Adinbitor Induces Apoptosis Of C6 Rat Glioma Cell

Adinbitor, a 9 KD soluble homogeneous protein, is obtained throughrecombinant DNA technology and is purified by His-Bind columnchromatography. This work was done by Dr. Wang Jihong in our lab. It hasthe characteristic motif of disintegrin which is called R-G-D sequence( Arg-Gly-Asp ). As a new kind of disintegrin, Adinbitor is proved to havethe function of restraining the metastasis of tumor cell, inhibiting theproliferation of ECV304, and inducing apoptosis of ECV304. Disintegrin isknown to play an important role in the area of cellular adhesion.Our experiment is on basis of Dr. Wang's work, and we observe theproliferation-inhibition function of Adinbitor in C6 (rat glioma cell),cellular morphology changing, DNA electrophoresis, decrease inmitochondrial transmembrane potential, and enzymology influence. Weprobe into the effect and mechanism of apoptosis induced by Adinbitor, sothat we can provide theoretic evidence that Adinbitor can exert tumortreatment function in clinic. C6 are cultured in DMEM (Dulbecco's modified essential medium)culture medium containing 10% heat-denatured fetal bovine serum. Thecells are treated in different concentrations of Adinbitor when they are ingood stage. Observe the cells with a microscope in order to find out thechange of morphology. Fluorescent microscope is also used to look into thecell after fluorescent dyeing. Effects of Adinbitor on the cell proliferationand survival were examined through MTT assay. DNA ladder is used todemonstrate the apoptosis of C6 cell treated by Adinbitor. Flow cytometry(FCM) is used to determine the cell death pattern. Mitochondrialtransmembrane potential is also measured by FCM. The activity ofcaspase-3 is measured by spectrofluorometer . As a result we observed that Adinbitor inhibits the cell proliferationand induces apoptosis depends on it's concentrations in a certainty range. Itis most effective in the range of 16~24μg/ml and in 24 hours. The functionof Adinbitor induced apoptosis is increased with concentration and time inthe range of 9~24μg/ml. However, superfluous Adinbitor behaves theinhibition effect of C6 adhesion., which enhanced with the increase ofAdinbitor's concentration. And that effect also leads to a kind of apoptosis:anoikis. In the apoptosis range, the mitochondrial transmembrane potentialpresents a trend of decrease. And the activity of Caspase 3/7 significantlyincreases in the apoptosis cells. From the experiment above we come to the conclusion that as a kindof disintegrin , Adinbitor mainly presents the effect of inhibiting theadhesion of C6 , before the inhibition concentration, it can inducesapoptosis of C6. The probably mechanism of apoptosis could be theactivation of caspase-3 in a cytochrome c-dependent manner. The effectthat Adinbitor inhibits C6 adhesion can be used to suppressed the migrationof tumor cell, and it can also block the proliferation and induce apoptosisso that it present a anti-tumor effect. Adinbitor could be a up-and-cominganti-tumor medicine in the future.