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

Abstract

Colorectal cancer is a common cancer with high mortality rate. Despite being the standard anti-colorectal cancer drug, 5-fluorouracil (5-FU) exhibits only limited therapeutic benefits. Herein, we investigated whether paricalcitol, a synthetic vitamin D analogue with potential antitumor properties, would enhance the chemopreventive efficacy of 5-FU on an intermediate-term (15 weeks) model of colorectal tumors induced by azoxymethane (AOM) in rats. After AOM injection, 5-FU was administered during the 9th and 10th weeks (12 mg/kg/day for 4 days, then 6 mg/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 15th week. At week 15, the animals were euthanized and their resected colons were examined macroscopically and microscopically. Quantitative RT-PCR was used to measure the transcription activities of Wnt, b-catenin, DKK-1, CDNK-1A, NF-kB, and COX-2 genes, and ELISA was used to quantify the protein levels of b-catenin, COX-2, HSP90, and VEGF. IHC was additionally used to measure b-catenin, HSP90, and inducible nitric oxide synthase (iNOS). Compared with their individual therapy, combination of 5-FU and paricalcitol showed more significant reducing effect on numbers of grown tumors and large aberrant crypts foci. Mechanistically, paricalcitol and 5-FU had cooperated together to repress the expression of procancerous Wnt, b-catenin, NF-kB, COX-2, iNOS, VEGF, and HSP-90 more, and to upregulate the expression of antitumorigenesis DKK-1 and CDNK-1A, compared with their monotherapies. Our findings suggest that combined use of paricalcitol with 5-FU exhibits an augmenting chemopreventive effect against colorectal tumors, and might potentially be useful for chemoprevention in colorectal cancer patients. Cancer Prev Res; 9(6); 491-501. Ó2016 AACR.

Vitamin D-Induced Molecular Mechanisms to Potentiate Cancer Therapy and to Reverse Drug-Resistance in Cancer Cells

“…Preclinical studies in animal models have demonstrated that vitamin D treatment may inhibit colorectal [119][120][121][122][123][124], prostate [15,[125][126][127][128][129] and breast cancer [126,[130][131][132] development, by reinforcing the concept that vitamin D plays a crucial role in carcinogenesis.…”

“…Vitamin D may also act in combination with different therapies, by inducing an antiproliferative additive effect. Indeed, in both mouse and rat chemically induced colorectal cancer models (AOM-induced, 1,2-dimethylhydrazine dihydrochloride (DMH)-induced and DMH-dextran sodium sulfate (DSS)-induced) and xenografts models, vitamin D analogs, such as paricalcitol and PRI-2191, in combination with 5-fluorouracile (5-FU) or metformin, diminished the risk to develop early colon neoplasia by controlling the early stage of carcinogenesis and reducing or delaying cancer growth through a specific cell cycle arrest and apoptosis induction, when compared to the 5-FU and metformin treatment alone [122][123][124]. The reported effects were triggered by several synergistic molecular mechanisms such as antiproliferative and antiinflammatory processes.…”

“…The reported effects were triggered by several synergistic molecular mechanisms such as antiproliferative and antiinflammatory processes. Indeed, the analysis of Wnt, β-catenin, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), cyclooxygenase-2 (COX-2), dickkopf-related protein (DKK-1) and cyclin dependent kinase inhibitor 1A (CDKN1A, also known as p21) messenger tissue expression revealed a significant reduction when rats with AOM-induced colorectal cancer received combined treatment of paricalcitol and 5-FU, compared to treatment with 5-FU alone [122]. Similarly, in DMH-induced and DMH-DSS-induced mouse models, the inhibition of insulin like growth factor 1 (IGF1)-mammalian target of rapamycin (mTOR) pathway was associated with the downregulation of the expression of c-MYC, a transcription factor implicated in multiple cellular processes, including proliferation, differentiation and apoptosis, and cyclin D1, an important regulator of cell cycle progression [124].…”

Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model

“…For instance, in some published articles, the accession number of the reference sequence used is not indicated or the qPCR is not well designed or described (i.e., the primer sequence is missing or contains mistakes, primers are not specific). On the basis of the aforementioned data, in this study we provide a well-designed and described qPCR protocol according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines (Bustin et al., 2009) for 17 genes routinely studied in DMH/AOM CRC rat model (El-Shemi et al., 2016; Kensara et al., 2016; Islam et al., 2016; Qie & Diehl, 2016; Rivera-Rivera & Saavedra, 2016; Rubio, 2017; Al-Henhena et al., 2015; Tan et al., 2015; Gamallat et al., 2016; Walter et al., 2010). Moreover, we also analyse two reference genes commonly used in this carcinogenesis model.…”

Effects of 1,25(OH)2D3 on Cancer Cells and Potential Applications in Combination with Established and Putative Anti-Cancer Agents

“…It is therefore not surprising that several groups have investigated the combination of 1,25(OH) 2 D 3 with 5-FU in different colorectal cancer models. Using an azoxymethane-induced colorectal cancer rat model, El-Shemi et al [ 20 ] and Refaat et al [ 21 ] demonstrated that the combination of paricalcitol (a synthetic VDR analog), in case of the former, and vitamin D 3 , in case of the latter, together with 5-FU, led to significant reductions in the number of tumors grown. Additionally, both studies illustrated enhanced interference of this combination with the Wnt signaling pathway, through decreasing the expression of Wnt and β-catenin, and the induction of the pathway’s inhibitor, Dkk1.…”