| Background and ObjectiveThe malignant tumor has badly been threatening the health and life of human being. In recent half of a century, tumor treatment with Chinese herbal medicine has been being accepted by more and more experts and patients, being the common way and one of the effective measures in the comprehensive treatment of malignant tumor. Clinical experiments prove that Chinese herbal medicine has the better effect of antitumor, not only decreasing the toxicity and increasing sensitivity or efficiency of radiotherapy and chemotherapy, but also preventing tumor from relapse, metabasis. In the treatment of malignant tumor , Chinese herbal medicine shows particular superiority for its less side effect, lower toxicity, aboundant resources ,lower price and being suitable to be used for a longer time. The researchs of the effective component and mechanism of Chinese herbal medicine have been being paid more and more attentions. Some drugs from herbs such as Vincristione (VCR) and Paclitaxel (PTX) have been used in the clinical treatment of tumor, which has gotten good clinical effect. However, effective components of Chinese herbal medicine used in clinic at the present time are still fewer, so exploiting and developing new effective antitumor drugs from Chinese herbal medicine becomes one of the important subjects of treatment of malignant tumor.Spike of Common Selfheal, a traditional Chinese herbal medicine has the effect of detoxifcation and detumescence. There are a lot of reports on its effect of antitumor.People had been using it to treat the carcinoma of thyroid gland, lymphoma, parotidean carcinoma, tonsillar cancer, nasopharyngeal carcinoma, breast cancer, cervical cancer, liver cancer etc so long .The modern pharmacology has proved that Spike of Common Selfheal has broad pharmacological activities. The experiments of cell apoptosis in vitro showed that it could induce the apoptosis of SGC-7901 (human gastric cancer cell line) cells and K562(human erythroleukemia cell line)cells .The experiments of restraining tumor in vivo reported that Spike of Common Selfheal has remarkable cytotoxicity. Therefore, extracting the effective component from Spike of Common Selfheal and studying on the mechanism of it has broadly applied value. However, the reports about the effect of antitumor of effective component of Spike of Common Selfheal still have been seen.The aim of this experiment is to exploit effects of extract of Spike of Common Selfheal on human esophageal cancer cell line Eca-109, human T-lymphoma leukemia cell line Jurkat, and then discuss the characteristic and possible mechanism of apoptosis of Jurkat cells by cell biologic methodsMethods1. Inhibition ratio of the extract from Spike of Common Selfheal on Jurkat cells and Eca-109 cells was tested by MTT assay. The experiment was divided into 3 groups:(l) experimental group: the extract from Spike of Common Selfheal at the different concentrations were administered to culture tumor cells;(2) positive control group:the Vincristione at the different concentrations were administered to culture tumor cells;(3) negative control group: the same volume of saline was administered to culture tumor cells. Every concentration of the extract from Spike of Common Selfheal which was administered to Jurkat cells and Eca-109 cells, respectively,was set for 4 parallel holes, after culture of 48 hours, the ratio of inhibition of cell growth was tested and IC50 was figured out. At the same time ,the growth curve line of Jurkat cells which was acted on by the extract from Spike of Common Selfheal at the two concentrations (50jUg/m\,80jug/ml) and that of Eca-109 cells acted on by the extract from Spike ofCommon Selfheal (300//g/ml, 60Qwg/ml) were drawn . Positive control group (Vincristione 5Qug/ ml, 20/Ug/ml) and negative control group (the same volume of saline) were set. The above experiment was repeated 5 times. The average value was figured out.2. The morphologic of cells was observed by invert microscope and HE staining. Jurkat cells apoptosis was observed by agarose electrophoresis. the expression of apoptosis related protein Bcl-2, Bax were measured by immunocytochemistry and the quantitative amalysis was made with figure analysis system.Results1. The effect of growth inhibition of the extract from Spike of Common Selfheal on two types of tumor cells.1.1 The extract from Spike of Common Selfheal can remarkably inhibit the growth of both Jurkat cells and Eca-109 cells. After dealing with the concentrations of 80/zg/ml, 66.67//g/m 1,55.56 fig/m\, 46.30 fig/ml, 38.56 fig/ml ,the ratio of inhibition of Jurkat cells growth was 87.14+1.08%, 77.82±1.65%, 53.20±2.91%, 33.08±2.65%, 18.37 ± 1.67% respectively ; After dealing with the concentrations of l000jug/m\, 500 ^g/ml, 250//g/ml, 125/*g/ml, 62.5^g/ml, the ratio of inhibition of Eca-109 cells growth was 91.66±1.48%, 76.91±2.90%, 35.84±2.59%, 30.37±2.65 %,5.34±0.82%, respect tively. The ratio of inhibition of two types of cells have remarkable dose-dependence, the correlative coefficient was 0.992,0.978 respectively, (jP<0.001).1.2 The extract from Spike of Common Selfheal dealed the two types of cells with, the IC50 was 53.59±3.10/ag/ml,268.82±2.21,Mg/ml, respectively. There are statistic differences between experimental group and control group of two types of cells1.3 From the curve lines, the extract from Spike of Common Selfheal of two concentrations (50,tfg/ml,8QMg/ml) dealed Jurkat cells with for 7 days ,which had stastic differences when three groups were compared with each other,(P<0.001). The livability of Jurkat cells dealed with the lower concentration of the extract from Spikeof Common Selfheal (50/ug/m\) was the lowest on the third day, and then there was a partial recovery of the growth. However, the growth of the cells dealed with the extract from Spike of Common Selfheal (80//g/ml) was remarkably inhibited on the second day and continued to the seventh day. The extract from Spike of Common Selfheal of both concentrations (SOQug/ml^OQug/ml) dealed Eca-109 cells with, which had stastic differences when three groups were compared with each other,(P<0.001).The livability of Eca-109 cells dealed with the lower concentration of the extract from Spike of Common Selfheal (SOOlug/ml) was the lowest on the fifth day, and then there was a partial recovery of the growth. However, the growth of the cells dealed with the extract from Spike of Common Selfheal (600/ig/ml) was inhibited remarkably on the third day and continued to the seventh day.2. The effect of the extract from Spike of Common Selfhea inducing Jurkat cells apoptosisAfter processed by the extract from Spike of Common Selfheal (5Q?g/ml) for 48 hours, the typical shape of Jurkat cells apoptosis was observed by invert microscope and HE staining. The DNA agarose electrophoresis showed obvious DNA ladder phenomenon in experimental group, however, there were no the ladder phenomenon in negative control group. The immunocytochemistry showed that the expression of Bcl-2 in cells decreased and the expression of Bax in cells increased. The differences between experimental group and control group are significant (P<0.001).Conclusion:1. The extract from Spike of Common Selfheal can remarkably restrain the proliferation of Jurkat cells and Eca-109 cells, and the effect of anti- proliferation has the relation of dose-dependence. From the IC50, the effect of anti - proliferation against Eca-109 cells are less than that against Jurkat cells2. It is possible that the extract from Spike of Common Selfheal restrains Jurkat cells proliferation by inducing apoptosis.3. The molecular mechanism of the extract from Spike of Common Selfhealinducing apoptosis of Jurkat may be that the expression of Bcl-2 was down-adjusted and the expresion of Bax was up-adjusted, which started-up the mechanism of apoptosis and induced cells apoptosis.
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