Characterization of Vitamin D insensitive prostate cancer cells

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Abstract

The antitumor effects of 1,25-dihydroxyvitamin D3 (calcitriol) are being exploited for prevention and treatment of prostate cancer (CaP). These studies examined the antiproliferative effects of calcitriol in primary cell cultures derived from transgenic adenocarcinoma of mouse prostate (TRAMP) mice chronically treated with calcitriol (20 μg/kg) or vehicle 3×/week from 4 weeks-of-age until palpable tumors developed. This is a report on the response of two representative control (Vitamin D naïve, naïve) and calcitriol-treated (Vitamin D insensitive, VDI) cells to calcitriol. VDI cells were less sensitive to calcitriol based on less cell growth inhibition and less inhibition of DNA synthesis as measured by MTT and BrdU incorporation assays. Similarly, VDI cells were less sensitive to growth inhibition by the vitamin analog, 19-nor-1α,25-dihydroxyvitamin D2 (paricalcitol). There was no change in apoptosis following treatment of naïve and VDI cells with calcitriol. Vitamin D receptor (VDR) expression was up-regulated by calcitriol in both naïve and VDI cells. In addition, calcitriol induced the Vitamin D metabolizing enzyme, 24-hydroxylase (cyp24) mRNA and enzyme activity similarly in naïve and VDI cells as measured by RT-PCR and HPLC, respectively. In summary, VDI cells are less responsive to the antiproliferative effects of calcitriol. Understanding Vitamin D insensitivity will further clinical development of Vitamin D compounds for prevention and treatment of CaP.

Introduction

Prostate cancer (CaP) is one of the leading causes of cancer and cancer-related mortality in American men [1]. 1,25-Dihydroxyvitamin D3 (calcitriol) is currently being developed for CaP prevention and treatment because it exerts antitumor activities in CaP [2], [3], [4], [5], [6]. The mechanisms of action of calcitriol in CaP include induction of cell cycle arrest [7], apoptosis [8], [9] and differentiation [2], [10]; and inhibition of invasion [11] and metastasis [4]. Clinical trials have been conducted in men with CaP using calcitriol alone or in combination with other antitumor agents [12], [13], [14]. Calcitriol is inactivated and degraded by 24-hydroxylase (cyp24) [15], and previous studies indicate that the availability of calcitriol in CaP cells is inversely proportional to cyp24 activity [16].

Evidence of insensitivity to the antiproliferative effects of calcitriol has been described in some malignant cells. Following chronic exposure to calcitriol, MCF-7 human breast cancer cells become resistant to the growth inhibitory effects of calcitriol and other Vitamin D analogs [17], [18]. The Vitamin D resistant chronic myelogenous leukemia cell line, JMRD3, is less sensitive to growth inhibition by calcitriol compared to parental RWLeu-4 cells [19]. However the molecular mechanisms of Vitamin D insensitivity in CaP is poorly understood. These studies are a report on primary cell cultures established from the transgenic adenocarcinoma of mouse prostate (TRAMP) model that were used to study Vitamin D insensitivity in CaP. The TRAMP model was developed using the minimal rat probasin promoter to target expression of SV40 early genes (T, t) specifically to the prostatic epithelium [20]. These mice progressively develop disease in the prostate ranging from prostatic intraepithelial neoplasia (PIN) to poorly differentiated cancer and metastasis. Prostate tumors of calcitriol-treated (Vitamin D insensitive, VDI) and vehicle-treated (Vitamin D naïve, naïve) mice were digested to generate primary cell cultures. This is a report on the response of naïve and VDI primary cells to calcitriol.

Section snippets

Chemicals and reagents

Calcitriol and paricalcitol (Abbott Laboratories, Chicago, IL) were dissolved in 100% ethanol and stored at −80 °C. Calcitriol was always handled with indirect light. Test compounds were diluted in DMEM media (Invitrogen, Frederick, MD) before use.

Animal studies

Experimental uses of laboratory animals were in accordance with IACUC and NIH guidelines. TRAMP animals were in the C57BL/6 X FVB 50:50 strain background. Breeding colonies were maintained at the Roswell Park Cancer Institute animal facilities. Four

Antiproliferative effects of Vitamin D compounds in naïve and VDI cells

Compared to naïve cells, VDI cells were less sensitive to inhibition of cell growth following treatment with calcitriol for 96 h (Fig. 1A). Calcitriol (3.6 nM) inhibited growth of naïve cells by 30%; however, there was no effect on growth of VDI cells. To determine whether VDI cells are cross-resistant to other Vitamin D compounds, cells were treated for 96 h with the Vitamin D analog, paricalcitol. VDI cells were less responsive to growth inhibition by paricalcitol than naïve cells which were

Discussion

Vitamin D resistance has been observed in some cancers and may limit the efficacy of Vitamin D therapies in the clinic. Progression to advanced CaP is associated with insensitivity to Vitamin D. There is a need to understand the molecular mechanisms that underlie insensitivity to Vitamin D therapies in order to improve the clinical efficacy of Vitamin D compounds. Previous studies have indicated that MCF-7 breast cancer cells and RWLeu-4 leukemia cells become insensitive to the antitumor

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