| Ovarian cancer is one of the common malignant tumors in the female reproductive system.The incidence of the disease was third in gynecologic tumors.However,Most of them are in the late stage of the clinic when they found them.Tumor cell subtraction combined with paclitaxel,cisplatin and other combined chemotherapy is an important method for the treatment of ovarian cancer.The primary response rate of ovarian cancer patients treated with combined chemotherapy based on platinum drugs was up to 80%.But most patients eventually fail in the appearance of chemotherapeutic drug resistance.The five year survival rate of ovarian cancer was around 30%.Drug resistance of ovarian cancer,in particular,the mechanisms and reversals of platinum resistance have become an extremely urgent and important research topic.Therefore,the mechanism and possible reversing pathways of ovarian cancer resistance are need to be studied.It is helpful to take specific measures to reverse chemotherapeutic drug resistance of ovarian cancer and to improve the patient’s sensitivity to chemotherapy drugs.Thus,the survival rate is improved and the prognosis is improved.Autophagy is proposed for the first time by Christian de Duve more than 40 years ago.Autophagy means that a membranous structure that forms a double or multilayered fold within a cell encapsulating a cell or macromolecule that is damaged in a cell under stress conditions such as starvation,hypoxia,and other undernutrition,and outside pressure.The autophagic corpuscle is formed,and the autophagosome is combined with the lysosome.Thus,the contents are digested and degraded,and the corresponding amino acids,nucleotides are circulated and reused.It satisfies the energy reuse of the cell itself and the renewal of the organelles.Autophagy is a conservative metabolic process in eukaryotes.It degrade intracellular damaged and degenerated proteins and organelles through lyase system,thrus maintaining cell homeostasis and genome stability.Under normal circumstances,autophagy maintains the balance of the intracellular environment at a basic level.In recent years,more research found that the function of autophagy may be involved in the occurrence and development of many diseases,such as diabetes,immune system disease,neurodegeneration and cancer invasion.In recent years,a variety of chemotherapeutic drugs have been found to induce autophagy in tumor cells.Tumor cells remove the damaged organelles caused by chemotherapeutic drugs and the wrong folding proteins to maintain their stability by autophagy and then evade the apoptosis induced by chemotherapeutic drugs and promote the survival of tumor cells.So autophagy may be a protective mechanism for cancer cells.The sensitivity of chemotherapeutic drugs was reduced.It leads to the tolerance of tumor cells to chemotherapeutic drugs.A number of studies have shown that autophagy has been involved in the process of chemotherapeutic drug resistance in a variety of tumors.The application of autophagy inhibitors may be one of the most important strategies for improving chemotherapeutic resistance and enhancing chemosensitivity.The effect of chemotherapeutic drugs on the autophagy of ovarian cancer cells and the role of autophagy in the chemosensitivity of ovarian cancer cells is rarely reported.Autophagy is a very complex process regulated by some special genes and multiple signal pathways.These regulatory genes are known as autophagy related genes(ATG).At present,more than 30 autophagy related genes have been found.Beclinl is a relatively mature autophagy related gene.It is combined with Class III P13K to form a complex.It plays an important role in the nucleation process of autophagy.Studies show that Beclin1 is also associated with the occurrence and drug resistance of tumors.Ambral(Activating molecule in Beclinl-regulated autophagy 1,Beclinl autophagy related activation molecule)is a new ATG gene.As the upstream factor of Beclinl,it controls the formation of autophagy by regulating the Beclinl-Class III complex.In mitochondria,Ambral regulates the autophagy and apoptosis of Beclin1 dependent Bcl-2 by combining with B lymphocyoma-2.Therefore,Ambra1 can determine the fate of tumor cells by regulating autophagy and apoptosis,but the role of Ambra1 in cisplatin treated ovarian cancer cells has not been reported.Studies have shown that in colon cancer cell line SW620,Ambral combined with Beclinl can positively regulate autophagy and negatively regulate apoptosis at the same time.The expression of Ambral upregulates in