 8 E 10 F a,d,e,f 7 8 6 b,c b,c 6 b,c 5 4 b,c 4 3 DKK-1 mRNA 2 mRNA CDKN1A 2 Relave expression Relave expression b Relave expression 1 b 0 0 Control AOM 5-FU Pcal 5FU/Pcal Control AOM 5-FU Pcal 5FU/Pcal

Figure 3. qRT-PCR ﬁndings show the modulatory effects of paricalcitol (Pcal), 5-FU, and their combinations on the relative mRNA expression of Wnt (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 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 versus AOM group.

without treatment, particularly in the glandular epithelium, Discussion and the combination treatment with 5-FU and paricalcitol was 5-FU, either alone or in combination with other chemothera- synergistically interacted to diminish the expression of these peutic agents, remains the standard drug in the treatment of procancerous proteins in the colorectal tissues of AOM-injected human colorectal cancer; however, low response rates and devel- rats(Fig.5;panels1Eand1F,2Eand2F,and3Eand3F, opment of resistance to 5-FU therapy still represent a major respectively). challenge (2–4). On the other hand, the potential antitumor properties of paricalcitol, a synthetic less calcemic vitamin D The biochemical findings analogue directly activates VDR, have recently attracted a specific The synthesis of vitamin D analogues, including paricalcitol, deal of attention (9, 10, 13–16). Herein, an intermediate-term (15 was initiated to achieve the therapeutic properties of calcitriol weeks) model of colorectal tumorigenesis was induced in rats by but precluded its hypercalcemic side effects (8, 9). In agree- AOM, a commonly used in vivo model for the experimental study ment, the biochemical analyses of the current study (Table 2) of human colorectal cancer at several aspects (18–20); our study did not show any significant differences in the serum levels of was designed to investigate the chemopreventive efficacy of calcium, liver function enzymes, and renal function parameters paricalcitol and 5-FU alone and in combination, and whether among the different animal groups, confirming the noncalce- their cotherapy resulted in an enhanced chemopreventive effect mic property of the applied paricalcitol dosage regimen, also than individual therapy on this model. Interestingly, in compar- suggesting the hepato-renal safety of paricalcitol/5-FU combi- ison with their monotherapy, cotherapy with paricalcitol and 5- nation therapy. FU had resulted in augmenting effects in inhibiting the formation

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A B 500 1 a 450 a 400 0.8 350 300 0.6 b,c b,c 250 b,c 0.4 200 b,c d,e,f

150 b,d,e,f HSP90 (ng/mL) -catenin (pg/mL ) b -catenin (pg/mL 0.2 100 50 0 0

Cont AOM AOM Control AOM/5-FU AOM/Pcal AOM/5-FU AOM/Pcal AOM/5-FU/Pcal C AOM/5-FU/Pcal D 35 500 a a 450 30 400 25 350 300 b,c 20 b,c b,c 250 15 b,c 200 d,e,f d,e,f (pg/mL) VEGF 150 COX-2 (pmol/mL) 10 100 5 50 0 0

