Research Of Xiaoaiping Injection On Tumor Cell Vivo And Vitro

1. Background and ObjectiveMalignant tumor has been seriously threatening human being's life. Its mortality is second only to cardiocerebrovascular disease and its incidence has been increasing year by year. Though combined chemotherapy, which is the main tumor therapy, has better anti-tumor effect; it has inevitably more side-effects. Moreover, Primary or secondary drug resistance in the process of treatment has become one of the tough problems in the clinical therapy of malignant tumor. It is time to seek new anti-tumor drugs which have high therapeutic effect and mild side-effect. During the recent 50 years, traditional Chinese medicine has been accepted by more and more experts and patients and is effective in the combined therapy of malignant tumor.Clinical experiments have shown that, the traditional Chinese medicine can not only decrease the toxicity and increase antitumor efficiency of radiotherapy and chemotherapy, but also prevent precancerous lesion development and has anti-tumor effect. With mild side effects, abundant resources, cheap price and long history of application, traditional Chinese medicine has become a hot spot through the development and research of new anti-tumor drugs. Nowadays, anti-tumor Chinese patent medicine is becoming more and more popular, however, the mechanism of anti-tumor is not clear.Marsdenia tenacissima, a traditional Chinese herb, has "QingReJieDu" effect. According to the vitro and vivo experiments, it has obvious anti-tumor effect, vitro experiment cells include live cancer Bel-7404, hep G2 cell, and gastric cancer SGC-7901 of human, and vivo experiment cells include W256, Ehrlich's ascites carcinoma, reticulum cell sarcoma S180, gastric cancer, P388, uterine cervix cancer U₁₄, lymphatic sarcoma of Mus musculus albus. However, up to now, it has no report about its anti-tumor effect on vitro esophageal carcinoma and internal melanoma experiment. The objective of this experiment is to observe the inhibition efficacy and mechanism of Xiaoaiping (XAP, commodity praeparatum of Marsdenia tenacissima) Injection on esophagus carcinoma cellular strains and melanoma with cytobiology and molecular biology technology.2. Methods2.1 Vitro experiment2.1.1 Inhibition ratio of XAP Injection on EC9706 cells was tested by MTT assay. The experiment was divided into 3 groups: (1) experimental group: Xiaoaiping Injections of different concentration were administered to cultured EC9706 cell; (2) positive control group: cisplatin of different concentration were administered to cultured EC9706 cell; (3) control group: same volume of physiological saline was. administered to cultured EC9706 cell. Every concentration which was administered to kill EC9706 cells was set for 4 paralleled poles, after 48 hours, the ratio of cell growth was tested and IC50 was figured out. At the same time the growth curve line of EC9706 cells which acted on by the extract from Xiaoaiping Injection of concentration 15mg/mL was drawn. Positive control group (cisplatin 10μg/mL) and control group was set. The above experiments were repeated 4 times respectively and the average value was figured out.2.1.2 The cellular shape was observed by invert microscope and acridine orange staining with fluorescent microscope, flow cytometry was applied to detect the diference of cell cycle, and immunohistochemistry was applied to the expression of CyclinD1, after XAP Injection (15 mg/mL) mixed with EC9706 cellular strains (1×10⁶ cells), EC9706 cells apoptosis was proved.2.2 Vivo experiment2.2.1 Mouse melanoma cells B-16 were subcutaneously injected into C57BL/6 mice. On the next day, tumor bearing mice were randomly divided into five groups and each group consisted of 10 mice: large,moderate,small three dosage groups of XAP Injection (0.8g/20g/d, 0.4g/20g/d, 0.2g/20g/d of XAP Injection was respectively injected intraperitoneally for 8 days); Positive control group(100mg/kg/d of CTX was injected intraperitoneally on day 1); Negative control group(the same volume of physiological saline was injected intraperitoneally for 8 days). Kinetics