A Nanosized Carcinogen Successifuly Induced Liver Cancer In Mice,and How It Works

Liver cancer is one of the diseases that severely damage the function of normal liver.The "World Cancer Report,2014" released by WHO ICRC statistics shows that there are almost 400 thousands new patients of liver cancer in China every year,accounting for 55% of the world new liver cancer patients.In our country,the mortality of liver cancer is the second place among all cancer mortalities.The mechanistic researches of HCC development have made significant progress and three stages(initiation,promotion,and progression)have been defined in HCC,during which both genetic and epigenetic changes accumulate and subsequently lead to injury,inflammation,steatosis,fibrosis or cirrhosis of the liver and finally turn liver into cancer.To further study the molecular mechanism of different liver cancer stages and the development of new anti-tumor drugs and therapies,an animal model similar to the natural process of human hepatocellular carcinoma should be established.Mice have been widely used in the laboratory,due to the advantages of cheap,easy to operate and similar to the process of the occurrence of human hepatocellular carcinoma.Diethylnitrosamine(DEN)is known as a representative chemical carcinogen.According to recent investigations,relative large dose of DEN(ranging from 50 to 100 mg/kg/week)and complicated protocols(orally,i.p.or intra-gastric application of DEN 2-5 times a week for 15 to 20 weeks)were used for the establishment of mice model of cancer.However,there is another disadvantage under-the-table: high death rate of mice during extensive application of DEN.Our previous study showed that an alternative administration method(DEN dissolved in sesame oil by oral administration),also called the long-term-low-frequency administration,can overcome the disadvantages of traditional modeling methods,but still takes a long experimental period.With the development of nano-technology,nano carrier(especially nano liposomes)has been widely used in tumor therapy in the field of medicine due to its positive biological effects(enhanced targetability,sustained release and reduced side effects compared with traditional chemotherapy drugs).Our early cooperative research showed that DOX/Cur-NPs can be effective in the treatment of primary liver cancer.However,to date,no research has been conducted on the ability of “carcinogenesis” of nano-carriers in the establishment of animal cancer model.So,we do exactly the opposite,established an mouse model of liver cancer efficiently by nano "poison"-lipid nano material coated diethylnitrosamine(nanoDEN),and the carcinogenic mechanism was studied.Objective: To prepare a stable and efficient nano-carcinogen(nanoDEN)and establish a good,fast and constant mouse liver cancer model through a long-term and low frequency oral administration of nano-carcinogen agent.In addition,the carcinogenic mechanism of nanoDEN has been studied as well.Methods:1.The nano-carcinogen(nanoDEN)was prepared by using high pressure microfluidics technique.The particle size,zeta potential and stability were measured by a Zetasizer.The morphology of nano-carcinogen was observed by FE-SEM and the release of nano-carcinogen in vitro test was evaluated.2.In vivo,we established a liver cancer model in mice with low dose(as low as 16.5 mg/kg/week)and frequency(once/week)application of nano-carcinogen,the change of body weight of mice was recorded and liver samples were observed grossly(whether appear tumor nodules),the biochemical function of liver including ALT and AST was detected,and liver stainning(H&E and Masson)was scored by microscope.Based on the experiments above,the oncogenic effects between nano-carcinogen and DEN can be compared clearly.3.From the molecular level,the expression changes of COX-2(an inflammatory indicator),?-catenin(an important factor during oncogenesis),and PCNA(a protein recently related with tumor formation)were detected by immunohistochemistry and the change of apoptosis was detected by tunnel staining method.By those ways,we analysed the effects of the nano-carcinogen to cause cancer.4.To detect the toxic effect of nano-carcinogen and DEN.We adopt MTT to observe the viability of HepG-2(a cell model of liver hepatocellular carcinoma)and L02(a cell model of normal hepatic cell),meanwhile HepG-2 and L02 cells were treated with nanoRhod B to simulate the endocytosis process of hepatoma cells and normal hepatocytes.5.To simulate the distribution of the nano-carcinogen in vivo,nanoRhod B was injected to the caudal vein of mice.Result:1.The characterization of nano-carcinogen showed that: nano-carcinogen with a stable particle size and potential,the smooth surface,uniform size under the electron microscope.In addition,the material can be released in asustained and stable way.2.Compared with oral administration of low frequency and trace amount of DEN,nano-carcinogen is better in inducing mouse liver cancer: During the entire period of establishing animal model,the amount of ALT and AST in nanoDEN group is higher than those in DEN group.In nanoDEN-treated mice,scores of pimelosis and fibrosis were much higher than that in DEN-treated mice.At week 20,the body weight of mice in the nanoDEN group showed a downward trend and the visible tumor nodules were observed.Those demonstrated an improved toxicity of nanosized DEN towards livers.3.At the molecular level,both nano-carcinogen and DEN could introduce liver cancer(the change pattern of expression level of ?-catenin,COX-2 and PCNA),although the oncogenic effect of nano-carcinogen was superior to DEN(the increase of PCNA expression in the nanoDEN-treated group is higher in the DEN-treated ones and more apoptosis could be observed in the liver of mice treated with nanoDEN at week 20).4.Cancer cells were more tolerant to nano-carcinogen compared with normal cells in vitro: Compared with almost equal toxicity of DEN or nanoDEN to HepG2 cells,nano-carcinogen is much more efficient in killing L02 cells than DEN.More nano-carcinogen was up-taken and restrained in normal cells.Therefore compared with hepatocellular carcinoma cells,the inhibitory effect of nano-carcinogen to normal cell is stronger.5.The nano-carcinogen showed a better targetability on liver.CONCLUSION: Compared with DEN,the nano-carcinogen has the advantages of targetability and sustained release behavior.Besides,the oncogenic effect of nano-carcinogen was superior to DEN.Therefore it is an easy while low-cost and efficient way of establishing liver cancer in mouse with nano-carcinogen.