Efficacy of a non-hypercalcemic vitamin-D2 derived anti-cancer agent (MT19c) and inhibition of fatty acid synthesis in an ovarian cancer xenograft model

Abstract

Background: Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models.

Methodology/principal finding: Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c-VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein.

Significance: Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis.

Figure 1. Chemotherapeutic properties of MT19c in vivo.

(A) chemical structure of MT19c. (B) Anti-cancer activity of MT19c in an EOC model in mice. Nude mice (20 treated and 10 controls) bearing SKOV-3 derived tumor xenografts were dosed (IP) with either vehicle control or MT19c (5 mg/kg bwt) on alternate days for 60 days. Tumor size was calculated (upper panel) using a caliper every 5 days and weight recorded (lower panel). (C) Kaplan-Meier survival analysis. Kaplan-Meier survival analysis for MT19c and vehicle-treated mice was performed using STATA 9 (StataCorp, College Station, TX) and SAS 9.1 software (SAS Institute, Cary, NC). (D) Efficacy of MT19c in a syngeneic EOC model in rats. Fisher 344 rats (3 animals/treatment group) were injected IP with rat EOC cells NuTu-19. After 3 weeks, either MT19c (100 or 500 µg/kg bwt) or vehicle were injected IP daily for 12 days. Tumor tissues were harvested and omental weight (D-1), ascitic volume (D-2) and body weight (D-3) recorded. Mean omental weight and volume were compared by Student's T-test with unequal variances. The lower panel depicts the response index (D-4).

Figure 2. Characteristics of MT19c as a vitamin-D3 derivative.

(A) Serum calcium levels in mice after MT19c treatment. 8 mice each were treated with MT19c(5 mg/kg bwt) or calcitriol (10 ug/kg bwt) or vehicle (EtOH) for 35 days, blood collected and serum calcium analyzed at day 35. Change in mean serum calcium was significant (P<0.05) compared between groups by Student's T-test with unequal variances (B) Acute toxicity study of MT19c. MT19c or vehicle was administered to nude mice and animals were monitored for any observable toxicity. (C) MT19c in a VDR-agonist or antagonist screening. VDR-over-expressing VDR-UAS-bla HEK 293T cells were treated for 5 h with calcitriol/vitamin-D3 (0.1 pM-1 nM; left panel) or MT19c (1 nM-1 µM; middle panel) and VDR-activation was analyzed. To analyze antagonistic effects the assay was carried out (SelectScreen® Cell-based Nuclear Receptor Profiling Services: http://www.invitrogen.com) after cell stimulation with calcitriol/vitamin-D3 (120 pM) and treatment with MT19c (1 nM-1 µM; right panel;) for 5 h. (D) Summary of salient features of MT19c and related compounds. Comparison of MT19c with to calcitriol and other clinically relevant vitamin-d derivatives.

Figure 3. Molecular Docking Simulation of VDR and MT19c.

(A) 3D structures of VDR/calcitriol and VDR/MT19c complexes. Left panel: VDR/calcitriol complex (Calcitriol in center, helix 11 = white color). Right panel: VDR/MT19c complex (MT19c in center, 11 = light green). MDS was carried out using the AutoDock 4.0 program with the structure of MT19 and of calcitriol-liganded VDR provided by the Protein Data Bank. Images of structures were generated using UCSF Chimera. (B) Sequence of VDR ligand binding site. Yellow color code represents helices in the structure. Green color code represents amino acids with direct interaction to the ligand (calcitriol). (C) Interaction comparison. Left panel: Interaction between Leu227 and calcitriol. Right Panel: Interaction between Leu227 and MT19c. (D) Comparison of helix 12 and helix 11 interactions with ligands. Left panel: Interaction between helix 11 (purple), helix 12 (yellow), and calcitriol. Right Panel: Interaction between helix 11 (white), helix 12 (yellow) and MT19c. In both panels interaction between His397 on helix 11 and ligand is depicted. (E) Distances between His 397 and ligands. Left panel: Interaction between His397 and calcitriol. Right Panel: Interaction between His397 and MT19c.

Figure 4. Cellular and biochemical effects in MT19c treated EOC cells in vitro.