of tumor growth and survival time of tumor-bearing mice were measured, tumor growth inhibition ratio was calculated.2.2.2 Tumor tissue samples were taken and examined by HE staining to observe pathomorphologic difference between experiment group and negative control group and assess the inhibitory effects of XAP Injection on tumor-bearing mice.2.3 Statistical treatmentSPSS11.5 software was used to carry out statistical treatment. T-test and x²-test were employed. The size of test was "α= 0.05".3. Results3.1 Vitro experiment: XAP Injection inhibited the growth of EC9706 cells3.1.1 XAP Injection can remarkably inhibit the growth of EC9706 cells. When 40mg/mL, 20mg/mL, 10mg/mL, 5mg/mL XAP Injection was administered with EC9706 cells, the ratio of cell growth inhibition were 90.15±2.74%, 68.02±3.56%, 48.73±1.79%, 22.52±2.08%. There was significant difference between every experimental group and control group (P<0.05). IC50 was 10.89±1.05 mg/mL.3.1.2 Based on the curve line, the results of XAP Injection (15mg/mL) dealing with EC9706 cells showed that it has remarkable anti-tumor effect. Comparing with the control group, there was statistic differences between the two groups every day (P<0.01). The survival rate of EC9706 cells dealed with cisplatin (10ng/mL) became the lowest on the fourth day, and then part of them recovered. However, the growth of the cells dealed with XAP Injection (15mg/mL) were inhibited remarkably and continuously for seven days.3.1.3 After EC9706 cells were cultured with XAP Injection (15mg/mL) for 48 hours, the typical shape of apoptosis was observed by invert microscope.3.1.4 The results of acridine orange staining with fluorescent microscope showed that the cells administered by XAP Injection for 48 hours appeared hyperchromic flavovirens staining in nucleus or intracytoplasm, even flavovirens frags typical apoptosis morphological character.3.1.5 Flow cytometry analysis showed that cellular period were 59.56±1.73%, 86.68±2.57 %, 83.26±1.86% in G0/G1 period, respectively, after XAP Injection (10 mg/mL, 20 mg/mL, 40 mg/mL) mixed with Ec-9706 cellular strains for 48 hours . The differences between experimental group and control group are significant (P<0.05)3.1.6 There is no staining in nuclear or intracytoplasm of EC9706, however, brown-yellow fine grain in nuclear of positive control group, staining in both nuclear and intracytoplasm part of them. Repeated for 4 times, the results are same.3.2 Vivo experiment: XAP Injection inhibited the growth of Mouse melanomacells B-163.2.1 The tumor inhibition ratios of groups injected with XAP with large, moderate and small dosage are 33.21%, 25.78%, 15.15% respectivly, while that of positive control group is 83.76%. Compared with negative control group, IR difference of large and moderate dosage groups are statistically significant (P<0.05), and that of small dosage group is not (P>0.05). IR difference among XAP injected groups are statistically significant, and inhibition effect is dosage dependent.3.2.2 The surviving time of the tumor-bearing mice of large, moderate, small three dosage groups of XAP lnjection(27.7d, 25.3d, 22.8d) and the positive control group(31.3d) were longer than that of negative control group(23.1d), the difference between every dosage groups and control group were significantly(P<0.05). The difference among moderate dosage and large, small groups was significant(P<0.05).3.2.3 More widespread necrosis domain of tumor cells and apoptosis of EC9706 cells was revealed by HE staining examination in the group of mice treated with moderate dosage of XAP Injection than in the negative control group.4. Conclusion(1) XAP Injection can remarkably restrain the proliferation of EC9706 cells, and IC50 is 10.89±1.05mg/ml.(2) XAP Injection can induce EC9706 cells to apoptosis.(3) XAP Injection restrained the proliferation of EC9706 cells probably by blocking cell cycle at G0/G1 period.(4) CyclinD1 does not express in protein level in Ec-9706 cellular strains.(5) XAP Injection has markedly inhibitory effect on the transplanted Mouse melanoma cells B-16 and can prolong the survival time of the tumor-bearing mice.