| Background and object:Primary liver cancer is a global common disease, especially in our country. Which is due to its rapid progress and insidious onset, and difficult early diagnosis, it's too late when more than 80% of patients were diagnosed. They had lost chance to have a surgery and local medical treatment. Their prognosis was poor. They need effective drug or method to improve the living urgently. Experimental research and clinical practice shows that as a rich vascular tumor, anti-angiogenesis is therapy is of great value for liver cancer. Elemene injection, modern Chinese medicine preparation, is one of terpene compounds, which extracted from zingiberaceous plants Wenyujin (curcuma). Recombinant human endostatin injection (Endostar) is a anti-angiogenesis drug, which targets mainly on vascular endothelial cells. Been marketed, both drugs have some anti-angiogenesis effect, mainly on Non-small cell lung cancer. The aim of this study is to explore the angiogenesis effect of the elemene injection and combination of Recombinant Human Endostatin (Endostar) on liver cancer, especially to find whether the combination can cause synergism and to explore the mechanism of synergism.Methods:1. in vivo study (1)The model of ICR mice bearing xenograft was made, firstly. Respectively 50mg/kg,75mg/kg, 100mg/kg dose of elemene injection alone and 5mg/kg, 10mg/kg,20mg/kg dose of endostar monotherapy were used on ICR mice, tumor size was observed and rate of tumor suppressor was calculated to determine the optimal dose of medicine. (2) Then, two different doses of drugs were used on ICR mice, tumor size was observed and rate of tumor suppressor was calculated to determine the single drug dose that could increase the antitumor effect of chemicals. (3) After we found the right dose of two drugs in combination, we used two drugs alone or combined on ICR mice which were divided into four groups:saline solution group, endostar group, elemene injection group, combination of two drug group. We calculated the tumor-inhibiting rates and observed the survival period of the ICR mice, and determined if the two drugs had synergistic effects. And then to examine two drug synergy mechanism with the following indicators:①microvessel density (MVD) by using immunohistochemical detection, VEGF expression in tumor tissues;②mice serum VEGF, VEGFR-2, bFGF, MAPK, ERK cytokine protein expression by using ELISA assay;③circulating endothelial cells in peripheral blood of mice percentage of nucleated cells by using flow cytometric method;④VEGF and bFGF mRNA expression in tumor tissues by using RT-PCR method;⑤ultrastructure of tumor cells by using transmission electron microscopy.2. in vitro study We used MTS/PMS to test the effect by combination of 50ng/ml,100 ng/ml,200ng/ml concentration of endostar, respectively, and 25μg/ml,50μg/ml,100μg/ml and 200μg/ml concentration of elemene in human hepatocellular carcinoma BEL-7402 cell absorbance values, and calculated the cell suppression rate. Results:1. in vivo study (1) We found best single-agent antitumor dose of elemene injection was 75mg/kg and best single-agent antitumor dose of endostar was 10mg/kg;(2) Our study found that the combination of 5mg/kg doses of endostar and 75mg/kg dose of elemene increased the antitumor effect of both.(3) We used saline solution,5mg/kg endostar,75mg/kg elemene monotherapy and combination of two medicines to treat ICR mice, the joint group was compared with saline solution group and monotherapy group:Inhibition rate significantly increased, joint group of inhibitory rate was 76.2%, endostar group was 16.9%, elemene alone group was 43.5%. Lifetime extension, joint group median survival was (23.8±2.7) days, endostar group median survival was (19.9±2.1) day, elemene group median survival was (20.6±2.1) day, saline group median survival was (16.9±2) days. The detection results showed:①Immunohistochemical study showed:MVD significantly decreased in the number. MVD joint group was (4.88±1.56), endostar group was (9.42±1.67), elemene group was (9.29±2.2), and saline solution group was (11.88±2.42). VEGF expression marked decreased in the number. VEGF joint group was (1.33±0.75), endostar group was (3.17±0.90), elemene group was (3±0.82) and the saline solution group was (3.67±1.37).②Circulating live of endothelial cell in the blood of the number of nucleated cells in peripheral percentage decreased. The joint group was (0.27±0.12)%, endostar group was (1.27±0.17)%, elemene Group was (1.15±0.37)%, and saline solution group was (2.97±1.17)%.③ELISA results showed:In mouse serum, the joint Group serum VEGF, bFGF, MAPK and ERK content significantly reduced, and VEGFR-2 had no significant change.④PT-PCR results showed:In combined group, the grayscale values of VEGF mRNA compared with the corresponding alone, showed no significant differences; but when these compared to the saline group all single-agent group showed significantly reduction relative expression(P<0.01). in combined group, the grayscale values of bFGF mRNA compared with the corresponding alone, showed no significant differences; but when these compared to the saline group all single-agent group showed significantly reduction relative expression(P<0.05);⑤Transmission electron microscopy results indicate that the joint group of cell injury considerably heavier than monotherapy group.2. in vitro study Calculate the rate of cell growth inhibition results:the rate of cell growth inhibition of combined group compared to that of all single drug endostar groups significantly increased(P<0.01). The rate of cell growth inhibition of the combined groups compared to that of monotherapy elemene groups, all groups increased. However, only elemene 100μg/mL, elemene 200μg/mL joint groups compared with the corresponding single-agent groups there was a significant statistically increased (P<0.05), elemene 25μg/ml, elemene 50μg/ml joint groups compared with the corresponding single-agent groups was not statistically increased (P>0.05).Conclusions:In vivo proof when elemene injection combined with endostar in appropriate doses, they could cause synergism,and increased effect of anti-angiogenesis of liver cancer, inhibited liver cancer, and extended the life time. In vitro experiments proved that elemene injection joint endostar in some concentration, could enhance the effect of inhibition of tumor cell destruction. Two drug joint application improved anti-liver cancer effect, and suppressed liver cancer vascular formation. The mechanism maybe:①. Combination of both can increase their direct suppression of tumor cell killing effects.②. Combination of both can effective reduce tumor organization micro-vascular density, suppress tumor organization VEGF of expression, suppress the VEGF, and bFGF, and MAPK and ERK in serum, promote vascular generated factor of expression.③. Combination of both can effective reduce the live of endothelial cell accounted for cycle blood in the nuclear cell of percentage in the serum.④. Combination of both can strengthen the suppression of liver cancer vascular formation and enhance directly inhibition of tumor cell killing effects, both complement each other.This experiment proved for the first time elemene injection jointed endostar can cause synergism, its mechanism is enhanced growth inhibitory effect on liver cancer cells and enhanced anti-tumor angiogenesis. Therefore, we can try to combine two drugs in practice and do further clinical researches. In addition, the findings has a significant enlightening meaning on other modern Chinese medicine preparation and molecular targeting drug combination. |
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