| BackgroundEpithelial ovarian cancer is the most fatal tumor in gynecological malignant tumor, mortality ranks first in gynecologic oncology. Although surgical treatment has a good effect on early-stage ovarian cancer, yet the vast majority of patients’ condition will advance in late stage.It is difficult to treat advanced ovarian cancer with existing chemotherapy drug, because advanced ovarian cancer is susceptible to have drug resistance and systemic toxic and side reactions to chemotherapy drugs, however, the advanced ovarian cancer has lost its conditions and chance for operation, therefore, chemotherapy still remains the most important treatment method for advanced ovarian cancer, that is to say, chemotherapy and surgery occupy the equal important position in the treatment of ovarian cancer.Thus it can be seen, It is very necessary to develop new type of antitumor drugs with high efficiency and low toxicity, the research focus of the new way to treat ovarian cancer is to seek new antitumor drugs.The research of new types of anticancer drugs are as follows:researchers favor target cell signaling molecules, such as protein tyrosine kinase inhibitors, proteasome inhibitors, histone deacetylase inhibitors, angiogenesis inhibitors antimetastatic drugs, telomerase inhibitors and so on. In recent years, researchers incline toward computer-aided design simulation anticancer drugs which target tumor-specific targets, such as chemical synthesis of small molecule anticancer drugs, a research focus of the new anticancer drugs. The compound used in this experiment is the analog compound designed by the structure of the manganese superoxide dismutase (MnSOD).The body’s normal metabolism will generate free radicals, but will produce a large number of free radicals in special circumstances, which through oxidation damage the body of the cell and tissue, including inactivating the enzyme, damaging cell membranes and leading to mutations and cancer. So the free radicals is the basis of the development of the pathology of many diseases. Intracellular superoxide dismutase (SOD) is an important enzymatic radical scavenger, which can be catalyzed O-2disproportionation of H2O2and O2. Superoxide dismutase in the human body is the most important manganese superoxide dismutase (MnSOD). Balance system between MnSOD gene expression and regulation of the body’s free radicals-radical scavenger is essential, and it is one of the most important enzymes in the antioxidant system.Currently, a large number of studies have confirmed that the MnSOD and growth, differentiation, invasion and resistance of tumor cell and other biological behaviors are closely related. MnSOD Generally speaking, MnSOD activity decreased in most tumor cells, but its activity increased in ovarian cancer. In ovarian cancer, high expression of MnSOD has a protective effect on tumor cells, and may contribute to tumor differentiation progress. However, when production of amount of protective MnSOD of oxidation in ovarian cancer restoration balance is broken, MnSOD expression further increased, causing the excess of H2O2which will inhibit the proliferation of tumor cells, and can lead to apoptosis of tumor cells. It is easily inferred that if MnSOD is more than the protective expression level of ovarian cancer, MnSOD but may inhibit the development of ovarian cancer.In recent years, the use of the MnSOD chemical stimulants to replace natural MnSOD becomes a research hotspot. Due to the large molecular weight of the natural MnSOD, short vivo half-life, easy access to the cell membrane and poor stability reasons, its medicinal value is greatly limited. Biochemistry sector researchers, with the help of synthesis and characterization of chemical, synthesize small molecules, of copper, manganese, iron and other metal ions, which have related structures complex to simulate the SOD in order to overcome the disadvantages of natural SOD, so that the analog compounds of small molecule could be applied clinically. Lanzhou University uses flexible aliphatic amines and preferably water-soluble biocompatible to synthesize water-soluble and fat-soluble preferably MnSOD complexes of o-vanillin, which has a higher activity that has been confirmed in study. The compound has a water grease and chief dissolved, high activity and good stability, with small molecular weight, easy transmembrane, low-cost, high purity, high yield and other characteristics, which can be seen to overcome the limitations of natural MnSOD, in order to apply the possibility of the clinical potential.MnSODm Inhibits the tumor, under the help of non-cytotoxic effect, and has no toxic side effects of chemotherapy drugs with high efficiency and low toxicity characteristics, making itself potential clinical application of anticancer drugs. Any drug used in clinical premise should be sure that its mechanism of action must be clear. The research about anti-tumor effect of MnSODm at home and abroad is rare, and research of its anti-tumor mechanism is still unmanned. The foreign reference figures that manganese superoxide dismutase mimetic compounds (MnSODm) combined with