breast cancer cells treated by cisplatin and the drug resistance of breast cancer cells are increased.This study conjectured that Ambral is an important regulator of autophagy and apoptosis in ovarian cancer cell line OVCAR-3,proposed treatment of ovarian cancer OVCAR-3 cells by cisplatin,through the assessment of microtubule associated protein 1 light chain 3(LC3)positioning,autophagy exists,LC3 level and Ambral,Beclinl protein expression,confirmed the expression of Ambral and cisplatin the treatment of ovarian cancer cell OVCAR-3 induced autophagy and autophagy,to confirmed that the autophagy induction and the expression of Ambral in autophagy induced by cisplatin in ovarian cancer OVCAR-3 cells.We determined the autophagy promoting effect of Ambral in cisplatin treated ovarian cancer cells,and then transfected Ambral gene silencing by shRNA.Re evaluate the effect of Ambral gene interference on cisplatin induced autophagy in ovarian cancer OVCAR-3 cells.Further verify the role of Ambral in promoting autophagy in cisplatin treated ovarian cancer cells.The effects of cisplatin on the growth,cell viability and apoptosis induction of ovarian cancer OVCAR-3 cells after Ambral knockout were determined.It is clear that the effect of Ambra1 knockout on the sensitivity of OVCAR-3 cells to cisplatin.It provides a theoretical basis for finding therapeutic agents that improve the chemosensitivity of ovarian cancer based on Ambral.Part I:Cisplatin reduces cellular viability,promotes autophagy and upregulates Ambral in OVCAR-3 cellsObjectives:1.To study the sensitivity of OVCAR-3 cells in ovarian cancer to cisplatin2.To explore the regulation of autophagy and expression of autophagy related molecules in ovarian cancer OVCAR-3 cells by cisplatinMethods:1.MTT assay was used to detect the cell viability and time dependence of OVCAR-3 cells treated with different concentrations of cisplatin.2.Using cisplatin to treat ovarian cancer OVCAR-3 cells,we observed the LC3 transformation of ovarian cancer cells,including autophagy,autophagy formation and the key molecule of autophagy.3.The autophagic inducer,rapamycin as positive control,was used to observe the changes in the expression of autophagy related protein Ambral and Belinl after cisplatin treatment of ovarian cancer OVCAR-3 cells.Results1.Changes of cell activity in ovarian cancer OVCAR-3 cells which werre treated with different concentrations of cisplatinBy 0,10 and 50μM concentrations of cisplatin treating of ovarian cancer cells 0,12,24,48 hours later,MTT was used to detect the ovarian cancer cells,and the results showed as follows:with 10 or 50uM cisplatin treatment of ovarian cancer OVCAR-3 cells(p<0.005,p<0.01)for 24 hours,48 hours(p<0.001,p<0.0001),the activity of OVCAR-3 cells were decreased,and the decreased with difference was statistically significant.Moreover,there was a significant difference in cell survival rates between the two time differences between 10μM and 50μM cisplatin treated cells 24(p<0.05)hours and 48(p<0.01)hours,and the difference was statistically significant.The results are detailed in figures 1A and 1B.2.The effect of cisplatin on the autophagy of ovarian cancer OVCAR-3 cellsOvarian cancer cells were treated with 10μM cisplatin for 24 hours(experimental group).Compared with the ovarian cancer cells(control group)that were not treated with cisplatin,After transfection of GFP-LC3 fusion protein particles,the number of autophagic vesicles in the ovarian cancer cells in the experimental group was more than that in the control group.The results are detailed in figure 1C and 1D.The difference was statistically significant(p<0.001).The results are detailed in figure 1E.3.Cisplatin induced autophagy in ovarian cancer OVCAR-3 cells,accompanied by increased expression of Ambral and Beclin1.Compared with the ovarian cancer OVCAR-3 cells treated with non chemotherapy drugs,rapamycin could significantly improve the level of LC3-I protein conversion to LC3-II protein(p<0.001)after treating ovarian cancer OVCAR-3 cells with 100nM as autophagy as an autophagic inducer.After 24 hours treatment of cisplatin with 10 and 50μM of cisplatin,it can also significantly increase the level of LC3-II protein converted from LC3-I protein(p<0.01,p<0.001)after 24 hours.This transformation is metrological dependence(p<0.001).The higher the concentration of chemotherapeutic drugs,The higher level of LC3-II protein converted form LC3-1 protein.The induction of autophagy of rapamycin in 100nM was stronger than that of 10,and 50μM cisplatin.The