| Background and Object:Primary liver cancer (PLC) is the most common malignant tumor in China, where the incidence and mortality is the first in world. About 90% of PLC is Hepatocellular carcinoma (HCC). Because of insidious onset, early diagnosis difficulity and rapid process, the HCC has been advanced when patients are clinically diagnosed, which results in lost opportunities for local treatment such as surgery and interventional therapy. Those patients can only be taken a sustermatic therapy as alleviative treatment. Due to limited ellicacy, in China, the median survival time for patients with advanced HCC is only 3-4 months and the prognosis is poor. So it needs to explore new drugs and methods. Elemene (Elemene) is an extractant from Chinese traditional medicine plants warm ginger turmeric (Curcuma) (Curcuma aromatica Salisbl) in terpenes. Both in vivo and in vitro studies have demonstrated that Elemene was able to inhibit tumor cell proliferation, induce cell apoptosis as well as inhibit tumor angiogenesis, in addition, induce tumor cells autophagy. Apatinib mesylate (N-[4-(cyano-cyclopentyl) phenyl] [2-[(4- methyl- pyridin-) amino] (3- pyridyl)] carboxamide methanesulfonate, C25H27N5O3S, MW 493.58 (mesylate)) is a new small-molecule inhibitor for VEGFR2 tyrosine kinase similar to Vatalanib(PTK787). Apatinib inhibits tumor angiogenesis mainly through highly-selective and potent inhibition of phosphorylation of VEGFR-2 and actication of the downstream ERK1/2-MARK and PI3K-AKT-mTOR pathways. Many in vivo and in vitro studies have demonstrated that apatinib have significant anti-tumor activity in different types of human and animal cancers, such as lung cancer, colon cancer and stomach cancer xenografts in nude mice. Currently, the phase II clinical studies of apatinib treatment of advanced HCC are active.This study firstly explores whether combination therapy of Elemene and apatinib have synergistic effect on HCC treatment. Secondly, further study of the anti-hepatoma characteristics and mechanism of action. In addition, this study ingestigates the influence of inhibition of autophagy on Elemene inducing HepG2 cell apoptosis, through Elemene induces HepG2 cell apoptosis and hydroxychloroquine combination with antophagy.Methods1. In vivo study (1) The model of ICR mice bearing H22 xenograft was generated. Then 50mg/kg,75mg/kg, or 100mg/kg dose of elemene was injected into the mice respectively. Tumor suppressing rate was calculated and the effective dose of drug was determined. (2) 50mg/kg, 100mg/kg,200mg/kg dose of apatinib and 200 mg/kg dose of PTK787 were used respectively on ICR mice with H22 xenograft. Tumor suppressing rate was calculated, the efficacy and toxicities of apatinib was evaluated and compared with PTK787. (3) The different dose of elemene and apatinib were used on ICR mice with H22 xenograft, Tumor suppressing rate was calculated, whether elemene and apatinib have synergistic effect By CDI was determined.2. In vitro study (1) Cytotoxicity assay of elemene on HepG2 cell:inhibiton on HepG2 cell growing was measured by MTT. (2) Influence of elemene on cleaved-caspase 3、 cleaved-caspase 9、PARP、GAPDH、Bcl-2 Bax、LC3 Ⅰ and LC3 Ⅱ expression was measured by Western Blot. LC3 is a specific marker protein of autophagy, the induction of elemene on HepG2 cell autophagy was determind by detecting LC3. (3) In starvation state, inhibition of elemene on HepH2 cell apoptosis:HepG2 cells were cultured in serum-free medium ofr 12 hours and treated with injections of elemene. (4) Effect of Elemene and hydroxychloroquine on HepG2 cells growth was observed under light microscope. (5)effect of Elemene and hydroxychloroquine on HepG2 cell apoptosis on was measured by flow cytometry.3. Statistical process:using SPSS 19.0 software, date are expressed as mean±standard deviation(X±s), using One-way ANOVA, using LSD assay for homogeneity of variance for multiple comparisons between groups, using Games-Howell assay for heterogeneity of variance for multiple comparisons between groups.Results1. In vivo study(1) Tumor suppressing rate of elemene is proportional to dosage intensity within a certain range of dosage.(2) Apatinib could inhibit the murine hepatocarcinoma H22 subcutaneous implanted tumor growth in a dose-dependent manner, the inhibitory effect is better than that of PTK787.(3) After the combination of apatinib (200mg/kg) and Elemene, with the increasing concentration of the Elemene Injection, the tumor inhibiting rate was declined.2. In vitro study(1) To observe the inhibitory effect of Elemene on the growth of HepG2 cells by MTT assay, the results show that Elemene could inhibit the proliferation of HepG2 cells in a dose-dependent manner.(2) The WB test shows in HepG2 cells,16h after Elemene treatment (0.1 mg/mL), the expression of Bcl-2 protein was significantly upregulated, no change for the expression of Bax protein was observed; LC3 Ⅰ protein expression was also increased significantly 16h after treatment and the autophagy marker LC3Ⅱprotein began to increase obviously 8h after treatment, suggesting elemene induced the accumulation of LC3 Ⅱ; Within the first 8h after treatment, Bcl-2/Bax protein ratio continued to decline which suggesting increase of the cell apoptosis, LC3 I/LC3 II protein ratio also continued to decline which suggesting the activation of the autophagy. But while within 16-24 hours after treatment, Bcl-2/Bax protein ratio and the LC3 I/LC3 II protein ratio were raised simutaneously, suggesting the balance was reached between cell apoptosis and autophagy during this period.(3) HepG2 cells were cultured in serum-free medium for 12 hours. The results showed that, compared with the control group, in a state of starvation, Elemene significantly decreased the apoptosis of HepG2 cells.(4) HepG2 cells were observed under the light microscope 24h after Elemene Injection plus hydroxychloroquine treatment. The control cells showed similar size, growing with epithelial adherent manner, round in shape, clear outline, intercellular tight structure, uniform and transparent cytoplasm, good refraction, and vigorous growth; Cells treated with chloroquine were smaller, round and detached from the bottom, loose, refraction and weak adherent ability; Cells treated with Elemene Injection were elongated, loose, and with more intracellular granules. Cells treated with a combination of the drug were thinner, looser, and with more particles than those in single drug group or the control group.(5) Compared with the control group, cell apoptosis was increased in either Elemene Injection or hydroxychloroquine group. While in the group with two drugs combination, HepG2 cell apoptosis was more than those in any single drug group. Inhibition of autophagy can promote the HepG2 cells apoptosois induced by elemene.Conclusions1 Apatinib mesylate (200mg/kg) and elemene have no synergistic effect, with increasing concentration of Elemene, the tumor inhibition rate is decreased. Elemene induced-autophagy may prevent tumor cell death. In vivo, apatinib mesylate inhibit the growth of tumor cells through inhibiting angiogenesis, while Elemene may reduce the effect of apatinib mesylate through the induction of autophagy.2 Elemene Injection can induce HepG2 cell autophagy, and inhibition of autophagy may promote Elemene Injection-induced HepG2 cell apoptosis. |
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