Journal of Undergraduate Research
Volume 2, Issue 2 - November 2000

Vitamin D Receptor Regulation Via Estrogen Administration: A Possible Link to Colon Cancer

Ted Lin

ABSTRACT

Colon cancer is the second most deadly type of cancer in the U.S. today. Using rat models and the Caco-2 colon cancer cell lines, we were able to observe the effects of estrogen treatment on vitamin D receptor (VDR) concentrations in vivo and in vitro. Our results suggest a direct correlation between the amount of estrogen administered to the concentration of VDR present in vivo. No such results were observed in vitro.

INTRODUCTION

Colon cancer is the second most deadly type of cancer in the United States. Several factors are believed to contribute to the risk of colon cancer, including sunlight exposure, dietary fat and fiber intake, and genetic predisposition. The importance of vitamin D relative to colon cancer is its function of regulating calcium absorption in the body, which may exert an anti-proliferative influence on colonic tumors. Because of the potential benefits calcium and vitamin D may possess, estrogen and the estrogen receptor (ER) are also important as it also can influence calcium uptake and possibly affect vitamin D and vitamin D receptor (VDR) concentrations.

Vitamin D triggers biological responses via regulation of gene transcription, mediated by the VDR and a nongenomic mode initiated at the cellular membrane. The primary biological role of vitamin D is the regulation of calcium levels and uptake via actions of the intestine, bone and kidney. Calcium absorption can occur by any of these three steps: 1) entry of calcium into the cell via a putative apical membrane transport protein or channel 2) intracellular diffusion of calcium through the cytosol via the ferrying action of calbindin D or 3) the extrusion across the basolateral membrane via an ATP-dependent calcium pump. The mechanisms by which calcium interacts with vitamin D are still not certain. It has been proposed that calcium binds to bile acids in the bowel lumen, inhibiting their proliferative and carcinogenic effects, but still more research is needed to fully understand its capabilities in terms of treating colon cancer.

Estrogen is a sex steroid hormone that modulates sexual development and reproductive functions as well as other effects associated with the cardiovascular and central nervous systems. More importantly, estrogen has been observed to promote calcium absorption, independent of vitamin D, via specific proteins, which mediate the stimulatory action of estrogen and convert estrogen to its active form. These receptor proteins, ER-a and ER-b, share several similarities and differences, with ER-b being more prevalent in this study because of high ER-b mRNA expression found in the epithelium of the stomach, duodenum, colon and rectum. Each receptor binds to estradiol with a high affinity and specificity and activate transcription of reporter genes containing estrogen response elements (ERE) in a hormone-dependent manner. The effect of 17-b E2, when bound to ER-b, on intestinal calcium absorption can be exerted via 1,25(OH)2D3 action or independent of 1,25(OH)2D3. Some mechanisms between 17-b E2, vitamin D and VDR, and the intestine have been formulated but are purely speculative.

With both estrogen and vitamin-D posing as possible treatments for colon cancer, it is important to observe the concentrations and expressions of these hormones and their receptors in the colon. Studies reveal a direct relationship between estrogen exposure, VDR-mRNA-transcript content, and VDR protein expression in the duodenal mucosa, suggesting that estrogen have a pivotal role in supporting VDR expression in duodenal mucosa. The effect of estrogen on intestinal VDR expression provides a pertinent mechanism for the stimulatory effect of estrogen replacement on intestinal calcium absorption observed in estrogen-deficient rats and in women. These experiments were conducted to confirm the effects of estrogen on VDR expression.

MATERIALS AND METHODS

RNA Extraction

Rat Duodenal Mucosa. Female OVX rats were treated with estrogen weekly for a total time of two months. The rat duodenal mucosa samples with and without estrogen treatment were homogenized and total RNA was extracted using a single-step RNA isolation based on acid guanidium-thiocynate-phenol-chloroform followed by precipitations in isopropanol and ethanol. Aliquots of total RNA from samples were prepared and quantified. 1.6 mg of mRNA was isolated from the total RNA using the Promega mRNA Miniprep kit and analyzed using a 6 % formaldehyde/ 1% agarose gel.

Caco-2 Cell Line. The Caco-2 cell lines with and without estrogen treatment were plated out in DMEM without phenol red. After 48 hours when cells were 70-80 % confluent, cells were treated with a control vehicle at the 0.5-hour and 24-hour time points to be used as control lanes. The Caco-2 cells were then treated with a final concentration of 100 nM of 17b-estradiol at 0.5, 4, 8, and 24-hour time points before RNA was extracted. After estrogen treatment, the Caco-2 cells were washed with PBS before being resuspended in guanidine thiocynate. Total RNA was isolated using a single-step RNA isolation procedure based on acid guanidium-thiocynate-phenol-chloroform followed by isoproponal and ethanol precipitations. The total RNA from the samples was aliquoted and quantified.

