| Backgroud:With the improvement of living standards and aged tendancy of population, the incidence of prostate cancer increased every year in China. Bone is the most common metastatic site of prostate cancer. Bone metastases can cause severe pain to the patient,easily lead to pathological fractures, resulting in paralysis and movement disorders,which is also one of the main causes of death. Its mechanism is not clear, and the treatment effect is not ideal.The prostate cancer patients9 biological rhythm will be disturbed because of anxiety,treatment, pain and the biological effects of tumor itself. It has been proved that biological rhythm disorder is very harmful to human health, which can promote the formation,development and metastasis of breast cancer and lung cancer. IL-6 plays a very important role in these processes However, there have no reports been found that the influence of biological rhythm disorder on bone metastasis of prostate cancer.At present, there are many methods to establish the animal model of bone metastasis,such as left ventricular injection, tibia injection and tail vein injection. These methods have many disadvantages, such as poor specificity of metastasis, high mortality rate of mice, and the pathophysiology of bone metastasis. Therefore, the establishment of an animal bone metastasic modle which is simple to operate, has a high specificity in transfer, and conforms to the pathogenesis of bone metastatic is very important to the study of tumor metastasis molecular mechanism and the reaserch on anti tumor drugs.Objective:In this reaserch,we intend to establish a specific and efficient mouse bone metastasis model, using mouse prostate cancer RM-1 cells and the mouse breast cancer cell 4T1-luc which expresses luciferase reporter gene to verify the effect of bone metastasis. In vitro,isoprenaline(ISO) and Norepinephrine(NE) are added into the RM-1 cell culture medium respectively to simulate the sympathetic excitation of the patients with biological rhythm disorder.Then we will detect the expression of IL-6 gene and explore the signal pathway through the forward and reverse experiments. Finally, animal experiments are conducted to verify the effect of biological rhythm disorder on bone metastasis of prostate cancer.Methods:This reaserch is divided into three parts.1. The RM-1 cells were cultured in 1640 medium containing 10% serum.We added ISO 10?mol/L and NE 10?mol/L(final concentration) respectively in the medium in the logarithmic phase of cell growth and detected the expression of IL-6 by using qPCR method .We added propranolol, adrenergic beta 1 and beta 2 signaling pathway inhibitor pretreated 1 hours subsequently and detected the expression of IL-6 after the cells stimulated by ISO for 30 minutes. Then, the expression of related signal pathways' proteins was detected by using of western blot after the cells were treated ISO and inhibitors which has been metioned. The effects of ISO and inhibitors on the proliferation and migration ability of RM-1 cells were determined by proliferation assay and scratch healing assay.At last,we tested the effect of propranolol on MMP2 using qPCR.2. C57BL/6J male mice were devided into two groups randomly, every group contains 15 mice. Another group contains 15 BALB/c female mice. Prostate cancer cell RM-1 and breast cancer cell 4T1-luc were injected into 15 C57BL/6J mice and 15 BALB/c mice,respectively, via femoral artery after the cells are digested, washed, counted, and suspended to the concentration of 2*105/ml. Each mouse was injected with 100 ?l cell suspension.The same concentration of RM-1 cells were injected into another group of C57/6J mice via external iliac artery. Then the operating time, postoperative survival rate, and the rate of tumor formation by two different injection methods were compared. The effectiveness of tumor metastasis was evaluated by means of in vivo imaging.3. We studied the effect of biological rhythm disorder on bone metastasis of prostate cancer by in vivo experiment, and explored the effect of propranolol and zoledronic acid on the inhibition of bone metastasis. We choose C57/6J male mice, which were divided into control group, propranolol group, stress group and stress + propranolol group randomly.The stress group and stress + propranolol group mice were continuously illuminated for a week,then the RM-1 cells were injected into mice via femoral artery according to the method described in the second part of the article. After the operation, the stressed mice were raised in a 12 hr light, 12 hr dark cycle. The control group and propranolol group mice were fed in the normal circadian rhythm. 