The Mechanism Study Of Antioxidants Decreasing5-Fu-induced Human Colon Cancer Cell Apoptosis By Regulating Src Activity

Colon cancer is one of the most common human malignancies today. According to statistics data, the incidence of colon cancer is in the fifth place of all kinds of cancers and the mortality rate is in the third place. It is a serious threat to human health and life. Currently the surgery combined with5-fluorouracil (5-Fu) based chemotherapy is the main treatment for colon cancer. For the advanced colon cancer, surgery alone can’t remove all the cancer cells in the body. Systemic chemotherapy is helpful for removing the residual tumor cells, preventing from tumor recurrence and metastasis and prolonging the lifetime of patients.5-Fu is the key chemotherapeutic drug in the colon cancer chemotherapy way, however the clinical data showed that patients with colon cancer frequently got resistance to5-Fu. How to reduce the incidence of5-Fu resistance has important significance to colon cancer therapy.Antioxidants are widely used as dietary supplements and clinical drugs such as vitamin C, vitamin E and glutamine all has antioxidative effects. Antioxidants could remove the reactive oxygen species (ROS) in the human body, protect the human normal cells from ROS-induced DNA damage and maintain the normal function of cells. Some researches show that endogenous or exogenous stimuli can induce reactive oxygen species in vivo and continuing DNA damage could cause function gene deletion or mutation which eventually lead to cancer. Therefore it is the common sense that antioxidants could prevent cancer formation and development. However a lot of chemotherapeutic drugs kill cancer cells by increasing ROS level in the human body and inducing cellular apoptosis. Colon cancer patients use many kinds of antioxidants such as vitamin C in the trea2tment process. However, it is still uncertain that whether antioxidants combined with5-Fu could induce the drug resistance.Src is known to be overexpressed in more than80%of colon cancers and is closely associated with cancer cell proliferation, survival, angiogenesis, adhesion and metastasis. However, the relationship between Src and apoptosis is controversial. Src has been reported to inhibit apoptosis by activating the phosphatidylinositol3-kinase/Akt pathway, which protects cells against pro-apoptotic stimuli through the phosphorylation and inactivation of many downstream death accelerators. Src could downregulate caspase-8activity either directly or indirectly. In addition, recently some publications show that Src could increase drug-induced cellular apoptosis. For example, inhibition of Src could suppress the aspirin-induced NF-κB activiton and the cell apoptosis is inhibited in the colon cancer. In the breast cancer cells, inhibition of Src expression and activation could induce phosphorylation missing of eIF2a and AMPK, block the estrogen-induced ROS production and cell apoptosis. However, it is still unknown that whether Src has effect on5-Fu induced cellular apoptosis in colon cancer cells.In this study, we show that5-Fu can induce reactive oxygen species-dependent Src activation in colon cancer cells. Mouse embryonic fibroblast cells that are deficient in Src showed a clear resistance to5-Fu, and knocking down Src protein expression in colon cancer cells also decreased5-Fu-induced apoptosis. We found that Src could interact with and phosphorylate caspase-7at multiple tyrosine sites. Functionally, the tyrosine phosphorylation of caspase-7increases its activity, thereby enhancing cellular apoptosis. When using5-Fu and antioxidants together, Src activation was blocked, resulting in decreased5-Fu-induced apoptosis. Our results systemically show the molecular mechanism as to why antioxidants decreased colon cancer cell apoptosis induced by5-Fu, which is important for clinical chemotherapy because too much antioxidants were used in clinical process. Our findings could provide guidance to enhance the effects of chemotherapy by decreasing5-Fu resistance in colon cancer patients.Chapter I Antioxidants decrease5-Fu-induced human colon cancer cell apoptosisMethods1. To detect the human colon cancer cell apoptosis induced5-Fu-induced when combining with antioxidants by flow cytometry.2. Western blotting to detect the expression and cleavage of PARP, caspase-3, caspase-7in colon cancer cell which induced by5-Fu and antioxidants.3. To detect the5-Fu-induced HT29colon cell apoptosis which combined with vitamin C or glutamine treatment by flow cytometry.Results1. N-acetylcysteine (NAC) decreased5-Fu-induced apoptosis in HT29colon cancer cells, and a hydrogen peroxide scavenging enzyme, catalase, decreased5-Fu-induced apoptosis in SW480colon cancer cells.Flow cytometry results indicated that if HT29colon cancer