Studies On The Antitumor Activities And Mechanisms Of Nortriterpene Saponins From Salicornia Bigelovii Torr

Salicornia bigelovii Torr, a leafless annual salt-marsh plant with green and succulent stem is a halophyte plant from Chenopodiaceae family, Salicornia genus, which originals from North America. It can improve the poor soil environment, protect the surface, optimize the environment, and its seed oil extraction can produce economic benefits. Salicornia is a folk medicine in China, with function of calming the Liver, diuretic and antihypertensive functions, and can be used to treat diseases such as hypertension, headache, and then has broad research value.Bigelovii A, Bigelovii B, pfaffine B and Boussingoside A2 are nor-olean triterpene saponins isolated from the whole plant of Salicornia bigelovii Torr. Among them, Bigelovii A and Bigelovii B are new structure compounds, while pfaffine B, and Boussingoside A2 are isolated from Chenopodiaceae for the first time. Based on the screening results of antitumor activity, Bigelovii A shows good development prospects. The results are as follows:1. MTT assay was used to determine the antiproliferative effect of the four compounds: Bigelovii A, Bigelovii B, pfaffine B and Boussingoside A2. The result showed that Bigelovii A had the most potent antitumor growth effect against three human tumor cell lines, and the IC50 for HL-60, HepG2, MCF-7 was 2.24,3.72,8.46μM, respectively. While Bigelovii B and Boussingoside A2 did not reflect significant anti-tumor activity.2. LDH assay was used to investigate whether Bigelovii A destroyed the cellular membrane. The result showed that the effect of Bigelovii A on the release of LDH was in a concentration-dependent pattern. Comparison of results of MTT and LDH indicates that cell death caused by Bigelovii A may be not only due to membrane permeabilization.3. Hoechst 33258 assay found Bigelovii A-induced cell death in HL-60 cells displayed typical characteristics of apoptotic cells including membrane blebbing, cell condensation and chromatin condensation.4. Annexin-V-PI staining assay confirmed that treatment with Bigelovii A increased the fraction of apoptotic cells and that this effect was concentration-dependent.5. To investigate the effects of Bigelovii A on cell-cycle status, HL-60 cells were treated with different concentration of Bigelovii A for 12h and 24h respectively and then analyzed for cell-cycle alteration by flow cytometry. We observed that Bigelovii A caused time-dependent and concentration-dependent increase in hypodiploid DNA fraction, indicating apoptosis due to fragmentation.6. Caspase-3 activity was measured by Colorimetric assay and Western assay. We found that Caspase-3 protease was activated by Bigelovii A, and the activation effect was completely inhibited by the pancaspase inhibitor, Z-VAD-FMK.7. To evaluate the effect of Bigelovii A on Bcl-2 and Bax, the RT-PCR and Western blot assay were employed. The two group results indicated that Bigelovii A caused dose-dependent decrease on Bcl-2 expression, while the effect on Bax expression is not obvious.In conclusion, present experiments suggested that Bigelovii A had the most potent antitumor activity. Bigelovii A induced HL-60 cell death by apoptosis and membrane permeabilization. Further studies showed that Bigelovii A treatment in HL-60 cells down-regulated the expression of anti-apoptotic gene bcl-2 and activated caspase-3.