Study On The Molecular Mechanisms Of Toosendain On Inducing Apoptosis In Cancer Cell Lines

ObjectiveToosendanin(TSN), a triterpenoid derivative isolated from Melia toosendan Sieb.et Zucc, possesses different pharmacological effects in human and important values in agriculture. As indicated by previous reports, TSN could confer anti-proliferative and pro-apoptotic effects on human cancer cell lines, But the mechanism by which TSN induced apoptosis remains poorly clarified.we investigated the inhibitory effects of TSN in vitro and in vivo with cell cultivating method, H22 xenograft tumor models estabilishing in BALB/C, and explored the probable mechanisms of TSN in solid carcinoma. And to further explore the mechanisms and side effects of TSN. As the difference methods in the treatment of hematologic malignancies and solid cancers, Taking the human chronic myeloid leukemia (CML) K562 cells as our experimental object, we observed on the proliferation, differentiation and apoptosis of K562 cells treated with TSN and discussed the probable mechanisms of anti-tumor effects. Methods1. Taking the SMMC-7721 and Hep3B cells as our experimental object, we observed the SMMC-7721 and Hep3B cells'proliferation inhibion treated with TSN and discussed the probable mechanisms of anti-tumor effects in vitro from the following aspects: the inhibitory rate of cell proliferation and cell growth curve were detected using MTT assay and cell counting method in SMMC-7721,Hep3B,IAR-20,LO2 cells; the morphlolgical changes of cells was observed by the optical microscope; cell apoptosis at the early stage was detected with AnnexinV-FITC/PI assay; The activities of Caspase-8, Caspase-9 and Caspase-3 were analyzed by spectrophotometry; the expression of Bcl-2,Bax and Fas were detected using immunohistochemistry assay.2. H22 xenograft tumor models were established in BALB/C. The tumor suppression effects of TSN was studied in vivo from the following aspects: the xenograft tumor were excised and weighted to calculate the tumor growth inhibitory rate; the tumor volume were measured; sections from BALB/C xenograft tumor was subjected to HE staining to reveal the changes of histomorphologism; transmission electron microscopy was used to test the ultramicro-structure alternations of BALB/C xenograft tumor cells, Pathological features of heart, liver, spleen, kidney, thymus and testis of cancer bearing mice were observed; the expression of Bcl-2,Bax and Fas were detected using immunohistochemistry assay. 3. Taking K562 cells as our experimental object, we observed the K562 cell's proliferation inhibion, differentiation, apoptosis treated with TSN and discussed the probable mechanisms of anti-tumor effects in vitro from the following aspects: The growth inhibition rate of K562 cells and Peripheral Blood Mononuclear Cell (PBMC) was measured by MTT; Morphology of K562 cells was detected by wright's stain; The ultrastructure changes of K562 cells were analyzed by transmission electron microscope; DNA fragmentation and the percentage of apoptotic cells were examined, respectively; benzidine staining and POX staining was detected. The activities of Caspase-8, Caspase-9 and Caspase-3 were analyzed by spectrophotometry; the expression of Bcl-2,Bax and Fas were detected using immunohistochemistry assay.Results1. TSN presented the striking proliferation inhibition, as well as apoptosis induction potency on SMMC-7721 and Hep3B cells in vitro in a time-and dose-dependent manner(P<0.05); the number of TSN-treated cells was significantly reduced accompanied by decreased adhesion ability,a fraction of cells was shrunk and round, without toxicity to IAR-20 and LO2 cells; Flow cytometry indicated the apoptosis rate of SMMC-7721 cells and Hep3B cells was 21.55% and 13.35%, respectively, the apoptosis of these cells induced by TSN could be partially blocked by z-VAD-fmk(P < 0.05); The activities of caspase 3, 8 and 9 in SMMC-7721 cells were markedly enhanced accompanied by decreased expression of Bcl-2 and increased expression of Bax as well as Fas, In Hep3B cells treated with 0.5μM TSN for 72 h, the activities of caspase 3 and 9 were markedly increased, but the activity of caspase 8 was not profoundly changed. Additionally, the expression of Bcl-2 was decreased accompanied by increased expression of Bax and no evident changes were found in the expression of Fas.2. The experiment in H22 tumor-bearing BALB/C documented the TSN had obvious effects on tumor inhibition. Its inhibitory rate were 66.23%,87.01% in the BALB/C administrated TSN with low dose (0.173 mg/kg),high dose (0.69 mg/kg) , respectively. The ultrastructure alterantions of tumor cells in BALB/C xenograft tumor treated with TSN exhibited apoptotic body appearance; HE staining of heart, liver, spleen, kidney, thymus as well as testis indicated no significant difference in the morphology of these tissues was noted between the control group and TSN group, but the number and area of thymus lobules in TSN group were decreased or even loss; The expression of Bcl-2 protein was down-regulated while expression of Bax and Fas protein was up-regulated in tumor tissue treated with TSN compared with the control groups.3. TSN was a potent inhibitor of proliferation of K562 cells, without toxicity to PBMC. It is shown that the inhibitory efficacy of TSN in K562 cell depended on the reaction time and dosage; The morphological features of apoptosis were observed by light microscopy and transmission electron microscopy in cell shrinkage, even appearance of apoptosis body. After toosendanin treatment at 10 , 30 and 50 nmol/L for 72 h, the apoptosis rate of K562 cells was 9.66%, 26.06% and 52.70%, respectively(P<0.01); A typical ladder pattern was identified in DNA electrophoresis; benzidine staining and POX staining could not singnifcantly change compared with the control groups; The enzyme activity changes of Caspase-3,Caspase-8 and Caspase-9 were detected. Immunocytochemistry technique showed that toosendanin could significantly down-regulate Bcl-xl protein expression and up-regulate Bax and Fas proteins expression (P<0.05)Conclusions1. Results indicated TSN in vivo and in vitro could inhibit proliferation and induce apoptosis in hepatocellular carcinoma cells. the toosendanin-induced apoptosis involved the mitochondrial pathway.2. TSN in vitro inhibit proliferation and induce apoptosis of K562 cells by mitochondrion and death-receptor dual pathways.but K562 cells can be induced in vitro to erythroid differentiation or granulocytic differentiation.3. TSN in vitro on human normal liver cell line L02, normal rat hepatocytes IAR-20 and PBMCs were almost no toxic effects.4. TSN could inhibit proliferation and induce apoptosis both in P53 positive SMMC-7721 cells and and P53 negative Hep3B,K562 cells, TSN can induce human cancer cell lines apoptosis via P53-independent mechanisms and play as a natural low toxicity anti-cancer drugs.