Mechanisms of preventive effect of tetrandrine on acquired multidrug resistance in K562 cells / 中国实验血液学杂志

This study was purposed to explore the mechanisms of preventive effect of tetrandrine (TTD) on doxorubicin (ADM)-induced multidrug resistance (MDR) in human leukemia cell line K562 from two aspects of the transcription control of MDR1 gene and cell apoptosis. The experiment was divided into 3 groups: group I-blank control; group II-ADM-induced drug-resistance; group III-ADM-induced drug-resistance after pretreatment with TTD. Reverse transcription-PCR (RT-PCR) was used to detect the mRNA expression levels of c-Jun, YB-1 and Survivin genes. Western blot was used to determine the nuclear protein expression levels of c-Jun and YB-1. Flow cytometry was used to assay the apoptosis of cells. The results showed that as compared with group I, the expression levels of c-Jun mRNA and nuclear protein decreased (p < 0.05), as well as the expression levels of YB-1 mRNA and nuclear protein increased in group II (p < 0.05). However, the expression of Survivin mRNA had no change (p > 0.05); the apoptosis rate of cells was 8.31%. As compared with group II, the expression levels of c-Jun mRNA and nuclear protein increased (p < 0.05), expression levels of YB-1 mRNA and nuclear protein as well as Survivin mRNA decreased in group III (p < 0.05). The apoptosis of cells was 97.2%. It is concluded that TTD can inhibit the expression of YB-1 and up-regulate the expression of c-Jun, thus inhibit the expression of MDR1 gene. TTD can also inhibit the expression of Survivin and increase the apoptosis of cells induced by ADM.

Cloning, expression and functional analyses of human platelet-derived growth factor-B chain peptide for wound repair of cat corneal endothelial cells / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application.</p><p><b>METHODS</b>Total RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively.</p><p><b>RESULTS</b>PDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells.</p><p><b>CONCLUSIONS</b>The construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.</p>

Effect of tetrandrine on the doxorubicin-induced expression of mdr1 gene in K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of tetrandrine (TTD) on doxorubicin-induced mdr1 gene expression and its mechanism.</p><p><b>METHODS</b>MTT assay was used to detect the cytotoxicity of TTD to K562 cells. K562 cells were treated with doxorubicin alone or 0.6 microg/ml doxorubicin combined with various concentrations of TTD. RT-PCR was used to detect the mRNA expression of mdr1 and NF-kappa B. Flow cytometry was used to assay the expression of P-glycoprotein (P-gp). Intracellular rhodamine 123 (Rho123) retention assay was applied to test the P-gp function.</p><p><b>RESULTS</b>After treatment with 0.6 microg/ml doxorubicin for 24 hours, the expressions of mdr1 mRNA, NF-kappa B mRNA and P-gp in K562 cells were increased from 0.171 +/- 0.012, 0.783 +/- 0.090, 7.85 +/- 0.15 to 0.428 +/- 0.012, 1.075 +/- 0.047 and 73.68 +/- 1.84, respectively. The intracellular Rho123 retention was decreased from 711.9 +/- 63.6 to 347.8 +/- 60.6, indicating up-regulation of P-gp function (P<0.05). Pretreatment of K562 cells with 2.0 microg/ml TTD for 24 hours and then incubated for another 24 h with doxorubicin, the expressions of mdr1 mRNA, NF-kappa B mRNA, P-gp and up-regulation of P-gp function induced by doxorubicin were prevented in K562 cells (0.148 +/- 0.006, 0.627 +/- 0.098, 7.18 +/- 0.38 and 799.7 +/- 45.8, respectively P<0.05). But 0.5 microg/ml and 1.0 microg/ml TTD had little effect.</p><p><b>CONCLUSIONS</b>TTD inhibits the expression of mdr1 mRNA, P-gp and up-regulated P-gp function induced by doxorubicin in a dose dependent manner. The mechanism of this effect may be down-regulation of NF-kappa B by TTD.</p>