| Deaths total ratio of malignant tumor in china was slightly lower than the average ratio in world, however, Deaths ratio of digestive organs in china was two times of the average ratio in world. There were four kinds of malignant tumor of digestive system among the most common nine kinds of tumors in china and the deaths ratio of liver cancer in china was top death ratio in the world. It was obvious that malignant tumor of digestive system was very harmful for our Chinese residents, Therefore it was very important for us to explore the development mechanism of malignant tumor of digestive system and take powerful intervention for prevention and treatment.Ursolic acid existed in kinds of nature plant as free or compound form, some study at home and abroad suggested that ursolic acid had multiple effects and great applied value as the anti-tumour drug.Kinds of study suggested that ursolic acid had kinds of anti-tumour effect for lung cancer, breast cancer, stomach cancer, colon carcinoma and liver cancer. In fact most research about the anti-tumour mechanism were focused on the apoptosis pathway.Recently some study suggested that the development of tumour could be contributed to its characteristic metabolic activity.Thus the anti-tumour effect of ursolic acid might correlate with intervening the metabolic activity of tumour cells, and this correlation was worth studing.In fact this was the emphasis of this research.In this research the whole experiment was divided into three parts, that is the contrastive analysis of human tissue specimens, the contrastive analysis of animal tissue specimens and cellular pharmacology experiment in vitro.The former two parts attach importance to reveal the abnormal change rule by analyzing the difference of related signaling proteins between the control group group and the positive group with tumours.The last part was the major content of this reseach and focused on revealing the anti-tumour pharmacodynamic action and the pharmacodynamic mechanism. Part I The contrastive analysis of signaling proteins at the Glycolysis pathway in The human gastrointestinal caner tissuesObjective:Aerobic glycolysis activity in tumour cells was the obvious characteristics of tumour tissues distinct from the normal tissue, PI3K/AKT/mTOR/HIF-1α/PKM2pathway was tightly correlated with the aerobic glycolysis activity in tumour cells.This part compared gastrointestinal tumour tissues with normal tissues and analyzed the difference of related signaling proteins so that the abnormal change rule of PI3K/AKT/mTOR/HIF-1α/PKM2could be elucidated.Methods:Collect the human gastrointestinal tumour tissues at the surgical gastroenterology department in the renmin hospital of wuhan university from January2012to May2013. All the tumour tissues were from surgical Procedures and under pathological diagnosis, and among these tissues,there were82samples of stomach cancer and105samples of colorectal cancer, meanwhile, normal gastrointestinal tissues were collected from digestive endoscopy center in the renmin hospital of wuhan university, all these normal tissues were from splanchnoscopy and under pathological diagnosis, there were92samples of normal gastrointestinal tissues among which48samples were gastric tissues and44samples were colorectal tissues. All the tissues were fixed at the4%paraformaldehyde solutions for three days after the collection and then under paraffin embedding for the following making of tissue sections. At last immunohistochemisty was employed to compare and analyze the expression difference between two groups.Results:(1).The average optical density value of PKM2and mTOR at gastric cancer group(4423.18±328.77) was significantly higher than that at the control group(135.84±15.36,813.55±61.26).p<0.05.(2).The average optical density value of PKM2(9736.04±28.73), mTOR (5165.64±391.79), HIF-1α(4474.39±255.93) and Ubiquitin(4107.99±726.33) was significantly higher than that at the control group(4031.78±477.09,1215.83±153.41,1633.00±115.55,411.08±72.18),P<0.05.Conclusions:(1).The relative expression amount of PKM2and mTOR at Chinese gastric cancer tissues were significantly higher than those at non-tumour Chinese people. (2). The relative expression amount of PKM2, mTOR, HIF-la and Ubiquitin at Chinese colorectal cancer were significantly higher than those at non-tumour Chinese people. Part Ⅱ The contrastive analysis of signaling proteins at the Glycolysis pathway in The colorectal cancer tissues of athymic miceObjective:Establish the colorectal cancer animal model with athymic mice of BALB/c-nu by the way of surgery.The relative expression amount of related signaling proteins was detected and compared between the control group and the bearing cancer group so that the change rules of these proteins could be revealed.Methods:Twenty8-week-old male athymic mice of BALB/c-nu were divided into two groups at random according to the experimental design:(l).the control group,(2). the orthotopic nude mouse model group of colorectal cancer.During the period of establishing and maintaining the mouse model the survival condition and related physiological index were observed and recorded per week. Meanwhile, The abdomen of the model mouse was examined to ensure whether that there was the formation of enclosed mass. At the end of the8th week, all the mice were sacrificed and colorectal tissues were separated and stored, one part was immersed in the4%paraformaldehyde solutions for the fixation in order to making the paraffin embedding for immunohisto-staining, the other part was immersed in the liquid nitrogen for the detection of proteins with western blot.Results:(1)From the date of the implantation of LOVO cells in situ to the date of establishment of mouse model of colorectal cancer, two mice at the model group died successively for some accidental reasons, the achievement ratio of the bearing cancer mice model at the model group was80%.