Anti-HCC Study Of Saponins From Schizocapsa Plantaginea (Hance) And Saponin Compound SSPH Infflbite Hela Cell Proliferation By Regulating ROS-ERK Signal

Objective:1.To analysis the in vivo and vitro against hepatocellular carcinoma (HCC) effect of saponins from Schizocapsa plantaginea (Hance) (SFSP).2. To study the in vitro anticancer mechanism of a saponin compound separated from the SFSP (SSPH).Methods:The anti proliferation and anti clonogenic effect of SFSP in human HCC SMMC-7721 and Bel-7404 cell line was measured by MTT assays. The morphology change and VEGF expression of SMMC-7721 cells were observed using immunofluorescence. The affect of SFSP to chemotherapy drugs were measured by MTT assays. The toxicity of SFSP was studied through the inhibition effect of human normal liver cell line HL-7702, rabbit erythrocyte hemolysis test and mice acute toxicity test. To evaluate the apoptosis induction of SFSP, Hochest 33258 stains was used to observe the nucleus chromosomes in SMMC-7721 cells and further analysis was carried out by flow cytometry, ROS concentration was also measured by flow cytometry. Caspase activation was measured by ELISA. Western Blot assay was performed to measure the MAPK expression and activation, special inhibitor or activation was used to verify the MAPK regulation. The in vivo study of SFSP was using a SMMC-7721 nude mice xenograft model, the in vitro anti-HCC mechanisms of SFSP was cross-checked with immunohistochemistry assays in xenograft tumors. The anti proliferation effect of SSPH in Hela cells was measured using Neutral Red assays and WST-8 stain. Preliminary evaluation of SSPH regulating tumor-related pathway in Hela cell was performed by luciferase assays. Special inhibitors and qRT-PCR assays were used for depth study the SSPH regulating tumor-related pathway. Hela cells apoptosis were measured using Annexin V/PI stain by flow cytometry. MAPK expression and phosphorylation were determined by WB assays. The influence of SSPH to ROS was determined by flow cytometry and verified by combined with NAC.Results:SFSP significantly inhibited the proliferation of SMMC-7721 and Bel-7404 with an IC50 of 3.75 and 3.93μg/ml, clonogenic inhibition also observed with an inhibition rate of 83.37 and 89.02%. Morphology changing and VEGF decreasing in SMMC-7721 cells was observed after SFSP 24h trearment. SFSP showed no influenced to Adriamycin and Daunorubicin, but reduced the anti-HCC effect of Taxol. SFSP showed a low toxicity with a IC50 of 47.6μg/ml to HL-7702 cell, LD50 of 4520mg/kg/D and a Hemolysis concentration of 60.2μg/ml. SFSP 2,5and 10μg/ml caused a 40.23,42,30, 46.83% apoptosis in SMMC-7721 cells,14.50,33.97 and 46.77% in Bel-7404 cells, Hochest 33258 staining also indicted an apoptotic. SFSP blocked cell cycle in G2/M phase, Caspase -3,-8 and -9 activation were detected in both cell lines. SPSF increased ROS concentration in SMMC-7721 cell line. WB assays showed that SFSP upregulated the phosphorylation level of JNK and Erk, downregulated the phosphorylation level of p38 without influenced the expression level of Erk, JNK or p38. JNK or Erk special inhibitor blocked the cytotoxicity of SFSP. High (p<0.01) and Middle (p<0.05) dosage groups of SFSP significantly inhibited SMMC-7721 xenograft tumor in nude mice. Immunohistochemistry verified that SFSP had a similar mechanism between in vivo and vitro. SSPH significantly inhibited the proliferation of Hela cell in vitro with an IC50 of 4.13μM. Further study showed that SSPH significantly inhibited Stat3, Smad, NF-κB, E2F, Myc, Ets and Wnt P pathway in Hela cell. SSPH also regulated genes in Hela cells by upregulated A20, AXIN2, BCL2, CCND1, PYGO2 and TERT genes to 2.53,2.50,2.15,2.14,2.05 and 3.00 folds. SSPH may promote its anti-proliferation effect through MEK, NF-κB and PI3K signal pathway. In cell WB assays showed SSPH caused a continuous phosphorylation in Erk after a slightly decreased in 15 min. SSPH 12 μM induced significantly apoptosis in Hela cell (p<0.01), SSPH caused 10.62% early apoptosis,38.14% necrosis and late apoptosis after 24 h treatment.6μM of SSPH caused a 30% increased of ROS in Hela cells in 24h; pre-treat with lOmM NAC could block this effect to 10%. ROS increasing leaded to an Erk phosphorylation was observed, NAC significantly block the SSPH induced Erk phosphorylation. SSPH and NAC showed no influenced in JNK and p38 signal pathway. SSPH caused c-PARP increasing and caspase-3, PARP decreasing in Hela cell, NAC could blocked these effect by reducing ROS level.Conclutions:SFSP inhibited HCC proliferation in vivo and vitro by regulating MAPK signal pathway. SSPH activating Erk by increased ROS in Hela cell and leads to a anti-proliferation and apoptotic effect. To sum up, SFSP and SSPH are potential anticancer drugss.