results are detailed in figures 2A and 2B.Compared with the ovarian cancer OVCAR-3 cells treated with untreated chemotherapy drugs,After treating OVCAR-3 cells with rapamycin in 100nM as autophagic inducer,the expression level of Ambra 1 and Beclinl protein was significantly increased(P<0.001).After treating ovarian cancer OVCAR-3 cells with 10 and 50μM cisplatin for 24 hours,the expression level of Ambral and Beclin1 protein can also be significantly increased(p<0.05,p<0.01).And the measurement dependence(p<0.05),The expression of Ambral and Beclinl protein induced by cisplatin at 50μM was higher than that induced by cisplatin at 10μM.The difference was statistically significant(P<0.05).The expression of Ambra1 and Beclin1 protein in rapamycin induced by 100nM was higher than that induced by cisplatin at 50μM.The difference was statistically significant(P<0.0001).The results are shown in figures 2C and 2D.Conclusion:1.The cell activity of ovarian cancer OVCAR-3 cells decreased with cisplatin in time and concentration dependent.2.Cisplatin can induce autophagy in ovarian cancer OVCAR-3 cells.3.When cisplatin induced autophagy in ovarian cancer OVCAR-3 cells,the expression of autophagy related protein Ambral and Beclinl protein was increased,suggesting that Ambral can promote autophagy in ovarian cancer cells treated with cisplatinPart Ⅱ The study of shRNA mediated Ambral gene silencing on autophagy induced by cisplatin and the Beclinl expression in ovarian cancer OVCAR-3 cellsObjective:1.Screening Ambral gene transfected and stable expression of ovarian cancer OVCAR-3 cell line.2.we investigated the effect of Ambral knockout(interference suppression)on the induction of autophagy in cisplatin in ovarian cancer OVCAR-3 cells.3.To investigate the effect of interference inhibition of Ambral gene on the expression of autophagy related protein in ovarian cancer OVCAR-3 cells induced by cisplatin.Methods:1 LCells transfected with Ambral specific shRNA and RT-PCR mRNA assay were used to detect the expression of Ambral mRNA after Ambral knockout2.The effects of Ambral gene expression on the formation of autophagic vesicles in ovarian cancer OVCAR-3 cells were detected by autophagic vesicles.3.The effect of interference inhibition of Ambral gene expression on the expression of autophagic protein in ovarian cancer OVCAR-3 cells was detected by Western Blotting.Results:1.Transfection efficiency of OVCAR-3 cells in ovarian cancerIt is observed that GFP(green fluorescence)can be regarded as transfection work under the fluorescence microscope.Fluorescence counts were carried out at 12 hours,24 hours,36 hours and 72 hours after transfection.Comparing the number of fluorescent cells,We find that as time goes on,the expression of GFP was highest at 72 hours after transfection and the transfection efficiency of ovarian cancer OVCAR-3 cells reached more than 90%.It can be used for the follow-up experiments.According to whether transfection of shRNA-Ambral,ovarian cancer OVCAR-3 cells were divided into two groups,ovarian cancer OVCAR-3(Ambral-)group and OVCAR-3(Ctrl)group.2.RT-PCR was used to detect the expression of Ambral mRNA in two groups of cells.The expression level of Ambra1 mRNA in the OVCAR-3(Ambra1-)group was lower than that of the OVCAR-3(Ctrl)group.The difference was statistically significant(P<0.01),and the result was shown in figure 3A.It indicates that shRNA-Ambra1 can significantly inhibit the expression level of target gene Ambral and achieve the interference effect.3.The effect of Ambra 1 knockout on autophagic vesicle formation in ovarian cancer OVCAR-3 cells was detected.After the same treatment of two groups of ovarian cancer cells with 10 M cisplatin,the expression of GFP positive autophagy was decreased in the OVCAR-3(Ambral-)cell group compared with the ovarian cancer OVCAR-3(Ctrl)cell group,and the difference was statistically significant(p<0.01).The results were detailed in figure 3C.4.Detect the effect of Ambra 1 knockout on the expression of Ambra 1,LC3-I protein,LC3-Ⅱ protein and Beclin 1 protein in ovarian cancer OVCAR-3 cells.Cisplatin in 10 μM were used to treat two groups of ovarian OVCAR-3 cells for 12 and 24 hours,the Western Blotting method was used to detect and find:After cisplatin treatment of two groups of ovarian cancer OVCAR-3 cells for 12 hours and 24 hours,the expression of Ambral protein in the OVCAR-3(Ambra1-)group of ovarian cancer was lower than that in the OVCAR-3(Ctrl)group of ovarian cancer,and the difference was statistically significant(p<0.01,p<0.05).The