Ilexgenin A Blocks The PI3K/AKT/mTOR Pathway, Inhibits Tumor Angiogenesis, And Inhibits H22 Cells Proliferation

ObjectiveHepatocellular carcinoma (HCC) is one of the most common cancers in the world. A high incidence of HCC with 55% mortality rate of the world occurs in China, and this estimation has been rising, indicating that HCC is a serious threat to public health in China. Since the 21st century, we have made important progresses in medical researches and clinical studies. However, we still couldn’t reverse the fact that HCC is a worse prognosis overall. It is of great significance to develop potent drugs with low toxicity for the treatment of advanced HCC patients.As one of the pentacyclic triterpenoids, Ilexgenin A is extracted from the active fraction of Ilex hainanensis Merr. Our previous study found that Ilexgenin A has potent antitumor activity in varous of cancers. Due to Ilexgenin A extensive source and mature extraction technologies, we aimed at the inhibition of Ilexgenin A on HCC proliferation, and identify its new target of anti-angiogenesis.The first part:The inhibition of Ilexgenin A on tumor growth in H22 tumor bearing mice MethodsH22 cells were inoculated to the left armpits of ICR mice to establish model of tumor bearing mice. The mice were randomly divided into model group (0.5%CMC-Na, i.g.), Sorafenib group (60 mg/kg, i.g.), Ilexgenin A group (15,30,60 mg/kg, i.g.). After 2 weeks, the serum VEGF level was determined by mouse enzyme-linked immunosorbent assay. All mice were sacrificed and tumor tissues were harvested and weighted. Take the photos of tumor and calculate the tumor inhibition rate. The tumor tissues were fixed for H&E staining and immunohistochemical staining of VEGF and CD31. In addition, we observed the ultrastructural tumor cells on the transmission electron microscope.Results(1) The tumor weight of model group was 0.50±0.18 g, whereas it were only 0.14±0.06 g in Ilexgenin A 60 mg/kg group (61.83% of decrease, p< 0.01),0.26±0.23 g in Ilexgenin A 30 mg/kg group (52.49% of decrease, p< 0.05); and 0.37±0.20 g in Ilexgenin A 15 mg/kg group (37.37% of decrease, p>0.05).(2) Ilexgenin A reduced the new blood vessels of tumor surface and tissues stained with H&E; Ilexgenin A at 60 mg/kg significantly inhibited serum VEGF level (p< 0.01) and decreased the expressions of tumor VEGF and CD31 (p< 0.001, p< 0.001, respectively) in H22 tumor bearing mice;(3) An amount of the focal necrosis and the vacuole formation in tumor were observed in Ilexgenin A groups on light microscopes; The condensed chromatin, and an mount of vacuoles, and membrane shrinkage were also observed in Ilexgenin A groups on transmission electron microscope.ConclusionsIlexgenin A promotes tumor cells to form necrosis, and ultimately inhibits tumor growth by inhibiting the serum VEGF level in H22 tumor bearing mice, and reducing the VEGF and CD31 expressions in tumor.The second part:Study of Ilexgenin A on prolonging of survival time in H22 tumor bearing miceMethodsH22 cells were inoculated to the left armpits of ICR mice to establish model of H22 tumor bearing mice. The mice were randomly divided into model group, sorafenib group (60 mg/kg, i.g.), Ilexgenin A group (15,30,60 mg/kg, i.g.). The mice were monitored daily for body weights and food intake during the administration. The administration lasted 4 weeks and went to withdrawal observation at the 8th week.Results(1) The tumor volumes were significantly inhibited in the Ilexgenin A groups compared with the model group on the 10th day;(2) All mice in model group died by the 27th day, and its median survival time is 14.5 days. Median survival time of the Ilexgenin A groups at doses of 60,30 mg·kg-1 are 91.8 days and 42.5 days, respectively (50%,16.7% of survival rate,p< 0.001,p< 0.001, respectively); All mice in Ilexgenin A group at dose of 15 mg/kg died by the 48th day, and its median survival time is 27 days (p< 0.001).(3) Signs such as significant weight and food intake changes, and impaired movements, were not observed in the Ilexgenin A groups during the whole treatment.ConclusionsIlexgenin A inhibits the H22 cell proliferation, and prolongs the survival time of tumor-bearing mice.The third part:The mechanism study of Ilexgenin A on angiogenesisMethodsTo evaluate the cytotoxic activity of Ilexgenin A, H22 cells were incubated with various concentrations of the Ilexgenin A for 24,48 and 72 h, respectively and detected by CCK-8 assays. In addition, we performed MTT assays to evaluate its cytotoxicity on human umbilical vein endothelial cell (HUVECs). The protein phosphorylation expressions of JAK, PI3K, AKT, mTOR, STAT3, MAPK were detected in the HUVEC cells by western blot analysis.Results(1) Ilexgenin A inhibited the H22 cells proliferation and its median inhibition concentration (IC50 values) at 24 h,48 h and 72 h were 136.8μM,52.79 μM and 36.14 μM, respectively;(2) The IC50 values of Ilexgenin A on HUVEC cells were 155.7 μM and 61.67 μM;(3) Ilexgenin A at concentration of 6.25μM significantly decreased the phosphorylated expressions of JAK, PI3K, AKT, mTOR, STAT3, and MAPK in HUVECs cells for 48 h, respectively.ConclusionsIlexgenin A inhibits the H22 cells proliferation. Ilexgenin A regulates the PI3K/AKT/ mTOR pathway via inhibition of JAK, PI3K, AKT, mTOR, and STAT3 phosphorylated expressions in human umbilical vein endothelial cells. The fourth part:Preliminary safety evaluation of Ilexgenin A in H22 tumor bearing mice MethodsH22 cells were inoculated to the left armpits of ICR mice to establish model of H22 tumor bearing mice. The mice were randomly divided into model group (5%CMC-Na, i.g), Sorafenib group (60 mg/kg, i.g.), Ilexgenin A group (15,30,60 mg/kg, i.g.). The mice were monitored daily for the body weights and food intake during the administration. After 2 weeks, blood was collected to detect biochemical parameters. All mice were sacrificed and major organs were harvested and weighted. The indexs of organs (heart, liver, spleen, lung, kidney) were calculated and tissue samples were fixed for H&E staining. The mRNA expressions of cytochrome P450 enzymes in liver were detected by real time-PCR.Results(1) Ilexgenin A inhibited the mRNA expressions of CYP1A2, CYP2E1, CYP3A1 in the liver;(2) Ilexgenin A did not remarkably affect the blood hematologic parameters of mice (WBC, RBC, Hb, MPV, and PLT, etc), and the parameters were within the physiological range.(3) There were no marked histological changes in the major organs in Ilexgenin A groups;(4) Signs such as significant weight and food intake changes, and impaired movements were not observed in the Ilexgenin A groups during the whole treatment.ConclusionsIlexgenin A inhibits the mRNA expressions of CYP 450 enzyme. In addition, there is no obvious adverse reaction of the blood and major organs in Ilexgenin A groups.