gentamicin treatment, can reduce ototoxicity, and tumor treatment of MnSODm, only could be found at home in An Xia、n Fan Pro Lan which study inhibition of MnSODm on the proliferation of leukemia K562cells and induction of cell apoptosis.The subject chooses MnSODm, a new research focus and research gaps of anticancer drugs. Considering human epithelial ovarian carcinoma cell line SKOV3and its xenograft tumor in transplanted tumor tissue as the research object, the experiment uses HE staining, electron microscopy drugs for the observation of pathological changes of the tumor tissue after treatment, with the help of the calculation of tumor weight inhibitory rate and measurement of tumor size, as well as the immunohistochemical assay tumor tissue cancer gene. The experiment observes expression changes of cell proliferation and activity of CCK-8assay, PI single stain, inverted microscope apoptosis, flow cytometry apoptosis cycle, PCR, NF-KB, BAX and BCL-2gene, the combination of in vitro and in vivo experiments, the experiment studies growth inhibition of MnSODm on SKOV3cell lines and xenograft tumor in vitro and in vivo, andexplores the molecular mechanism to provide new ideas and new methods for the treatment of ovarian cancer.Chapter1Inhibition on proliferation OF MnSODm on human epithelial ovarian cancer SKOV3cell and induction of apoptosisObjective:The observation of growth inhibition.of MnSODm on human epithelial ovarian cancer SKOV3cellMethod:1. By means of CCK-8, take the logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml, lOug/ml,50ug/ml) to process cell proliferation and activity changes in the control group whose cisplatin concentration is10ug/ml in Oh,24h,48h,72h,.before and after the treatment of MnSODm,2.Take the logarithmic phase of SKOV3cells with different concentrations MnSODm (Oug/ml,10ug/ml,50ug/ml) to process for72h, and observe the morphological changes inverted the microscope MnSODm treated cells.3. Take the logarithmic phase of SKOV3cells, for72h with different concentrations MnSODm (Oug/ml,10ug/ml,50ug/ml) to observe the morphological changes of cell apoptosis PI single staining before and after the process of MnSODm.4.Take logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml,10ug/ml,50ug/ml) to process for72h, and use transmission electron microscope to observe the ultrastructural changes SKOV3cells before and after MnSODm treatment.5.Take SKOV3cells in logarithmic growth phase, with of different concentrations MnSODm (0ug/ml,10ug/ml,50ug/ml) to process for72h, flow cytometry the apoptosis rate changes before and after MnSODm treatment.6.Statistical analysis:use SPSS17.0statistical analysis software to analyze measurement data which were expressed as mean±standard deviation, and use single-factor analysis of variance (One-way ANOVA) to compare the groups with the help of multiple comparison LSD method. P<0.05was considered statistically significant.Results:1.Compared with the control group, after MnSODm treatment, SKOV3cell proliferation activity was significantly inhibited in a time-dependent and concentration-dependent. Compared with cisplatin the MnSODm concentration10ug/ml for24,48,72h, the inhibition of the cells were less than cisplatin, and the MnSODm concentration50ug/ml24h cell inhibition is less than smooth platinum, but at48and72hours, cell inhibition is stronger than cisplatin.2.After Treatment of MnSODm, SKOV3cell survival cells were significantly reduced under inverted microscope.3.After Treatment of MnSODm, PI staining showed that the SKOV3apoptotic cells increased.4.After Treatment of MnSODm, transmission electron microscopy under SKOV3cells appear obvious ultrastructural changes of apoptotic cells.5.After Treatment of MnSODm, flow cytometry displays that the SKOV3cell apoptosis was significantly higher.Conclusion:MnSODm can inhibit the proliferation of SKOV3cell in human epithelial ovarian cance.Its effect on inhibition of tumor cell proliferation is dependent on the time and concentration and also MnSODm can induce apoptosis of SKOV3cells. Chapter2Study on the mechanism of inhibition of MnSODm on proliferation of epithelial ovarian cancer SKOV3cell and induction of apoptosisObjective:Explore the mechanism of inhibition of MnSODm on proliferation of epithelial ovarian cancer SKOV3cell and induction of apoptosis.Method:l.Take SKOV3cells in logarithmic growth phase, with of different concentrations MnSODm (0ug/ml,10ug/ml,50ug/ml) to process for72h, using flow cytometry to detect changes in the SKOV3cell cycle distribution before and after MnSODm treatment.2.Take the logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml, lOug/ml,50ug/ml) to process for72h, using fluorescent chemiluminescence to detect SKOV3cells of apoptosis-related factor Caspase-3/7activity before and after MnSODm treatment.3.Take the logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml,10ug/ml,50ug/ml) to process for72h, using RT-PCR assay to detect the expression changes of nuclear factor-KB/p65、apoptosis-related factors bcl-2,and bax mRNA in SKOV3cells in cells before and after MnSODm treatment.4.Take the logarithmic growth phase