AOM AOM Control Control AOM/5-FU AOM/Pcal AOM/5-FU AOM/Pcal

AOM/5-FU/Pcal AOM/5-FU/Pcal

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 versus AOM group. of preneoplastic large ACF and the grade of cellular dysplasia, and with their target plasma membrane receptors/coreceptors, Wnt in reducing the number of grown colorectal tumors, which proteins induce a series of downstream signaling events resulting collectively are the main macroscopic and microscopic features in b-catenin dephosphorylation and stabilization. This allows during the short- to intermediate term of AOM model (18–20). b-catenin, the main effector protein of this signaling, to translo- Moreover, paricalcitol and 5-FU had also synergized to modulate cate into the nuclei wherein it stimulates the transcription of a number of molecular pathways and candidate molecules that several oncogenes, particularly in cells derived from the intestinal their dysregulations play crucial roles in the development and crypts (25, 40). At this point, approximately 80% of all human progression of human and experimental colorectal cancer disease colorectal cancers have aberrant overactivations in Wnt/b-catenin such as Wnt/b-catenin pathway (24, 25), DKK-1 (26–29), signal and its downstream components in the colorectal cells CDKN1A (30, 31), NF-kB (32–34), COX-2 (35), iNOS (33), (25). On the other hand, the expression pattern of Dkk-1, an VEGF (35), and HSP-90 (2, 36). In turn, our findings not only inhibitor of Wnt/b-catenin pathway, by blocking Wnt signaling support the importance of paricalcitol as an adjuvant agent in receptor complexes and contributing to colon cancer suppression, cancer therapy (10, 12–16), but also are in harmony with the is remarkably downregulated in the colonic biopsies of colorectal hypothesis that vitamin D or its analogues improve tumor cell cancer patients, and its downregulation is disclosed as a biomark- sensitivity and tumoricidal efficacy of 5-FU (6, 7). er of chemoresistance and poor clinical outcome (25–29). Development and progression of colorectal cancer is multigen- Although the overall available data still have discrepancies, ic and heterogeneous in origin, and also has clinical importance as Dkk-1 has been found to not only act as an inhibitor of Wnt/ predictors of disease prognosis and treatment response (37). In b-catenin signaling but also has additional b-catenin–indepen- this view, aberrant activation of Wnt/b-catenin signaling pathway dent tumor suppressor, antiangiogenesis, and antimetastasis is highly implicated in the induction and dissemination of most actions in colorectal cancer disease (27, 41). Likewise, CDNK- human cancers, including colorectal cancer (24), and significant 1A, a tumor suppressor gene encoding a potent cell-cycle inhib- allocation in the development of 5-FU and multidrug resistance in itory factor (CDKN1A, p21, or CIP1), is downregulated or even human cancer therapy (38, 39). Mechanistically, upon binding lost in most colorectal cancer cases (30), and some colorectal

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El-Shemi et al.

β-catenin iNOS HSP90

Cont. 1A 2A 3A

AOM

1B 2B 3B

Figure 5. AOM Findings of immunohistochemical assays + 5-FU demonstrate the modulatory effects of paricalcitol (Pcal), 5-FU, and their combinations on the protein 1C 2C 3C concentrations of b-catenin (A), HSP90 (B), and iNOS in the colorectal tissues of azoxymethane (AOM)-induced rat colorectal tumors (C). Data are represented as mean SD. a, P < 0.01 AOM versus normal controls; b, P < 0.05 versus + normal controls; c, P < 0.05 versus AOM Pcal group; d, P < 0.05 versus AOM/5-FU group; e, P < 0.05 versus AOM/Pcal 1D 2D 3D group; and f, P < 0.01 versus AOM group.

AOM + 5-FU + Pcal 1E 2E 3E

1F400 2F 500 3F a a 350 a 400 300 400 350 b,c 300 b,c 250 300 200 b,cb,c 250 b,c 200 b,c 150 b,d,e,f iNOS 200

b,d,e,f HSP-90 150 d,e,f

β-Catenin 100 100 50 100 50 0 0 0

Cont AOM 5-FU Pcal Cont AOM 5-FU Pcal Cont AOM 5-FU Pcal 5-FU+Pcal 5-FU+Pcal 5-FU+Pcal

cancer patients have anti-CDKN1A autoantibodies in their colo- colorectal tissues of the chemically induced colorectal cancer rectal tissues (31). On the basis of these facts, it is conceivable that model, suggesting a cooperative mechanism between the two targeting these crucial colorectal tumorigenesis pathways, drugs that, in part, might be behind their observed tumoricidal through repression of Wnt/b-catenin activity and/or stimulation effect. In support of our observations, paricalcitol therapy has of Dkk-1 and CDNK-1A, may hold tremendous therapeutic previously shown to mediate blockade of Wnt/b-catenin signaling potential in treating colorectal cancer and other cancers, and (40) and upregulate CDNK-1A (p21; ref. 14), and 1,25(OH)2D3 in enhancing the cytotoxic effects of chemotherapeutic agents (the most active form of vitamin D) is a multilevel repressor of (25, 41). Interestingly, data of the current study are in agreement Wnt/b-catenin signaling pathway and its downstream target and showed that cotherapy with paricalcitol and 5-FU signiﬁ- proinﬂammatory and oncogenes (42). Furthermore, Aguilera cantly interacted to repress the overexpressed Wnt and b-catenin, and colleagues (28) reported that in a dose- and VDR-dependent and upregulated the decreased Dkk-1 and CDNK-1A in the manner, 1a,25-dihydroxyvitamin D3 activates the human DKK-1

