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<p><b>OBJECTIVE</b>To investigate the apoptosis inducing effects of ponicidin (PON) on leukemic K562 cells and its mechanisms of action.</p><p><b>METHOD</b>K562 cells in culture medium in vitro were given different concentrations of PON (10-50 micromol x L(-1)) for 24, 48 and 72 h. The inhibitory rate of the cells was measured by MTT assay, cell apoptotic rates were detected by flow cytometry (FCM) using Annexin V staining after K562 cells were treated with different concentrations of PON for 72 hours, and cell morphology was observed by Wright-Giemsa staining. Western blot was used to detect caspase-3 and poly(ADP-ribose) polymerase (PARP) expression, and the protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) as well as p-AKT and p-P85 in PI3K/AKT signaling pathways were also detected.</p><p><b>RESULT</b>PON (over 30 micromol x L(-1)) could inhibit the growth of K562 cells in both time- and dose-dependent manner. FCM analysis revealed that apoptotic cells were gradually increased in a dose-dependent manner after treatment for 72 hours, and that marked morphological changes of cell apoptosis such as condensation of chromatin was clearly observed by Wright-Giemsa staining after treatment by 50 micromol x L(-1) PON. Western blot showed cleavage of the caspase-3 zymogen protein (32 kD), with the appearance of its 17 kD subunit, and a cleaved 89 kD fragment of 116 kD PARP was also found. Furthermore, Western blotting also showed that expression of p-AKT and p-P85 in PI3K/AKT signaling pathways was downregulated dramatically whereas the expression of p-P38 as well as p-ERK and p-JNK remained unchanged after the cells were treated by PON for 48 h.</p><p><b>CONCLUSION</b>The results demonstrate that PON exhibits in vitro anti-leukemia effect by induction of apoptosis in K562 cells, and that PON induced apoptosis in K562 cells mainly related to activation of caspase-3 as well as inactivation of PI3K/AKT signaling pathway via down regulation of the expression of p-AKT and p-P85 protein levels. These results provide strong laboratory evidence for further anti-leukemia trials of PON.</p>
Subject(s)
Humans , Apoptosis , Blotting, Western , Caspase 3 , Metabolism , Cell Line, Tumor , Diterpenes , Pharmacology , Poly(ADP-ribose) Polymerases , Metabolism , Signal TransductionABSTRACT
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 effect of peroxisome proliferator-activated receptor r(PPARr)agomst on leukemic K562 cells and its mechanisms of action.Methods K562 cells in culture medium in vitro were given different concentrations of PPARragonist rosiglitazone(RGZ)(20-80 umol/L)for O,24,48 and 72h The inhibitory rate of the cells were measured by MTT assay,cell apoptosis was detected by Annexin V/PI staining,and the expression of P53 protein as well as the activity of caspase-3 were also detected.Results RGZ(over 40umoL/L)could inhibit the growth of K562 cells and cause apopto-sis remarkably,the suppression Was both in time-and dose-dependent manner.The expression of P53 pmtein was upregulated and the activity of caspase-3 Was increased concomitantly after the cells werle treated by RGZ.Conclusion PPARr agonist RGZ(over 40 umol/L)can induce apoptosis on K562 cells signifieantly,upregu-lation the expression of P53 protein as well as increasing caspase-3 activity may be one of its most important mechamisms.
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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.