Effect Of Trauma-stress On Transplanted Walker-256Tumor Growth And Proliferation Activity In Rats

Background and ObjectivesSurgical resection, which could be divided into curative and palliative type, is a very important therapeutic procedure for the patients with malignant tumor and has a decisive significance for the survival and prognosis of the patients which significant influenced by the tumor recurrence postoperatively. Surgical procedures is a strong traumatic stress for the body, may cause various responses (eg., neuroendocrine responses, cytokine responses, metabolic responses, and probably other unknown biologic responses). Such a complicate relationship existed among these various responses and their influence in the body may eventually change the body’s internal environment. It is known that the growth of tumor depend not only on the whole body regulation but also microenvironment condition. The correlationship among microenvironment concerning tumor cells, capillaries, microlymphatic vessels, a number of cytokines, tissue fluid and infiltrating cells is not fully clarified. But changes of microenvironment may significantly affect cell proliferation, migration and angiogenesis of tumor. A series of biological reactions after traumatic stress, which resulted in homeostasis damage, could affect the tumor microenvironment and furtherrmore affect the occurrence and development of the tumor. It’s believed that surgical procedures no matter curative or palliative could cause body response after traumatic stress which may make the impact on residual tumor lesions growth, accelerate the growth and spread of the residual tumor lesions. Though the influence to the prognosis lack in long-term clinical observation, it is well-known that the induction of apoptosis of thymocytes by endogenous glucocorticoids and the reduction of natural killer activity are associated with the enhancement of cancer metastasis. But it would not be satisfied that basic immunosuppression make the acceleration of tumor growth postoperatively. It is reported that the surgical stress may promote tumor metastasis through the mechanism of acute traumatic stress on tumor growth has not been fully clarified. As the surgical procedure remain to be the major manner in the tumor therapy, it would be meaningful to explore the relationship between trauma-stress and tumor growth and proliferation which would furthermore play roles in the prevention of tumor occurrence.Burns as a typical trauma, which offered the similar stress response and release of inflammatory cytokines caused by surgical trauma, is an easier and controllable experiment method as the burn area and degree could be standardized designed. Wistar rats which commonly used in burn researches, would be suitable for trauma stress studies, as the report suggested, rats are able to withstand the burn stress. Walker-256cells, which is a spontaneous carcinosarcoma come from the rat mammary gland, have a strong invasiveness and weak immune rejection in rats, therefore this tumor cells had been often used in tumor transplant models. In the cytokines after trauma stress, serum and tumor tissue changes share the same responsibility for tumor growth and proliferation. TNF-alpha, which is typical inflammatory marker, offers the severity of trauma stress and body response extent. VEGF, as the most important factor involved in tumor angiogenesis. is not only involved in tumor angiogenesis, proliferation, but also in tumor invasion and metastasis. At the same time VEGF can be a indicator that reflect the proliferative activity in serum and tumor tissue. PCNA, A polymerase accessory proteins required for DNA replication, is a marker reflecting the proliferative activity of cells. PCNA is changed with the cell proliferation and is a objective index to reflect the state of cell proliferation. Combined detection of VEGF and PCNA expression in tumor tissue can be a reliable reference for the evaluation of tumor proliferative activity and prognosis. The purpose of this study is to explore the relationship between burn trauma stress (surgical trauma) and tumor growth through observe the change in weight of tumor, serum TNF-alpha and serum VEGF levels, expression of PCNA and VEGF in tumor tissue after trauma. Furthermore to explain the possible mechanism for tumor recurrence after surgical procedures.Methods1. The production of subcutaneous Walker-256transplanted tumor model on rats: Walker-256cancer cells cultured to logarithmic phase,1ml of cell suspension injected into abdominal cavity of the rat, extracted the malignant ascites after a week, centrifuged and took the lower tumor cells, joined the PBS solution,made the tumor cells count in suspension must be more than1×10/ml. Then took0.15ml of the cell suspension injected into the subcutaneous in the inguinal of rats, a total of45. A diameter of about0.5cm subcutaneous tumor nodules can be touched after3-5days, the transplanted tumor model was produced successfully.2.36Wistar rats with transplanted Walker-256tumor were randomly divided into3groups(n=12):group A was control group,didn’t for burn treatment;group B was minor burn group, the back of rats were burned with100C boiling water for Ss,the area is3×3cm;group C was the severe burn group, the rats were burned for10s,4×4cm. All operations were conducted under the anesthesia.the rats were housed separately after regained consciousness, and provided adequate food and water. 