Expression Influnce Of Cox-2, Hmlh1, Hmsh2,bcl-2,caspase-3 In Colon Cancer Lovo Cell Lines Be Treated With NSAIDs And The Mechanisms Of The Anti-neoplastic Effect Of NSAIDs

Objective : Currently,With the lifestyle and diet changes in the structure,the incidence of colorectal cancer is increasing, about 1 / 3 of patients are found in late already , and transferred after post-operation.It is a key to explore the mechanism of occurrence,and findeffective measures , early detection ,early diagnosis, early treatment of cancer will help reduce the morbidity and mortality.In the mechanism study of colorectal cancer ,cyclooxygenase -2 (COX-2) and mismatch repair (MMR) systems are two of important target . In the preliminary studies [1,9]We found there's almost no lack of mismatch repair enzymes in gastric cancer and colon cancer specimens where COX-2 protein expressed; and the lack of mismatch repair enzymes mainly occurred in the low expression of COX-2 protein in the samples.This prompts us that the mechanisms is different.COX-2 which can be involved in regulation of cell proliferation, differentiation, inhibiting apoptosis and other ways to promote tumor's occurrence is the rate-limiting enzyme of prostaglandin synthesis. The inhibition of COX-2 on apoptosis is relevent to high expression of apoptosis inhibitory protein B-cell lymphoma 2 ( Bcl-2).Caspartate-specific cysteinyl proteinase (Caspase) is important to the Bcl -2 family proteins in enzyme modification especially,and Caspase-3 is a key part in the process of apoptosis.There is a high incidence of tumors in some people who have mutation or deletion in Some hereditary DNA repair regulatory gene. DNA mismatch repair is one of the DNA respair system,which can correct the mismatch in DNA bases that occurs during the replication to keep the genome stably . hMLH1, hMSH2 genes are the two of the most important members in MMR system,whose encoded proteins plays an important role in DNA mismatch repair process. The reduction and loss of function in protein expression of hMLH1, hMSH2 lead to genomic microsatellite instability ( MSI), which will decrease the stability of the whole genomeand increase the random mutation rate, and then lead to tumors with a series of tumor associated target genes changing.We found that long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk in colorectal, esophageal, gastric and pancreatic cancer and other digestive tract tumors in Epidemiological study[2] ,it Prompts us that NSAIDs have a certain chemically preventive effect on gastrointestinal tumors. Substantial research show that induction of apoptosis and affect cell growth cycle are the the main ways of NSAIDs to prevent tumors during the COX-mediated or non-COX- mediated pathway, of which the most significant role is apoptosis. Many studies have shown that the anti-tumor effects of NSAIDs primarily practiced by inhibiting COX-2 expression, inhibiting the activity or quantity of the prostaglandin product, changing the apoptosis protein and anti-apoptotic protein ,and finally, increaseing apoptosis of cell, this is the COX-2 -mediated apoptosis pathway. NSAIDs can also achieve the effect on promotion of apoptosis during affecting the nuclear factorκB signal transduction pathway, precursoring protein increases apoptosis,or inhibiting angiogenesis,this is the non-COX-2-mediated apoptosis pathway. some studies have reported[3] recently that Aspirin and Sulindac can significantly reduce the MSI phenotype, prevent the transformation of colorectal polyps to cancer, and even reverse the early stage of colorectal cancer after adding to the colon cancer cell lines which is defective in mismatch repair enzymes.It may also prompt us that NSAIDs prevent tumors during reducing the cell MSI phenotype and affecting protein expression of MMR system.We choose selective COX inhibitor valdecoxib and the non-selective COX inhibitor Aspirin act on person colon cancer cell lines lovo,whose COX-2 is high