Toxoplasma Gondii Excreted/secreted Antigen(ESA)Induces Apoptosis In Ovarian Cancer SKOV-3 Cells Via Mitochondrial Apoptosis Pathway

Objective:Conventional therapy for treating tumors can cause a greater degree of damage to the body's immune system while achieving therapeutic effects.Therefore,in order to improve the effect of tumor treatment and reduce the degree of damage to the body,biological therapy has become a new method for anti-tumor.Studies have found that the metabolites of Toxoplasma gondii can cause apoptosis in tumor cells.Therefore,this study extracted the excreted/secreted antigen(ESA)of Toxoplasma gondii to ovarian cancer SKOV-3cells.Observing ESA to SKOV-3 which the effects of cell proliferation,migration and apoptosis and explore its mechanism of apoptosis,which lays a theoretical foundation for the study of anti-tumor biological therapy.Methods:1.Using CCK-8 method to detect the effect of Toxoplasma gondii ESA on the proliferation inhibition of SKOV-3 cells.2.The direct observation method is used to observe the morphological changes of SKOV-3 cells in the treatment of SKOV-3 cells,and the changes of nuclear morphology of SKOV-3 cells are observed by DAPI staining.3.The effect of Toxoplasma gondii ESA on the migration ability of SKOV-3 cells is observed by scratch method.4.To investigate the effects of Toxoplasma gondii ESA on cell cycle,apoptosis,mitochondrial membrane potential and reactive oxygen species of SKOV-3 by flowcytometry.5.The expression of apoptosis-related proteins: Cleaved Caspase 9,Cleaved Caspase3,Bax and Bcl-2 in SKOV-3 cells is detected by Western blot.Results:1.CCK-8 results showed that Toxoplasma gondii ESA could inhibit the proliferation of SKOV-3 cells at 24 h and 800?g/ml,and inhibited the proliferation of SKOV-3 cells in a certain time and concentration range.The effect and the inhibitory effect increased in time-concentration.Compared with the control group,the difference is statistically significant(P<0.05).2.Morphological observation showed that: 800?g/ml,1600?g/ml of Toxoplasma gondii ESA intervention in SKOV-3 cells for 48 h,the adherent cells were observed to be detached to varying degrees,the cell volume was reduced,the nucleoplasm ratio was decreased,and the nuclear fixation was observed.Shrinkage,boundary,and expression of apoptosis such as folds.3.The results of nuclear staining showed that 800?g/ml and 1600?g/ml of SKOV-3cells could reduce the nuclear volume of the nucleus,increase the blue fluorescence,irregular nuclear morphology,chromatin condensation and aggregation.Nuclear pyknosis;as the concentration of intervention increases,the nucleus shrinks,the nuclear chromatin edge collects,the nucleus cleaves,and apoptotic features such as apoptotic bodies containing nucleic acid fragments and organelle components are formed.4.The results of cell scratching showed that Toxoplasma gondii ESA could decrease the migration ability of SKOV-3 cells after 48 hours of treatment with SKOV-3 cells at a concentration of 800?g/ml and 1600?g/ml.Compared with the control group,the difference is statistically significant.(P<0.05).5.Cell cycle results showed that compared with the control group,the concentration of Toxoplasma gondii ESA at 800?g/ml and 1600?g/ml for SKOV-3 cells increased significantly the proportion of cells in G0/G1 phase and the proportion of cells decreased significantly in G2/M phase,the difference is statistically significant(P<0.01).6.The results of apoptosis showed that the apoptosis rate of SKOV-3 cells increased with the concentration of Toxoplasma gondii ESA at 800?g/ml and 1600?g/ml for 48 h.Compared with the control group,the difference is statistically significant(P <0.05).7.The mitochondrial membrane potential of the cells showed that the mitochondrial membrane potential of SKOV-3 cells treated with 800?g/ml and 1600?g/ml of Toxoplasma gondii ESA was significantly decreased after 48 h intervention,and the difference is statistically significant(P <0.01).8.The results of cell ROS showed that compared with the control group,the concentration of ROS in the SKOV-3 cells was significantly increased after 48 h of Toxoplasma gondii ESA at 800?g/ml and 1600?g/ml,and the difference is statistically significant(P<0.01).9.Western blot analysis showed that 800?g/ml and 1600?g/ml of Toxoplasma gondii ESA could up-regulate the expression of proapoptotic proteins Cleaved Caspase 9,Cleaved Caspase 3 and Bax after 48 hours of SKOV-3 cells,and down-regulate the inhibitoryprotein.expression of Bcl-2.Conclusion:1.Toxoplasma gondii ESA can inhibit proliferation of SKOV-3 cells in a certain time and concentration range and the inhibitory effect is increasing in time-concentration.2.Toxoplasma gondii ESA can change the morphology of SKOV-3 cells: the cell volume shrinks,the nucleoplasm ratio decreases,the nucleus shrinks,the nuclear chromatin edge collects,and the nucleus cleaves.3.Toxoplasma gondii ESA acts on SKOV-3 cells to block SKOV-3 cells in G0/G1 phase,and the inhibitory effect is directly proportional to the concentration of ESA.4.Toxoplasma gondii ESA can attenuate the migration ability of SKOV-3 cells,and the inhibitory effect is directly proportional to the concentration of ESA.5.Toxoplasma gondii ESA can induced apoptosis in SKOV-3 cells and the inhibitory effect is directly proportional to the concentration of ESA.6.Toxoplasma gondii ESA intervention in SKOV-3 cells decreased intracellularmitochondrial membrane potential and increased reactive oxygen species may initiate apoptosis in early signaling molecules.7.Toxoplasma gondii ESA may induce apoptosis in SKOV-3 cells through mitochondrial apoptosis pathway,which may be induced by up-regulating the expression of proapoptotic protein Bax and down-regulating the expression of Bcl-2 protein.