| Objectives 1) To explore the expression patterns of CYP1A1 andCYP1B1 in human medulloblastoma cell line (UW228-3). 2) To analyzethe effects of resveratrol on the expressions of CYP1A1 and 1B1 and therole(s) of AhR in regulating the expressions of these two genes. 3) To studythe status of CYP1A1 expression in human medulloblastoma cells in vivoand its relation with AhR expression. 4) To elucidate the potential actionmechanism of resveratrol on medulloblastoma cell line. 5) To provide anew laboratory evidence and theoretic basis for better treatment of humanmedulloblastoma. Methods Human medulloblastoma cell line UW228-3 was cultured inDMEM (Dulbecco's Modified Eagle's Medium) culture medium containing10% Fetal Bovine Serum. The cells were treated with CYP1A1 selectiveinhibitor (alpha-naphthoflavone, α-NF,15μM), CYP1A1 selective inducer(beta-naphthoflavone, β-NF,15μM) and their combination with resveratrol(100μM) respectively when cells were in good state, then observe theeffects of α-NF and β-NF on resveratrol-induced differentiation andapoptosis of medulloblastoma cells. Putting sterilized cover slips in thedishes before cell seeding whenever cell shape observation orimmunocytochemical analysis was performed. After above treatments,cover slips were collected and fixed with 100% cold acetone. The cellproliferation situations of experimental groups were examined throughTrypan Blue counting method. Immunocytochemistry (ICC) and RT-PCRwere used to analyze the expression levels of CYP1A1, CYP1A2, CYP1B1,AhR, GFAP and synaptophysin of individual experimental groups. Thecorrelations of CYP1A1, CYP1A2, CYP1B1 expressions with celldifferentiation and apoptosis were examined. Paraffin embedded tissueblocks of eleven medulloblastoma cases and their surroundingnoncancerous counterparts were collected from the PathologicalDepartment, the First Affiliated Hospital of Dalian Medical University. Thetissue microarrays (TMA) in a density of 42 spots/1cm2 were constructed,followed by immunohistochemical (IHC) stainings of CYP1A1, AhR,GFAP and synaptophysin. Results 1) Resveratrol not only suppresses the growth ofmedulloblastoma cell line UW228-3 but also induces cell differentiationand apoptosis in dose and time dependent fashions;α-NF and β-NF do notoppose apparent effects on UW228-3 cell shape and growth;No statisticaldifference (P>0.05) can be found between the treatments ofα-NF+resveratrol, β-NF + resveratrol and resveratrol only in terms ofgrowth suppression, apoptosis and differentiation induction. 2) α-NFinhibits resveratrol-induced CYP1A1 expression in UW228-3 cells, andβ-NF really induces CYP1A1 expression. 3) α-NF inhibits expression ofCYP1B1, and β-NF enhances expression level of CYP1B1 in UW228-3. 4)CYP1A2 is undetectable in the cells irrespective to the treatments. 5) Arylhydrocarbon receptor (AhR) is absent in UW228-3 cells under normalculture and treated with resveratrol but can be induced by both α- and β-NF.6) Among the 11-medulloblastoma tissues, CYP1A1 is weakly positive inthree and diminished in the remaining eight cases; in contrast, all of elevennoncancerous cerebellar tissues are positive in CYP1A1; Neithernoncancerous nor medulloblastoma tissues show AhR expression. Conclusions 1) CYP1A1 and 1B1 expressions in humanmedulloblastoma cells do not oppose apparent effects on the anti-cancereffect of resveratrol. 2) Unlike the findings from other tumor systems, theanti-medulloblastoma effect of resveratrol is not mediated by CYP1B1 inUW228-3 cell line. 3) These in vitro and in vivo findings suggest for thefirst time that up-regulated CYP1A1 expression and down-regulatedCYP1B1 expression may reflect a re-differentiation tendency ofmedulloblastoma cells and therefore can be regarded as new positivebiomarkers for this kind of tumor. 4) Resveratrol regulated- CYP1A1 and1B1 expressions in human medulloblastoma cells are throughAhR-independent pathway, which has been further confirmed inmedulloblastoma tissues.
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