expressed and MSI is positive (hMSH2 loss of expression). We are using flow cytometry, immunocytochemistry, Western blot and other methods to observe the impact of two kinds of drugs on the proliferation, apoptosis and cell cycle and COX-2, Bcl-2, caspase-3, hMLH1, hMSH2 protein expression , in order to study the different mechanisms of NSAIDs in prevention and treatment preliminarily.We hope provide a theoretical and experimental basis on the occurrence , prevention and appropriate treatment for colorectal cancer.Methods :1 Materials: lovo (COX-2 high expression an, MSI is positive ,hMSH2 loss of expression)are permanent cell lines derived from colon adenocarcinoma cell line.Valdecoxib (COX-2 selective inhibitor) and Aspirin (COX-2 non-selective inhibitor) are used as intervention elements.2 Methods:2.1 we choose methyl thiazolyl tetrazolium (MTT) to determine proliferation of cell in each group lovo and to determine the half inhibitory concentration (IC50): established of the control group (control), solvent control group (solvent)and drug group. Set up the drug concentration: 25μmol.L-1,50μmol.L-1,100μmol.L-1,200μmol.L-1, 400μmol.L-1 and 800μmol.L-1. Action time: 72h, measure IOD values, calculate the rate of cell growth inhibition, and determine IC50 according to the formula.2.2 Experimental groups2.2.1 Concentration of group:three different concentrations of the two drugs were selected according to IC50, Aspirin: 90μmol.L-1, 180μmol.L-1, 360μmol.L-1; Valdecoxib: 20μmol.L-1, 40μmol.L- 1,80μmol.L-1 and acted on lovo cell lines for 72 h.2.2.2 Time Group: We selected IC50 concentrations of the two drugs to act on lovo cell lines for 24 h, 48 hand72 h ,and then collected cells.2.3 Detecting index2.3.1Detected the apoptosis and cell cycle changes in various group for a quantitative detection by flow cytometry (FCM).2.3.2 Western blot was used to detect the expression of COX-2, hMLH1, hMSH2, Bcl-2, caspase-3 protein after using drugs for24h,48h,72h. we also observed protein expression of COX-2, hMLH1, hMSH2 in location and changes by Immunocytochemistry (SP) .Results:1 The effects of Aspirin and Valdecoxib on the proliferation in lovo The IC50 Valdecoxib and Aspirin acting on lovo human colon cancer cell lines for 72h were 86.30μmol.L-1 and 382.46μmol.L-1.The drugs can inhibite proliferation,and the more concentration of drug we add to,the stronger effect on inhibition is shown. The effect on inhibition of Valdecoxib is stronger than Aspirin.Solvents show no significant effect on cell growth(p>0.05).2 The effects of Aspirin and Valdecoxib on apoptosis in lovo It is shown that the two drugs can increase apoptosis of lovo cells,and the characteristic sub-diploid peak in apoptotic cells were seen in the diploid DNA histogram-peak ,but we couldn't see an obvious apoptotic peak in the control group. lovo's apoptosis after using Valdecoxib for24,48 and 72h are (4.26±0.71)%, (29.03±0.64)% and (39.96±1.40)%,which are higher than control group. lovo's apoptotic rates after using different co for 72h were (8.39±0.33)%, (8.08±0.42) and (28.9±1.62)%.and when the ncentrations of Valdecoxib is 80μmol.L-1 ,the apoptosis rate increased significantly. The apoptosis rates in Aspirin group for 24,48and72h were (6.41±0.33)%, (28.73±0.45)% and (32.41±0.69)%,which were higher than control group(p<0.05). The effects on apoptosis changed after adding Aspirin of different concentrations for 72h, of which 180 and 360μmol.L-1 group was significantly increased ,the resurlts were (23.6±0.85)% and (28.0±1.28)%, which were higher than control group (0.92±0.35)%. we added IC50 drugs into the cell for 48h and 72h,apoptosis of lovo cells Valdecoxib induced was higher than Aspirin,and the differences were statistically significant (p<0.05). The two drugs of different concentrations on cell apoptosis was no significant difference (p> 0.05). (Table.2, 3)3 The effects of Aspirin and Valdecoxib on cell cycle of lovo cells When we added the two drugs as different