Ability Of MDR Reversing Effects Treated With Thallus Laminariae PE In Human Endometrial Carcinoma

Objective: MDR (Multidrug resistance) is defined as tumor cells resist to not only previously used cytostatic agents, but also cross-resist to other drugs having different structure and function. MDR is a major factor leading to the failure of many forms chemotherapy. To search a sort of safe and effective reversal agents for multidrug resistance from Chinese traditional medicine. To study the reversal effect and molecular mechanism of Thallus Laminariae PE (TLPE) in carcinoma uterus. To provide the scientific evidence for developing and manufacturing new reversal agents.Methods: 1 MDR multiple was investigated by using cell culture technique and MTT colorimetric method and to determine the experiment conditions.2 The expression of P-gp on B-MD-C1(ADR+/+)and B-MD-C1(wt)cells by cytoimmunochemistry method and flow cytometry method .3 MTT methods were used to determine the cytotoxicity of TLPE and the sensitivity of two cell lines, and then selected the no-cytotoxicity doses and low-cytotoxicity doses to the next experiments. 4 MTT methods were used to determine the changes of MDR of the cell lines treated by TLPE, and tested TLPE can reverse MDR or not. Cytoimmunochemistry was used to investigate the impacts on P-gp,s expression of cell lines treated by TLPE.5 Flow cytometry was used to the changes of ADM concentration in cells treated by TLPE to investigate the P-gp′s abilities.6 Flow cytometry was used to determine the cell cycle and apoptosis of cell lines treated by TLPE to investigate the impacts of TLPE on it.7 Gimsa staining methods studied the morphological changes of B-MD-C1(ADR+/+)cells treated by TLPE.8 RT-PCR was used to observe the expressions of mdr1 RNA in B-MD-C1(ADR+/+)cell lines treated by TLPE.9 Flow cytometry was used to determine the expressions of PKC-αand GST-πon cell lines treated by TLPE.10 Colorimetry was used to detect the changes of ATPase on B-MD-C1(ADR+/+)cells treated by TLPE to study the mechanism of impacting P-gp′s abilities.Results: 1 The MDR of B-MD-C1(ADR+/+)cells was higher 31 times than B-MD-C1(wt)cells, and expressed P-gp higher especially .2 The value of IC50 that detected B-MD-C1(ADR+/+)and B-MD-C1(wt)cells′by MTT showed:⑴. The value of IC50 between two cell lines had no significant difference by T-test (24h;48h;72h:P>0.05 );⑵.The dose of no-cytotoxicity and low-cytotoxicity of TLPE to two cell lines were both 70μg/ml and 125μg/ml in the same time range (72h);there was no difference in dose range. This experiment discovered that B-MD-C1(ADR+/+)and B-MD-C1(wt)cells has the same sensitivity to TLPE.3 TLPE had the obviously MDR effects on B-MD-C1(ADR+/+)cells, but only partially reversed. The value of IC50 of B-MD-C1(ADR+/+)cells reversed was degressed, but higher than B-MD-C1 ( wt ) cells′.The reversing ability and deregulating the expression of P-gp were related to the dose and acting time of TLPE, these appeared dose and time dependence.4 The concentration of ADM in cells was detected by FCM and the results showed the concentration of ADM increased. This experiment showed TLPE could decrease the effects of P-gp pump.5 After interfered by TLPE, the cell cycle and apoptosis were detected by FCM and the results showed the changes as below:①B-MD-C1(ADR+/+)cells grew well, every period distribution in cell cycle and apoptosis rates had no difference in every time range;②TLPE could block cell cycle at S period which appeared dose-dependence and time-dependence, and the apoptosis had no obviously changes.6 After interfered by TLPE, the morphous of B-MD-C1(ADR+/+)cells had significant changes. The volum of cell became smaller, chromatin condensed. These results appeared that the cells were blocked at S period, which were accordant with the results of FCM.7 After interfered by TLPE, the expression of PKC-αand GST-πin cells became fewer, which had obviously difference to control group. The degree of decreasing was related to the dose and acting time of TLPE. These results showed that TLPE maybe decrease the expression of PKC-αand GST-π, and then reverse MDR partially.8 RT-PCR was used to detect the changes of mdr1RNA level in cells and these results showed that TLPE could decrease the expression of mdr1 gene and the drugs pumped out of cells.9 After interfered by TLPE, there was no significance difference in the expression of ATPase in cells among the groups. These results showed that TLPE impacted the ability of P-gp maybe not impact the ATPase.Conclusion: 1 Human carcinoma uterus cell line B-MD-C1(ADR+/+)showed resistance to ADM and one of mechanisms is over-expression of P-gp.2 There were the same sensitivity in B-MD-C1(ADR+/+)and B-MD-C1(wt)cells to TLPE, TLPE could block the cell cycle at S period, but did not induce apoptosis, which effects appeared dose-dependence and time-dependence.3 TLPE can reverse MDR of B-MD-C1(ADR+/+)cell by down-regulating the expression of P-gp and decreasing the ability of P-gp, by the way of dose-dependence and time dependence; The reversing effects of TLPE had related to the degradation of mdr1′expression and decrease the expression of PKC-αand GST-πin cells; perhaps had no related to the changes of ATPase.