Vitamin D Receptor ( VDR ) polymorphism and antiproliferative activity of cholecalciferol in cancer cells

Vitamin D (VD) is a steroid hormone with multiple biological functions in the body and its activity requires the binding to the receptor named VDR. VDR polymorphisms seems to be involved in the development of several types of cancer. Herein we performed the genotyping of two VDR polymorphisms (Fok I and Taq I) in MCF-7 breast cancer and U87-MG glioblastoma (GBM) cell lines and investigated the antiproliferative effect of the VD analog cholecalciferol. Polymorphisms were identified by PCR-RFLP and the effect of VD was determined by viability and clonogenic assays. VD inhibited the growth of both tumor cells in Research, Society and Development, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810 2 vitro. MCF-7 cells were more sensitive than U87-MG cells at concentrations ranging from 0.1nM to 1000nM. The same primer pairs used for PCR amplification of VDR gene in MCF7 failed to amplify a fragment of expected size in the U87-MG cell line. VDR Fok I and Taq I polymorphisms in breast cancer MCF-7 cells were characterized as FF (CC) and TT respectively. The absence of amplification of VDR gene fragment in U87-MG suggests a possible chromosomal rearrangement and/or impairment of gene expression of VDR which could interfere in the sensitivity of this cell line to vitamin D.


Introduction
Vitamin D is a steroidal hormone derived from 7-dehydrocholesterol with more than 50 described metabolites, presenting two main biochemical forms: vitamin D3 or cholecalciferol, produced in the skin by ultraviolet B radiation, and vitamin D2 or ergocalciferol obtainable from diet (Zerwekh, 2008;Garland et al., 2011). The VD has a wide variety of biological functions in the organism. In addition to its classic action in bone mineralization and calcium absorption by the intestine (Boneti & Fagundes, 2013), VD also present immunomodulatory property (Evans et al., 2018), anti-inflammatory activity (Hardiman et al., 2016, Mousa et al., 2018 and participates in cell differentiation and apoptosis processes (PAN et al., 2010) angiogenesis modulation (Grundmann, et al., 2012), tumor invasion and metastasis (Bao et al., 2006,10).
The biological actions of VD are mediated by the VD steroid nuclear receptor (VDR) which is found in different cell types and tissues of the organism (Wang, Zhu & De luca, 2012). The gene encoding the VDR receptor is located on the long arm of chromosome 12 (12q12-14) and encompasses a region of 100 kb. About 200 polymorphisms have been identified in different regions of VDR gene and some of them is considered risk variant for some diseases. One of these polymorphisms is the Fok I (T / C) (rs2228570) located in exon-II and results in the use of one of two alternative translation initiation start site leading to an altered VDR receptor protein. Restriction Fragment Length Polymorphism -RFLP assay can Research, Society andDevelopment, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810 4 identify the base exchange in Fok I polymorphism where "f" denotes the presence of Fok I restriction site and "F" denotes its absence. Thus, the f allele contains both ATGs start sites whereas the F allele has only the second ATG and predicts a shorter VDR protein (Gross et al., 1998). Additionally, the Taq I (T/C) (rs731236) polymorphism is located in exon-IX and leads to a synonymous change at codon 352 (isoleucine) (Lu, Jing & Zhang, 2016;Rai et al., 2017).
The correlation between VDR polymorphisms and carcinogenesis or cancer risk is not well stablished, and many contradictory results can be found in the literature (Lu, Jing & Zhang, 2016;Rai et al., 2017). Some of the reasons for the conflicting results include the differences in study design and ethnic populations investigated, the requirement for VDR to associate with retinoid X receptor (RXR) and vitamin D response elements (VDREs) in order to induce several genes, the consumption and the levels of active vitamin D circulating in the blood and the different polymorphisms in other genes involved in the complex metabolic pathway of vitamin D (Giovannucci, 2005).
Glioblastoma is the most common malignant brain tumor in adults with five-years survival rate less than 5% (Dolecek, 2012). Despite the advances in the treatment of these tumors, currently available therapies do not improve the survival significantly. On the other hand, breast cancer is the most commonly occurring cancer in women and the second most common cancer worldwide and leading cause of death following lung cancer (Bray et al., 2018). These statistics demonstrate the need for new alternatives of treatment to decrease mortality and improve the quality of the life of patients. Therefore, the present study investigated the antiproliferative activity of the VD metabolite cholecalciferol in the glioblastoma U87-MG (native protein p53) and breast cancer MCF-7 (hormone sensitive) cell lines and determined the presence of two Fok I and Taq I polymorphisms.

