| Objective: Through the test that TSN extraction induce K562 cell apoptosis and inhibit tumor growth of tumor-bearing mice,we approached antineoplasmic activity and molecule mechanism of TSN extraction.Methods: 1. To separate and detect toosendanin in Melia toosendanin extractive by an improved transonic extraction - high performance liquid chromatography (HPLC) method. The method is simple,rapid,stable and producible , which can be used as a method for preparation and identification toosendanin of Melia toosendanin extractive in laboratory.2. MTT colorimetric assay was used to examine effect of toosendanin extraction on the growth inhibition of K562 cells. Cell morphological was detected by Wight'stain and light microscope. Flow cytometry detected alteration of cell cycle and apoptosis rate. DNA agarose gel electrophoresis was used to observe cell apoptosis.Chromatometry was used to detect relative activity of caspase. Expression of P21,H-ras and Bcr/abl on mRNA level were analyzed by RT-PCR. 3. Hepatocellular carcinoma cells H22 were subcutaneously injected into BALB/c mice and toosendanin extraction was administered to the tumor-bearing mice.The kinetics of tumor formation and tumor growth were measured , tumor growth inhibition rate(IR)was calculated,to select spleen,thoracic gland and lung,to calculate spleen index,thoracic gland index and noeud inhibition ratio,tumor tissue and Hepar tissue samples were taken an examined by light and electron microscopy to assess the inhibitory effects of toosendanin extraction on tumor growth in the mice.Results: 1. The linear regression equation for peak area - toosendanin concentration (4.64～88.2μg/ml) was: A= 7.6162C-2.5677,r=0.9996; mean recovery of standard substance was 100.83%;RSD of precision test was 1.42%; RSD of stability test was 2.26%.2. Toosendanin extraction significantly inhibited the growth of K562 cells in IC50 of 72 hours was 2×10-8mol/l.The K562 cells treated with toosendanin extraction showed morphological characteristics of apoptotic cells. Cell cycle and Annexin V/PI double labeling test indicated that toosendanin extraction induced apoptosis of K562 cells in a concentration-depedent and time-dependent manner.The typical DNA ladder on agarose gel electrophoresis for analysis of cellular apoptosis were significan appeared treated K562 cells. Toosendanin extraction induced K562 cells apoptosis involved in the activation of caspase. The mRNA exspression of P21,H-ras were up-regulation,The mRNA exspression of Bcr/abl was down-regulation . 3. Marked inhibitory effect of toosendanin extraction on the transplanted hepatocelhular carcinoma H22 was observed in the rumor-bearing mice.The inhibitory rates were 84.4% and 50.4% in the groups treated with high and low dosage of toosendanin extraction,respectively(P<0.01 VS.control group).The tumor formation was significantly retarded and tumor growth was inhibited in toosendanin extraction -treated groups compared with those in control mice.spleen index and thymus index were obviously degraded compared with those in control mice(P<0.01),and noeud inhibition ratio was markedly ingraded ( P<0.01 ) .Histopathological examination revealed widespread necrosis with massive accumulation of infiltrating plasmacytes in the tumors.Numerous apoptotic cells and apoptotic bodies were observed in the tumors under the electron microscope.Conclusions: TSN extraction have better antineoplasmic activity in vivo and vitro. Mechanism is concerned with caspase pathway, especially mitochondria pathway. |
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