| BackgroundThe main methods of treating ovarian cancer is to induce the apoptosis of cancer cells and to enhance the sensitivity of cancer cells to the chemotherapy medication. The main mechanism of chemotherapy drug treating ovarian cancer is to induce the apoptosis of cancer cells. Apoptosis is a kind of programmed cell death, including two kinds of pathway:the extrinsic pathway and the intrinsic pathway. In extrinsic pathway, named death receptor pathway, death ligands(TRAIL) bind their respective membrane receptors, then active death receptors signaling with DISC (death inducing signaling complex) and initiator caspase8/3, in the end trigger the apoptosis. In intrinsic pathway named mitochondrial apoptosis pathway, cytochrome c releasing from mitochondria to cytosol composes a apoptosome with caspase9and Apaf1, and induce cell apoptosis consequently. Caspase3activity is the common end response of both the two kinds of apoptosis pathway.Smac is a kind of mitochondrial protein,which can be released with cytochrome C. Residue AVPI of Smac bind with domain BIR of IAPs by which IAPs bind with caspase to suppress apoptosis. IAPs is the main antiapotosistic protein which determine the tolerance and sensitivity of carcinoma to chemotheraoeutiscs. As IAPs is the new target and its suppressing protein Smac become a new method of curing carcinoma. As a result smac simularums come out which contain three kinds: polynucleotide, polypeptide and mimetic. Mimetic has the advantages of high penetrability of cell membrane, long time of metabolism and so on.In2004the United States of Texas Southwestern Medical Center synthesize a kind of smac mimetics named compound3. Cisplatin is the first chemicotherapic medication of ovarian carcinoma. Cisplatin is the main chemotherapic medication which can work with DNA into a complex disturbing the synthesis of DNA, and that is the main mechanism of anti-carcinoma. Cisplatin also can work with nuclear protein and cytoplasm protein, do great damage to the structure of cell membrane, and destroy the physiology function of the cell. Both of all the terminal pathway is the induction of apoptosis.ObjectiveDetect the capacity of a small Smac mimic molecule(compound3) to induce the growth inhibition, cell apoptosis and the protein expression of ovarian cancer cell lines when added alone or in combination with cisplatin. Discuss that Smac mimic molecule compound3can enhance the sensitivity of ovarian cancer cell lines to cisplatin.Materials and MethodsMaterials:Human ovarian cancer cell line SKOV3is purchased from cell bank of Chinese Academy of Sciences in Shanghai. Cisplatin is purchased from Shandong Qilu pharmaceuticals company. Smac mimetic compound3is a kind of small molecule compound that is synthesized by the United States of Texas Southwestern Medical Center according to the domain of AVPI by computer.Methods:SKOV3is cultured in MACOY5A culture medium containing12%FBS. When the cells are in logarithmic phase, they are divided into groups:negative group(culture medium), compound3group (the concentration of compound is100nmol/L, which is recommended by the United States of Texas Southwestern Medical Center.), cisplatin group(the concentration of cisplatin is4mg/L,which is the lethal concentration50of cisplatin to human ovarian cancer cell line SKOV3detected by MTT before), compound+cisplatin group(the concentration of compound3is100nmol/L and the concentration of the cisplatin is4mg/L.). Measure the cell viability and the growth inhibition ratio of ovarian cancer cell line SKOV3of all the groups by MTT. Apoptosis of human ovarian cancer cell line SKOV3of all the groups was detected by Annexin V-FITC/PI staining followed by flow cytometry. Proteins expression of XIAP and caspase3(actived and unactived fragments) was determined by immunoprecipitation followed by the western blot analysis.Results1.The effect of compound3to SKOV3on the viability and the growth cure:The viability of SKOV3in cisplatin group in12,24,48h is (96.38±2.0)%,(88.50±1.38)%,(45.78±0.66)%. The livability of SKOV3in compound3group in12,24,48h is (98.67±0.88)%,(89.88±0.39)%,(64.18±0.92)%.That in compound3combining with cisplatin group in12,24,48h is (92.2±0.99)%,(53.59±1.17)%,(25.56±0.6)%.Cellular viability was inhibited when SKOV3was added compound3alone and in combination with cisplatin, and the inhibition depends on time. The difference has statistical significance.2. The growth inhibition of compound3to SKOV3:The growth inhibition ratio of SKOV3in24h treated with compound3combining with cisplatin is (46.4±1.17)%,that of cisplatin group is (11.5±1.38)%. The growth inhibition ratio of SKOV3in48h treated with compound3combining with cisplatin is (74.03±0.6)%, that of cisplatin group is (54.22±0.66)%. The growth inhibition ratio of SKOV3when treated with compound3combining with cisplatin exceed that of treated with cisplatin alone, and the difference has statistical significance. Compound3can enhance the growth inhibition of cisplatin on SKOV3.3. The effect of compound3to SKOV3on apoptosis:Apoptosic rate of SKOV3added compound3combining with cisplatin in24h was (28.32±1.09)%, that advanced to that of added cisplatin which is (20.8±2.09)%and compound3which is (19.44±1.89)%. That of SKOV3added compound3combining with cisplatin in48h was (84.48±0.98)%, that also advanced to that of added cisplatin which is (54.4±1.32)%and compound3which is (23.59±2.04)%. The difference of apoptosic rate between the compound3combining with cisplatin group and the cisplatin group. Compound3can promote the proapoptosis of cisplatin to SKOV3.4. The effect of compound3to SKOV3on the expression of XIAP and caspase3: Expression of XIAP of compound3combined with cisplatin was depressed depending on time. Expression of XIAP of SKOV3was depressed in compound3 group and compound3combined with cisplatin group. After48hours of combining compound3and cisplatin in SKOV3, actived fragment of caspase3was detected. Compound3can depress the expression of XIAP. It also can depress the expression of actived fragment of caspase3when combine with cisplatin.Conclusion1.Treatment with compound3enhances growth inhibition of SKOV3induced by cisplatin depending on time.2.Treatment with compound3enhances the apoptosis of SKOV3induced by cisplatin.3.The process of proapoptosis of compound3was in connection with expression of XIAP. |
PDF Full Text Request