Methodology
This is an experimental study that used molecular and cellular methods to address the hypothesis that vitamin D present antitumoral activity.

Genotyping of VDR gene polymorphisms
The genomic DNA of the cell lines was extracted by the phenol: chloroform: isoamyl alcohol method (25: 24: 1), according to Koh, (2013). The Fok I and Taq I polymorphisms in the VDR gene were determined by PCR-RFLP. For Taq I, it was used the primer oligonucleotides described by Yaylım-Eraltan et al., (2007), and for Fok I, the primers   (2007), ** Goknar et al., (2016). Source: Authors.

Cell viability
The U87

Clonogenic assay
The clonogenic assay allows to evaluate the capacity of individual cells to divide and form colonies in a culture plate, and it can be used to determine the effectiveness of cytotoxic agents (Franken, 2006). The assay was performed in triplicates and repeated four times. U87- Cells were then incubated for 8-10 days under humidified atmosphere containing 5% CO2 at 37ºC. The MCF-7 cells were plated at a density of 500 cells per 35 x 10mm Petri dishes in Research, Society and Development, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810 medium already supplemented with cholecalciferol at concentrations of 100nM, 1000nM and 10000nM of VD in 0.1% ethanol/DMEM. After the incubation period, the colonies formed were stained with Wright's solution (0.06%). Analysis of the colony size and number was performed using Image J ® software (NIH, Bethesda, MD, USA).

Statistical analysis
The differences between treated and untreated cells were performed using the GraphPad prism statistic software (version 5.0). The results were compared by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test, two-way analysis (ANOVA), followed by Bonferroni post hoc test with significance level p <0.05.

VDR polymorphism genotyping
After PCR with the set of primers designed to amplify a region encompassing the Fok I and Taq I polymorphisms it was expected to obtain products with band sizes of 272 bp and 745 bp respectively. As shown in Figure 1, DNA extracted from MCF-7 cell lines worked as template for both set of primers allowing the amplification of VDR gene with the expected sizes. After digestion with the restriction enzymes Taq I and Fok I the presence of polymorphism could be determined. Based on the size of the bands produced after digestion, MCF-7 demonstrated the absence of Fok I restriction site, producing a band the same size as the undigested PCR product (272 pb). Therefore, it was genotyped as CC also known as (FF) in the first start codon of VDR gene, which changes the nucleotide sequence from ATG to ACG. For Taq I polymorphism MCF-7 was genotyped as TT (normal homozygous), showing bands of 494 and 251 pb after restriction digestion (Figure 1 A and B). Interesting, the same set of primers under the same conditions failed to generate PCR products with the expected size even with different DNA extractions obtained from the U87-MG cell line. Then we tried different PCR conditions and still could not obtain a PCR product as we got with DNA from MCF-7 cells. Research, Society and Development, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810    Source: own study.
The figure shows the rate of growth of MCF-7 cells exposed to different concentrations of cholecalciferol compared to the vehicle (ethanol-EtOH) alone used to solubilize the compound. Results represent the mean ± standard deviation of three independent experiments (four replicates for each experimental set). * Statistically significant difference, p<0.05. Two-way ANOVA and Bonferroni post hoc test. Research, Society and Development, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810  Source: own study. Research, Society andDevelopment, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810 Rate of growth of U87-MG cells exposed to different concentrations of cholecalciferol compared to the vehicle (ethanol-EtOH) alone used to solubilize the compound. The results represent the mean ± standard deviation of three independent experiments (four replicates for each experimental set). *Statistically significant difference, p<0.05. Two-way. ANOVA and Bonferroni post hoc test.

Anticlonogenic effects of cholecalciferol
The clonogenic assay is able to demonstrate the ability of individual cells proliferate and form colonies after a specific period of time. The initial appropriate number of cells as well as the plate efficiency for each cell line was previously determined as 100 cells per plate and 500 cells per plate for U87-MG and MCF-7 respectively. A representative set of plates are shown in Figure 7A. Cholecalciferol was able to decrease the clonogenicity of MCF-7 Research, Society andDevelopment, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810 13 and U87-MG as demonstrated by the number of colonies formed after 8-10 days exposure to different concentrations of the vitamin D metabolite. MCF-7 was more susceptible to cholecalciferol than U87-MG and at 10.000 nM these cells showed a tendency to resume growth ( Figure 7B).