(2).The average optical density value of PKM2(5223.99±742.60) and HIF-la(1601.65±224.54) at the bearing cancer group were significantly higher than those of homologus proteins at the control group(2215.85±184.28,466.77±31.34),P<0.05.(3).The average gray density value of PKM2(1252.51±51.90),PI3K(1109.69±19.87),mTOR(917.46±48.98) at the bearing cancer group were significantly higher than those of homologus proteins at the control group(298.35±32.54,383.72±20.80,398.28±14.68), P<0.05.Conclusions:(1).The expression of PKM2and HIF-la at the model group were significantly higher than those at the control group by the contrastive analysis of results from the immunohistostaining. (2).The expression of PKM2, PI3k and mTOR at the model group were significantly higher than those at the control group by the contrastive analysis of results from the western blot. Part Ⅲ The inhibition effects of Ursolic acid for the proliferation and migration of tumour cells at digestive system and the possible effects of Ursolic acid on the related glycolysis pathwayObjective:Ursolic acid was added into the cell culture fluid by the way of specific concentration gradient to affect three kinds of tumour cells including Gastric cancer cell line HGC-27, Hepatocellular carcinoma cell line SMMC7721and Colorectal cancer cell line LOVO.And the final objective was to elucidate systematically the pharmacody-namic action and the pharmacodynamic mechanism of ursolic acid on the tumours of digestive system.Methods:Make the group with the dose of Oumol/1of ursolic acid as the control group and choose four concentration gradient(5μmol/l,10μmol/l,20μmol/l,40μmol/l) as the means of intervention.That was to say that each cell line was divided into five groups from the dose of Oμmol/1to40μmol/l. The related indexes were detected by the following methods:(1)The MTT colorimetric assay was employed to detect the inhibition effect of ursolic acid with different concentration gradient for the three kinds of tumour cells;(2).The flow cytometry was employed to detect the rate of apoptosis and the cell cycle arrest at different groups with different concentration gradient;(3).The scarification test was employed to detect the immigration activity of three kinds of tumour cells at different groups with different concentration gradient;(4).Western blot was employed to detect the expression difference of PI3k,Akt,mTOR, HIF-lαand PKM2among different groups with different concentration gradient;(5).Real time PCR was employed to detect the expression difference of mRNA of PI3k,Akt,mTOR,HIF-1α and PKM2among different groups with the concentration gradient.Results:(1).Ursolic acid reflected the inhibitory effect of the cell proliferation for three kinds of tumour cells when the dose of ursolic acid overcame1Oμmol/1, the inhibitory concentration50of HGC-27cell at the point of12hours,24hours and48hours was respectively51.89μmol/L,37.07μmol/L and28.19μmol/L; the inhibitory concentration50of SMMC7721cell at the point of12hours,24hours and48hours was respectively66.66μmol/L,57.42μmol/L and35.58μmol/L, and the inhibitory concentration50of LOVO cell at the point of12hours,24hours and48hours was respectively63.69μmol/L,46.02μmol/L and38.39μmol/L. (2).The apoptosis rate of three kinds of tumour cells increased continuously when the dose of ursolic acid overcame10μmol/l, the group of high dose exhibited showed obvious cell cyle arrest of G1stage and G2stage, and the apoptosis rate and cell cycle arrest at the point of24hour were higher than those at the point of12hour when the compared groups were at the same dose.(3).After24hours, the migration activity of three kinds of tumour cells were inhibited at different degree with the increasing dose.(4).After three kinds of tumour cells were treated wth four kinds of concentration gradient, the groups wth the dose of20～40umol/L showed the significant downregulation of the expression fo PKM2,however, the expression of PI3k, Akt, mTOR and HIF-lawere significantly upregulated at different degree according to the difference of tumour cell lines when compared with the control group,P<0.05.(5).The amount of mRNA of Akt and PI3K at the group of40μmol/L were obvious higher than the control group as for the HGC cell line, and the amount of related signaling proteins were increased at a certain degree at the group of40μmol/L in the SMMC cell line and the LOVO cell line when compared wth the control group,however, the trend was not obvious.Conclusions:(1).Ursolic acid could effectively inhibit the proliferation of human gastric cancer cells, human liver cancer cells and human colon cancer cells.(2). Ursolic acid could promot the apoptosis of three tumour cells of digestive system and have the obvious effect of cell cycle arrest for G2stage, and these effects showed the dependence of time and dose.(3). Ursolic acid could effectively inhibit the migration activity of tumour cells of digestive system.(4). Ursolic acid could specifically inhibit the expression of PKM2so that the proliferation of tumour cells of digestive system could be effectively inhibited and the apoptosis was induced, meanwhile, Ursolic acid could also excessively activate the activated signaling pathway of PI3K/Akt/mTOR/HIF-lα so that it could disturb the homeostasis of tumour cells and induce the apoptosis and necrosis of tumour cells. |
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