| Background and PurposeOvarian cancer, as the second most common malignancy of the reproductive system in women, onset of occult, remains extremely difficult to diagnose in its early stages. Most women with ovarian cancer have no, or only non-specific, symptoms. And the highly malignant, easy to recurrence and metastasis. Therefore, ovarian cancer is still the leading cause of mortality among gynecological cancers. Development and progression of ovarian cancer is a complex process involving multiple genes and signaling pathways, may be related to genetic, immunological, environmental and psychological factors. Clinicians and researchers at home and abroad alike now pay close attention to the effects of psychosocial factors on tumor patients. It has become conclusive about long-term living in the poor state of psychological stress to promote occurrence/growth of malignant tumors, including ovarian cancer. However, its mechanism of action remains unclear because a comprehensive study of the relationship between psychology and tumor development, has yet to be performed. Studies suggest that psychosocial factors affect the immune system primarily through dysfunction of the hypothalamus-pituitary-adrenal axis. Thus, these factors affect the occurrence and prognosis of cancer. But, the mechanism requires further exploration. It is a powerful tool to study cancer using animal model to simulate the development of clinical oncology, and, domestic and foreign scholars commonly to study the effect of psychological stress on tumor development using unified control animal models of chronic stress. The purpose of this study was designed to investigate the possible molecular mechanism of the relationship between psychology and tumor development using a adverse psychological stress model of nude mice bearing human ovarian epithelial carcinoma, and comparative proteomic methods have been used.Methods:1. Nude mice were randomly divided into four groups:normal growth group (A), simple stress group (B), simple tumor group (C) and tumor-bearing stress group (D), each group contained six.2. To establish the nude mice xenograft models employed human ovarian epithelial carcinoma SKOV3 cell lines.3. To establish the model of adverse psychological stress used chronic restraint.4. To observe the nude mice’s body weight, and other general living conditions in each group, The xenografts volume and pathological changes of simple tumor group and tumor-bearing stress group nude mice. To confirm that xenografts tissues were ovarian cancer and confirmed the histological type empolyed HE staining.5. To detect the levels of serum norepinephrine (NE), interleukin-10 (IL-10) and CA125 antigen (CA125) in each mouse of four groups.6. After extracting total protein of every xenograft of simple tumor group and tumor-bearing stress group nude mice, we obtained the total protein profiles of every group by two-dimensional gel electrophoresis (2DE), and then analyzed by naked eye and image analysis software to identify differentially expressed protein spots in each group. Further, the differentially expressed protein spots were selected to identificated by mass spectrometry (nanoUPLC-ESI-MS/MS).7. Five differentially expressed proteins (SOD1, Nm23, Prdx1, GSTO1, CKM) were selected to assays proteomic results by immunoblotting (Western blotting).8. The expression of nm23 protein and NDRG1 protein were detected by an immunohistochemical SP method in xenograft tissues including 25 cases of tumor-bearing stress group (stress group) and 20 cases of simple tumor group (control group), aiming at verify the proteomic results further. To analysis of the relationship about the expression of Nm23 protein and NDRG1 protein with psychological stress and the tumor.Results:1. Established successfully a adverse psychological stress model of nude mice bearing human ovarian epithelial carcinoma used SKOV3 cell lines by subcutaneous injection and restraint stress.2. The first few days began to stress, simple stress group and the tumor-bearing stress group nude mice show irritability, they manifested as hyperactivity and biting tied tubes, and then adapt in a few days later. Them have a poorer growth state compared with the mice of normal growth group and simple tumor group, and them eating less but drinking more. The subcutaneous nodules began to emerge four days after SKOV3 injection, gradually grew up, and xenografts began to grow rapidly ten days after SKOV3 injection, and we can find, xenografts in tumor-bearing stress group grow more rapid than in simple tumor group. No mice death throughout the trial, and the success rate of tumour transplantation is 100%. There was no difference in mouse body weight among the four groups prior to the experimen(P＞0.05), whereas, mouse body weight were increased in each group. However, them was increased more obvious in the normal growth group and the simple tumor group than in the simple stress group and tumor-bearing stress group. There were significantly different in body weight between the stressed and no-stressed groups after the experiment (P＜0.01).3. Subcutaneous tumor tissue confirmed by HE staining, poorly differentiated adenocarcinoma, which consistent with the SKOV3 tumor. Average xenograft weight in the tumor-bearing stress group was significantly higher than in simple tumor group (P＜0.05).4. NE, IL-10 levels ranked as A<C<D, A<B<D, There were significantly different (P＜0.05). CA125 levels ranked as A<B (P>0.05), A<C<D (P＜0.05), B<D (P＜0.05).5. Xenograft tissues were processed for 2D gel electrophoresis and mass spectrometry (nanoUPLC-ESI-MS/MS), we found that 20 protein spots were expressed at significantly different levels in the tumor-bearing stress group to the respective spots from the simple tumor group,14 were up-regulated (Prdxl, SOD1, Laminin-binding protein, Prdx3, MRPL12, GSTO1, Esterase D/formylglutathione hydrolase, SOD2, RPS12,14-3-3-ε, BPGM, Nm23, NADPH, PGK1),5 were down-regulated (CKM, Fast skeletal myosin alkali light chain 1 isoform If, CCT8, NDRG1, Myosin light chain 2) and one protein spot (SET) was found only in the tumor-bearing stress group.6. The Western blotting results showed that Prdxl, SOD1, GSTO1 and Nm23 protein expression in the tumor-bearing stress group xenograft tissue was significantly higher than that in the simple tumor group, while CKM protein expression level was significantly reduced, in agreement with the results of the proteomic analyses.7. The results of immunohistochemistry showed that the positive rate of Nm23 in the stress group and control group were 100% and 70%, respectively, while NDRG1 protein were 56% and 85%, respectively, the differences were significantly (P<0.05). Results were consistent with those of proteomic and Western blotting analyses.Conclusions:1. Adverse psychological stress model of nude mice bearing SKOV3 tumor is a ideal model to study the relationship between psychological stress and tumor progression.2. Adverse psychological stress can make mice growth restriction, and can contribute significantly to tumor growth and invasion.3. Adverse psychological stress can increase the serum NE, IL-10, CA125 levels of nude mice, indicating that adverse psychological stress can interfere with the function of hypothalamus-pituitary-adrenal axis and lead to weakened immune system defenses against some risk factors in a susceptible condition. When the stress occurs in cancer patients, may promote tumor occurrence and development and affect the prognosis.4. We have screening and identified a number of differentially expressed proteins between the stressed and no-stressed nude mice xenograft specimens by proteomic analysis, and our results demonstrated that the use of proteomics will aid in identifying and understanding the molecular mechanism underlying the relationship between psychological stress and tumor progression.5. The results of immunohistochemical analysis were consistent with those of two-dimensional electrophoresis and Western blotting, and further validate the proteomics is a feasible method in the study of molecular mechanisms about the relationship between psychological stress and tumor.6. Adverse psychological stress may be influence the development of tumors by activation of oncogenes and inactivation of tumor suppressor genes, nm23 and Ndrgl can be used as genetic research and gene therapy target. |
PDF Full Text Request