| Background and ObjectiveMalignant ascites is a sign of end-stage events in various cancers, which deteriorates patients' quality of life and shortens their survival. The established approaches occasionally relieve symptoms but only for a short while and the effects are variable and unreliable. Therefore, novel methods are required for its treatment in clinic. As angiogenesis and increased peritoneal microvascular permeability are the two factors leading to the formation of ascites, the block of VEGF could be a promissing treatment of malignant ascites.Endostar is a novel recombinant human endostatin (rh-Endostatin)-an endogenous angiogenesis inhibitor, which can significantly inhibit VEGF-induced endothelial cell proliferation and downgrade angiogenesis. Elemene injection (short for elemene), which is extracted from traditional Chinese medicinal herb Curcuma wenyujin Y. H. Chen&C. Ling (Wenyujin), has been applied to treat different kinds of cancers and malignant ascites. In this research, the combination therapy of endostar and elemene was applied in the treatment of malignant ascites to verify the treatment efficacy via the H22mouse ascites model and explore the further mechanism, which may provide theoretical basis for the application in clinic.Methods1. In vivo:(1) The safety of combination treatment of endostar and elemene was evaluated on normal mice.(2) To determine the most effective dose of endostar and elemene, the model mice were intraperitoneal injected with normal saline.4mg/kg,8mg/kg,16mg/kg dose of endostar alone and50mg/kg,75mg/kg,100mg/kg dose of elemene alone, the ascites volume and survival time was observed to determine the optimal dose.(3) To determine the optimal dose combination of endostar and elemene, we combined the most effective dose of endostar with other two or three doses of elemene treat malignant ascites mice, ascites volume and survival time was observed to determine the optimal dose combination.(4) The malignant ascites mice were treated with endostar and elemene respectively and combinedly to observe the effects and explore the mechanism:①The mean ascites volume was measured and the CDI value was calculated to determine the synergistic effects:②The number of tumor cells and red blood cells was counted;③The protein and lactic dehydrogenase (LDH) in ascites fluid and sera were detected;④The survival time of each mouse was record to draw survival curves;⑤The subcellular structure of tumor cells treated with endostar combined with elemene was observed by transmission electron microscope:⑥The rates (%) of CECs was analyzes by flow cytometric;⑦To determine whether drugs decreased peritoneal microvascular permeability, the leakage of Evans' blue dye into the ascites fluid was detected;⑧The levels of VEGF, MMP-2, CD44V6and HIF-1α were determined with ELISA assay;⑨The expressions of VEGF, MMP-2, p-p38MAPK and p38MAPK in tumor cells were analysised with western blot assay;⑩The levels of VEGF mRNA in tumor cells were detected with RT-qPCR.2.1n vitro:(1) The proliferation of H22cells treated with endostar and elemene injection was evaluated by CCK-8test.(2) The levels of VEGF, CD44V6, MMP-2and HIF-1α in supernatants were detected by ELISA assay.(3) The expressions of VEGF and MMP-2in tumor cells were analyzed by Western blot.Results1.In vivo:(1) The endostar (16mg/kg) and elemene (100mg/kg) did not induce hematologic toxicity, hepatotoxicity, as well as liver and kidney injury.(2) The most effective doses of endostar and elemene were8mg/kg and100mg/kg respectively.(3) The optimal dose combination was8mg/kg endostar plus100mg/kg elemene.(4) The mice with malignant ascites were randomly divided into four groups:control group (treated with NS), endostar group (treated with endostar8mg/kg), elemene group (treated with elemene100mg/kg) and combination group (treated with endostar8mg/kg plus elemene100mg/kg), they were treated for8days and the results of related examinations were as follows:①The mean ascites volume of control, endostar, elemene and combination group were8.93±0.79,7.34±0.76,4.79±0.47and3.12±0.23ml, and the CDI value was0.79which indicated the synergistic effects;②Endostar and elemene reduced the number of tumor cells and the effects were enhanced when they combined. Elemene did not influence the number of red blood cells, but enhanced the effects of endostar;③Endostar and elemene decreased protein and LDH in ascites fluid, and the reduction was more obvious when they combined;④The mean survival time of mice in control, endostar, elemene and combination groups were11,14,15and18days, the prolonged survival rate of mice in endostar, elemene and combination groups were34.74%,43.67%and66.77%;⑤The transmission electron microscope found that the tumor cells in combination group were damaged severely;⑥The rates (%) of CECs in control, endostar, elemene and combination groups were6.82±0.91,1.95±0.17,6.16±0.42and0.57±0.09;⑦The optical density (OD) value of ascites fluid in control, endostar, elemene and combination groups were1.71±0.13.1.45±0.09,1.05±0.08and0.88±0.07;⑧VEGF. CD44V6, MMP-2and HIF-la were reduced in endostar and elemene groups, and the decrease was more obvious in combination group;⑨The combination of endostar and elemene enhanced the down-regulation of VEGF and MMP-2proteins in tumor cells. Although elemene injection did not influence the phosphorylation of p38MAPK, it enhanced the inhibition of p38MAPK signaling pathway when combined with endostar;⑩The relative expressions of VEGF mRNA in control, endostar, elemene and combination groups were2.28±0.28,0.94±0.11,1.58±0.15and0.63±0.06.2. In vitro:(1) The inhibition rates of endostar and elemene on H22cells were about4～8%and10～40%respectively, endostar could not enhance the inhibition effect of elemene.(2) VEGF, CD44V6, MMP-2and HIF-1α were decreased in endostar and elemene groups, and the decrease was more obvious in combination group.(3) The combination of endostar and elemene enhanced the inhibition of VEGF and MMP-2proteins in tumor cells.ConclusionThis study revealed that8mg/kg endostar combined with100mg/kg elemene could synergetically suppress ascites formation, as well as prolonging survival time of mice. The mechanism might be following:(1) Endostar enhances the effect of elemene to directly suppress tumor cells;(2) They reduce the live of endothelial cell accounted for cycle blood in the nuclear cell of percentage in the serum;(3) They effectively inhibit the expression of VEGF in both protein level and gene level, which decreases the peritoneal microvascular permeability and reduces the extravasation of protein and red blood cells from vessels;(4) They suppress the levels of CD44V6, MMP-2and HIF-1α to inhibit the invasion and metastasis of tumor cells;(5) They inhibite the p38MAPK signaling pathway. Taken together, endostar and elemene cooperatively inhibit the growth of tumor cells and microvascular permeability in complementary relationship.Our findings provid the theoretical basis and practical experience in carrying out multicentric clinical research for endostar combined with elemene in the treatment of advanced cancer associated with malignant effusion, and has a significant enlightening meaning on combining other modern Chinese medicines with molecular targeting drugs to treat cancer. |
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