| Part 1Inhibition and mechanism of human p66Shc recombinant adenovirus on HeLa cells and MCF-7 cellsObjective:p66Shc is a gene that regulates the level of reactive oxygen species (ROS), apoptosis induction, and lifespan in mammals.In recent years, its effect on tumor cells also attracted the attention of researchers. However, the effect is to promote or inhibit tumor cells still remains controversial. In order to observe the biological effect of p66Shc, we constructed human p66Shc recombinant adenovirus expression vector (AdenoX-p66Shc). Human cervical cancer HeLa cells and breast cancer MCF-7 cells were used to observe p66Shc’s effect on proliferation and the mechanism after treated the cells with AdenoX-p66Shc.Methods:Cell viability was examined in HeLa cells and MCF-7 cells by MTT method. Human umbilical vein endothelial cells (HUVECs) were used as a control group of normal cells. Intracellular reactive oxygen species (ROS) level was measured by DCFH-DA fluorescent probes. The levels of 8-OHdG were detected by ELISA assay. Hoechst 33342 staining was used to observed the nuclear morphology, the proportion of apoptotic cells and the cell cycle of each group were assayed by flow cytometry. Western blot assay was used to detect associated proteins.Results:1. Ad-p66Shc could significantly suppress the proliferation of HeLa cells and MCF-7 cells compared with the blank control, but the proliferation of HUVECs was not inhibited by Ad-p66Shc.2. The intracellular ROS of HeLa cells and MCF-7 cells was significantly increased, as well as the level of oxidative DNA damage product 8-OHdG.3. The nuclear morphology and chromatin of HeLa cells in each group showed normal appearance by Hoechst 33342 staining. The apoptotic cell rate showed no significant difference between the groups.4. The ratio of the cell phase G2/M of Hela cells and MCF-7 cells showed a significant increase, and the ratio of the cell phase G1 showed a significant decrease on contrast.5. The expression of p66Shc strongly increased in HeLa cells and MCF-7 cells infected by Adeno X-p66Shc, as well as the expression of p53 protein and p-p53.The expression of cell cycle-related proteins CyclinBl and CDK1 also showed significant increase. However, the expression of proteins regarding apoptosis including Bax and Bcl-2 and Caspase-3 were not changed.Conclusion:Ad-p66Shc has a inhibitory effect on the proliferation of HeLa cells and MCF-7 cells, but can not inhibit the proliferation of normal cells. Infected with Ad-p66Shc makes a significantly higher expression of p66Shc, induces ROS generation and DNA oxidative damage, and upregulates the expressions of p53. Ad-p66Shc induces cell cycle arrest at G2/M phase, thereby inhibits the proliferation of cancer cells. infected by Adeno X-p66Shc, as well as the expression of p53 protein and p-p53.The expression of cell cycle-related proteins CyclinBl and CDK1 also showed significant increase. However, the expression of proteins regarding apoptosis including Bax and Bcl-2 and Caspase-3 were not changed.Conclusion:Ad-p66Shc has a inhibitory effect on the proliferation of HeLa cells and MCF-7 cells, but can not inhibit the proliferation of normal cells. Infected with Ad-p66Shc makes a significantly higher expression of p66Shc, induces ROS generation and DNA oxidative damage, and upregulates the expressions of p53. Ad-p66Shc induces cell cycle arrest at G2/M phase, thereby inhibits the proliferation of cancer cells.Part 2Study on the antitumor activity of Gambogic acid lysinate in vitro and the mechanismObjective:Previous studies have shown that gambogic acid (GA),a major active component of gamboge, possesses potent antitumor effect. GA is soluble in organic solvents such as alcohols and ketones, but is insoluble in water. Because of its water insolubility, the antitumor efficacy of gambogic acid is limited. In the present study, we prepared gambogic acid lysinate (GAL), and investigated the antitumor activity of GAL.Methods:Human breast cancer MCF-7 cells were used to observe the effect of GAL on the cells’proliferation.GAL was determined by Mass Spectrometry. MTT assay was used to detect the effect of GAL on MCF-7 cells.Intracellular reactive oxygen species (ROS) level was measured by DCFH-DA fluorescent probes. Hoechst 33342 staining was used to observed the nuclear morphology, and the proportion of apoptotic MCF-7 cells was measured using an Annexin V/PI-FITC apoptosis detection kit. Western blot was used to observe associated protein expression.Results:1. The molecular weight of Gambogic acid is 629, and lysine is 146. Gambogic acid and lysine combine together through ionic bond. The Theoretical molecular weigh of gambogic acid lysinate is 775. It can deduce that the peak value (755.4Da) is gambogic acid lysinate.2. MCF-7 cells showed a decreased cell proliferation in the presence of various concentrations of GAL, and the inhibition of GAL to MCF-7 cell proliferation was comparable to that of GA. In addition, the proliferation inhibitory effect of GAL in MCF-7 cells was blocked, when NAC was added in combination with GAL.3. GAL could increase ROS level in dose dependent manner, however NAC could decrease the level of ROS induced by GAL.4. By Hoechst 33258 staining the nuclei of untreated cells were normal in appearance and showed diffused staining of the Chromatin. After exposure to GAL for 24 h, most cells presented typical morphological changes of apoptosis such as chromatin condensation, cell shrinkage, or apoptotic bodies. Induction of apoptosis by GAL was further confirmed by annexin V-FITC/PI staining.5. GAL inhibited the expression of SIRT1 but increased the phosphorylation of FOXO-3a and the expression of p27Kip1. In addition it enhanced the cleavage of Caspase-3.Conclusion:The inhibition of GAL to MCF-7 cell proliferation was comparable to that of GA. GAL inhibited MCF-7 cells proliferation and induced MCF-7 cells apoptosis by increasing ROS level which could induce cell apoptosis by both SIRT1/FOXO3a/ p27Kip1 and Caspase-3 signal pathway.
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