ABSTRACT
Using beta-sitosterol as a starting material, (6E)-hydroximino-24-ethylcholest-4-en-3-one (1), a natural steroidal oxime from Cinachyrella alloclada and C. apion, was synthesized in four steps with a high overall yield. First, beta-sitosterol (5a) is transformed into the corresponding 24-ethylcholest-4-en-3,6-dione (6a) via oxidation with pyridinium chlorochromate (PCC). Selective reduction of 6a by NaBH(4) in the presence of CoCl(2) gives 24-ethylcholest- 4-en-3beta-ol-6-one (7a). The reaction of 7a with hydroxylamine hydrochloride offers the oxime 8a and the oxidation of 8a by Jones reagent gives the target steroid 1. (6E)-Hydroximinocholest-4-en-3-one (2) and (6E)-hydroximino-24-ethylcholest-4,22-dien-3-one (4) were synthesized by a similar method. The cytotoxicity of the synthesized compounds against sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells were investigated. The presence of a cholesterol-type side chain appears to be necessary for the biological activity.
Subject(s)
Antineoplastic Agents/chemical synthesis , Cholestadienes/chemical synthesis , Cholestenones/chemical synthesis , Oximes/chemical synthesis , Porifera/chemistry , Steroids/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Cell Line, Tumor , Cholestadienes/toxicity , Cholestenones/toxicity , Humans , Oximes/chemistry , Porifera/classification , Steroids/chemistryABSTRACT
BACKGROUND/AIMS: Biliary epithelial cells are exposed to highly concentrated oxysterols. Therefore, oxysterols may play a role in pathogenesis of biliary tract diseases. We investigated the cytotoxic effect and apoptosis inducing effect of oxysterol on gallbladder epithelial cells. METHODS: We studied the cytotoxic effect of 3,5- cholestadien-7-one, 5 beta-cholestan-3-one and 5,24-cholestadien-3 beta-OL which are identified in human bile and pigment gallstones on dog gallbladder epithelial cells (DGBE) and mouse gallbladder epithelial cells (MGBE). We used model bile to dissolve oxysterols as in vitro experiment and also used MTT, cell count, Diff-Quick stain, and flow cytometry to investigate cytotoxicity and apoptosis. RESULTS: Oxysterols dissolved in model bile have cytotoxic effects in a dose dependent fashion. In oxysterol containing model bile, viable cells are 51% in 500 microM 5 beta-cholestan-3-one (cholesterol:oxysterol 50:50) and 47% in 5 mM 3,5-cholestadien-7-one (90:10) on MGBE, and are 129% and 38% in 500 microM (50:50) 3,5-cholestadien-7-one and 5 beta-cholestan-3-one on DGBE, and are 74% and 71.5% in 5 mM (90:10) 3,5-cholestadien-7-one and 5 beta-cholestan-3-one on DGBE, respectively. 500 microM (50:50) 3,5- cholestadien-7-one, 5 beta-cholestan-3-one, and 5,24-cholestadien-3 beta-OL treated on DGBE increase the apoptotic cell number as 22.0+/-8.8, 30.2+/-12.6, and 45.5+/-13.2%, respectively, compared with control (14.6+/-10.0%). 500 microM (50:50) 3,5-cholestadien-7-one, 5 beta-cholestan-3-one, and 5,24-cholestadien-3 beta-OL also affect the changes in cell cycles compared with the control. CONCLUSIONS: We concluded that oxysterol containing model bile is useful as an in vitro experiment as model to analyze the effects of oxysterols on biliary epithelial cells and that adequate concentration of oxysterols can induce the cytotoxic effect and the apoptosis on gallbladder epithelial cells.
Subject(s)
Apoptosis/drug effects , Cholestadienes/toxicity , Cholestadienols/toxicity , Cholestanes/toxicity , Epithelial Cells/drug effects , Gallbladder/cytology , Animals , Bile/chemistry , Dogs , Dose-Response Relationship, Drug , Gallbladder/drug effects , In Vitro Techniques , RatsABSTRACT
BACKGROUND/AIMS: Biliary epithelial cells are exposed to highly concentrated oxysterols. Therefore, oxysterols may play a role in pathogenesis of biliary tract diseases. We investigated the cytotoxic effect and apoptosis inducing effect of oxysterol on gallbladder epithelial cells. METHODS: We studied the cytotoxic effect of 3,5- cholestadien-7-one, 5beta-cholestan-3-one and 5,24-cholestadien-3beta-OL which are identified in human bile and pigment gallstones on dog gallbladder epithelial cells (DGBE) and mouse gallbladder epithelial cells (MGBE). We used model bile to dissolve oxysterols as in vitro experiment and also used MTT, cell count, Diff-Quick stain, and flow cytometry to investigate cytotoxicity and apoptosis. RESULTS: Oxysterols dissolved in model bile have cytotoxic effects in a dose dependent fashion. In oxysterol containing model bile, viable cells are 51% in 500 microM 5beta-cholestan-3-one (cholesterol : oxysterol 50:50) and 47% in 5 mM 3,5-cholestadien-7-one (90:10) on MGBE, and are 129% and 38% in 500 microM (50:50) 3,5-cholestadien-7-one and 5beta-cholestan-3-one on DGBE, and are 74% and 71.5% in 5 mM (90:10) 3,5-cholestadien-7-one and 5beta-cholestan-3-one on DGBE, respectively. 500 microM (50:50) 3,5- cholestadien-7-one, 5beta-cholestan-3-one, and 5,24-cholestadien-3beta-OL treated on DGBE increase the apoptotic cell number as 22.0+/-8.8, 30.2+/-12.6, and 45.5+/-13.2%, respectively, compared with control (14.6+/-10.0%). 500 microM (50:50) 3,5-cholestadien-7-one, 5beta-cholestan-3-one, and 5,24-cholestadien-3beta-OL also affect the changes in cell cycles compared with the control. CONCLUSIONS: We concluded that oxysterol containing model bile is useful as an in vitro experiment as model to analyze the effects of oxysterols on biliary epithelial cells and that adequate concentration of oxysterols can induce the cytotoxic effect and the apoptosis on gallbladder epithelial cells.