21 days later, the mice were taken the blood via orbital artery blood after they were anesthetized. Serum IL-6 level was detected by ELISA.The limb bones which were injected were treated by HE staining. Bone metastasic cavity area ratio was calculated. Subsequently, 36 mice were randomly divided into control group,propranolol group, 20ug/ml zoledronic acid group, propranolol + 20ug/ml zoledronic acid group, 40ug/ml zoledronic acid group, propranolol + zoledronic acid group 40ug/ml.We operated these mice as described in the second part of this article.21 days later,the injected limb bones were got out and were treated by HE staining. The effect of propranolol and zoledronic acid on bone metastasis was varified by the compairation of bone metastasis area ratio.Results:1. The IL-6 expression in RM-1 cells was 3-6 times higher than that of control group after ISO 10 ?mol/L treated 30min.The IL-6 expression was significantly decreased when the cells were pretreated 1h by propranolol.The similar phenomenon was observed after the cells were treated by ICI118551,the ?2 singalinging pathway inhibitor. However, the inhibitory effect of ATEN,the ?1 inhibitor ,on the expression of IL-6 was weaker. The protein expression of ?2 downstream signaling pathway was detected by Western blot. Only the expression of P-AKT protein was increased after the 30 min treatment of ISO, and there was no significant change in the expression of other ?2 downstream signal pathway. The P-AKT expression was significantly decreased after pretreatment with P-AKT specific inhibitor GDC0941, and the expression of IL-6 was obviously inhibited at the genetic level.It can be observed through the wound healing assay and cell proliferation experiment that ISO can promote migration of the cells,and GDC0941 and propranolol can inhibit the ability of migration and proliferation. The propranolol had obvious inhibitory effects on MMP2 expression.2. The average hemostasis time is 2.53±1.75 min in femoral artery injection group,but is 4.70±1.63 min in external iliac artery infection group(P<0.05); the average operating time is 14.67±2.16 min, and 22.4713.50 min (P<0.05).After 21 days of the operation, the survival rate is 93.3% and 66.7% in femoral artery and external iliac artery injection groups,respectively (P<0.05). The tumor metastasis rates of both groups are 100%.3.By the use of ELISA,it can be found that the IL-6 level in the mice serum was significantly increased in stress group (P<0.01), howerver it was evidently decreased in the stress + propranolol group compaired with stress group.We analysed the mice limb bone slice and found that in the stress group the metastasis area raio was higher than the control group (P<0.01), it was significantly reduced in the propranolol + stress group compared with the stress group (P<0.01). In the second batch animal experiment,it is more effective on the inhibition of bone metastasis with the use of propranolol combined with zoledronic acid than using propranolol alone (P<0.05). There was no significant difference in the inhibitory effect of zoledronic acid in high dose group and low dose group.The use of Zoledronic acid alone had no significant effect on bone metastasis.Conclusions:1. ISO can stimulate the genetic expression of IL-6 in RM-1 cells through the P-AKT pathway.It can be inhibited by use of propranolol, ICI118551 and GDC0941.At the same time, GDC0941 and propranolol can reduce the RM-1 cells' migratory and proliferating ability .As the qPCR results show,the longer time propranolol contact with RM-1 cells,the stronger inhibition to the expression of MMP2.2. The tumor cells can specifically transfer to the leg via the femoral artery injection.This method has the advantages of short operating time, high survival rate and high success rate of tumor metastasis. Femoral artery injection is more suitable for establishment of model of cancer bone metastasis.3. Biological rhythm disorder can make level of IL-6 expression elevate in the serum of mice, and it can promote bone metastasis.This phenomenon can be inhibited by propranolol. The combination of propranolol and zoledronic acid can reduce the degree of bone metastasis.The use of propranolol alone can also inhibit bone metastasis occurred, but the effect is weaker.There is no obvious inhibiting effect by use of zoledronic acid alone. |
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