Study On Reversal Of Multidrug Resistance In Tumor By

Cancer is the most common malignant disease worldwide, and the occurrence is increasing rapidly. Chemotherapy is an effective way of cancer treatment. However, the resistance of tumor cells against chemotherapy drugs is a significant limitation to successful chemotherapeutic treatment of cancer. Therefore, searching low toxic and effective agents against MDR has become a research hotspot. The flower of Chrysanthemum indicum has been used traditionally by humans as food and medicine, with the characteristics of sources and low toxicity. Earlier research of our lab has spotted that the CIE could increase the accumulation of Rho123 in tumor cells. Studies have found that CIE which contained apigenin and luteolin can increase the sensitivity of tumor cells to chemotherapy drugs. In this research, we chose CIE and EACI as experimental drug, researched its reversal effect on human gastric cancer cell line SGC7901/ADR, and then explored the possible mechanism. The results of this study could provide the heoretical basis for the CIE and EACI clinical application.Methods and results:(1)The preparation of CIE and EACI.(2)MTT assay was used to determine the cytotoxicity of CIE and EACI on SGC7901/ADR cells. The results showed that the IC50 and IC20 of CIE were 1.36,1.0 mg/mL, respectively, while the IC50 and IC2o of EACI were 990.07,57.31μg/mL, respectively. Then we chose concentration below IC20 as the non-cytotoxicity concentration for the following experiments.(3)MTT assay was applied to detect the revesal effect of CIE and EACI on SGC7901/ADR cells. The results showed that After SGC7901/ADR were exposed to 0.8,1.0mg/mL of CIE, the reversal fold to adriamycin were 1.93,2.54, respectively, while when the cells were treated with 25,50μg/mL of EACI, The reversal fold to adriamycin were 1.82,6.05, respectively. The results indicated that the CIE and EACI can reverse the multidrug resistance of SGC7901/ADR cells.(4)Flow cytometry(FCM) was applied to detect the accumulation of adriamycin(ADR) in SGC7901/ADR cells. The results showed that, compared with control groups, the ADR accumulation were 1.59-,1.77-fold when the cells treated with 0.8,1.0mg/mL of CIE, respectively, while the accumulation of ADR were 1.7-,2.17-fold when the cells were treated with 0.8, 1.0mg/mL of CIE. While the accumulation of adriamycin were 1.70-,2.17 fold when the cells were treated with 25,50ug/mL of EACI, respectively. The results indicated that both CIE and EACI can remarkably increase the accumulation of adriamycin in SGC7901/ADR cells.(5)Utilizing Semi-quantitative reverse transcription PCR to determine the influence of CIE and EACI on MDR1 gene transcription in SGC7901/ADR cells. The results of RT-PCR illustrated that the transcription level of MDRl gene were declined compared with the control group. The transcription of MDR1 gene were decreased by 28.57%, 32.14% when the cells were treated with 0.8, 1.0mg/mL of CIE, respectively. While, the transcription of MDR1 gene were decreased by 59.26%,73.33% when the cells were treated with 25,50μg/mL of EACI, respectively. The results demonstrated that both the CIE and EACI can decrease the transcription of MDR1 gene in SGC7901/ADR cells.(6)Immunofluorescence technique was used to determine the influence of CIE and EACI on P-gp expression in SGC7901/ADR cells. The results showed that both CIE and EACI could significantly decrease the expression of P-gp in a concentration-dependent manner. The expression of P-gp were decreased by 13.93%, 27.06% when the cells were treated with 0.8, 1.0mg/mL of CIE, respectively.While, the expression of P-gp were decreased by 21.40%,34.15% when the cells were treated with 25,50μg/mL of EACI.(7)Western blot was applied to detect the regulation of CIE and EACI on IKBa and phosphorylation of IκBα in SGC7901/ADR cells. The results showed that IKBa protein levels was increased, while, the level of p-IKBa was degradated when the cells were treated with CIE and EACI.(8)Western blot was used to define the effect of CIE and EACI on NF-κB activity in SGC7901/ADR cells. The results showed that NF-κB activity were induced in SGC7901/ADR cells when the cells were treated with CIE and EACI.Conclusions:Both CIE and EACI have the effect of reversing MDR. The reversal mechanism may be related to decrease the activity of NF-κB and down-regulated the transcription of MDR1 gene, then the expression level of P-gp was decreased.