concentrations into lovo for 72h,we could see that the number of lovo cells in G0/G1 phase cells increased (74.61±1.63)% and (67.38±1.64)%in Valdecoxib80μmol.L-1 group and Aspirin360μmol.L-1 group, S phase and G2 / M phase cell reduced in the number, and proliferation index (PI) declined,which was statistically significant compared with the control group(P <0.05). When we added the two kinds of drugs for different time as IC50,we found that the number of G0/G1 phase cells and PI in Valdecoxib72h group increased significantly,the number of S phase and G2 / M phase cell decreased too.The same to Aspirin 48h and 72h group. Valdecoxib 's effect was stronger than Aspirin, and the two drug group were significantly different compared with the control group(P <0.05). It was seen that lovo cells could be arrested in the G0/G1 phase by Aspirin and Valdecoxib (Table.2,3).4 The impact on protein expression of COX-2, hMLH1, and hMSH2 after using Aspirin and Valdecoxib by ImmunocytochemicalCOX-2 protein was located in cytoplasm and nuclear membrane,hMLH1 mainly nuclear and hMSH2 is negative under the light microscope . (Fig. 5,6) With the extensing of the time of Aspirin and Valdecoxib, the expression of COX-2 protein reduced gradually,and the role of Valdecoxib was stronger than Aspirin. The expression of hMLH1 protein was increased significantly in 72h group.The effect of Aspirin was stronger than Valdecoxib.The expression of hMSH2 protein was negative.5 The results of Western blotThe proteins molecular weights of COX-2, hMLH1, hMSH2, Bcl-2, caspase-3 were 72kD, 85kD, 110kD, 26kD, 32kD,and the corresponding positive bands will be found position after Western blot . The hybrid stripe was analysed by Gelimaging analysis system,the protein content was shown by IOD,andβ-actin protein was chose as an internal,compared the relative integral optical density in order to show the level of protein expression. The inhibitory effect of Valdecoxib on COX-2 expression increased gradually with time extending,especially in the 48h and 72h (p<0.05). However,the expression of COX-2 protein was inhibited significantly only after using Aspirin for 72h(p<0.05).The effect on COX-2 protein inhibition of Valdecoxib was stronger than Aspirin.There was no effect on expression of hMSH2 protein in lovo cells after using two drugs. The expression of hMLH1 protein increased significantly when Valdecoxib acted on lovo for 72h and Aspirin in the role of 48h(p <0.05).The role of Aspirin was stronger than Valdecoxib. The expression of Bcl-2 protein had a gradual reduction as the extension of time after using Valdecoxib and Aspirin.Caspase-3 protein expression increased with the time extending, effect in 48h and 72h group was obvious (P <0.05).The effect on bcl-2 protein of Valdecoxib is stronger than Aspirin,but the inpact on expression of caspase-3 protein had no significant difference(p>0.05).Conclusions:1 NSAIDs could inhibit the growth of lovo human colon cancer cell,the inhibition role which was dependented with time and dose was related to inducting apoptosis and extending cell cycle.2 NSAIDs could increase the expression of hMLH1 protein, but there was no change on the expression of hMSH2 protein in lovo which was lack of hMSH2.3 NSAIDs could increase apoptosis through inhibiting the COX-2 protein, reducing the expression of anti-apoptotic protein Bcl-2 ,and increasing the expression of caspase-3 primarily. It can also be through affecting MMR to the maintenan DNA repair and regulatory functions ,to promote apoptosis of tumor cells.4 Comparing non-selective COX inhibitor(Aspirin) to selective COX inhibitor (Valdecoxib), the effect of Valdecoxib on inhibiting growth,inducing apoptosis,blocking cycle and inhibiting the expression of COX-2 was stronger than Aspirin. But the effect of Aspirin on increasing the expression of hMLH1 protein was stronger than Valdecoxib. NSAIDs could affect the occurrence and development in colorectal cancer by influencing MMR.