| Research BackgroudMalignant tumor is one of major diseases that threat to human health in nowadays.In China,1,400,000 to 1,500,000 people dying of cancer each year.Drug treatment,surgical treatment and radiation therapy are the major treatments of tumors.Tumor treatment involving chemotherapy, biological therapy,gene therapy,targeted therapy,traditional Chinese medicine and the effective natural product of anti-cancer therapy.Nearly half a century active ingredients come from Chinese medicines has been made remarkable breakthroughs in using in clinical anti-cancer treatment. Application of effective Chinese medicine in anti- tumors treatments,has been more and more accepted by scholars and the majority of patients.It is an effective means of Comprehensive treatment in malignant tumors. Therefore,there has been the consensus that we shoule do some research and develop the anti-tumor drugs from Chinese herbals by a vast number of medical workers.A large number of clinical and experimental studies have shown that Chinese medicine in cancer prevention,treatment and rehabilitation has played an important role.Chinese medicine in addition to have a significantly attenuated toxic effect on chemotherapy and radiation therapy,have a effect on anti-recurring and anti-transferring after operation on tumor,with good effect on tumor symptoms treatment,there is a certain role in the inhibition of tumor cells.Chinese medicine and traditional Chinese and western medicine in the new century on the eve has become indispensable force in anti-cancertreatment.Finding anticancer drugs from natural substances with small toxic side effects and small,safe and effective effects has become the research hot-spot worldwide.The anti-tumor molecular mechanism of Chinese medicines and its active ingredients have made a larger progress.That variety of mechanisms such as anti-tumor effects may be through inducing apoptosis, cytotoxicity,regulation of cell signal transduction,induction of cell differentiation,reversal of multidrug resistance,inhibition of telomerase activity.Triptolide is the root of Celastraceae plant Tripterygium Triptreygium Wilferdii Hook.f.The major components are alkaloids, diterpenoid,triterpenoid and sesquiterpenoid.It has been isolated from more than 100 kinds of active ingredients,which tripterine,triptolide in Schisanlignone,triptolide(triptolide),tripterygium B are the highest activity.Triptolide isolated diterpenoid lactone from Tripterygium,which is one of the most representative components of tripterygium.Pharmacological and clinical trials show that it has some biological activity on immuno-suppressive,anti-inflammatory, anti-tumor.It used as the main active ingredient,which in clinical application of various tripterygium.The content of triptolide also used to assess the number of good quality ingredients in natural medicine and are widely used to treat rheumatoid arthritis,nephritis,hepatitis,skin diseases and tumors,such as a variety of common and difficult disease.Triptolide MC002,a derivative of triptolide,is catalyzed into triptolide by esterase in the body.lt has advantages in the preparation and clinical administration because of its water-soluble.This paper studies is the tasks of creation of new drugs in Beijing.According to State Food and Drug Administration requirements issued in 2007 <>,in vitro MTT assay using the MC002 for 12 kinds of human tumor cells;in vivo test using four kinds of human tumor ceils in nude mice xenograft tumor model of anti-cancer effect of MC002.On this basis, we used the detection of apoptosis TUNEL and immunohistochemical method, EMSA method of MC002 on human lung adenocarcinoma cell line A-549 to detect anti-tumor mechanism.In this paper,in vitro and in vivo tests on three kinds of human tumor cells:human lung adenocarcinoma cell line A-549, human fibrosarcoma cell line HT-1080,human colon cancer cell line HCT-8 and use preliminary test to explore its anti-tumor mechanism of action.Research ContentResearch 1 MC002 serum incubation of tumor cells in vitro studyA certain concentration of MC002 has incubated for 2 hours with human serum in 37℃.Cisplatin as a positive control.Application of methyl thiazolyl tetrazolium(MTT) method to observe the cell activity of MC002 on human lung adenocarcinoma cell line A-549,human fibrosarcoma cell line HT-1080,human colon cancer cell line HCT-8 cytotoxicity,and calculate the IC50.We used six 10-fold dilution of the concentration of MC002(1×10-5M,1×10-6M,1×10-7M,1×10-8M,1×10-9M,1×1010M) to deal with three kinds of tumor cells 24h,after that OD values were detected by microplate reader.The results showed that:compared with the cells in the control group,MC002 after incubated by human serum,three kinds of human tumor cell proliferation inhibitory effects were significantly(P＜0.05),and has a good dose-effect relationship,IC50 were 1.45×10-7M,6.81×10-7M, 3.07×10-7M,repeat the test IC50 of 9.64×10-8M,2.88×10-7M,1.16×10-7M.Research 2 MC002 in human tumor xenograft model in nude mice inhibited the growth of researchUsing BALB / C nude mice inoculated human lung adenocarcinoma A-549 tumor line,the HT-1080 fibrosarcoma tumor line,HCT-8 human colon tumor line to establish tumors in nude mice xenograft model,set up MC002 large, medium and small dose group(1.5mg·kg-1,1.0mg·kg-1,0.75mg·kg-1),,and cyclophosphamide as a positive control,5%glucose solution as a solvent control,by intraperitoneal injection,once the next day and administered seven times,eleven times and nine times.Last 24 hours after administration of each group,animals were put to death,stripped of the tumor and taken tumor weight measurements,and then calculate the average tumor weight and inhibition rate.To observe the tumor growth inhibition of MC002,relative tumor proliferation rate,tumor weight,inhibition rate used as the evaluation index.The results showed that:For the three different tumor strains,the tumor volume of MC002 large,medium and small three-dose group tumor size is smaller than the model control group,tumor weight were significantly lower,and has a good dose-effect relationship.Research 3 MC002 tumor tissues of nude mice induced apoptosis and nuclear factor-κB activationReported in the literature,anti-tumor effects of Tripterygium wilfordii has a multi-target effect.Triptolide can induce apoptosis with wild-type p53 in gastric cancer cell line AGS cell and inhibite its nuclear transcription factorκB(NF-κB) and activator protein -1(AP-1) of the trans-activation,and with mutant p53 of the MKN-28 and SGC-7901 cells can not induce apoptosis;Inhibit the expression of p53 or over-expression of p53 can inhibit the activity of TP activation of NF-κB inhibition; Prompted triptolide induced apoptosis may be related to the promotion of p53 expression and inhibit activation of NF-κB.In order to further clarify the triptolide MC002 of semi-synthetic derivatives of the anti-tumor mechanism of action,the subject of the use of TUNEL method,immunohistochemistry,gel shift experiment tried to detect the induction of apoptosis in human lung adenocarcinoma may be the mechanism.Treatment group results suggest that compared with model control group,treatment group of MC002 large,medium and small dose induced tumor cells apoptosis,the apoptotic index are 46.65%,27.58%,13.32%;Expression of NF-κB positive rates were 2.75±1.51%,3.52±1.79%,5.64±1.60%,7.28±2.53%,and showed good dose-effect relationship;At the same time,NF-κB activation was inhibited,indicating that MC002 might induce apoptosis on tumor cells through inhibiting NF-κB activation. |
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