SKOV3cells with different concentrations of MnSODm (Oug/ml,10ug/ml,50ug/ml) to process for72h, using Western blotting MnSODm to detect the change of bax protein expression.of SKOV3cells of nuclear factor-κB/p65, cells wither apoptosis associated factor bcl-2,.5.Statistical analysis:use SPSS17.0statistical software to analyze data expressed as mean±standard deviation. Measurement data are compared, using independent samples t test, and P<0.05was considered statistically significant.Results: 1. Compared with the control group, after MnSODm treatment, the SKOV3cell cycle distribution changed significantly, the performance increase in cells in G0/G1phase, and S phase cells decreased cell cycle arrest in G0/G1phase.2.After MnSODm treatment, SKOV3cell apoptosis-related factors Caspase-3/7activity did not change significantly.3After MnSODm treatment, SKOV3cells nuclear factor-κB/p56, apoptosis-related factor bcl-2mRNA expression was significantly lowered, and bax mRNA expression was significantly increased.4.After MnSODm treatment, SKOV3cells nuclear factor-κB/p56, apoptosis-related factor bcl-2protein expression was significantly reduced, while bax protein expression was significantly increased.Conclusion:MnSODm, through affecting cell cycle distribution, and more importantly, through nuclear factor-κB/p56way, regulates apoptosis-related factors bcl-2and bax in order to achieve inhibition on proliferation of human ovarian cancer SKOV3cell and induction of apoptosis. Its inhibition on proliferation of ovarian cancer cell has nothing to do with factor of Caspase-3/7.Chapter3MnSODm’s inhibition on growth of transplanted allogeneic Tumor cells in SKOV3nude ratObjective:Set up the model of transplanted tumor cells in SKOV3nude rat, to observe inhibition of positive drugs, cisplatin and different doses of MnSODm on growth of transplanted Tumor cells.Method:1. Cell cultivation:SKOV3cells were cultivated in incubation box containing10%fetal calf serum, the RPMI1640culture radical penicillin and streptomycin, at 37℃,5%CO2gas mixture as well as95%humidification. Medium was changed every2-3days.2. Plantation of tumor:Take cells with the logarithmic growth phase, trypsinize dubbed2.5X106cells/ml, and take the cell suspension0.2ml per mice from tumor-bearing rat subcutaneously. After stable growth of the tumor, the tumor-bearing rats with strong tumor growth and ulceration were sacrificed and dislocate cervical, the skin and the tumors.3. Allograft:Tumor tissue was cut into small pieces (about1.5mm3trocar, subcutaneously) and then was inoculated in into nude rats of SPF level BALB/c-nu (4-6weeks old, female, weight20-22g). Use a ruler to measure the diameter of the transplanted tumor free. Set tumor subcutaneous nodules greater than0.5mm as the standard.4. Grouping:After the completion of xenograft model of the nude rats, randomized experimental group was divided into saline Control group, the chemotherapy drug positive control group (Cisplation), high dose group (MnSODm high), medium dose group (MnSODm medium) and low dose group (MnSODm low). And all groups were given saline10mg/kg, cisplatin10mg/kg, MnSODm high dose of20mg/kg, medium doses of10mg/kg and low dose of5mg/kg. Drug treatment lasted for seven days.5. With7days’dosing, drug treatment withdrew the next day and the rats were weighed. Nude rats were killed and the author collected orbital blood with anticoagulant, took300ul to check the number of blood cells and platelet in the blood analyzer leukocyte, then took0.5ml blood stand for30min, centrifuged to obtain serum, and detected changes in serum alanine aminotransferase (ALT), blood urea nitrogen (BUN), and serum creatinine (Ccr) in each group.6. Nude mice were sacrificed, and the author removed the tumor, measured tumor volume and calculated tumor inhibition rate (greater than30%inhibition rate to be valid) with the formula:T/C=(TRTC/CRTV)×100%detection, in which TRTV stands for the treatment group, RTV, CRTV for the negative control group RTV, RTV=VT/V0.VT is the tumor volume after administration of7days. V0is the measurement of tumor volume when administered and results of less than40%T/C is valid.7. Nude rats were sacrificed, and the author took the vehicle control group (saline), the chemotherapy drug positive control group and the experimental drug with MnSODm dose group fixed with10%formalin, dehydrated, embedded in paraffin and HE staining. After all these operations, the above tumors were observed under light microscope and organizations were stained immunochemically.