The Study Of Anti-Tumor Activities In The Shikonin Analogue J Of High Unsaturated Fatty Acids Of Isatis Tinctorial L

Objecitve:To investigae the anti-tumor activity of the compound J in vitro and vivo,and to research the activity of drug resistance,as well as its biological mechanism of anti-tumor.In order to offered the basic experimental evidence to its clinical application.Methods:①Anti-tumor in vitro:The anti-tumor activity of compound J was observed by MTT. The cell cycle and apoptosis were detected by flow cytometry. Experimentation on animals:establish the subcutaneous tumor model in nude mice of TCA8113 tongue cancer and investigate inhibition of tumor cells of compound J.The expression of Ki-67 was detected by immunohistochemical technique. The cell cycle and apoptosis were detected by flow cytometry.②Reversal of multidrug resistance in vitro:the resistant cell line of BEL-7404 and TCA8113 were induced by increasing the concentration gradient.The reversal effcet of compound J on resistant cell line was detected by MTT. After the combine compound J and ADM, the cell cycle and the expression of the expression of ABCG2 were detected by flow cytometry.Results:①Ⅰ:In vitro,compound J had no inhibiting effect on HUV-EC-C when its concentration was under 100μg/ml;<250μg/ml,its inhibiting rate to BEL-7404 and TCA8113 was under zero; the action that compound J affected TCA8113 was detected by flow cytometry. At 250μg/ml, the cell ratio was 52.28% at G1 stage,6.47% at G2 stage,41.25% at S stage,but in negative group, the cell ratio was 49.35% at G1 stage,7.12% at G2 stage,43.53% at S stage;when the concentration of compound J was 50μg/ml, the early apoptotic cells ratio was 0.46%,the late apoptosis or already dead cells ratio was 3.7%; when the concentration was 10μg/ml, the early apoptotic cells ratio was 0.26%,the late apoptosis or already dead cells ratio was 3.36%. II:Through the experimentation of the subcutaneous tumor model in nude mice in vivo,while the dose of compound J was 5mg/kg,the inhibiting rate of tumour-tissue's weight was 33.47%, the inhibiting rate of volume was 31.33%; while the dose was 2.5mg/kg,the inhibiting rate of tumour-tissue's weight was 12.75%, the inhibiting rate of volume was 9.87%; while the dose was 1.25mg/kg, the inhibiting rate of tumour-tissue's weight was 78.49%, the inhibiting rate of volume was 70.39%, the effcet had no positive correlation with concentration;by immunohistochemical technique, the expression of Ki-67 was present in each group.The expression ratio of negative group was higher (47.36±1.53%). It reduced obviously (7.93±1.42%) in J 2.5mg/kg group, and it reduced in positive group (28.64±2.06%) too.The results of cell cycle detection display: the cells ratio of G1,G2 and S stage were similar to negative group,there was no difference compared each other; the dose of compound J was 2.5mg/kg, the early apoptotic cells ratio was 3.045±0.525%,the late apoptosis or already dead cells ratio was 17.96±1.84%,they were higher than negative group (the early apoptotic cells ratio was 1.24%,the late apoptosis or already dead cells ratio was 1.52%).②Ⅰ:The 7404/ADM and TCA/ADM were established successfully. Compound J reversed TCA/ADM cells that resisted 0.1μg/ml ADM, the reverse fold (RF) was 3.17,the relative reversal effect was 68.59%,and in positive group RF was 62.69, the relative reversal effect was 98.54%; by flow cytometry, after ADM, the cell ratio was 3.22% at G1 stage,72.59% at G2 stage, 24.19% at S stage; after compound J and ADM, the cell ratio was 0.62% at G1 stage,45.31% at G2 stage,54.08% at S stage. Compared with the group dealed with ADM, it was broadly unchanged in G1 stage,reduced in G2 stage,and rised in S stage, in the group dealed with VRP and ADM, the cell ratio was 49.42% at G1 stage,16.07% at G2 stage,34.51% at S stage;The expression ratio of ABCG2 reversed by compound J before and after were very low,0.65% and 1.86% respectively.Conclusions:①The anti-tumor effect of compound J was not obvious in vitro,but obvious in vivo.It was not concerned with the regulation of cell cycle,but it was closed relationship with inducing apoptosis.And it reduced the expression of Ki-67.②The compound J has significant reversal effect on the resistant cell line of TCA8113, and it has disturbed the cell cycle.However,it has no obvious effect on expression of ABCG2.