Diastereotopic and Deuterium Effects in Gemini

Changing the geminal methyl groups on 1α,25-dihydroxyvitamin D₃ and its analogues to the deuterio versions generally improves the bioactivity. Derivatives of 1α,25-dihydroxyvitamin D₃ with two chains emanating at C20, commonly referred to as gemini, are subject to the same phenomenon. Additionally, gemini with different side chains are susceptible to bioactivity differentials where the C17–C20 threo configuration usually imparts higher activity than the corresponding erythro arrangement. In an effort to analyze the deuterium effect on gemini with minimal diastereotopic distortion, we synthesized gemini with equal side chains but introduced deuterium diastereospecifically on either chain. We solved the crystal structures of these compounds in the zebra fish zVDR ligand binding domain as complexes with NCoA-2 coactivator peptide and correlated the findings with growth inhibition in a breast cancer cell line

CD44 knockdown inhibits cell invasion and down-regulates MMP-9, MMP-14 and uPA.

<p>(A) The protein levels of CD44v, CD44s and pSTAT3 were markedly repressed in DCIS-shCD44 cells. (B) DCIS-shLuc or DCIS-shCD44 cells (2,000 cells/well) were incubated for 48 h and cell proliferation was determined by thymidine incorporation. Two separate experiments with triplicates were conducted (** p<0.01). (C) MCF10DCIS (DCIS), DCIS-shLuc or DCIS-shCD44 cells were incubated for 48 h in BME-coated invasion assay chambers. The number of cells that penetrated the BME layer was counted by Calcein-AM staining. Two separate experiments with triplicates were conducted (**<i>p</i><0.01). (D) DCIS-shLuc or DCIS-shCD44 cells were incubated for 48 h in Fluoroblok biocoat invasion assay chambers. Since both cells were labeled with green fluorescence, the cells that penetrated matrigel layer were detected as green pixels in the image. The green pixels were counted using Image-J program for quantitative evaluation. Two separate experiments with triplicates were conducted (*<i>p</i><0.05). (E) The mRNA expression levels of CD44 (20, the approximate qPCR cycle number of DCIS-shLuc cells at 24 h), MMP-2 (24), MMP-9 (29), MMP-14 (23) and uPA (21) in DCIS-shLuc and DCIS-shCD44 cells were determined after 24 h and 48 h of incubation. Three separate experiments with duplicates were conducted (**<i>p</i><0.01).</p

CD44 knockdown inhibits MCF10DCIS xenograft tumor growth and invasion marker expression.

<p>DCIS-shLuc or DCIS-shCD44 cells (1.0×10⁶ cells) were injected into the mammary fat pad of nu/nu mice (n = 5 per group), and mammary tumor size was measured twice a week. (A) The xenograft tumors from DCIS-shCD44 cells showed significantly slower growth rate than that of DCIS-shLuc xenograft tumors (*<i>p</i><0.05, **<i>p</i><0.01). (B) The average tumor weight from DCIS-shCD44 cells was significantly smaller than that from DCIS-shLuc cells (*<i>p</i><0.05). (C) The mRNA expression levels of CD44 (21, the approximate qPCR cycle number of DCIS-shLuc tumors), MMP-9 (22) and uPA (22) were significantly down-regulated in DCIS-shCD44 xenograft tumors (n = 5) (*<i>p</i><0.05, **<i>p</i><0.01). (D) The protein levels of CD44v, CD44s, pSTAT3, and MMP-9 were markedly decreased in DCIS-shCD44 xenograft tumors. Three xenograft tumors from each group were combined for pooled samples. β-Actin was used as a loading control. (E) The protein levels of CD44 and pSTAT3 in DCIS-shLuc and DCIS-shCD44 xenograft tumors were determined by immunofluorescent staining of CD44 (red) and pSTAT3 (green). Nuclei were stained with TO-PRO-3 (blue).</p

The repression of CD44-STAT3 signaling by BXL0124 in basal-like breast cancer cells.

