Study Of Anti-tumor Effect Of Saponin From Tupistra Chinensis Baker And The Underlying Mechanisms

OBJECTIVE: To study the anti-tumor effect of saponin extracted fromTupistra chinensis Baker (STCB) on S-180 sarcoma in vivo and the primarymechanism of action. To investigate the underlying mechanisms of Saponin fromTupistra chinensis Baker and Sapogenin from Tupistra chinensis Baker(SGTCB) onthe apoptotic induction of diverse cell lines.METHODS: Kunmin mice bearing S-180 transplant tumor were used to test theinhibitory efficacy of STCB. Cytotoxic effects of STCB and SGTCB on multiple celllines were evaluated by MTT colorimetry. Light microscope was applied to examinemorphologic changes and transmission electron microscope was applied to examineultrastructural changes of cells after treated with the saponin in vitro. Treated withdifferent concentrations of STCB, the S-180 cells were first stained with propidiumiodide (PI) and then subjected to flow cytometry (FCM) analysis in an attempt to testthe cell-cycle distribution and the apoptosis rate. Additional DNA ladder analysis wasused to determine cell apoptosis. Haemolysis experiment was conducted to study theinfluence of STCB on the cell membrane. Expression of bax and bcl-2 genes wasmeasured by RT-PCR. Using Laser confocal scanning microscopy was exploited toinvestigate the effects of STCB on the intra-cellular concentration of free cytosoliccalcium and the hydrogen ion concentration of U251 cells.RESULTS: STCB could markedly inhibit the cell proliferation in vitro and the50 percent inhibitory concentration was 8.5-54.85μg/ml, showing selectively inhibitory effects on tumor cell lines. SGTCB could also inhibit the cell proliferationin vitro and the 50 percent inhibitory concentration was 17.20-132.05μg/ml, whichwas less effective than STCB. STCB given by intragastric administration showedremarkably inhibitory effect on the growth of S-180 solid tumor. The inhibitory rateexceeded 30% at the dose of 0.5 g/kg body weight and reached 54.86% at the dosageof 2 g/kg body weight. Electron microscopy observations and FCM detectionsrevealed that tumor cell apoptosis rate increased along with the increment of thesaponin concentration. An increase in the percentage of cells in S phase and adecrease in proportion of cells in G₂/M phase were observed when 10 or 30μg/ml ofSTCB was applied. However the fraction of cells in G₀/G₁ phase was reduced and theamount of cells in G2/M phase was raised as the concentration of STCB reached60μg/ml. It was, therefore, presumed that STCB might hamper S-180 cells from Sphase entering G₂ phase at low concentration, and interfere with mitosis at highconcentration. STCB does not cause hemolytic crisis but induce the apoptosis ofU251 cells by declining expression of bcl-2,raising expression of bax and decreasingof the intra-cellular concentration of free cytosolic calcium and the hydrogen ionconcentration of U251 cells.CONCLUSION:1. Both STCB and SGTCB could inhibit the cell proliferation in vitro. And they hadselectively inhibitory effects on tumor cell lines. SGTCB was less effective thanSTCB.2. STCB given by intragastric administration showed remarkably inhibitory effecton the growth of S-180 solid tumor. Low dose and middle dose of STCB would notcause the lose of the body weight.3. The highest inhibitory dose of STCB on the growth of S-180 solid tumor wasfour times effective than that of the lowest dose.4. The death of tumor cells caused by STCB might be apoptosis interrelated.5. STCB might induce the apoptosis of tumor cells through following ways: exerteffect on the cell-cycle distribution, decline the expression of bcl-2, increase theexpression of bax, and decrease the intra-cellular concentration of free cytosolic calcium and the hydrogen ion concentration.