|J Tradit Chin Med. 2014 Dec;34(6):678-83. |
|Reversal effect of bufalin on multidrug resistance in K562/VCR vincristine-resistant leukemia cell line.[Pubmed: 25618972]|
| To probe insights into the reversal effect of Bufalin on vincristine-acquired multidrug resistance (MDR) in human leukemia cell line K562/VCR. |
METHODS AND RESULTS:
Proliferative inhibition rate and the reversal index (RI) of Bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin (ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry (FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein (P-gp), multidrug-associated protein-1 (MRP1), Bcl-xL and Bax protein were measured by immunocytochemistry. The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to Bufalin. The RIs of Bufalin at concentrations of 0.0002, 0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77, respectively. Preincubation of 0.001 μmol/L Bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07% (P < 0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with Bufalin. When exposed to 0.01 μmol/L Bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-xL and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.
Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-xL/Bax ratio.
|J Biol Chem. 1996 Jun 14;271(24):14067-72. |
|The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells.[Pubmed: 8662906]|
|Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells.|
METHODS AND RESULTS:
When U937 cells were treated with 10(-8) M Bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with Bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with Bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to Bufalin.
Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to Bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.