RT-PCR. The Caco-2 cells were harvested and RNA was isolated and analyzed for VDR RNA levels and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA levels by semiquantitative RT-PCR. 5 mg of RNA was q.s.to 11 mL and 1 mL of 1:10 dilution random hexamer-primers(Boehringer Mannheim , Mannheim, Germany)were added. RT reactions were carried out at 42º C for 1 hour and boiled for 5 minutes.

The cDNA product from the RT reactions was then amplified using polymerase-chain reaction (PCR) in a final volume of 10 mL containingTaqpolymerase (Promega) in Taq buffer, 5 mM primers for VDR, and dNTP's. using the air thermocycler (Idaho Technology). The cycling settings are as follows: intial denaturing at 94º C for 30 sec., 30-45 cycles of denaturing at 94º C for 0 sec., annealing between 52-55ºC for 0 sec., extension at 72ºC for 15-30 sec., and final extension at 72ºC for 30-45 sec. The negative control sample lacking cDNA was run concurrently. 8.5 mL of each sample was loaded into a 1-2 % agarose gel for analysis and run at 150V for 30 min, then 22V for 10 min.

Southern Analysis

The RT-PCR products were transferred to nylon membranes and hybridized for VDR with a 32P-labeled probe followed by autoradiography. The agarose gel from the RT-PCR procedure was trimmed, rinsed in 10 x SSC for 10 min, blotted using BM paper and 10 x SSC for 2 hours up to overnight, crosslinked, and hybridized with the 32P-labeled cDNA probe for VDR and GAPDH (to show equal loading.)

Northern Blot Analysis

After denaturation, 1.6 mg of RNA from the rat duodenal mucosa was submitted to electrophoresis for analysis using a 6 % formaldehyde / 1 % agarose, 2.2 M formaldehyde gel, transferred to nylon membranes using capillary blotting in 20 x SSC, crosslinked to the membrane with UV light, and hybridized with a 32P-labeled probe for VDR and GAPDH (Random-primed labeled 32P). The membranes were hybridized and washed in 1 % SDS, 150 mM NaCl, and 10 mM Tris in 3-20 min. intervals. The membranes were exposed to film for 1.5 hrs.

RESULTS

By Southern Analysis of the breast cancer cell line MCF-7 and the colon cancer cell lines HT29, Caco-2, T84, and SW1116, expressions for VDR, ER-a, ER-b, PR, and GAPDH were observed (Figure 1). A band for ER-a was present in each cell line, with the MCF-7 and SW1116 cell lines exhibiting the most intense bands, indicating the presence of greater expression. Relatively equal expression for ER-b was present in all cell lines examined, with the exception of the HT29 colon cancer cell line, which showed very little expression. Intense bands for PR were present only in the MCF-7 and Caco-2 cell lines, indicating higher expression for PR for these two samples relative to the other cell lines. VDR expression was fairly equal for MCF-7, HT29, T84, and SW1116. Lower VDR expression was observed in the Caco-2 cell line (Figure 2). High expression for all receptor types was present in the MCF-7 breast cancer cell line. Each of the different colon cancer cell lines showed varying expressions for each of the receptor types. The equal GAP band intensities in each cell line sample are used as a basis for comparison of the amounts loaded into the gel. Densitometry readings are needed to verify the equality of sample loading.

Figure 1. Shows bands, from RT-PCR and Southern Analysis, for various breast cancer and colon cancer cell lines probed.
Figure 1. Shows bands, from RT-PCR and Southern Analysis, for various breast cancer and colon cancer cell lines probed for ER-a (Estrogen Recptor-a), ER-b (Estrogen Recptor-b), PR (Progesterone Receptor), VDR (Vitamin-D Receptor), and GAP (glyceraldehydes-3 phosphate). Each cell line shows varying expressions for the different receptors probed for. The HT-29, Caco-2, T84, and SW1116 lanes all represent different colon cancer cell lines. The RT lane represents a negative control lane whereas MCF-7, a breast cancer cell line, serves as a positive control, exhibiting high expression for all receptor types. The GAP bands are used to ensure equal amounts of each cell line were used in the experiment.

Figure 2. Shows VDR expression for various breast cancer and colon cancer cell lines using an ethidium bromide gel and Southern Analysis.
Figure 2. Shows VDR expression for various breast cancer and colon cancer cell lines using an ethidium bromide gel and Southern Analysis. The RT sample lane was used as a negative control lane. The Caco-2 cell line exhibited the lowest expression for the VDR compared to the other cell lines. The remaining cell lines produced relatively the same intensity band for the VDR.