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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 0.45 ALP (IU/L) 122.6 11.2 125.7 9.7 120.8 12.4 117.3 13.5 119.3 12.4 ALT (U/L) 67 2.4 71.2 6.7 68.7 4.1 67.3 4.1 65.8 3.4 AST (U/L) 92.4 24.2 105.8 26.7 109 21.6 101 18.6 97 12.1 Creatinine (mg/dL) 0.22 0.03 0.2 0.06 0.2 0.03 0.19 0.03 0.21 0.05 Urea (mg/dL) 48.6 5.1 50.3 4.3 50.6 9.5 48.3 5.8 49.0 3.7 BUN (mg/dL) 22.2 2.4 24.4 1.9 26.3 4.4 22 2.7 21.4 2.1 Body 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.

gene promoter with subsequent high expression levels of DKK-1 and stimulation/stabilization of the NF-kB inhibitory protein a RNA and protein in different phenotypes of human colon cancer (Ik Ba), all of which are responsible for blocking NF-kB binding to cells. Furthermore, in vivo therapy with a less calcemic vitamin D DNA and inhibition of its canonical activation pathway (47). analogue in a xenografted model of colorectal cancer in immu- Similarly, earlier in vitro studies on cancer cells revealed that 5-FU nodeficient mice has resulted in antitumor action associated with exerts direct inhibitory effect on NF-kB and on nitric oxide significant induction of DKK-1 gene transcription and inhibition production mediated by NF-kB (48, 49). Administration of of Wnt/b-catenin signaling (28). selective inhibitors of NF-kB (34), or COX-2 (50) had previously Indeed, human colorectal cancer is a life-threatening compli- seen to augment the antitumor effects of 5-FU on colon cancer, cation of inflammatory bowel diseases with a complex patho- and was also associated with suppression of VEGF and the tumor genesis, in which NF-kB, COX-2, and iNOS may provide a crucial vessel density (50). In short, we may speculate that there was a mechanistic link between inflammation and carcinogenesis direct and indirect crosstalk between paricalcitol and 5-FU in (32, 33). With this concept, aberrant activation of NF-kB has inhibiting NF-kB and concomitantly in repressing the colorectal been shown to regulate the expression of many tumorigenesis tumorigenesis effects of iNOS, COX-2, and VEGF. genes involved in cellular transformation, proliferation, inflam- Notably, the potential carcinogenic role of HSP-90 in activation mation, angiogenesis, invasion, metastasis, and numerous other of various oncogenic proteins and growth factors is currently of potentially carcinogenic processes (43). NF-kB induces COX-2 intense interest, particularly in colorectal cancer, which is why gene expression and drug resistance genes (10), and COX-2 specific HSP90 inhibitors are currently being investigated as upregulation directly correlates with colorectal cancer progression potential anticancer drugs (36). More importantly, HSP90 depen- and poor prognosis in human patients (35). iNOS levels and dence acquired resistance to 5-FU therapy, as well as its potential activities are also increased in human adenocarcinomas and in role as a proangiogenic factor for the induction of VEGF and iNOS colon tumors chemically induced in rats, to induce chronic for de novo angiogenesis has been reported recently (2). With inflammation and to create a microenvironment that favors colon this concept, treatment of 5-FU–resistant colon cancer cells carcinogenesis (33). Moreover, angiogenesis mediated by VEGF with selective HSP90 inhibitors had repressed primary colorectal and other pivotal angiogenic facilitators plays a crucial role in tumor growth, circulation in the blood, and metastatic tumor development, neovascularization, progression and metastasis of development (2). Remarkably, our data are consistent and colorectal cancer and other human cancers, and COX-2 appears to showed a significant increase in HSP90 expression in the AOM be importantly involved with multiple aspects in this phenom- group compared with normal controls, and cotreatment with enon, particularly by overexpressing VEGF (35). Coherently, paricalcitol and 5-FU resulted in a more significant repression suppression of NF-kB, iNOS, COX-2, and VEGF could represent on HSP90 expression compared with their monotherapy. On the therapeutic potential in colorectal cancer therapy (35, 43). In basis of our findings and on the given potential carcinogenic role harmony, data of the current study showed that paricalcitol and 5- of HSP-90 in colorectal cancer, we therefore reasoned to hypoth- FU alone or in combination significantly decreased the expression esize that paricalcitol could promote the efficacy of 5-FU and the of NF-kB, iNOS, COX-2, and VEGF in the colorectal tissues of regression of the chemically induced colon cancer partly by diseased rats, and their combination was cooperatively acted to downregulating the expression of HSP90, although the current further downregulate these tumorigenesis molecules. In agree- available data on the possible effects of vitamin D or its analogues ment with our findings, several lines of evidence have suggested on this protein in colorectal cancer or other cancers are limited. the robust capacity of either vitamin D or its analogues, including In conclusion, this study suggests that paricalcitol augments paricalcitol, to disrupt NF-kB, COX-2, VEGF, and/or iNOS-depen- the therapeutic efficacy of 5-FU on colorectal cancer disease by dent inflammation, tumor promotion, and carcinogenesis. For modulating several pro- and anticancerous molecules that instance, paricalcitol treatment had suppressed COX-2 and NF-kB have important roles in the regulation of colorectal tumor expression in colon, leukemic, and gastric cancer cells (10, 44), cells' DNA damage, growth, differentiation, inflammation, and attenuated VEGF in renal diseases (45). In a constant line, angiogenesis, and metastasis. These molecular pathways vitamin D has intrinsically shown to inhibit NF-kB activity, reduce include Wnt/b-catenin pathway, NF-kB, COX-2, iNOS, VEGF, NF-kB protein levels, and downregulate a variety of NF-kB target and HSP90. In this context, our findings have therapeutic genes in a variety of inflammatory and cancer cell types (46, 47). impact considering ongoing clinical development of paricali- Mechanistically, activation of VDR by vitamin D or its analogues tol/5-FU combination therapy for the treatment of colorectal results in VDR/IkB kinase beta protein (IKKb) physical interaction cancer patients; however, further studies are warranted to