(see chapter Ⅱ for details).8. The author used SPSS17.0statistical software to process measurement data which are presented as mean±standard deviation expressed, normality and homogeneity of variance test, and the mean between the two groups used independent samples t-test, and the two groups before and after treatment compared with the help of paired t test, One-way ANOVA is used in comparison among groups while LSD is used in comparison between different groups,with a=0.05level of inspection. The difference was statistically significant when P<0.05, while the difference was not statistically significant when P>0.05.Results:1. General observation of experimental animal:7days after tumor transplantation, growth of xenograft tumor in the general situation is better, with no sample loss. After observation of each intervention group of nude rats with transplanted tumor, each treatment group were able to significantly improve the nude rats’ spirit, activity, response, tumor growth.2Change of volume of different treatment groups with the SKOV3xenograft tumor:with comparison of tumor volume the SKOV3xenograft in nude rats found in each group before and after treatment, the tumor volume difference was significant (P <0.05).7days after treatment at different drug concentrations, the author measured of tumor length diameter, and calculated the volume change of the volume and the group. The results suggested that there are statistically significant (P<0.05). T/C ratio results indicated that cisplatin group T/C ratio was36%, MnSODm high dose group was33%, middle dose group was41%, and the low dose group was45%. T/C less than40%of the treatment is effective, and the cisplatin MnSODm high-dose group less than40%is effective. Naked eye stripped transplanted tumors tumor found in the different concentrations MnSODm treatment group and negative control group, significantly reducing the volume and the cisplatin group which differed obviously from volume of MnSODm high dose group.3. Difference in weight and volume of tumor of tumor xenografts and nude body:nude rats were killed after stripping tumor and weighed tumor, and calculated the rate of tumor suppressor. Between each treatment group and the control group, tumor weight was significantly reduced, except in low dose group MnSODm. The rest of the treatment group and the control group (P<0.05) have significant difference:66.30%tumor inhibition rate for MnSODm high-dose group,54.02%tumor inhibition rate for middle dose group, and32.98%tumor inhibition rate for the low-dose group. Weighing nude rats before execution, the author found that under drug intervention the weight of unde rats did not change significantly and each treatment group, compared with the control group, was statistically significant (P>0.05).4Observation of tissue morphology:as for control group, the performance had characteristics of poorly differentiated adenocarcinoma. The tumors were solid group pieces like intensive cancer cells, cellular atypia, and a large deeply stained chromatin lumps with obvious nucleoli, abundant cytoplasm, showing more mitotic little interstitial. The performance of HE staining in MnSODm high dose group was: sparsely arranged cells, visible necrotic area of large tracts of red dye structure, deeply stained tumor nuclei, pyknosis. the medium dose group of MnSODm had a larger necrotic area while low-dose group of MnSODm changed significantly, with the physiological stained piece similar to the control group.5. Effect on Blood cells and function of Liver and kidney:as statistical results suggest with the help of LSD, the difference between treatment group with different dose of MnSODm and saline group (P>0.05) was not statistically significant; the comparison between the treatment group with cisplatin and the saline group (P< 0.05), has significant differences.The comparison between the treatment group with cisplatin and high dose group of MnSODm as well as medium dose group of MnSODm has no significant differences. The comparison among high dose group of MnSODm, medium dose group of MnSODm and low dose group of MnSODm has no statistical difference.Conclusion:1. Successfully established animal model of epithelial ovarian cancer SKOV3nude rats xenograft and laid the foundation for further research MnSODm.2.Experiments confirmed growth inhibition of MnSODm on epithelial ovarian cancer SKOV3nude mice xenograft, and has the advantages to lower the impact on kidney functions.Chapter4The study of the mechanism of the inhibitory effects of MnSODm on human epithelial ovarian cancer SKOV3cells in nude rats xenograftObjective:Using immunohistochemistry, the author detect effects of MnSODm on human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor of bcl-2and bax protein, and explored the mechanism of growth inhibition of MnSODm on human epithelial ovarian carcinoma cell line SKOV3xenograft tumor.Method:1. Set up the transplantation model of nude rats;2. Randomized grouping as Chapter One, excluded the positive control group of chemotherapy drugs (Cisplation Group), and other treatment groups were measured like the first chapter;3. Terminated to observe next day after withdrawal, and the mice were sacrificed, and the tumor specimens by immunohistochemistry (SABC) were sectioned and stained,with bcl-2and bax immunohistochemistry reagents;4. After completion of the stained piece, with high-definition digital microscope radiography, the author collected pictures and analyzed system for processing data, exported data for statistical analysis, and detected bcl-2and bax protein in human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor tissue and strength of expression in each dose group after the intervention MnSODm.5. Statistical