Synthesis Of Higher Fatty Acids Analogue J And Its Mechanism Of Reversing Drug Resistance

Objective:1. To synthesize the higher fatty acids analogue J.2. To observe the reversal effect of the analogue J on the resistant cells and to explore its possible mechanism of drug resistance reversal.Methods:1. Analogue J was synthesized with typical reaction and cheap raw materials erucic acid. It was identified by TLC and its physical constants were determined by UV. And its hemolytic effect on red blood cell was examined.2. BEL-7404/ADM and TCA8113/ADM were induced by impacting of high-dose combined increasing the concentration gradient. The resistance index was measured by MTT colorimetric assay and the parent cells and the resistant cells were observed under optical microscope,respectively.3. MTT colorimetric assay was used to detect the reversal effect of analogue J on resistant cells line and reversal times were calculated. Experiment is divided into three groups, including cancer drug positive control group (parental cells or resistant cells + ADM), cancer drug and analogue in the combination group (resistant cells + ADM + J), cancer drug and the combination group the positive reversal (resisitant cells + ADM + VER).4. Experiment is divided into three groups, including parental cells in the control group, resisitant cells in the control group and group of resistant cells treated with analogue J. To explore its possible mechanism of drug resistance reversal, Flow cytometry was used to analyze the accumulation effect of Rho123 and the activity of GST on cells influenced by analogue J was detected. 5. Gas chromatography was used to detect the intracellular concentration of J.Results:1. The higher fatty acids analogue J was synthesized. It was confirmed by TLC and the two developing systems showed a single spot, Rf values were 0.51, 0.41. Measured by UV spectrophotometry, the pKa of the analogue J was 5.56. And analogue J does not produce the effect of hemolysis on red blood less than 100mmol/L.2. BEL-7404/ADM and TCA8113/ADM cells were induced successfully,IC50 were (194.7±4.82)μg/ml and (87.94±0.78)μg/ml,respectively,multidrug resistance were 82.61 and 28.83 times. Observed under the inverted microscope parental cells and resistant cells showed epithelial-like adherent monolayer growth. Resistant cells than parental cells of different size, part of the cell deformation, and the increased volume shaped cells can be seen. Cells were observed to 90% of bottom area covered the length of time.3. Analogue J on two resistant cells were significant reversal effect,the relative efficiency of J were 85.30% and 61.49% respectively. 4. Flow cytometry was used to measure the accumulation of intracellular Rho123 case. The experimental results showed that BEL-7404 and TCA8113 cells Rho123 had strong fluorescence, and the intensity reached to (2260.94±125.25) and (2911.16±54.29). The BEL-7404/ADM and TCA8113/ADM Rho123 fluorescent cells significantly decreased. Their intensity were (868.90±71.32) and (481.31±11.16). After adding analogue J, BEL-7404/ADM and TCA8113/ADM fluorescence intensity of cells were (659.63±99.83) and (233.71±78.57).5. The activity of GST on BEL-7404 and TCA8113 cells were (90.8±20.04) U/mgprot and (94.1±18.68) U/mgprot, while BEL-7404/ADM and TCA813/ADM were (246.3±27.16) U/mgprot and (222.1±22.85) U/mgprot. After analogue J added,the activity of the two resistant cells were (132.5±3.85) U/mgprot and (142.6±6.20) U/mgprot.6. Gas chromatography was used to detect the intracellular concentration of J. The results showed that the intracellular and medium amount of J were 8.99μg and 108.25μg respectively. Calculated into the percentage of cells in the tested drugs was 8.30%.Conclusions:1. Analogue J can be synthesized successfully. And it dose not produce the effect of hemoylsis on red blood cell on the downturn concentration.2. Analogue J in vitro can significantly improve the liver and tongue cancer drug resistance cell BEL-7404/ADM and TCA813/ADM sensitivity to chemotherapy,with significant reversal effect.3. Accumulation of intracellular Rho123 and the GST activity of cells showed that analogue J in vitro reversal of drug resistance mechanisms and P-gp in the role of independent. May be reduced the GST activity of cells,reducing its toxic effects of chemotherapy drugs.4. Established analogue J intracellular drug concentration experiments by gas chromatography.