RHAMM And CD44 Expressions In Human Medulloblastoma Cells And Their Relevance With Anti-medulloblastoma Effects Of Resveratrol

Objectives: 1) To observe the expression of RHAMM and CD44 in human medulloblastoma cell lines UW228-2 and -3, and tissues, 2) To analyze the effects of resveratrol on the expression of RHAMM and CD44. 3) To study the roles that RHAMM and CD44 played in the processes of apoptosis, differentiation and cell cycle blocking of UW228-2, -3 induced by resveratrol. 4) To further study the potential correlation between RHAMM, CD44 and the high tendency of invasion.Methods: Human medulloblastoma cell lines UW228-2 and -3 were cultured in DMEM (Dulbecco's Modified Eagle's Medium) culture medium containing 10% Fetal Bovine Serum. The cells were treated with resveratrol (100μM) for 48 hours. Put sterilized cover slips in the dishes before cell seeding whenever cell shape observation or immunocytochemical analysis was performed. After above treatments, cover slips were collected and then fixed with 100% cold acetone. At the same time, cell were diluted with cold PBS and centrifuged to extract RNA and proteins. Immunocytochemistry (ICC), Immunofluorescence, RT-PCR, Western-Blot were used to analyze the expression levels of RHAMM and CD44 and their distribution in normal cultured and resveratrol treated cells. Paraffin embedded tissue blocks of eleven medulloblastoma cases and their surrounding noncancerous counterparts were collected from the Pathological Department, the First Affiliated Hospital of Dalian Medical University. The tissue microarrays (TMA) in a density of 42 spots/cm~2 were constructed, followed by immunohistochemical (IHC) stainings of RHAMM.Results: 1) RHAMM was expressed in both medulloblastoma tissues and surrounding noncancerous counterparts, and the difference in distribution is not observerd between the two groups. 2) The changes in expression of RHAMM were undetectable in UW228-2 and -3 cell lines before and after resveratrol treatment. 3)RHAMM was distributed in the cytoplasm and nucleus of medulloblastoma cells, and the overall distribution of RHAMM is almost the same withα-tubulin. 4) CD44s was expressed in medulloblastoma cell lines, and no CD44v variants were found. 5) Being consistent with RHAMM, CD44 underwent no obvious change after resveratrol treatment in both two cell lines.Conclusions: 1) The consistent expression of RHAMM in both medulloblastoma tissues and surrounding noncancerous counterparts, suggested RHAMM may be not involved in the high tendency of invasion of medulloblastoma. 2) Resveratrol-induced differentiation, apoptosis and cell cycle blocking of medulloblastoma cells holded no direct relation with the expression of RHAMM. 3) CD44s may be an important factor in promoting meduloblastoma cells invasion. 4) Though resveratrol exerted anti-medulloblastoma function by inducing differentiation and apoptosis, it may be unable to suppress the invasion of medulloblastoma cells.