cells were pretreated with NAC10mM for2h and then combined with5-Fu50μM for an additional48h, the early period apoptosis rate was5.8%. It sigficiantly decreased compared with5-Fu treatment alone for48h which early apoptosis rate is11.6%(P<0.001). When SW480colon cancer cells were pretreated with catalase200mU for2h and then combined with5-Fu50μM for an additional48h, the early apoptosis rate was5.5%. It sigficiantly decreased compared with5-Fu treatment alone for48h which early period apoptosis rate is12.3%(P<0.001).2. Combining NAC or catalase with5-Fu dramatically decreased HT29and SW480colon cancer cell apoptotic signal.Western blotting results showed that combining NAC with5-Fu dramatically decreased cleavage of PARP, caspase-3and caspase-7in HT29cells compared with cleavage induced by5-Fu alone. Similar effects were observed in SW480cells treated with both catalase and5-Fu. These results indicated that antioxidants might decrease the apoptotic effects of5-Fu in colon cancer cells.3. Combining L-glutathione (GSH) or vitamin C (VC) with5-Fu dramatically decreased HT29colon cancer cell apoptotic effect.HT29cells were pretreated with VC50μg/ml or GSH40μM for2hours and then combined with5-Fu50μM for an additional48hours. The early apoptosis rate was6.2%and4.9%respectively. It sigficiantly decreased compared with5-Fu treatment alone for48hours which early period apoptosis rate is11.2%(P<0.001).Chapter ⅡROS production and Src activation in colon cancer cells is regulated by antioxidants and5-FuMethods1. Western blotting to detect H2O2effect on Src activation in colon cancer cells.2. Western blotting to detect5-Fu effect on ROS production and Src activation in colon cancer cells.3. Western blotting to detect the combination effect of antioxidants and5-Fu on ROS production and Src activation in colon cancer cells.Results1. ROS production mediated Src activation in colon cancer cells.Western blotting results showed after treated with various concentration of H2O2in HT29and SW480cells, Prx-SO3expression dramatically increased, which represented ROS production in cells. P-Src (tyr416) also increased dose dependently after H2O2treatment, which was a direct marker for Src activation.2.5-Fu promoted ROS production and Src activation in colon cancer cells.Western blotting results showed when HT29and SW480cells were treated with5-Fu50μM, Prx-SO3expression went up from1h, arrived peak at6h, and maintained in high level at24h. P-Src(tyr416) expression increased time dependently.3. Antioxidants inhibited5-Fu induced ROS production and Src activation in colon cancer cells. Western blotting results showed combination treatment24h or48h of NAC/catalase and5-Fu decreased Prx-SO3and p-Src (tyr416) expression in colon cancer cells.ChapterⅢ Down regulation of Src expression inhibits5-Fu-induced cell apoptosisMethods1. Down regulate Src expression by infecting HT29and SW480with sh-Src lentivirus particles. Western blotting to identify Src expression in colon cancer cells after infection and in Src knockout mouse embryonic fibroblasts (MEF).2. Treat Src low expression cells and their control cells with5-Fu, MTS assay to detect cytotoxicity of5-Fu. Flow cytometry to detect early apoptosis in cells. Western blotting to identify expression and cleavage of PARP, caspase-3, caspase-7in cells. Caspase-3/7Colorimetric Activity Assay Kit detected caspase-3/7activity in cells.Results1. Lentivirus particles containing sh-Src#1and Sh-Src#2sigficantly down regulate Src expression in HT29and SW480cells. No Src expression is observed in Src knockout MEFs.Western blotting results showed HT29and SW480cells infected by lentivirus particles containing sh-Src#1and Sh-Src#2exhibit lower Src expression compared to control cells. Src expression was totally no deleted in Src knockout MEFs.2. Src low level cells exhibit decreased sensitivity to5-Fu.Compared to each control cells, in Src knockout MEFs and Src knockdown colon cancer cells5-Fu showed lower cytotoxicity and less cellular apoptosis. Cleavage of PARP, caspase-3, caspase-7in Src low level cells also decreased, accompanied with decreased caspase-3/7activity.Chapter Ⅳ Src phosphorylates caspase-7in vitro and interacts with caspase-7in cells Methods1. Wildtype (Wt) caspase-3, caspase-7and caspase-9protein purification in Escherichia coli BL21and Escherichia coli Rosetta.2. In vitro kinase assay to detect if Src can phosphorylate caspase-3and caspase-7.3. Endogenous immunoprecipitation to detect binding between Src ans caspase-3or caspase-7in cells.4. Immunofluorescence to detect co-localization of Src and caspase-7.5. Flow cytometry and Western blotting to detect the influence of caspase-7deletion on MEFs cellular apoptosis induced by5-Fu.Results1. Optimize the purification condition for caspase-3, caspase-7and caspase-9.The best