CCND1 silencing promotes chemosensitivity of glioblastoma cell lines to temozolomide / 第二军医大学学报

Objective To screen chemotherapeutic drugs that can synergistically inhibit the proliferation of glioblastoma cells with CCND1 by constructing CCND1-silenced and overexpressed human glioblastoma cell lines SHG-44. Methods SHG-44 cells with CCND1 silenced or overexpressed were constructed, and the expression of P-glycoprotein (P-gp, expression product of multidrug resistance geneMDR1) and apoptotic factors CBcl-2, Caspase-3) in the cells were detected by Western blotting. The SHG-44 cells with CCND1 silenced or overexpressedwere cultured with carmustine GBCNU), lomustine (CCNU) and temozolomide (TMZ), respectively; and then the available chemotherapeutic drugs were screened, which could synergistically inhibit the proliferation of tumor cells with CCND1 sliencing. Human glioblastoma cell lines U251 were used toverify the findings in SHG-44cells. Results Western blotting analysis showed that CCND1 silencing significantly down-regulated the expressions of MDR1 and Bcl-2, and up-regulated the expression of Caspase-3 (all P<0. 01). There were no significant differences in cell growth curves between the CCND1-silenced cells treating with BCNU (0. 05 µg/mL, 0. 25 µg/mL) and CCNU (20 µg/mL, 80 µg/mL) for 2, 3, 4, and 5 days; However, the proliferation of CCNDUsilenced SHG-44 cells was significantly inhibited by TMZ (9. 1 µg/mL) compared with parent SHG-44 cells on the 4th and 5th day after treatment (P<0. 05). The findings that CCND1 silencing promoted chemosensitivity of TMZ were confirmed by U251 cellexperiments. Conclusion CCND1 silencing combined with TMZ is more effective in inhibiting the proliferation of human glioblastoma cell lines SHG-44 than CCND1 silencing or TMZ alone, suggesting that CCND1 may be involved in chemotherapeutic resistance of glioblastoma cells to TMZ.

Effect of RNA interference targeting for cyclin D1 gene on growth,cell cycle and apoptosis of K562 / 中国基层医药

Objective cyclin D1 gene plays a significant role in regulating cell cycle progression.Suppression of cyclin D1 protcin expression can effect on cellular proliferation,distribution of cell cycle and apoptosis.This study was to determine whether this effect also existed in chronic leukemia ceil line K562 by inhibiting the expression of cyclin D1 protein through RNA interference in vetro.Methods Plasmid vectors expressing small hairpin RNA (shRNA) targeting at cyclin D1 gene were constructed and transfected into K562 cells by chitosan,cyclin D1 protein was examined by using Western blot analysis.Inhibition of cellular proliferation was evaluated hy soft agar colony formation assay.The cell cycle and apoptosis were determined by flow cytometry.Results Expression of cyclin D1 protein was markedly down-regulated and capability of colony formation was suppressed after transfection with pshRNA-419 and pshRNA-575 at 48h.Down-regulation of cyclin D1 protein could effect on distribution of cell cycle arrested at G_0/G_1 phase and markedly induce apoptosis of K562 cells.But there had no above biological effects ob- served after transfection with blank vector and control vector of m-pshRNA-790.Conclusion Down-regulation of cyclin D1 expression can inhibit growth of K562 cells,and effect on distribution of cell cycle arrested at G_0/G_1 phase.The primary results suggest that cyclin D1 gene might serve as an effective target for the treatment of leukemia.