analysis:with SPSS17.0statistical software, the measurement data are presented as mean±standard deviation expressed. Single-factor analysis of variance (One-way ANOVA) groups were compared using multiple comparison LSD method, P<0.05, and results had statistically significant difference.Results:1.Expression of nude rats’tumor cells bcl-2protein after drug treatment: immunohistochemistry results of control group suggested that the expression of Bcl-2protein in human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor tissue were located in the cytoplasm nuclear membrane at more positive control group cytoplasmic or nuclear membrane in coloring the number, showing deeply stained brown,and indicating higher levels of bcl-2protein expression in human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor tissue; in the different gradient administration group of MnSODm, coloring cytoplasm gradually reduced faded pigmentation, and the bcl-2protein expression gradient was non-linear correlation. Only a handful of coloring was seen in MnSODm high dose group, and was mainly located in the cytoplasm.The nuclear membrane is rare, indicating the low expression of bcl-2protein MnSODm high dose intervention.2. Expression of bax protein in nude rats’tumor cells after drug treatment:the expression of positive control group bax protein could be seen, which is located in the cytoplasm. The weak reflected in its expression in human epithelial ovarian cancer SKOV3cells in nude rats with transplanted tumor tissue with rendered brown light staining in the cytoplasm. The expression of bax protein is rare, in which almost no expression could be found with only a handful of coloring; in MnSODm different gradient administration group, cytoplasmic and nuclear membrane coloring gradually increased coloring of the deepened cytoplasm and Bax protein expression was gradient upward trend. MnSODm high dose group was stained brownish yellow, and presented with high expression of bcl-2protein in MnSODm high dose intervention.3. Comparison between the intervention group and Control group MnSODm different concentrations (P<0.05):the expression of down-regulated bcl-2in MnSODm different concentrations of the intervention group could be visible, expression of raised Bax protein could also be found;4.comparison between MnSODm Medium Group (10mg/kg) MnSODm Low Group (5mg/kg) and MnSODm High Group (20mg/kg)(P<0.05):results suggested that as MnSODm dose increased, expression of Bax protein bcl-2decreased. The expression of down-regulated bcl-2in MnSODm different concentrations of the intervention group could be visible, expression of raised Bax protein could also be found.Conclusion:1. bcl-2and bax were involved in the process of growth inhibition on the epithelial ovarian cancer SKOV3cell in tumor tissues;2. Confirmed that the MnSODm high dose group, by increasing bax protein and decreasing bcl-2protein, inhibited the growth of human epithelial ovarian cancer cells in SKOV3nude mouse xenograft.Summary1. Successfully establishing animal model of epithelial ovarian cancer SKOV3nude mice xenograft lays the foundation for further research MnSODm. After the second passage of the lines of the model, the tumor grows in vivo. The stable growth rate of tumor and the short experimental period make it easy to implement interventions. The sample has the homogeneity and reproducibility and it can truly reflect the evolution process of the tumor in vivo.2.Observing the weight of the tumor-bearing mice and general state of change, and detecting the changes in the white blood cells, platelets, transaminases, creatinine and urea nitrogen in mice after drug treatment can truly reflect the low toxicity characteristics of MnSODm in vivo.3.Firstly applied the MnSODm in vivo test providesa possible clinical application of MnSODm for a reliable basis of pre-clinical laboratory studies.4.MnSODm can inhibit the proliferation of SKOV3cell, which is dependent on time and concentration, and MnSODm can induce apoptosis in SKOV3cells.5.Observing from the vivo tests, the highest dose of MnSODm has the highest rate of tumor suppressor and the highest rate of apoptosis of tumor cells in72h. The in vitro and in vivo experiments confirmed that the role of MnSODm on SKOV3was time-dependent and concentration-dependent but no corresponding increase in toxicity.6.MnSODm acts on vivo test. Taking an intraperitineal injection that the local skin tissue does not appear ulceration, red, swelling, heat, pain, and other phenomena, diarrhea, bloating and other gastrointestinal symptoms did not appear. All of this show the compound in vivo metabolic cause toxicity directly organized the irritation is also very small, it is worthy of further research and development.7.MnSODm, through affecting cell cycle distribution, and, more importantly, through regulating nuclear factor-κB/p56way of apoptosis-related factors bcl-2and bax, can achieve inhibition on proliferation of human ovarian cancer SKOV3cell, induce apoptosis, and inhibit ovarian cancer cell proliferation factor of Caspase-3/7. |
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