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El-Shemi et al.

evaluate this therapeutic combination and also to explore its Administrative, technical, or material support (i.e., reporting or organizing precise antitumor mechanisms. data, constructing databases): B. Refaat, O.A. Kensara, S. Idris, J. Ahmad Study supervision: A.G. El-Shemi, B. Refaat, O.A. Kensara

Disclosure of Potential Conﬂicts of Interest Acknowledgments No potential conﬂicts of interest were disclosed. The authors thank Mr. M. Al-Malki and Mr. H. Alshareef (Faculty of Applied Medical Sciences, Umm Al-Qura University) for processing the samples.

Authors' Contributions Grant Support Conception and design: A.G. El-Shemi, O.A. Kensara, A.M. Mohamed This project was funded by the National Science, Technology and Innovation Development of methodology: A.G. El-Shemi, B. Refaat, A.M. Mohamed, Plan (MARRIFAH)-King Abdul Aziz City for Science and Technology (KACST), S. Idris, J. Ahmad the Kingdom of Saudi Arabia (award number 12-MED2965-10). A.G. El-Shemi, Acquisition of data (provided animals, acquired and managed patients, B. Refaat, O.A. Kensara, and A.M. Mohamed are the recipients of the grant. provided facilities, etc.): A.G. El-Shemi, B. Refaat, O.A. Kensara, The costs of publication of this article were defrayed in part by the payment of A.M. Mohamed, S. Idris, J. Ahmad page charges. This article must therefore be hereby marked advertisement in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. computational analysis): A.G. El-Shemi, B. Refaat Writing, review, and/or revision of the manuscript: A.G. El-Shemi, Received December 31, 2015; revised February 2, 2016; accepted March 17, A.M. Mohamed 2016; published OnlineFirst March 28, 2016.

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Paricalcitol Enhances the Chemopreventive Efficacy of 5-Fluorouracil on an Intermediate-Term Model of Azoxymethane-Induced Colorectal Tumors in Rats

Adel Galal El-Shemi, Bassem Refaat, Osama Adnan Kensara, et al.

Cancer Prev Res Published OnlineFirst March 28, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1940-6207.CAPR-15-0439

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