Effect of 5-aza-2'-deoxycytidine combined with trichostatin A on RPMI-8226 cell proliferation, apoptosis and DLC-1 gene expression / 中国实验血液学杂志

This study was aimed to investigate the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) on DLC-1 gene transcription regulation and molecular biological behaviours in the human multiple myeloma RPMI-8226 cells. The cells were treated respectively with 5-Aza-CdR and TSA alone, or the both combination; the cell proliferation and apoptosis, DLC-1 expression, the protein expression of Ras homolog family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) were examined by CCK-8 method, RT-PCR and ELISA, respectively. The results showed that the 5-Aza-CdR and TSA had cell growth inhibitory and apoptosis-inducing effects in dose-dependent manner (P < 0.05). Compared with a single drug (5-Aza-CdR or TSA alone), the effects were significantly enhanced after treatment with the combination of 5-Aza-CdR and TSA (P < 0.05). DLC-1 was weakly expressed in the control group; the treatment with 5-Aza-CdR alone enhanced its re-expression dose-dependently (P < 0.05). Compared with 5-Aza-CdR alone, 5-Aza-CdR plus TSA enhanced DLC-1 re-expression significantly.Compared with the control, 5-Aza-CdR and TSA significantly decreased RhoA and Rac1 protein expression (P < 0.05). It is concluded that 5-Aza-CdR and TSA can effectively reverse DLC-1 expression of RPMI-8226 cells; TSA has a synergistic effect on its re-expression. 5-Aza-CdR and TSA have significant cell growth inhibitory and apoptosis-inducing effects on RPMI-8226 cells. These effects may be related to the inhibition of Rho/Rho kinase signalling pathway.

Expression and significance of melanoma antigen gene-3 in endoplasmic reticulum stress-induced apoptosis of K562 cells / 中国实验血液学杂志

This study was aimed to explore the expression and significance of melanoma antigen gene-3 (MAGE-3) in endoplasmic reticulum stress-induced apoptosis. After the treatment of leukemia cell line K562 and its multidrug-resistant cell line K562/A02 by thapsigargin, intracellular calcium concentrations ([Ca(2+)]i) in K562 and K562/A02 were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM; expression changes of glucose-regulated protein 78 (GRP78) were detected by Western blot; morphological change of apoptotic cell was investigated by AO/EB fluorescent staining under fluorescent microscope; apoptosis rate was determined by terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) staining; the expression of MAGE-3 gene mRNA was detected by RT-PCR. The results showed that (1) thapsigargin induced the enhancement of [Ca(2+)]i with different extent in K562 and K562/A02 cells, and the enhancement of [Ca(2+)]i was dose-dependent in experiment range. At the same time, thapsigargin induced upregulation of GRP78 protein expression and typical apoptotic changes of K562 and K562/A02 cells, apoptotic rate was also dose-dependent in experiment range. The [Ca(2+)]i in K562/A02 cells were higher than that in K562 cells. (2) in the course of endoplasmic reticulum stress-induced apoptosis by thapsigargin, the expression of MAGE-3 gene mRNA was remarkably downregulated. Moreover, the expression of MAGE-3 gene mRNA in K562/A02 cells was higher than that in K562 cells. It is concluded that (1) thapsigargin induces endoplasmic reticulum stress-induced apoptosis of K562 and K562/A02 cells in experiment range, and this may be associated with downregulation of MAGE-3 mRNA expression or MAGE-3 gene may participates in the regulation of endoplasmic reticulum stress-induced apoptosis. (2) MAGE-3 gene may possess anti-apoptotic activity, multidrug resistance in K562/A02 cells can be associated with [Ca(2+)]i increase and upregulation of MAGE-3 expression.