Figure 4 shows that no change in VDR expression was observable after the Caco-2 cell line was treated with estrogen. Relatively equal expression for VDR in each at each time point sample and the control vehicles for 0.5 hrs and 24 hrs produced was observed (Figure 5). No observable change in VDR expression was present among the different estrogen treatment time points for the Caco-2 cell line sample. The GAPDH band intensities for each sample were also relatively equal, indicating that the amounts of total RNA loaded were roughly equal. The densities for VDR and ER-b were adjusted for GAPDH. The density of VDR and ER-b bands (ER-b bands not shown) were recorded and graphed, showing that an inverse relationship between the densities of ER-b and VDR was present. ER-b density decreased between the 0.5-hr and 4-hour estrogen treatments and the 8-hour and 24-hour estrogen treatments, but increased sharply between the 4-hour and 8-hour treatments. VDR density decreased only between the 4-hour and 8-hours estrogen treatments and increased during the remaining treatments. At the 4-hr. time point, both the peak VDR density level and the lowest ER-b density level were present. The peak ER-b density was observed at the 8-hr. time point.

Figure 4. Shows the densitometry for VDR band in the Caco-2 cell line.
Figure 4. Shows the densitometry for VDR band in the Caco-2 cell line. The cell line was treated with estrogen over a 24-hr. time period. Lane 1 and 6 represent cells treated with a control vehicle at 0.5 hrs. and 24 hrs. respectively. Lane 2-5 represent the Caco-2 cells treated with estrogen at 0.5 hrs, 4 hrs, 8 hrs., and 24 hrs. respectively. The cell line was also probed for GAPDH to ensure equal amounts of the samples were used. The VDR band intensities for each sample were nearly equal.

Figure 5. Graph showing the changes in ERb and VDR density in the Cac0-2 cells over the 24-hour time period.
Figure 5. Graph showing the changes in ERb and VDR density in the Cac0-2 cells over the 24-hour time period. Estroegn was administered to the cell line at 0.5 hrs, 4 hrs., 8 hrs., and 24 hrs. The graph shows an inverse relationship between the VDR and ERb density in the Caco-2 cells. At 4 hrs., peak VDR density and lowest ER-b density is observed. At 8 hrs., peak ER-b density is present.


 Northern Analysis

Data from the Northern Blot Analysis for mRNA transcripts of VDR indicated that the rat duodenal mucosa samples treated with estrogen exhibited a higher expression for VDR (seen as ~4.9Kb band) compared to the rat duodenal mucosa sample without estrogen treatment (Figure 3). The samples were also hybridized and probed for GAPDH to show even sample loading (data not shown).

Figure 3. Shows the Northern Blot Analysis of mRNA isolated from the estrogen-treated and untreated rat duodenal mucosa sample.


Figure 3. Shows the Northern Blot Analysis of mRNA isolated from the estrogen-treated and untreated rat duodenal mucosa samples (from female OVX rats), probed with 32P-labeled rat VDR cDNA. Blot was exposed for 1.5 hrs. Lane 1 represents the VDR band from rat duodenal mucosa with no estrogen treatment. Lane 2 represents the VDR band from estrogen treated rat duodenal mucosa.

DISCUSSION

From these results we concluded that in the rat duodenal mucosa, estrogen treatment does upregulate VDR expression. Recent studies also showed a marked increase in VDR mRNA content and VDR protein expression in the normal rat colonic mucosa after estrogen treatment. Possible mechanisms by which estrogen influences VDR expression are still being observed and tested, in particular the estrogen response element and its possible indirect effects an increased VDR mRNA transcription. Others include direct molecular interaction between estrogen and vitamin-D and its receptors. From our results we can then assume that estrogen indirectly affects calcium absorption in the colon since vitamin-D regulates calcium uptake. Since increased calcium uptake has been observed to decrease tumor proliferation and increase cell differentiation in tumors, estrogen seems to have an indirect influence on tumor growth and development in the colon; however, this cannot be taken as definitive evidence yet. 

Other questions also develop concerning possible effects of estrogen on other molecules. Since estrogen can only exert its effects while attached to its receptor, vitamin-D could have the same effect on estrogen as estrogen had on VDR. Also, besides influencing the concentration of VDR, estrogen could also facilitate the binding of vitamin-D to its receptor, thus activating vitamin-D. It can be assumed from these results that estrogen has an indirect influence on calcium uptake and possibly tumor development in vivo. However, no assumptions can be made about the effects of estrogen on VDR expression in vitro. Some suggested effects estrogen has on VDR expression involve its influence in CpG DNA methylation in the colonic mucosa, preventing the silencing of the VDR gene. A more general question is the possibility that estrogen and its receptors both influence and are influenced by vitamin-D and VDR. The specific mechanisms and molecular interactions between these hormones could be an essential part of learning how to influence tumor growth and development in the colon.

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