results are detailed in figure 3B.After 12 and 24 hours of treatment,the ratio of LC3-II/LC3-I protein in the OVCAR-3(Ambral-)cell group decreased with the ratio of LC3-II/LC3-I protein in the OVCAR-3(Ctrl)cell group of ovarian cancer,and the difference was statistically significant(p<0.01,p<0.01).It is indicated that the induction of autophagy is inhibited in the ovarian cancer cell line OVCAR-3,when the expression of Ambral gene is inhibited.The results are shown in figures 3D and 3E.After 12 hours of treatment,the expression of Beclinl protein in the OVCAR-3(Ambra1-)cell group was lower than that in the OVCAR-3(Ctrl)cell group of ovarian cancer,and the difference was statistically significant(p<0.05).It was also found that after 24 hours of treatment,the expression of Beclinl protein in the OVCAR-3(Ambral-)cell group was lower than that in the OVCAR-3(Ctrl)cell group of ovarian cancer,and the difference was statistically significant(p<0.01).It is indicated that the expression of autophagy related Beclinl protein is inhibited in ovarian cancer OVCAR-3 cells after Ambral gene expression is inhibited.The results are detailed in figure 3F.Conclusions:1.shRNA-Ambral can significantly inhibit the expression level of target gene Ambral,and achieve the interference effect.2.silencing Ambral gene expression inhibits cisplatin induced autophagic vesicle formation in ovarian cancer cells.3.after silencing the expression of Ambral gene,the ratio of autophagy related protein LC3-II/LC3-I decreased,and the expression of Ambral and Beclin 1 protein decreased.4.Ambral plays a positive role in the induction of autophagy in cisplatin treated ovarian cancer OVCAR-3 cells.Part III The effect of shRNA mediated Ambral knockout on the apoptosis of ovarian cancer OVCAR-3 cells treated with cisplatinObjective:The effects of cisplatin on the growth,cell activity and apoptosis of ovarian cancer OVCAR-3 cells were determined after Ambral knockout,and the effect of Ambral knockout on the sensitivity of cisplatin in OVCAR-3 cells was determined.Methods:1.Colony forming assay was used to detect cell colony formation after treatment with 10μM of cisplatin in two groups of OVCAR-3(Ctrl)and OVCAR-3(Ambra1-).2.MTT test was used to detect the changes in the activity of OVCAR-3(Ctrl)and OVCAR-3(Ambral-)cells treated with cisplatin in 10μM.3.Flow cytometry was used to detect the apoptosis rate of OVCAR-3(Ctrl)and OVCAR-3(Ambral-)cells treated with cisplatin in 10μM.Result:1.Changes in colony formation of OVCAR-3 cells induced by cisplatin in ovarian cancer after the Ambra1 genes are knockout2.As shown in figure A:10μM of cisplatin in ovarian cancer cells can significantly reduce colony formation in the OVCAR-3(Ctrl)cells,differences are statistically significant(p<0.01 in Figure4B,the third and the first columns),the similar decreased colony formation also appeared in OVCAR-3(Ambral-)cells,and the differences are statistically significant(p<0.01,in Figure4A,the fourth and the second columns).More significant is that in OVCAR-3(Ctrl)and OVCAR-3(Ambral-)group,cell colony formation is different,the cell colony decline in OVCAR-3(Ambral-)group is more significant(p<0.05,in Figure 4B,the fourth column and the third column).3.Changes in cell activity after cisplatin treatment of ovarian cancer OVCAR-3 cells after Ambral genesare knockoutThere is no difference in cell activity between OVCAR-3(Ctrl)and OVCAR-3(Ambral-)two groups.As shown in figure 5A,however,when the cells were treated with 10μM cisplatin,the cell viability of OVCAR-3(Ambral-)group was significantly lower than that of OVCAR-3(Ctrl)group,and the difference was statistically significant(in Figure 5B,24 hours after treatment,p<0.05 and 48 hours after treatment,p<0.01).4.Changes in apoptosis after cisplatin treatment of ovarian cancer OVCAR-3 cells after Ambral genes are knockoutAfter treating two groups of cells with cisplatin in 10μM,the apoptotic rate of OVCAR-3(Ambral-)group increased significantly than that of OVCAR-3(Ctrl)group,and the difference was statistically significant(in Figure 5C,24 and 48 hours after treatment,all p<0.01).Conclusions:1.Silencing Ambral gene can improve the sensitivity of OVCAR-3 to cisplatin and increase the rate of apoptosis.2.Ambral may be the key regulator to maintain the balance between autophagy and apoptosis in ovarian cancer cells.Ambral targeting inhibition may be an effective method for ovarian cancer cells to be sensitive to chemotherapeutic drugs. |
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