(A) Genomic analysis of naïve or MT19c treated xenograft tumors. SKOV-3 xenografts were treated with vehicle or MT19c (5 mg/kg bwt) and tumor tissue were harvested on day-8, 16 and 30. The mRNA from tumors was analyzed by Affymetrix microarray chips in triplicate and expression of genes was clustered by GSEA analysis. The difference in expression of the genes from MT19c treated tumors was expressed as % of the control (±). (B) Expression of nuclear EGFR (nEGFR) in SKOV-3 xenograft tissues treated with vehicle or MT19c. SKOV-3 xenografts were treated with MT19c or vehicle. Paraffin embedded tissues were processed and stained with Alexafluor-conjugated phosphor-EGFR antibody and analyzed by confocal microscopy as described in Material and Method section. EGFR is shown in the green along with nuclear stain DAPI. Magnification: 40×2 (C) Western blot analysis of phospho-EGFR and phospho-PI-3K and expression of nuclear EGFR (nEGFR) in SKOV-3 cells. (Left panel): SKOV-3 cells were treated with 250 nM MT19c. PAGE and Western blot analysis of cell lysates was carried out. Activated phospho-EGFR and phospho-PI-3K was visualized by immunoblotting using primary antibodies recognizing cleaved fragments. As an internal standard for equal loading (50 µg total cell protein/lane) blots were probed with an (-tubulin antibody. (right panel): expression of nuclear EGFR (nEGFR) in vehicle (upper panel), calcitriol (2 uM, middle panel) and MT19c (250 nM, lower panel) treated SKOV-3 cells by immunofluorescence microscopy. EGFR is shown in green and right columns shows DNA (blue). Magnification: 40. (D) MT19c suppressed PI-3k kinase activity in SKOV-3 cells. SKOV-3 cells were treated with MT19c (0, 250 nM) for indicated time intervals. Cell lysates were immunoprecipitated with an antibody specific for phospho-tyrosine and PI3K activity was determined with in vitro lipid kinase assay. PIP-3 (phosphoinositide 3-phosphate), the phosphorylated end-product is shown. The bar graph shows the densitometric scanning results of a representative experiment. (E): Identification of interaction between MT19c and PPARγ using fluorescence polarization. Disruption of binding between PPARγ-LBD and fluorescent DRIP2 peptide by MT19c was investigated in the ▪ presence and • absence of agonist GW929. Controls were vehicle DMSO (▴ with agonist, ○ without agonist), ▾ unlabeled DRIP2 peptide (positive control) and ♦ fluorescent DRIP2 peptide by itself (positive control).

Figure 5. MT19c disrupts mitochondrial functions and fatty acid synthesis machinery in ovarian cancer cells or xenograft tissues.

(A-C) Western blot analysis of lipogenetic proteins in SKOV-3 cells. SKOV-3 cells were treated with 250 nM MT19c or vehicle for 24 h. Analysis of the expression of proteins by western blotting of lysates with primary antibodies against fatty acid synthase (FASN), Acetyl co-A carboxylase (ACC), phosphorylated ACC and AMPA was carried out (Material and Methods). Representative experiments are shown. As an internal standard for equal loading (50 µg total cell protein/lane) blots were probed with an anti-(-tubulin antibody. (B) Mitochondrial transmembrane depolarization-potential (ΔΨm) analysis after MT19c treatment. SKOV-3 cells were treated for 3 or 24 h with 1 µM MT19c fixed and stained with DiOC₁₈(3) and FACS analysis carried out. The bar diagram depicts the number of non-fluorescing cells (%) with ΔΨm loss. A representative experiment is shown. (C) Effect of fatty acid synthase substrates on cytotoxicity of MT19c. SKOV-3 cells, preincubated with citrate (1 mM), acetyl co-A (200 µM) for 1 hr and varying concentration of MT19c (0, 1 µM) were added and cells were incubated for 24 hrs and cell viability was determined by MTS assay. (D) LDH release in SKOV-3 cells. SKOV-3 cells were treated with varying concentration of MT19c (0, 1 µM) were added and cells were incubated for 24 hrs and LDH release estimated using cytotox kit (Promega). (E) expression of fatty acid synthesis proteins in naïve and MT19c treated xenograft tumors. Expression of Fatty acid synthase (FASN) and phospho-acetyl CoA carboxylase (ACC) in vehicle (left panel) and MT19c (5 mg/kg bwt, right panel) treated SKOV-3 xenografts was determined by a confocal immunofluorescence microscopy. FASN is shown in green and phospho-ACC is shown in red. DNA is shown in blue. Magnification: 40×2. (F) HPLC-MS quantification of malonyl-co-A content in MT19c treated SKOV-3 cells. SKOV-3 cells were treated with vehicle or MT19c (500 nM) in serum free DMEM media. Acid soluble extracts were analyzed by HPLC-MS. The area integrals and retention time of the malonyl CoA in control and treatment group was compared to the reference standard (Sigma-Aldrich). (left panel): retention time of reference standard (Sigma Alrich, USA); (middle panel): retention time of malonyl CoA in the vehicle treated SKOV-3 cells; (right panel): retention time of malonyl CoA in the MT19c (500 nM) treated SKOV-3 cells.