| [Background]Colorectal cancer which is behind lung, gastric and liver cancer is the fourth most common malignancy in China, With the improved changes of living standards and dietary, the incidence of colon cancer is significantly increased.Currently,the surgery is the major treatment options,but there is still a considerable number of patients in the surgery that has been transferred through the blood to the liver, lungs and so on.Cancer is essentially a genetic disease.A variety of factors can induce the cause of cancer,there are mainly two factors. exogenous and endogenous factors, exogenous factors including physical, chemical, biological factors, endogenous factors including genetic, immune, and many others. Varieties of endogenous and exogenous factors as a synergistic or sequential way cause cancerization,they activate the proto-oncogenes and (or) inactivate tumor suppressor genes,or change the apoptosis regulatory genes and (or) DNA repair genes to cause the abnormal levels of protein expression.Reactive oxygen species (ORS) play the mainly role in the process of tumor. Regulation disorders of cell proliferation and apoptosis lead to uncontrolled cell proliferation, and the adhesion changes between the tissue matrix and tumor cells as well as their biological characteristics lead to tumor cells metastasis.So,the mechanism of most anti-tumor drugs are inhibiting the tumor cell proliferation and promoting tumor cell apoptosis. Traditional Chinese medicine(TCM) holds that the cause of tumor nothing more than the external and internal injuries, internal injuries related to the weakness of human righteousness and emotional factors, external injuries included the diet, environment, geography, other insect poison and disease-causing evil. In traditional chinese medicine. the pathogenesis of cancer is mainly summarized as blood stasis, phlegm pool, hot intrinsic toxicity, organs disorders, upright weakness, Cure measure is made with justifing the symptom-complex,so strategy for the treatment of cancer are mainly circulating the blood, dispelling phlegm and dampness, heat-clearing and detoxicating, strengthening and restoring, etc, the dispelling phlegm and dampness, invigorating blood circulation and eliminating stasis, heat-clearing and detoxicating and tonic medicine are commonly used.There are some drugs to inhibit tumor cell proliferation and induce apoptosis of tumor cells, such as purple glycerol, arsenic trioxide, etc.Radix trichosanthis is the root of the Cucurbitaceae Trichosanthes,which is commonly used in clinc.In traditional Chinese medicine,it has the effect of clearing heat-fire, helping produce saliva and slaking thirst and relieving swelling and discharging pus. Trichosanthin (TCS),which is extracted from Radix trichosanthis, is a type I ribosome-inactivating proteins(RIPs) with247amino acids,which molecular wieght is27610and inactivates eukaryotic ribosomes via its N-glycosidase activity.TCS has long been used to induce mid-term abortion and treat ectopic pregnancies,and in recent years,some researchs found that TCS had other activities,such as anti-HIV and anti-tumor.And the present studies of TCS are focused on the effect of anti-tumor and mainly concentrated in the cervical cancer, choriocarcinoma,but the researchs about colorectal cancer are much less.In this experiment,we choice the highly metastatic human colon cancer SW-620cell lines in vitro test to observe the effect of TCS on colon cancer cell line SW-620cell line about cell proliferation, adhesion and migration, and to explore the mechanism.[Objective]The purpose of the study is to investigate the growth inhibition and adhesion as well as migration of SW-620cells induced with TCS.In order to observe thrchosantin(TCS) inhibiting proliferation,adhesion and migration of human colorectal carcinoma SW-620,we used the highly metastatic SW-620cells.Furthermore, we investigated the adhesion abilities between the vascular endothelial cell(HUVEC) and SW-620which was dealed with TCS.[Methods]1.The antiproliferative effect of TCS on SW-620and cells was measured with MTT.2.The apoptosis of SW-620cells were detected by fluorescence staining with hochest33258.3.The FCM analysis was carried out to examine the effect of TCS on the cycle and apoptosis distribution of SW-620and cells.4.The changes of adhesion abilities with HUVEC and SW-620cells were examined by using cell adhesion assay.5. The changes of migration abilities and of SW-620cells were examined by using wound healing assay.[Results]l.The proliferation of SW-620and cells was significantly inhibited by TCS in a concentration-and time-dependent manner.Dealed with0.625,1.25.2.5,5,10,20and40μg/ml TCS for24,48and72hours,the survival rate of experimental group decreased gradually. When the concentration of TCS was increased the and the time was prolonged, the growth inh ibitory rate increased gradually. the median inhibitory rates of TCS for SW-620cells were56.8,33.96,22.18μg/ml after12,23and48hour,respectively. Statistical analyses by MTT indicated that the inhibition rate increased signficantly in all experiment groups except the0.625μg/ml group after24hours(P<0.05).2. Use Hoechst33258fluorescent staining to observe the morphology changes of SW-620cells.After cells were treated by TCS (20,40μg/ml) for48hours,respectively.some apoptosis of cells could be seen by a fluorescence microscope.The apoptosis of cells presented pyknotic (shrunken and dark).and some apoptotic bodies appeared,but the control group did not have the phenomenon.3. After cells were treated by TCS(20.40(μg/ml) for48hours. flow cytometry indicated that the early apoptosis percentages is (3.53±1.07)%and (6.7±0.5)%,respectively, compared with the control group which the early apoptosis percentages is (0.77±0.75)%,Statistics and analyses proved that TCS(20μg/ml) could not induce early cell apoptosis (P>0.05),but TCS(40μg/ml) had the function.The percent of S and G1phase was25.15%and64.56%in control group,respectively, after cells were treated by TCS(20,40μg/ml) for48hours,the percent of S and G1phase was32.91%,58.09%and59.21%.33.91%, respectively,and the percent of G2/M had no statistical significance.The result showed that the TCS could block the SW-620in S phase in a concentration dependent.4. MTT assay detected the adhesion test between SW-620cells and vascular endothelial cell. The result showed that SW-620cells which were treated with different concentrations of TCS and HUVEC cells were cultured after2hours, compared with the control group, cell adhesion rate decreased, and20μg.g/ml and40μg/ml TCS compared with the control group, P values were P=0.03and P=0.01,respectively.5.Wound healing assay detected the migration ability of SW-620.After24and48hours,the migration lenth of control group was (154.8±18.3) and (222.0±12.2) pixel,and after treated with TCS(2.5μg/ml) for24and48hours, the migration lenth was (58.40±15.7) and (97.0±16.4)pixel,respectively.The result showed that TCS could inhibite the migration of SW-620.[Conclusion]1.The proliferation of SW-620and cells was significantly inhibited by TCS in a dose-and time-dependent manner.2.Hochest33258staining method showed TCS can induce SW-620cells apotosis.3.TCS led a S-phase arrest in cell cycle and induced cell early apoptosis.4.TCS inhibited the adhesion abilities of SW-620cells.5. TCS changed the migration abilities of SW-620cells. |
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