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[ABSTRACT]AIM:ToinvestigatetheeffectoforidoninontheinvasionandmigrationofhumanlungcancerNCI-H460 cells.METHODS:NCI-H460 cells were divided into high-dose (HD), middle-dose (MD) and low-dose (LD) oridonin groups (cultured with 40, 20 and 10μmol/L of oridonin, respectively, as experimental groups), and normal (N) group ( treated without oridonin as control ) .The cell growth was observed .The cell proliferation was detected by MTT as-say.Boyden chamber was used to determine the cell invasive capacity .The cell migration was also measured .The levels of MMP-2 and MMP-9 were assayed by Western blotting .RESULTS:The cell counts in the experimental groups were lower than that in N group .The cell proliferation was inhibited as the inhibitory rates were 48.94%, 36.17%and 19.15% for HD group, MD group and LD group, respectively.The numbers of the invasive cells were 26.67 ±5.16 for HD group, 36.17 ±5.08 for MD group, and 44.33 ±5.50 for LD group.The migration rates in the experimental groups were lower than that in N group .The expression of MMP-2 and MMP-9 decreased dependent on the oridonin dose as follows: HD group
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Objective To investigate the anti-proliferation effect of peroxisome proliferator-activated receptor γ(PPARγ)agonist troglitazone(TGZ)on leukemic Raji cells and its mechanisms.Methods Raji cells in culture medium in vitro were given different concentrations of TGZ(0～60 μmol/L)for 24,48 and 72 h.The inhibitory rates of the cells were measured by MTT assay,cell apoptotic rate was detected by flow cytometry(FCM),agarose gel electrophoresis was used to observe the DNA ladder,and western blotting was used to analyzed the variation of apoptosis related proteins bcl-2,Bax and Survivin.Results TGZ(over 20 μmol/L)could inhibit the growth of Raji cells and cause apoptosis remarkably,the suppression was both in time-and dose-dependent manner.DNA ladder was observed after the cells treated by TGZ for 72 h,and western blotting analysis revealed that anti-apoptotie proteins Survivin and bcl-2 were decreased remarkably while pro-apoptotic protein Bax increased significantly after the cells were treated by TGZ for 48 h.Conclusion PPARγ agonist TGZ can inhibit the growth and induce apoptosis on Raji cells significantly,downregnlating the expression of Survivin and bcl-2 as well as upregulating of Bax expression of Raji cells may be one of its most important mechanisms.
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Objective To investigate the apoptosis inducing effects of oridonin on leukemic THP-1 cells and its mechanisms of action. Methods THP-1 cells in culture medium in vitro were given different concentrations of oridonin (16～56 μmol/L) for 24, 48 and 72 h. The inhibitory rate of the cells were measured by MTT assay, apoptotic morphology was observed by Hoechst 33258 staining, and Annexin V/PI staining was used to detect cell apoptosis by flow cytometry (FCM). Caspase-3 and poly (ADP-ribose) polymerase (PARP) expression were detected by Western blotting. Results Oridonin (over 32 μmol/L) could inhibit the growth of THP-1 cells and cause apoptosis remarkably, the suppression was both in time-and dose-dependentmanner. Marked morphological changes of cell apoptosis such as condensation of chromatin was clearly observed by Hoechst 33258 staining, and Annexin V/PI staining showed that apoptotic cells gradually increased after the cells treated with oridinon. Western blotting showed cleavage of the caspase-3 zymogen protein (32×103), with the appearance of its 20×103 subunit, and a cleaved 89×103 fragment of 116×103 PARP was also found. Conclusion Oridonin can inhibit cell growth and induce apoptosis in THP-1 cells via activation of caspase-3. The results indicated that oridonin might be an important potential anti-leukemia reagent.
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Objective To investigate the mechanisms of oridonin inducing apoptosis on acute leukeamia NB4 cells and its mechanism. Methods NB4 cells in culture medium in vitro were given with different concentrations (8, 16, 24, and 32 μmol/L) of oridonin. The inhibitory rate of the cells was measured by MTT assay, cell apoptotic rate was detected by flow cytometry (FCM), morphology of apoptosis was observed by Hoechst 33258 fluorescence staining, DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3 expression was detected by Western blotting, and caspase-3 activity was assayed with colorimetric assay kit before and after apoptosis occurred. Results Oridonin (over 16 μmol/L) could inhibit the growth of NB4 cells and cause apoptosis significantly, the suppression was both in a timeand dose-dependent manner. Marked changes of apoptosis including condensation of chromatin and nuclear fragmentation were observed very clearly by Hoechst 33258 fluorescence staining and a characteristic "ladder" of DNA fragments was elicited by agarose gel electrophoresis; Western blot analysis revealed that caspase-3 was activated by the loss of caspase-3 proenzyme (32 kDa) and the appearance of its 20 kDa subunit, and that along with the apoptotic process caspase-3 activity was increased concurrently. Conclusion Oridonin can induce apoptosis in NB4 cells via activation of caspase-3. These results will provide laboratory evidence for the clinical treatment of acute leukemia with oridonin.
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Objective: To explore the mechanism of orindonin induced apoptosis on NB4 (Human acute promylocytic leukeamia cell line) cells. Methods: The variation of both morphology and inhibitory rate of NB4 cells were observed in culture medium with various concentrations of orindonin at different time in vitro. The activity of Caspase3 was detected before and after apoptosis occurred. Results: orindonin could increase the activity of Caspase3, inhibit the growth and cause apoptosis on NB4 cells significantly. The variations were both in time and dose dependent manner. Conclusion: Orindonin can inhibit the growth of NB4 cells and induce apoptosis. Increasing the activity of Caspase3 may be one of the most important mechanism.
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AIM: To investigate the differentiative and apoptotic effect of CpG-oligodeoxynucleotides on HL60 cells and its mechanism. METHODS: After HL60 cells were exposed to synthetic CpG-oligodeoxynucleotides, non-CpG-oligodeoxynucleotides or ZpG-oligodeoxynucleotides for 72 hours, respectively, the inhibition of HL60 cells were detected using MTT method, NBT test was used and CD14 expression were determined. Apoptosis of HL60 cells were mensurated with flow cytometry and transmission electron microscope, and caspase 3, Bcl-2 and Bax expression of HL60 cells treated with oligodeoxynucletides were detected using immunohistochemistry. RESULTS: Treatment with CpG-oligodeoxynucleotides induced the differentiation and apoptosis in HL60 cells, but non-CpG-oligodeoxynu- cleotide and ZpG-oligodeoxynucleotide had no effect on HL60 cells. CONCLUSION: CpG-oligodeoxynucleotides can induce the differentiation and apoptosis in HL60 cells. It may provide a new approach for the immunological treatment of leukemia.