The Vitamin D endocrine system of the gut—Its possible role in colorectal cancer prevention

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Abstract

While Vitamin D insufficiency in the US and European population is rising, epidemiological studies suggest an inverse correlation between low serum levels of 25-hydroxyvitamin D3 (25-OH-D3) and colorectal cancer incidence. The antimitotic, prodifferentiating and proapoptotic active metabolite 1α,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) is synthesized also by colonocytes, since these possess Vitamin D synthesizing (CYP27B1) and catabolic (CYP24) hydroxylases similar to the kidney. Early during colon tumor progression, expression of CYP27B1 and of the Vitamin D receptor increases, suggesting an autocrine/paracrine growth control in colon tissue as a physiological restriction against tumor progression. However, in human adenocarcinomas expression of the catabolic CYP24 is also enhanced when compared with adjacent normal mucosa. Therefore, to maintain colonic accumulation of 1,25-(OH)2-D3 its catabolism needs to be restricted. Our studies in mice show that low nutritional calcium causes hyperproliferation of colon crypts and significant elevation of CYP24 expression, which can be completely abrogated by soy feeding. We suggest that phytoestrogens in soy, known to be estrogen receptor modulators, are responsible for decreased CYP24 expression. These results and our observation that 17β-estradiol can elevate CYP27B1 expression in rectal tissue of postmenopausal women, may underlie the observed protective effect of estrogens against colorectal cancer in females.

Introduction

Already in 1980 Garland and Garland [1] proposed that Vitamin D might be a protective factor against colorectal cancer. They based this hypothesis on the observation that colon cancer mortality in the USA was highest in regions where the population was least exposed to solar radiation. UV-B is responsible for Vitamin D production in the skin and serum levels of 25-hydroxyvitamin D3 (25-OH-D3) are a direct reflection of sunlight exposure and of protective skin pigmentation [2]. The latter could lead to enhanced incidence of colorectal, breast and prostate cancer that has been observed in African Americans [2]. The link between colorectal cancer incidence and solar radiation was later confirmed by several large studies comparing southern and northern parts of the USA (see, e.g. Ref. [3]). Recently, Grant and Garland suggested that actually 20–30% of colorectal cancer incidence is due to insufficient exposure to sunlight [4].

Such associations suggest that there is a correlation between reduced colorectal cancer incidence and sunlight exposure, low skin pigmentation, nutritional Vitamin D intake and high serum levels of 25-OH-D3. A recent meta-analysis demonstrated convincingly the beneficial effect of high 25-OH-D3 serum levels with respect to colorectal cancer occurrence [5]. In a human pilot study, Holt et al. [6] demonstrated for the first time that rectal crypt proliferation was inversely correlated with 25-OH-D3 levels in serum. 1α,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), however, which has been recognized as an antimitotic, prodifferentiating steroid hormone in in vitro (see, e.g. Ref. [7]) and also in very few in vivo animal studies [8], did not show a negative correlation with colorectal tumor incidence, not even at the highest physiological serum range. In 1997, we [9] demonstrated that conversion of the precursor 25-OH-D3 into 1,25-(OH)2-D3 occurs in human colon cancer cells. We found constitutive expression of the 25-hydroxyvitamin-D3-1α-hydroxylase (CYP27B1) in almost any growth phase of Caco-2 cells, and the sequential metabolism/catabolism of the secosteroid occurred along the C-24 and C-23 oxidative pathways. This was suggestive that human colon cells can control their growth via 1,25-(OH)2-D3 in an autocrine/paracrine manner dependent upon presence of the Vitamin D receptor (VDR). Evidence is accumulating from studies on human colon tissue that intestinal cells express Vitamin D metabolic and catabolic hydroxylases as well as the VDR [10], [11], [12] which supports our hypothesis first outlined in 2001 [10] that human colon tissue has the capacity to accumulate the active hormonal metabolite. Therefore, serum levels of the precursor 25-OH-D3 could be limiting for extrarenal synthesis of 1,25-(OH)2-D3 which in turn may function as an autocrine/paracrine cell cycle regulator in the colon while high or low serum levels of 1,25-(OH)2-D3 within the picomolar range would not contribute to colorectal cancer incidence.

While adequacy of the precursor is of essence, regulation of expression and activity of Vitamin D hydroxylases will primarily determine levels of 1,25-(OH)2-D3 eventually accumulating in colonic tissue. Also, this regulation should be different from that of renal Vitamin D hydroxylases, since only distinctly separate regulation will guarantee prevention of tumor progression in the colon without disrupting renal control of calcium homeostasis.

In this paper, we present evidence in cell cultures as well as in human colon tumors that synthesis of 1,25-(OH)2-D3, i.e. protein expression and activity of CYP27B1, is dependent upon a relatively high differentiation status of colonic tissue. In contrast, CYP24 expression and activity is increasing with decreasing differentiation level of colonocytes. In addition, we observed in a mouse model that already hyperproliferation of colon crypt cells caused by low nutritional calcium intake is paralleled by elevated expression of CYP24.

Section snippets

Human colon tissue

Colon tissue was immediately snap frozen in liquid nitrogen after surgical removal and viewing by pathologists. All tumors were of adenocarcinoma type and were graded according to WHO criteria [13]. A total of 25 randomly chosen patients with moderately differentiated (G2), respectively, undifferentiated (G3) tumors as well as five diverticulitis patients after stoma reoperation (non-malignant tissue) were investigated for CYP27B1 expression by immunoblotting. We also screened these tissues for

High performance liquid chromatography (HPLC) analysis of 25-OH-D3 metabolism

0.5 μCi/ml 25-OH-[26,27-methyl-3H]-D3 (specific activity 30 Ci/mmol) as tracer were added to each 100 mm dish at a total concentration of 16.6 nM for 6 h. Incubations were stopped by addition of methanol. Ten microlitre of 25-OH-D3 and 1,25-(OH)2-D3 (each at 0.1 mM) were added as internal standard to evaluate efficiency of the extraction procedure. Lipids were extracted from cultured cells and medium as described previously ([14]).

The HPLC system encompassed the following components: one pump (Model

CYP27B1 and VDR protein in human colon tissue

We have demonstrated previously at the mRNA level ([10], [11]) that CYP27B1 as well as VDR expression are enhanced during early tumor progression. Recently, we also evaluated tissue from tumors during early progression with respect to CYP27B1 protein expression [16]. In the present study, we quantified CYP27B1 protein expression by immunoblotting in 24 patients including G2 (medium differentiated) and G3 (undifferentiated) tumor tissue (Fig. 1). While colon mucosa from non-cancer patients (NM)

Discussion

During the last decade evidence has been accumulating for prostate [19], [20] and also for colon cells [9], [17], which are derived from organ sites particularly prone to sporadic cancer occurrence, that they express the active Vitamin D synthesizing and degrading P450 enzymes. Since 1,25-(OH)2-D3 is well recognized to have antimitotic, prodifferentiating and proapoptotic activity in cancer cells by binding to its receptor [21], this supported the hypothesis, that non-renal synthesis of

Acknowledgements

Financially supported by grants nos. 9850 (to HSC) and 9335 (to EK) from the Austrian National Bank, and by a grant from the American Institute of Cancer Research, Washington DC (to HSC).

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