purification condition for caspase-3:choose E.coli BL21,0.5mM IPTG induction at25℃for4h.The best purification condition for caspase-7:choose E.coli Rosetta,0.5mM IPTG induction at25℃for4h. The best purification condition for caspase-9:choose E.coli BL21,0.5mM IPTG induction at30℃for1h.2. Src could phosphorylate caspase-7in vitro, but not caspase-3In vitro kinase results showed after reaction of Src active kinase and caspase-3or caspase-7protein, there was obvious phosphorylation signal by32P radioactive autography in Src and caspase-7group, but not in Src and caspase-3group, which indicated active Src could phosphorylate caspase-7but not caspase-3. Western blotting showed that p-tyrosine expression signal was seen in caspase-7site, which indicated Src could phosphorylate caspase-7at tyrosine sites.3. Src could bind with caspase-7in HT29and SW480cells.Immunoprecipitate caspase-3or caspase-7in whole cell lyasates of HT29and SW480cells, Src expression was only detected in caspase-7group by Western blotting.4. Src could co-localize casapse-7in SW480cells.Immunofluorescence results showed Src and caspase-7co-localized in nucleus and cytoplasm under normal condition. In5-Fu48h treatment group, Src and caspase-7co-localized only in nucleus.5. Caspase-7knockout MEFs are resistant to5-Fu-induced cell apoptosis. Flow cytometry results showed the early apoptosis rate dramatically decreased in caspase-7knockout MEFs (7.6%) induced by5-Fu48h treatment, compared to Wt MEFs (48.9%). Western blotting results showed cleavage of PARP and caspase-3significantly decreased in caspase-7knockout MEFs.Chapter V Src phosphorylates caspase-7at multiple tyrosine sites and enhances its activityMethods1. Predict possible tyrosine phosphorylation sites of caspase-7by Netphos2.0programme.2. Synthetize peptides containing possible tyrosine phosphorylation sites, identify the real phosphorylation sites by in vitro kinase assay.3. Point mutation of caspase-7and expression of Mutant caspase-7protein. Further identification of phosphorylation sites by in vitro kinase assay.4. Caspase-3/7Colorimetric Activity Assay Kit to detect the influence of Src phosphorylation on caspase-7activity.5. Transfect293T cells with Wt or Mutant pCMV-Myc-caspase-7, detect cellular caspase-7activity. Co-transfect293T cells with pcDNA4-his-Src and Wt or Mutant pCMV-Myc-caspase-7, detect cellular caspase-7activity.Results1. There are nine possible tyrosine phosphorylation sites of caspase-7.The prediction results from Netphos2.0programme showed there are nine tyrosine sites of caspase-7that may be phosphorylated by Src.2. Src phosphorylates caspase-7at tyrosine51,151,229and230.Synthetize peptides containing possible tyrosine phosphorylation sites according to the prediction results. Detect the reaction of Src with each peptideby in vitro kinase assay. Autography results showed peptides containing tyrosine51,151,229and230can be phosphorylated by Src. 3. Phosphorylation signal dramatically decreasd in Src and Mutant caspase-7group compared to Src and Wt caspase-7group.Mutate all the four tyrosine residues to phenylalanine and purify Mutant caspase-7protein. In vitro kinase assay results showed the Mutant caspase-7phosphorylation signal by Src dramatically decreasd compared to Wt caspase-7.4. Src enhances Wt caspase-7activity in vitro, but not Mutant caspase-7activity.Caspase-7activity assay results showed Wt caspase-7can be activated by caspase-9. If Wt caspase-7was phosphorylated by Src first, then activated by caspase-9, the Wt caspase-7protein showed a dramatically higher activity. While Mutant caspase-7activity was not influenced by Src.5. Src enhances caspase-7activity in293T cells.24h after transfected with Wt or Mutant pCMV-Myc-caspase-7in293T cells, the Wt group cells showed dramatic increase of caspase-7activity compared to cells in Mock transfection group. However, Mut group cells did not show any change of caspase-7activity. If co-transfected with pcDNA4-his-Src and Wt or Mutant pCMV-Myc-caspase-7in293T cells, caspase-7activity increased to1.5fold in Src and Wt group compared to only Wt group. However, caspase-7activity did not change in Src and Mut group compared to only Mut group. Conclusions1.5-Fu up regulates Src activity in colon cancer cells by inducing ROS production. Antioxidants down regulate Src activity by inhibiting ROS production. Down regulated Src expression and activity could inhibite5-Fu-induced cellular apoptosis. Because of the inhibition of Src,5-Fu-induced colon cancer cell apoptosis decreased which combine with antioxidants treatment.2. Src could bind with caspase-7in cells. Src phosphorylates caspase-7at multiple tyrosine residues and enhances caspase-7activity. Caspase-7plays an important role in5-Fu-induced cellular apoptosis, which can be not substituted by caspase-3.