Molecular Mechanisms Of Lappaconitine Sulfate Inducing Apoptosis Of HepG2 And HeLa Cells

Tumor is a multigene and multicellular disease.The incidence and mortality of malignant tumors have been on the rise.Lappaconitine(LA)is a diterpenoid alkaloid extracted from the root of Aconitium sinomontanum Nakai.Lappaconitine hydrobromide(LH)has been used in clinical as a nonaddictive analgesic.While,the solubility and bioavailability of LH were low,lappaconitine sulfate(LS)exhibited a better solubility and analgesic activity.Some studies have shown that LA,LH and LS could inhibit the proliferation and induce cell apoptosis in tumor cells,but the mechanisms of LA-,LH-or LS-induced apoptosis remain unclear.In this paper,we explored the mechanisms of LS-induced apoptosis.The cell viability and proliferation were evaluated using the CCK-8 and EdU assay,respectively.The cell cycle distribution was detected by PI staining,flow cytometry analysis.The cell apoptosis was expressed by DAPI staining and Annexin V-FITC/PI double staining,flow cytometry analysis.The cell cycle arrest related-proteins(p53?p21?Cyclin D1),apoptosis-related proteins(Bcl-2?Bax?cleaved caspase-9?cleaved caspase-7?cleaved caspase-3),the PI3K/AKT/GSK3? pathway-related proteins(PI3K?p-PI3K?AKT?p-AKT?GSK3??p-GSK3?)and the MAPK pathway-related proteins(ERK?p-ERK?JNK?p-JNK?p38?p-p38)were estimated by western blot analysis.The mitochondrial membrane potential(MMP)was determined through the JC-1 staining assay.The reactive oxygen species(ROS)was monitored by DCFH-DA staining assay.A HepG2 xenograft mouse model was established to investigate the in vivio antitumor effect and the toxic effect of transplanted mouse heart,liver,spleen,lung and kidney of LS.The rusults are showed as follows:(1)LS significantly inhibited the proliferation of HepG2 and HeLa cells in dosedependent manner.Compare to uniform concentration of LA and LH,LS exhibited a best growth inhibitory effect.The IC50(48 h)of HepG2 and HeLa cells are 362 ?g/mL and 571 ?g/mL,respectively.(2)LS induced cycle arrest in HepG2 and HeLa cells.In HepG2 cells,24 h LS treatment induced a S cell cycle arrest,48 h LS treatment induced a G0/G1 cell cycle arrest.While in HeLa cells,24 or 48 h LS treatment induced a G0/G1 cell cycle arrest.LS-induced cell cycle arrest was associated with up-regulation of p53,p21 and downregulation of Cyclin D1.(3)LS induced apoptosis in HepG2 and HeLa cells.After LS treatment,HepG2 and HeLa cells all showed obvious apoptosis characteristics,such as chromatin condensation and nuclear fragmentation.With the concentration of LS increased,the apoptosis ratio increased,the MMP decreased,the cellular ROS increased,the expression of Bcl-2 decreased and the expression of Bax,cleaved caspase-9,cleaved caspase-7,cleaved caspase-3 increased.(4)LS down-regulated the PI3K/AKT/GSK3? pathway.After LS treatment,the expression of PI3K/ AKT/GSK3? pathway-related proteins p-PI3 K,p-AKT,and pGSK3? decreased.(5)LS regulated the MAPK pathway.In HepG2 cells,after LS treatment,pJNK1/2 and p-p38 MAPK were up-regulated.In HeLa cells,after LS treatment,except of the upregulation of p-JNK1/2 and p-p38 MAPK,the expression of p-ERK1/2 was down-regulated.(6)LS significantly inhibited tumor growth in nude mice bearing HepG2 xenografts.Compared with the vehicle group,the tumor growth of LS and 5-FU group significantly slowed down,and the overall tumor suppressive effect was close to 5-FU.LS had no obvious toxic effects on the heart,liver,spleen,lung,and kidney tissues of mouse.Additionally,the expression of p-AKT was down-regulated,the expressionof pp38 and cleaved caspase-3 were up-regulated in tumor tissues.Collectively,LS inhibited the proliferation and induced apoptosis through the mitochondrial pathway in HepG2 and HeLa cells.LS-induced apoptosis was involved in the PI3K/AKT/GSK3? pathway and MAPK pathway.LS significantly inhibit tumor growth and had no marked toxic effect on heart,liver,spleen,lung and kidney tissues of transplanted mouse.The overall tumor suppressive effect is close to 5-FU.