Discussion
The hypothesis that vitamin D may have anticancer benefits was suggested several decades ago based on the fact that higher rates of total cancer mortality is seen in regions with less UV-B radiation, associated with lower levels of circulating vitamin D in the body (Giovannucci, 2005). Although some data on the effect of vitamin D and cancer are promising others show contradictory effects, adding a challenge to support a definite conclusion.
Herein we demonstrated that cholecalciferol inhibits proliferation of MCF-7 breast cancer cells and glioblastoma U87-MG brain tumor cells at different degrees. MCF-7 cells showed more sensitivity to cholecalciferol than U87-MG. Polymorphisms in the VDR gene have been considered one of the factors that could help understand the putative anticancer activity of vitamin D and cancer risk prevention, however the relationship between the polymorphism and the function of the VDR is still not clear. Genotyping of the Fok I (exon II) and Taq I (exon IX) polymorphisms, showed homozygose of the wild alleles (TT) for Taq I and polymorphic CC (FF) alleles in the Fok I analysis in MCF-7 cell line.
According to Alimirah et al., (2011), Fok I polymorphisms can alter the structure and consequently the function of VDR in human breast cancer cells, where the VDR FF variant is more efficient than VDR ff in modulating Vitamin D action. MCF-7 as shown here has FF genotype and demonstrated sensitivity to treatment with cholecalciferol. The ff variant of VDR gene has both translation initiation codon intact and the protein expressed is longer than its FF genotype. The alternate start codon (SCP) used in the FF genotype is located three codons downstream the original start site, leading to a shorter protein. It is not clear how the Research, Society and Development, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810 15 short version of VDR could lead to a sensitivity of MCF-7 cells to cholecalciferol, but it is known that VDR FF protein is more stable and VD increases its half-life. Also, once associated with its ligand, VDR ff upregulates antiapoptotic and pro-inflammatory genes in contrast with VDR FF cells (Alimirah et al., 2011). Then, the inhibitory growth effect observed in MCF -7 may be associated in part to the Fok I FF alleles.
Nevertheless, other factors must be better understood to translate these findings into a definite conclusion. Vitamin D is involved in different biochemical pathways and polymorphisms in other genes associated to VDR may also influence the activity of the vitamin D. Fok I polymorphism alone or in combination with other VDR polymorphisms has been investigated in breast cancer with contradictory results. In one study it was shown that Fok I FF allele with other polymorphisms increased breast cancer risk in a Caucasian population in United Kingdom (Guy et al., 2004). However, two other studies showed that women with ff genotype were more susceptible to breast cancer (Sinotte, et al., 2008;Chen, et al., 2005). Yet, another study showed no correlation between Fok I polymorphism and increased breast cancer risk in postmenopausal women (McCullough et al., 2007). In a study by Raza et al., 2019, evaluating VDR polymorphism  One of the hallmarks of cancer is the genetic instability that can lead to loss or rearrangement of chromosomes (Vargas-rondón, Villegas & Rondón, 2018). We tried to determine the status of Fok I and Taq I polymorphisms in the U87-MG cell line using the same set of primers able to amplify the fragment of the VDR gene in MCF-7 cells. However, we were not able to get PCR product with the expected size. Interestingly, U87-MG cells demonstrated less sensitivity to cholecalciferol than MCF-7 cells. We believe that one explanation for the lack of amplification may be due to some rearrangement, deletion or mutation in the region of annealing of the primers. We are currently investigating if this may be the case. Interestingly, Zou et al., (2000) reported different sensitivities to vitamin D in different glioma cell lines. They discovered a 220 kDa protein in glioma cells that were sensitive to VD. This protein was not a classical VDR but was recognized by anti-VDR monoclonal antibody. On the other hand, it has been shown that VDR is important in cellular Research, Society andDevelopment, v. 9, n. 12, e8991210810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10810 16 survival and migration in the T98-G glioma cell lines, which has mutated p53 tumor suppressor protein (Salomon et al., 2014).
As shown in Figure 6, cholecalciferol inhibited about 85% of cell growth of the MCF-7 strain at the end of the experiment. For the U87-MG, the inhibition was only 40%. The higher sensitivity of breast cancer lineage to VD suggests that in these cells the classic VD pathway may be active, which would not be the case in U87-MG. Since it was not possible, in our hands, to amplify regions of the VDR receptor in U-87 cells, some sequence rearrangement may be occurring and deserve to be better investigated. On the otrher hand, the effects of cholecalciferol on these tumor cells could not be dependent on VDR, which could explain the low inhibition observed in this cell line.
