A Rat Model Of Metastatic Bone Cancer Pain Produced By Walker256 Mammary Gland Carcinoma Cells And The Mechanisms Of Ibandronate

Objective: Breast cancer is the most common malignancy in women, which is prone to distant metastasis. Bone is the common site of distant metastases in breast cancer. There are some common complications with bone metastases in breast cancer, such as pain, bone injury, skeletal related events (SREs), and the loss of quality of life and so on, which seriously affect patient quality of life.Bone cancer pain is the most serious of cancer pain, which mainly attributed to primary bone tumor or bone metastases in lung cancer, breast cancer and prostate cancer, which seriously affects patient quality of life. Before Schwei first reported bone cancer pain model in mice in 1999, the research about cancer pain had been on the model of non-cancer chronic pain. Actually, the pathogenesis of the two are not the same, the effective drug in neuropathic pain can not alleviate cancer pain. In recent years, using the method of injecting different tumor cells into bone marrow cavity of tibia, successfully established tibia cancer pain model, which could be regarded as a metastatic bone cancer pain model, and which provided an effective means to the clinical research on the mechanism, prevention and treatment of bone cancer pain.At present, the diagnosis of bone metastasis in breast cancer mainly depends on imaging methods, such as X-ray, CT, ECT and so on. The diagnosis rate is low. Bone-carb-oxyglu- tamic acid-containing protein (BGP) is a vitamin K-dependent protein, which is synthesized and secreted by osteoblasts. It is reported that, the serum BGP may change in bone metastases of malignancy, which has diagnostic value in bone metastasis of malignancy. And it is reported that tumor-induced bone destruction activity produce some cytokines (such as ET-1, PGE₂), which not only lead to osteolysis, but also sensitize peripheral nerve endings, thus increase the body's sensitivity to pain stimuli. Ibandronate belongs to the third generation of bisphosphonate, which can significantly reduce bone-related incidents of bone metastases in breast cancer, particularly alleviate pain, and improve quality of life.Therefore, we use Walker256 rat mammary gland carcino- ma cells to establish tibia cancer pain model. At the foundation of which, we studied the changes of pain behavior, bone pathological changes in histology; measured serum BGP levels, detecting whose diagnostic value in bone metastases of breast cancer, so as to achieve the early diagnosis in bone metastases of breast cancer; observed the analgesic effect of ibandronate; observed the changes of cytokines ET-1,PGE₂ in the formation of bone cancer pain and after treatment, so as to explore the possible molecular mechanisms of the formation of bone cancer pain, as well as the role of bisphosphonate plays, and lay the foundation for the future development of molecular targeted therapy of bone cancer pain.Methods:60 Wistar rats (160-180g) were randomly divided into three groups, which were PBS group, observation group and treat- ment group, 20 per group. For the each rat of observation group and treatment group, we used micro-injector to inoculate 4ul walker256 mammary gland carcinoma cells (4*104 cells) into the bone marrow cavity of left tibia to establish rat tibia cancer pain model; for the each rat of PBS group, we used micro-injector to inoculate the same volume of sterile PBS solution into the bone marrow cavity of left tibia. From post implantation day 8 (PID8), we used ibandronate by subcutane- ous injection into rats of treatment group, 4ug/d, once a 10 days; meanwhile we used the same volume of normal saline by subcutaneous injection into rats of the other two groups, once a 10 days. On PID 0, 6, 8, 11, 14, 17, we carried out the observation of general state, freely walk pain score, as well as paw withdrawal reaction time. On PID 17, we measured the concentration of serum BGP, plasma ET-1, and plasma PGE₂, as well as rat left tibia HE staining. All data was showed by Mean±SD. The data was analyzed with the SPSS13.0 software.Results:1 The general condition of rats in each groupThroughout the observation period, no rat died. All rats had no significant difference in mental condition, consumption of food and water in each group before and after establishing models, the weight of all rats was slowly increasing in the observation period.2 The changes in pain behaviors2.1The score of freely walk painAfter establishing models, the score of rats in PBS group gradually increased, up to 1, but from PID 6 gradually decreased back to normal; the score of rats in observation group and treatment group also gradually increased after establishing models, but increased more rapidly than PBS group, up to 4; which was significantly different from PBS group on PID 6 (F = 12.83, P<0.01); there was no significant difference between observation group and treatment group within PID 14, the score of treatment group decreased significantly compared with observation group on PID 14 and PID 17, but which was still significantly higher than PBS group.2.2 The reaction time of paw withdrawalThe reaction time of observation group and treatment group decreased significantly on PID 8 (P<0.01), compared with PBS group, there was significantly statistical significance.The reaction time of treatment group extended on PID 14, which was significantly longer than observation group (P<0.01), but which was still significantly shorter than PBS group (P<0.05); there was no significantly statistical significance between treatment group and PBS group on PID 17 (P> 0.05). 2.3 Bone pathological changes in histology (HE staining)PBS groups: A variety of normal bone marrow cells could be seen in the bone marrow cavity, no changes in bone structure; Observation group: Bone marrow cavity was almost filled with tumor cells with large deeply stained nuclear, most of which were active, some of which were necrotic, bone trabecula was rare; Treatment groups: We could see a few active tumor cells, most of tumor cells were necrotic, and bone trabecular was seriously damaged.2.4 Comparison of serum BGP concentration among each groupThe concentration of serum BGP in observation group was significantly higher than PBS group (P<0.01), there was significantly statistical significance between the two. The concentration of serum BGP in treatment group decreased significantly compared with observation group (P<0.05), but which was still significantly higher than PBS group (P <0.05).2.5 Comparison of plasma Endothelin-1 (ET-1) concentrat- ion among each groupThe concentration of plasma ET-1 in observation group was significantly higher than PBS group (P<0.01), there was significantly statistical significance between the two. The concentration of plasma ET-1 in treatment group decreased significantly compared with observation group (P <0.01), and there was no significantly statistical difference compared with PBS group. 2.6 Comparison of plasma prostaglandinE₂ (PGE₂) concen- tration among each groupThe concentration of plasma PGE₂ in observation group was significantly higher than PBS group (P<0.05), there was significantly statistical significance between the two. The concentration of plasma PGE₂ in treatment group decreased significantly compared with observation group (P<0.01), and there was no significantly statistical difference compared with PBS group.Conclusion:1 This study successfully established an animal model of bone cancer pain, which is similar to the pain attributed to bone metastases in human breast cancer, which lays the foundation to further study on the mechanism of bone cancer pain.2 Serum BGP has diagnostic value for bone metastasis in breast cancer.3 Bone cancer pain is associated with the increased secretion of ET-1, PGE₂; ET-1 and PGE₂ may become the targeted spots for future development of molecular targeted drugs in bone cancer pain.4 Ibandronate on the Walker256 cell-induced bone cancer pain model has analgesic effect in rats, which may be associated with the reduced secretion of ET-1, PGE₂.