Anti‐proliferative and Pro‐apoptotic Properties of 3‐Bromoacetoxy Calcidiol in High‐risk Neuroblastoma

Abstract

The cytotoxic, anti-proliferative and apoptotic effects of 3-Bromoacetoxy Calcidiol (B3CD), a derivative of vitamin D 3 precursor calcidiol, on human neuroblastoma (NB) cells were examined. NB, predominantly a tumor of early childhood, is the most common extracranial solid tumor. Despite aggressive treatments, survival for advanced stages remains low and novel treatment strategies are needed. B3CD-induced apoptosis in various neuroblastic cells via caspases-3 and -9 activation. B3CD upregulated mitochondrial pro-apoptotic Bax and anti-apoptotic Bcl-2 expression, caused cytochrome c release, downregulated N-Myc expression and activated pro-survival marker Akt. Accordingly, B3CD treatment dose dependently reduced the viability of NB cells with IC 50 values between 1 and 3 μM. The cytotoxicity of B3CD was significantly higher than for the calcemic parentcompound vitamin D 3 (IC 50 between 10 and 30 μM). Further studies revealed that B3CD treatment inhibits the proliferation of NB cells at low concentrations (IC 50 between 30 and 100 nM). Cell cycle analysis showed a dramatic increase in the apoptotic sub-diploidal population along with a cell cycle block. In summary, the present study shows that B3CD is toxic to NB cells via suppression of cell proliferation and cell viability by caspase activation and regulation of survival signals. These results suggest that B3CD could be developed as a treatment for NB. Keywords chemical biology; drug design; kinases; signal transductionNeuroblastoma (NB), predominantly a tumor of early childhood, is the most common extracranial solid tumor. Two-thirds of the cases occur in children below the age of five. NB account for 7-10% of all childhood cancers; in the majority of patients older than 1 year of age, the disease is fatal (1). There are approximately 500-1000 new cases of NB in the USA each year (2). Treatment methods currently available include surgery, radiation therapy, chemotherapy, and autologous stem cell transplantation (3-5) either alone or in combination, depending on the location and biological characteristics of the cancer cells, stage and the risk group to which the patient belongs. However, despite intensive multimodality treatment, more than 50% of children with high-risk disease relapse, because of drug-resistant residual disease *Corresponding author: Laurent Brard, lbrard@wihri.org; Laurent_Brard_MD@Brown.edu Thilo S. Lange and Rakesh K. Singh contributed equally to the manuscript. [6][7][8]. Eradication of refractory microscopic disease remains one of the most significant challenges in the treatment of the high-risk NB and innovative treatments need to be designed. NIH Public AccessCalcitriol/vitamin D 3 (1,25-dihydroxy-vitamin D 3 ) (Figure 1), an endocrine hormone responsible for calcium and mineral homeostasis, inhibits cell proliferation and induces differentiation, via binding to the vitamin D receptor (VDR) (9). Its clinical use in cancer therapy is limited by its hypercalcemic side-effects (10-12). Synthetic analogs of calcitriol/ vitamin D ...

Description of the cytotoxic effect of a novel drug Abietyl-Isothiocyanate on endometrial cancer cell lines

“…Western blot analysis ECC-1 cells (1.5×10 6 per dish) were seeded into 100 mm cell culture dishes and treated with 3 μM ABITC for 6 or 24 h. Preparation of cell lysates, PAGE and immunoblotting was carried out as described previously [18] in Cell Extraction Buffer (BioSource International, Inc., CA.) supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO) according to the manufacturers' recommendations.…”

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“…Preparation of cell lysates, PAGE and immunoblotting was carried out as described elsewhere [13]. Primary antibodies were all purchased from (Cell Signaling Technology, Beverly, MA).…”

“…The p38 inhibitor itself was slightly cytotoxic to the cells but could almost completely counteract 7-MeIEITC cytotoxicity at lower concentrations and significantly at higher concentrations (89% viability at 0 μM, 81% at 2.5μM, 65% at 5μM 7Me-IEITC) ( Figure 5B). As a positive control for the maximal potential effect of inhibition of p38 activity on drug-induced cytotoxicity, SKOV-3 cells were treated with the calcidiol derivative B3CD (13). Here, at the same inhibitor concentration (40μM) the drug effect (8% viability) could be completely suppressed by the p38 inhibitor (93%).…”