Effect of a vitamin D(3) derivative (B3CD) with postulated anti-cancer activity in an ovarian cancer animal model

Abstract

The objective of the present study was to test the hypothesis that Calcidiol derivative B3CD qualifies as a potential anti-cancer drug in vivo employing an ovarian cancer xenograft model in mice. In addition, the selectivity of B3CD on viability and proliferation of platinum-resistant human ovarian cancer cell lines in comparison to control cell lines was analyzed in vitro. B3CD displayed cell line-specific cytotoxicity screened against a panel of ovarian and other carcinoma cell lines, endothelial and control cells. B3CD, at sub-cytotoxic concentrations, revealed stronger effects on the proliferation of SKOV-3 ovarian cancer cells vs. primary fibroblasts as determined by BrdU incorporation analysis. Treatment with B3CD at 0.5 microM resulted in highly condensed chromatin and fragmented nuclei in SKOV-3 cells but not in primary fibroblasts. B3CD induced cell death at low drug concentrations (< or = 0.5 microM) in SKOV-3 ovarian cancer cells is mediated by the p38 MAPK signaling pathway: B3CD induced p38 MAPK expression and activation in SKOV-3 cells and inhibition of p38 signaling counteracted B3CD induced cell death in vitro. An ovarian cancer cell animal model (human SKOV-3 cell derived xenografts in nude mice) revealed that tumor growth in few B3CD treated mice accelerated while the majority of B3CD treated mice displayed delayed tumor growth or full tumor regression. B3CD possesses anti-ovarian cancer properties in vitro and in vivo. We propose the further development of non-calcemic bromoacetoxy derivatives of vitamin D(3) as potential anti-cancer therapeutics.

Fig. 1

Comparative analysis of the cytotoxic effect of B3CD and calcitriol/vitamin D₃ on ovarian cancer and control cell lines. Panel a: Structure of Calcidiol, Calcitriol/Vitamin D₃ and derivative B3CD. Panel b: Effect of B3CD on cell viability. The cytotoxicity of B3CD and calcitriol/vitamin D₃ in ovarian cancer cell lines (SKOV-3, OVCAR-3) was compared to the effect in pancreatic cancer cells (BxPC-3), prostate cancer cells (PC-3, LNCaP), endothelial cells (HUVECs) and primary fibroblasts (BJ). The cells were treated for a total of 24 h with various drug concentrations (0–10 μM). The MTS viability assay was carried out as described (Materials and Methods). Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±SD) of a representative experiment in percent cell viability of untreated cells [=100%]

Fig. 2

Effect of B3CD on morphology and proliferation of ovarian cancer cells and fibroblasts. Panel a: Effect of B3CD on cell morphology. Ovarian cancer cells (SKOV-3), endothelial cells (HUVEC), or primary fibroblasts (BJ) were treated for 24 h with B3CD and calcitriol/vitamin D₃ at a concentration of 0.5 μM before microscopic analysis by phase contrast (PC) or fluorescence analysis after chromatin staining (DAPI) as described (Materials and Methods). Images obtained from a representative experiment are shown. Bar= 10 μm. Panel b: Effect of B3CD on cell proliferation. Ovarian cancer cells (SKOV-3) or primary fibroblasts (BJ) were treated with various concentrations (0–0.6 μM) of B3CD. A colorimetric assay (based on BrdU incorporation detected by an BrdU-antibody peroxidase conjugate) was carried out as described (Materials and Methods). The color intensity at 450 nm correlates directly to the amount of BrdU incorporated into the DNA, which in turn represents proliferation. Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±SD) in percent of absorbance by triplicate samples of untreated cells [=100%]

Fig. 3

Expression and activation of pro-apoptotic MAPK p38 in SKOV-3 cells after B3CD treatment, effect of p38 inactivation on cell viability. Panel a: p38 inhibition after B3CD treatment restores cell viability. SKOV-3 cells were pre-incubated with two different specific inhibitors against p38 MAPK (A: SB203580, B: SB202190) or a derivative of these inhibitors as control for 2 h and treated with 0.5 or 1.0 μM B3CD in the continued presence of the inhibitors (40 μM) for an additional 24 h. The MTS viability assay was carried out as described (Materials and Methods). Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±SD) of a representative experiment in percent cell viability of untreated cells [=100%]. Panel b: Activation of p38 MAPK. SKOV-3 cells were treated with 1 μM B3CD for 5 min, 1 h, or 6 h. Western Blot analysis was carried out as described (Materials and Methods) using primary antibodies against pro-and activated/phosphorylated (P-) p38 MAPK. As an internal standard for equal loading the blots were probed with an anti-beta-Actin antibody

Fig. 4

Anti-cancer activity of B3CD in an ovarian cancer model. SKOV-3 cells were inoculated subcutaneously in one flank of immunodeficient nude mice. After tumor development, mice were treated intraperitoneally every other day with either B3CD (0.15 mg/Kg) (mice B1-16; panels c, d) or vehicle control (mice C1-9; panels a, b). Mice were weighed (panels b, d) and tumor size calculated (panels a, c) using a caliper every 4–6 days. The length of the study was 8 weeks. Mice were euthanized if tumors were greater than 20 mm in diameter (B7, B9, B15 in treatment group) or if ulcers formed (C2, C4, C5, C6, C8 in control group; B8, B10, B12, B14 in treatment group). There was no loss of ≥15% body weight at any time point during the evaluation period. For animals with complete response to treatment (full tumor regression; B5, B13, B16) the observation period was extended by 14 weeks; no tumor recurrence was observed

Fig. 5

Kaplan-Meier analysis of tumor growth in an ovarian cancer animal model. Kaplan-Meier estimates of time until tumor surpassed 15 mm in diameter (by treatment group). Animals that developed ulcers were treated as censored events. P-value=0.3 by log rank test of equality of survival curves