3. All rats of three groups were killed at the10day after the burn. Cardiac blood was collected, centrifuged and collect the upper serum. The serum VEGF, TNF-a were detected, detection methods referenced to the kit test instructions.Then stripped the tumor tissue along the tumor capsule, measured its weight, stored in formalin solution.4. VEGF and PCNA expression of the tumor tissue were detected by immunohistochemical method:method SABC, the operations accordance with the instructions of the kit. Brown granular staining in the cytoplasm for the VEGF-positive cells, defined the positive cell count less than10%of total cells as (-),≥10%as (+). the number of positive and negative cases were recorded in each group. PCNA-positive staining was limited in the nucleus, showed brown granules or a diffuse distribution. Counted the number of PCNA-positive cells in each field and the total number of cells, the percentage of the PCNA-positive cells was the proliferation index.5. Statistical method:The obtained data were analyzed by SPSS13.0software in computer.The metrology data was analyzed by One way ANOVA analysis, including tumor weight, serum levels of TNF-a, VEGF, PCNA proliferation index. The difference of every group was tested by SNK method. Positive rate of VEGF was compared with the χ2test method. P<0.05means that there was statistical difference of experimental groups.Results1. Compared with group A (control group), the quality of tumors in the burn groups increased (group A:2.81±0.48g, group B:3.28±0.61g, C group,3.40±0.47g, P=0.022<0.05). Between B,C groups,there was no significant difference (P=0.589).2. Compared with the control group, level of serum TNF-α in burn groups increased (group A31.59±4.73pg/ml, group B41.72±4.80pg/ml, group C42.47±5.32pg/ml,P<0.01).The serum VEGF also increased in burn groups (group A189.04±27.43pg/ml, B221.53±26.55pg/ml, group C214.73±37.98pg/ml, P<0.01).There was no significant difference between the B, C groups both in the two indicators (P=.713and P=0.388).3. Expression of PCNA in tumor tissue in burn groups were higher than that in the control group (group A58.30±6.61%, group B77.08±4.53%, groupC81.58±4.56%, P<0.01), group C was higher than group B (P=0.024<0.05).4.The VEGF-positive cases in each group:group A were4cases,9cases of group B, group C were10cases,χ2=7.465, P<0.05.DiscussionIt is believed that the local microenvironment of the tumor plays an essential role in tumor growth and the tumor growth is accompanied with the changes of its microenvironment. Trauma-stress initiate the body reaction which activate the Hypothalamus-Pituitary-Adrenal System and Renin-Angiogensin Systems, followed by norepinephrine, cortisol and other stress hormones released into the blood and resulting in vasospasm, aggravating tissue ischemia and hypoxia. In addition, trauma-stress suppress the immune system, the anti-tumor effect of immune cells infiltrating in the tumor microenvironment are reduced, conducive to the progress of the malignant tumors, angiogenesis. and metastasis to specific parts. And large amounts of proinflammatory cytokines such as TNF-alpha, IL-6released after trauma-stress, may be an impact on the growth of tumor cells and lymphocytes in the blood circulation, as the results showed. VEGF as an important growth factor involved in tumor angiogenesis is elevated after trauma. Therefore, the results which showed a variety of factors caused by trauma could create a favorable environment for the growth of tumor. The results also showed that trauma-stress significantly accelerated the tumor growth which is correlated with the severity of trauma. The serum levels of TNF-alpha and VEGF elevated higher in the severer trauma animals and the expression of PCNA and VEGF in tumor tissue offered the similar results in histochemical studies. The experiment results suggest that trauma-stress could promote the proliferation and growth of the transplanted tumors in rats through releasing inflammatory cytokines and VEGF in the way of microenvironments changes. Regulation and changes of these cytokines might be useful approach in the preventing tumor recurrences after operation procedures which need further research.