Isolation And Anti-tumor Activity Of Caudatin From The Roots Of Cynanchum Bungei

Objective: In this study, we investigated the anti-tumoractivities of caudatin, one species of C21 steroidal aglycones. Caudatin was isolated from the root of Cynanchum bungei. The effects of caudatin on the cell apoptosis and cell proliferation were determined in HepG2 and A549 cells. The inhibition of cell migration for caudatin was detected in C6 cells. The anti-angiogenesis of caudatin were detected in the human umbilical vein endothelial cells(HUVECs). We also perform this experiment to elucidate the mechanism that caudatin inhibits the cell proliferation, metastasis and angiogenesis by regulating the signaling pathway of MAPK and Wnt/β-catenin.In addition, whether caudatinenhanced tumors necrosis factor-related apoptosis-inducing ligand(TRAIL)-induced HepG2 cell apoptosis was determined. Methods:(1)The concentration of C21 steroids was determined by spectrophotometric methods, and HPD-100 macroporous adsorption resinswere used to purify the C21 steroids. The technology of C21 steroids extraction from the root of Cynanchum bungeiand optimum conditions for acid hydrolysis of 90% ethanol elution were optimized byorthogonal test.(2) Antitumor constituents were isolated from 90% ethanol elute by high speed counter current chromatography, and the structure of antitumor constituent was determined on the basis of NMR.(3) MTT assay was used to determine the cell inhibition effect of different ethanol elutes purified by macroporous resins. The effect of caudatin on the cell viability of HepG2 cell, A549 cell and C6 cell were also detected by MTT assay. Furthermore, the enhancement effect of caudatin on TRAIL-inhibited cell viability was also confirmed by this method.(4) Flow cytometry assay was used to detect the cell cycle distribution of caudatin-treated HepG2 cells, A549 cells, C6 cellsand HUVECs.(5) Annexin V-FITC and PI double staining was used to detect cell apoptosis of HepG2 cells, A549 cells and C6 glioma cells, as well as the effect of combination of caudatin with TRAIL on the HepG2 cells apoptosis were also determined by this method.(6) Western blotting analysis was performed to exam the expressions of apoptosis-relative proteins.(7) C6 cells migration was explored by scarification test and transwell assay.(8) Tube formation assay was used to determine the anti-angiogenic activities of caudatin, and the secreted VEGF levels was determined by ELISA assay. Results:(1) The proposed method to determine the content of C21 steroids in the root of Cynanchum bungei is a stable, simple, and fast method that can provide high accuracy. The determination of C21 steroids was carried out at a wavelength of 444 nm by spectrophotometry. According to single factor test and orthogonal experiments, the suggested optimum extraction processwas to be extracted with 8 times amount of 95% ethanol for 2 times, 1.5 hour each time. MTT assay showed that 90% ethanol elute, purified by macroporous adsorption resins, could more strongly inhibit HepG2 cell viability than other elutes, but with weaker effects than its hydrolysate. Thus we explored the optimum hydrolysis process, determined by orthogonal experiments,was as follows: with 100 mL of 15% acetic acid hydrolyzing for 6hin water-bath heating to 100℃. Under the optimum hydrolysis condition, C21 steroidal glycosides can be hydrolyzed completely.(2) Antitumor constituent, identified as caudatin on the basis of NMR, was isolated by high speed counter current chromatography from hydrolysates of 90% Ethanol elute.(3)MTT results indicated that caudatin inhibited cell viability of HepG2, A549, C6 cell, and HUVECs in a dose-dependent manner. Moreover,combination of caudatin with TRAIL reduced cell proliferation of HepG2 cells.(4)FACS analysis results showed that caudatin treatment resulted in the accumulation of cells in the G0/G1 phase, and cell numbers in S-phase were decreased significantly, suggesting that caudatin inhibits cell growth by blocking the G0/G1 to S phase transition in the cell cycle. Furthermore, western blotting results indicated that caudatin inhibited the expression of cyclinD1 and increased the levels of p53 and p21 protein, activators or inhibitors of the cell cycle, in HepG2 cells.(5)AnnexinV-FITC/PI double staining results showed that caudatin induced HepG2 and A549 cell apoptosis with mechanism of cleavaging apical pro-caspase-9 and-3, PARP into the characteristic activate fragments, at the same time decreasing the expression of Bcl-2, an antiapoptotic protein, and increasing the expression of Bax, an apoptotic protein.(6) The anti-tumor activities of caudatin are related with the regulative effects of caudatin on the Wnt/β-catenin and MAPK signaling pathway. Caudatin inhibited theexpression of β-catenin and phosphorylation of GSK3β at Ser 9, and upregulated the expression of phosphor-β-catenin. In addition, caudatin activated MAPK signaling pathway by increased phosphorylation of JNK and ERK.(7) Caudatin could inhibit the C6 cell migration of in vitro with a dose dependent way,and the potential mechanism might be related to the inhibiting the expression of β-catenin and survivin in C6 cells.(8) Conditional medium of A549 cells-induced or growthfactors-induced tube formation of HUVECs was markedly inhibited by caudatintreatment, which was associated with the inhibiting VEGF secretion from A549 cellsby caudatin.(9) Caudatin synergizes HepG2 cells to TRAIL induced apoptosis by promoting the cleavages of caspase-3, caspase-7, caspase-9, PARP and inhibiting the expression of survivin. Caudatin sensitizes HepG2 cells to TRAIL-induced apoptosis through the upregulation of DR5. Conclusions:We isolated caudatin from the root of Cynanchum bunge, which showed antitumor activities in many cell lines. Caudatin inhibitedcancer cells proliferation by blocking G1 phase to S phase transition, induced cancer cell apoptosis by cleaving key factors of caspase, regulating the expression of Bcl-2 family. Moreover, caudatin also remarkably inhibited cancer cell metastasis and angiogenesis. The antitumor effect of caudatin is related to inhibiting Wnt/β-catenin signaling pathway and activating MAPK signaling pathway. In addition, caudatin sensitizes TRAIL-induced cancer cell apoptosis through the upregulation of DR5.