| BackgroundTelomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG repeats at the ends of linear chromosomes. Telomerase activity is strong in most cancer cells, whereas it is not detected in normal cells except for stem cells and germ cells. Telomerase activation is essential for the stabilization of chromosomes in proliferation cells, leading to cellular immortality and oncogenesis . The human telomerase contains human telomerase RNA (hTR), telomerase associate protein (TP1) and human telomerase reverse transcriptase (hTERT). It appears telomerase play an important role in the development of malignancy tumors, therefore telomerase is an obvious tumor marker for cancer diagnosis and a promising tumor therapeutic target. The regulatory mechanisms are very implicated and may correlate with telomerase composition, cell cycle , cell differentiation and apoptosis gene. Recent studies have shown that up-regulation of hTERT expression by oncogene C-myc may be a critical step of telomerase activation and cancer development.Oridonin is a diterpenoid isolated from Rabdosia Rubdosia Hemsl of medicine herb. Experiments in vitro and in vivo proved that oridonin have strong anticancer activity, and it had been used to treat esophageal cancer and liver cancer with specific curative effect. Therefore, oridonin is a new promising anticancer drug .Studies showed that oridonin can inhibit DNA transcriptase and arrest cell cycle in G2-M stage. Recent studiessuggested that oridonin can induce leukemic cells k562 and HL-60 cells to apoptosis. However, the correlation between telomerase activity and its possible anticancer molecular mechanisms has not been reported. The aim of this study was to investigate the effect of oridonin on telomerase activity in K562 cells and to further clarify its anticancer molecular mechanism through using the cytological and molecular biological methods.MethodsThe human leukemic cell line k562 cell was adopted in the experiments. MTT(3-(4,-5-dimethylthiazol)-2,-5-diphenylte-2H-tetrazolium bromide ) assay was used to analyze the cytotoxicity of oridonin alone and ORI combined with Adimycin(ADR) in k562 Cells. Immunohistochemistry (IHC) technique was applied to determine by flow cytometry (FCM) on the 24 hours and 48 hours respectively after adding the different concentration of ORI.Results1. Cytotoxicity of ORI in k562 cellsAccording to the result of MTT assay, ORI could inhibit the proliferation of K562 cells in concentration-dependent way. There was statistical significance (P<0.05) between experimental groups and the normal control. The IC50 of ORI in K562 cells is 639O.29uinol.L-.2. Cytotoxicity of ORI combined with ADR in k562 cellsCytotoxicity of ORI or ADR alone in k562 cells was weak , The inhibitory rate of k562 cells treated with 6.86umol.L ORI was ( 53.06?.83 ) %; Treated with 13.72nmol.L' ORI, the IR was ( 65.90?.93 ) %; Treated with 0.34umol.L ADR ,the IR was ( 44.50?.81 ) %;Treated with 0.34umol.L'' ADR combined with 6.86umol.U' ORI, the IR was ( 67.19? .71 ) %; Compared with that of 0.34nmol.L ADR alone or that of 6.86umol.L ORI alone, there was statistical significance in each group . 01). Treated with 0.34umol.L'' ADR combined with 13.72nmol.L~1 ORI, the IR was ( 90.921.29) %; Compared with that of 0.34umol.L"' ADR alone or that of 13.72umol.L-' ORI alone,there was statistical significance in each group (PO.01).3. Effect of OKI on expression of hTERT or C-MYC in k562 cellsThe expression of hTERT or C-MYC in k562 cells was measured with IHC and results were analyzed with HIPAS -1000 computer-assisted image analyzed system (IAS) to achieve semi quantitative data. The value of light density of hTERT in control group was 8.80?.64; If k562 cells was treated with 3.43umol.L~' ORI for 48 hours, the value was 3. 17?1.45, compared with control group, there was significance difference (PO.Ol). The value of C-MYC in control group is 7.48?.74; If K562 cells was treated with 3.43wmol.L~' ORI for 48 hours, the value was 2.28?.92, there was statistic...
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