| BackgroundColon Cancer is one of the most serious diseases that can threaten human's life and health currently.The morbidity and mortality of colon cancer comes the second and third of all malignant tumors respectively.In recent years,the incidence of colon cancer and mortality were increased year by year in our country and increasingly younger,especially between the age of 40-50.Colon cancer has extreme stronger invasion and metastasis potential.It metastasizes predominantly to liver.Operation is the most popular therapy nowadays.However,because of the extensively diffuse infiltration in advanced cancer patients,wide resection cased serious wound limits the surgical operation.Therefore,we would choose chemotherapy torelieve symptoms and prolong lifetime of patients.In the 1980 s,5-Fluorouracil(5-Fu)was already used to treat colon cancer for chemotherapy.To 1999,5-Fu plus leucovorin combination therapy has became the most important chemotherapy for colon cancer and be used for a long time.Until early this century,5-Fu combine with a range of other chemotherapeutics for clinical treatment gradually take the place of montherapy and it has be continued ever since.But as time goes on,the dose of chemotherapeutics increased,the growing drug concentration lead to serious damage to the organ of lesion and adjacent organs.Therefore,it is necessary to clarify the mechanism of tumor development and develop new medicines with good targetability and low toxicity to fundamentally control the development of cancer,prolong the lifetime of patients and improve the quality of life.Cancer stem cells(CSCs),as a part of cancer cells,took a very small proportion of cancer cells.But it has strong proliferation and differentiation abilities.Moreover,it's migratory capacity is far ahead of ordinary cancer cells.So it was considered as the fundamental cause of recurrence and distant metastases of cancer.The theory of CSCs originates in embryonic stem cells(ESCs).Nowadays,CSCs has be separated and cultivated from various of human malignant tumor tissues such as breast and brain cancer.After researched and repeatedly demonstrated,CSCs are the source of growth and development of cancer.The traditional chemotherapy suppress cancer's growth in the early days.Once the inhibitory effect reach a point,the stemness of cancer cells would be activated possibly.As a result,the proliferation and differentiation capacities of cancer cells would be promoted.Finally,it result in proliferation,differentiation and metastasis of cancer cells.Although CSCs took a small proportion in cancer cells,its' oncogenic capacity is about hundred times greater than ordinary cancer cells which result in cancer is difficult to be completely removed fundamentally.Therefore,based on theory of CSCs,researchers should clarify the characteristics of CSCs,survival area and oncogentic mechanism in order to inhibit the development of cancer.Epithelial-Mesenchymal Transition(EMT)refers to epithelial cells go through specific process translate into mesenchymal cells to achieve the characteristics of mesenchymal cells during the processes such as embryogenesis,inflammation,tumorigenesis and tumor development.Especially in tumor development,EMT was considered as one of the characteristics of CSCs.CSCs have strong invasion and metastasis capacities to be achieved by EMT.During this process,cancer cells' migrate ability can be promoted by losing of the adhesive function of E-cadherin and vanishing of tight junctions between cells.Keratin transformed into vimentin morphologically to make epithelial cells achieve the characteristics of mesenchymal cells;As the expression of adhesion molecules be inhibited,the expression of transcription factor Twist is increased.Snail,especially Snail1 and snail2 are the important markers of EMT.As for this,EMT is the important process of reccrence and distant metastasis of cancer.To control this process,based on clinical chemotherapeutics,we should try our best to develop a new chemotherapeutics,targeting CSCs,to inhibit EMT of CSCs.Pluronic was consisted by two embedded polymer nanomaterials consist of two polyoxyethylene chains and a poly propylene oxide chain(PEO-PPO-PEO),which can spontaneously form copolymer micelles.When the concentration of copolymer micelles higher than critical micelle concentration,it can form spherical micelle structure in water by head-tail connection.In addition,polymer nanomaterials have the hydrophobic characteristic which can form as the kernel of the copolymer micelles and touch with the drugs.Furthermore,poly propylene has the hydrophilia characteristic which can touch with water and