Considering the VDR polymorphism status related to brain tumor patients, VDR Fok I-ff genotype has been shown significantly higher in Turkish patients with meningioma, however, there was no association of this polymorphism in gliomas patients (TOPTAŞ et al., 2013). Another study has been shown that VDR expression is increased in GBM brain tumor tissues compared to non-malignant tissue and VDR expression is associated with improved outcome in patients with GBM (TOPTAŞ et al., 2013). In contrast, a study performed by Anic et al., (2012) demonstrated no association between the Taq I or Fok I polymorphism with lower survival in patients with Glioblastoma.
It is known that although different tumor cells can be classified as the same type of tumor, the genetic background can be very heterogeneous, which could explain differences in the sensitivity and response to therapy. In fact, it has already been demonstrated that the anticancer effects of VD may be dependent on VDR but tumors from different patients can show different sensitivities (Salomon et al., 2014, Ferronato et al., 2018.
Although cholecalciferol induced a decrease in the clonogenicity of U87-MG cells, we found that high concentrations of cholecalciferol was ineffective in inhibiting the growth at 24 and 48 hours. Similar results have been observed (Deberardinis Lemieux & Hadden, 2013), where VD synthetic analogues had no antiproliferative effect in U87-MG at concentrations of 1000, 5000 and 10000nM. On the other hand, the GBM T98-G strain showed sensitivity to concentrations of 1000nM only after 96h exposure (Salomon et al., 2014). This may indicate that the effect of VD on glioblastoma cells is time, cell line and VD metabolite dependent. The antiproliferative activity of VD in Glioblastoma strains is attributed to the activation of p21 and p27 protein expression and a decrease in Cyclin D1, thereby interrupting the cell cycle (Salomon et al., 2014, Ferronato et al., 2018. The high sensitivity of MCF-7 cells to cholecalciferol reported in this work corroborates similar studies that have shown that VD modulates the growth of breast cancer cells. Saracligil et al., 2017 demonstrated inhibition of MCF-7 cell proliferation in response to calcitriol, the active form of VD, in a dose and time dependent manner, with an IC50 of 145nM and apoptosis levels of 28% in 48 hours (Saracligil, et al., 2017). Antiproliferative activity of VD has also been demonstrated in the MCF-10 strain of breast cancer (Zheng et al., 2013). It has been also suggested that in response to calcitriol cancer cells may present a less aggressive or normal phenotype, indicating the influence of VD on cell differentiation and proliferation (Gocek & Studzinski, 2009). Another work demonstrated that treatment with calcitriol inhibited the growth of MCF-7 in a dose and time dependent manner and the cells were arrested in the G0/G1 phase and this effect was associated with inflammatory COX2/ PGE2 pathway (Yuan, Jiang, Yang, Ding & Deng, 2012).
Vitamin D has a pleiotropic effect and participates in a myriad of pathways related to cell multiplication and differentiation. Several proteins interact with VDR and once the complex is formed different set of genes may be induced. However, the complexity of these interactions is still not well understood. The conflicting results observed in the literature may be explained at least in part by the type of VDR expressed in the tumor cells which may be different from the genotype performed in DNA extracted from blood cells, considering the genetic instability of tumors. Also, the metabolism of vitamin D in the organism involves the expression of many genes that in turn may present functional polymorphisms as well. This demonstrate the need for further studies to better understand the key players in the activity of vitamin D and how this activity can be driven to kill tumor cells.

5-Final Considerations
We demonstrated that VDR gene in MCF-7 presents FF genotype for Fok I polymorphism and TT genotype for Taq I. These cells are sensitivity to cholecalciferol corroborating the results found by other authors. Also, our data suggest that the effect of VD is dependent on tumor cell type and the genetic background may influence in the activity of vitamin D. Considering the impossibility to amplify the VDR gene in U-87-MG cell line by the methodology used here our next step would be to sequence the region encompassing this gene in the genome of these cells to identify possible deletion or rearrangement of this gene.
We also will investigate the expression of VDR at protein level in MCF-7 and U87-MG to elucidate the mechanism of action of cholecalciferol in these cells.