<p>(A) MCF10CA1a and MDA-MB-468 cells were incubated with BXL0124 (10 nM) for 24 h and the protein levels of VDR, CD44v, CD44s, pSTAT3 and STAT3 were determined by Western blot analysis. β-Actin was used as a loading control. (B) A schematic diagram of proposed mechanism of action of BXL0124 on CD44-STAT3 signaling and breast cancer cell invasion in basal-like breast cancer.</p

1α,25(OH)₂D₃ and Gemini vitamin D analog BXL0124 repress proliferation, metabolic activity and invasion of MCF10DCIS breast cancer cells.

<p>MCF10DCIS cells were incubated with 0.01, 0.1, 1, 10 or 100 nM of 1α,25(OH)₂D₃ or the Gemini vitamin D analog BXL0124 for 72 h. (A) The cell proliferation of MCF10DCIS cells was measured by thymidine incorporation rate. Two separate experiments with triplicates were conducted (*p<0.05, **p<0.01). (B) The metabolic activities of MCF10DCIS cells were determined by the MTT assay. Two separate experiments with quadruplicates were conducted (*p<0.05, **p<0.01). (C) MCF10DCIS cells were incubated in the basement membrane extract (BME)-coated invasion chambers in the presence or absence of 1α,25(OH)₂D₃ (1, 10 or 100 nM) or BXL0124 treatment (1 or 10 nM) for 48 h. The cells that penetrated through BME layer were detected from the bottom of chamber, and counted using Calcein-AM staining. Two separate experiments with triplicates were conducted (*p<0.05, **p<0.01). (D) MCF10DCIS cells were incubated in 3D culture with or without 1α,25(OH)₂D₃ (1, 10 or 100 nM) or BXL0124 (1 or 10 nM) for 10 days, with replenishing medium every 2 days. Representative images are shown, and the cells with invasive outgrowth are indicated with arrows.</p

BXL0124 represses invasion markers and decreases protein levels of CD44 and pSTAT3 in MCF10DCIS cells.

<p>(A) MCF10DCIS cells were treated with BXL0124 (10 nM) for 24 h and 48 h. The mRNA expression levels of CD44 (20, the approximate qPCR cycle number of 24-h control), MMP-2 (24), MMP-9 (29), MMP-14 (23) and uPA (21) were determined. Three separate experiments with duplicates were conducted (*p<0.05, **p<0.01). (B) MCF10DCIS cells were treated with BX0124 (0.1, 1 or 10 nM) for 24 h. (C) MCF10DCIS cells were treated with BXL0124 (10 nM) for 6 h, 12 h and 24 h. (D) MCF10DCIS cells were transfected with non-targeting siRNA or VDR siRNA and treated with BXL0124 (10 nM) for 24 h. The protein levels of indicated molecules were examined by Western blot analysis, and β-actin was used as a loading control.</p

BXL0124 inhibits STAT3 activation and CD44-STAT3 interaction in MCF10DCIS cells.

<p>(A) MCF10DCIS cells were treated with BXL0124 (10 nM) for 24 h. Cells were fixed using 4% paraformaldehyde and stained with antibody against pSTAT3 (green). Nuclei were stained with To-PRO-3 (blue). (B) MCF10DCIS cells were treated with BXL0124 (0.1, 1 or 10 nM) for 24 h. Each cell lysate was incubated with oligonucleotides containing STAT3 binding sequences. The amount of STAT3 bound to the oligonucleotides was measured as chemiluminescent intensity value by luminometer. The fold change of chemiluminescent intensity value in each sample from control was determined (*p<0.05). (C) and (D) MCF10DCIS cells were treated with BXL0124 (10 nM) for 24 h, then the cell lysates were immunoprecipitated with STAT3 or JAK2 antibodies. The amounts of given proteins interacting with STAT3 or JAK2 were determined by Western blot analysis. STAT3 and JAK2 were used as loading control for each immunoprecipitation experiment, respectively.</p