form as the shell of the copolymer micelles.The copolymer micelles formed by pluronic spontaneously not only have the characters of general copolymer micelles,as increase solubility,prolong the role of drugs and targeting property,but also can decomposed into small molecular and obviously inhibited the synthesis of P-glycoprotein which reduce the drug tolerance of tumor cells in some degree.In many studies,pluronic was named as the most promising drug carrier in clinic chemotherapy and will be widely used in the study of carrier of antitumor drugs.Based on the state of pluronic in room temperature,it can be divided into three types: liquid(L),pasty(P)and solid(S).In addition,according to different length of pluronic,it has a variety of types,as L61?F127?P407 and ect.Many studies has demonstrated that,based on the shape,security or its enhanced drug susceptibility of pluronic,the type of P85 has obvious advantages than the type of L61 and other branches.Give those theories and background,at the initial stage of this project,we come up with this hypothesis that combining P85 and 5-FU together by using chemical process to forming 5-FU/P85 copolymer.Though this way,we want to inhibit the growth and metastasis of cancer by targeting CSCs,inhibiting the process of EMT and control the proportion of CSCs.AimsOur study aims to further explain the mechanism of colon cancer liver metastases,and then block the colon cancer liver metastases at the initial stage by copolymer micelle achieved by chemical synthesis method and based on the present chemotherapy drugs.Furthermore,it will provide a new strategy for curing colon cancer liver metastases in clinic.Methods1.5-Fu/P85 copolymer micelle was synthesized by thin-film hydration method,and then two different concentrations were prepared for further study in vivo and vitro.2.We used 5-Fu/P85 copolymer micelle to treated HCT116 cells to analyzed the effect of growth and proliferation by scratch assay,cell clone forming experiment,transwell invasion assay and CCK-8 testing.3.HCT116 cells were injected into the BALB/c nudemice splenic vein to build splenic vein metastasis model.5-Fu/P85 copolymer micelle was injected into BALB/c nudemice tail vein to observe the effect of 5-Fu/P85 copolymer micelle on HCT116 cells invasion and metastasis.4.The liver function(ALB and ALT)and survival time detected to determinate whether 5-Fu/P85 copolymer micelle could ameliorate the toxic and side effect and improve the lifetime and life quality.5.Cell clone forming experiments and flow cytometry was performed to analyze the ratio of CD133+CXCR4+ HCT116 cells influenced by 5-Fu/P85 copolymer micelle,the result indicated that whether 5-Fu/P85 copolymer micelle can specially target to cancer stem cells and kill them.6.In vivo and vitro study,the gene and proteins related to EMT were detected by real time-PCR,immunohistochemistry staining and immunofluorescent staining.The results will explain the mechanism of 5-Fu/P85 copolymer micelle in inhibiting colon cancer liver metastases.Results1.In vitro study,the result showed that 5-Fu/P85 copolymer micelle significantly inhibited the ability of proliferation and colon forming on HCT116 cells.Moreover,in vivo study,the result showed that 5-Fu/P85 copolymer micelle obviously ameliorated the size and serve degree of liver tumor formed by HCT116 cells,in addition,5-Fu/P85 copolymer micelle could decreased the toxic and side effect,and prolong the survive time of mice.2.The ratio of CD133+CXCR4+HCT116 can be obviously down-regulated by 5-Fu/P85 copolymer micelle.3.5-Fu/P85 copolymer micelle can decrease the ratio of CD133+CXCR4+HCT116 and the processing of EMT in mice splenic vein liver metastasis model.Conclusions5-Fu/P85 copolymer micelle,synthesized by thin-film hydration method,has obvious antitumor effect which can achieve the same or even better antitumor effect under the same conditions to reduce the concentration of chemotherapeutic drugs 5 – fluorouracil.At the same time,5-Fu/P85 copolymer micelle significantly reduced the side effects of chemotherapy drugs 5 – fluorouracil,which decreased the risk and damage degree in liver and other important viscera,and could prolong life and improve the quality of survival.Our study also demonstrated that,the mechanism of 5-Fu/P85 copolymer micelle in inhibiting anti-tumor was through targeting to tumor stem cells and inhibited the process of EMT.Consequently,5-Fu/P85 copolymer micelle can be used in clinic as a new chemotherapy.Furthermore,it can provide new method and new strategy for clinical treatment of colon cancer and inhibiting the distal metastasis. |
PDF Full Text Request