The Experimental Study About The Disodium Cantharidinate’s Anti-Carcinoma

Primary liver carcinoma (PLC) is a frequent malignant tumor doing great harm tohuman’s health. There is a high occurrence rate of PLC in China. The mortality is the 3rdhighest of digestive malignant tumor following to gastric and esophageal cancer. Sincethere is no effective treatment to PLC at middle and late stage till now,combined therapy,especially the way by combination of TCM with Western medicine is utility way to elevatethe curative effect of PLC. Some one advocated treating the malignant with poisonousagents to heal PLC in recent year, in it using blister and its ramification to heal PLC.Disodium Cantharidinate anticancer spectra is wider and its side effect is little. But themechanism of action and effect pathway is still not clear. Someone had reported DisodiumCantharidinate could lead hepatoma carcinoma cell apoptosis in vitro,but there has not beenreported about control liver cell and vascular endothelial cell (VEC) in vitro at present. Sowe approach Disodium Cantharidinate cure the PLC’s mechanism and safety using themodel of HepG2, L-02 and VEC at the help of found of the emphasis laboratory of JiangSu province.Objectives:(1) In order to find the evidence of clinic,we observe the effects of DisodiumCantharidinate on the growth of human liver carcinoma cell line HepG2,L-02 and VECat various concentration in different time,to find the best dose and time.(2) To study the effects of Disodium Cantharidinate on the growth of human livercarcinoma cell line HepG2, at the same time to observe the effects of DisodiumCantharidinate on the growth of L-02 and VEC, and study the harmside effect.(3) To study the mechanism of Disodium Cantharidinate anti- carcinoma.Methods:(1) Cell culture reference(2) Cell proliferation and inhibition test Cell proliferation and inhibition test wasdetected by MTT method, to calculate the inhibition rate and survival rate,at the sametime to observe the change in form of cell by inverted microscope and takephotographs.(3) Detect of apoptosis Using FCM,AO/EB fluorescent microscope,TEM and AG electrophoresis method,to detect the information of apoptosis at various concentrationsin different time.(4) Detect of the NO concentration Using classics Griess Reagent to detect the NOconcentration of cell supernate fluid.Results:(1)The effect of tumor cells treated with disodium cantharidinate. HepG2 and L-02,VECwere inhibited after these cells were treated with Disodium Cantharidinate at variousconcentration after 24h and 48h,especially middle dose. The inhibitionof HepG2 was betterthan L-02 and VEC,the inhibitionof the later two is corresp. In cell morphology we couldsee the cell become round and crenation gradually,shrinkage and decreasing of adherencedcells when these cells treated with Disodium Cantharidinate. Tumor ceils had becomeswelling and discrepancy in size,the gap between cells had become wider, and there weregenerous cell debris.(2) Detect of apoptosis①It had induced apoptosis to HepG2 with DisodiumCantharidinate at various concentration in FCM,and we could see the typical apoptoticpeak in DNA bar chart. The apoptosis rate of middle dose was 54.86％at 36h; It had nearlyno apoptosis effect on L-02; The apoptosis rate of middle dose was 24.29％at 24h on VEC.②With fluorescent microscope,we couldn’t see apoptosis cells on control and L-02medication group,but We could see viable apoptotic cells on HepG2 in littledose,displaying cell nucleus excursion,budding; non-viable apoptotic cell became more,and we could see apoptotic body,and became more and more with the raise of dose andtime.③We could see liver carcinoma cell crenulation,microvilli reduce and evendisappear,endochylema cavitation,karyopycnosis and apoptotic body is thus at variousconcentration in different time by electron microscope.④We could see the ladder-formstrap of DNA of liver carcinoma cell by AG electrophoresis method,but it was not clear onL-02 and VEC.(4) Detect of the NO concentration After Disodium Cantharidinate affect 24h,theexternalization of NO were mult on three cells,and [HepG2]＞[VEC]＞[L-02]. The contentsof NO in middle dose was highest,had statistical significance.Conclusion:(1) It has obviously inhibited effect on HepG2 with Disodium Cantharidinate,but theinhibited effect is little on L-02 and VEC.(2) It had induced apoptosis to HepG2 and VEC with Disodium Cantharidinate,especially obvious to HepG2,It had nearly no apoptosis effect on L-02.This has reference value inclinical application.(3) Disodium Cantharidinate can promote cell secretion of NO,then inhibiting thecomposition of DNA,inducing cell apoptosis.