The Effect Of PTHrP(1-34) On Bone Metastasis Of Mouse Breast Cancer Cells

Objective Bone metastases can occur in many types of cancer, such as breast carcinoma, lung canser and renal cell carcinoma. The interaction of tumor cells, bone-forming osteoblasts, bone-resorbing osteoclasts and the bone matrix stimulate the establishment and development of cancer osteolytic bone metastases. Tumor cells secrete factors that stimulate bone metastases and make bone destruction, The bone matrix contains a vast storehouse of growth factors which are synthesized by osteoblasts and released by osteoclastic bone resorption, attain high concentrations within the bone microenvironment and can attract tumor cells to bone and promote metastatic growth. Many of the factors involved in this vicious cycle such as PTHrP, TGF-β, RANKL remain areas of active investigation. Parathyroid hormone-related protein (PTHrP) is secreted by a variety of cells and holds extensive biological functions. Some studies show that PTHrP is overexpressed by a variety of tumors, it is one of the main mediated factor of osteoclast activity, inducing osteoclast differentiation and maturation, playing an important role in the establishment and development of breast cancer osteolytic bone metastases. Take breast canser as an example, Most studies show that PTHrP is a major candidate factor responsible for the osteoclastic bone resorption present at sites of breast carcinoma metastatic to bone. However,some studies show that PTHrP-positive tumors were independently predictive of improved patient survival, PTHrP production by breast cancer cells confers on them a less malignant phenotype, one that is less invasive and less likely to result in metastasis formation at all sites, including bone.In this experiment we establish mouse breast cancer model to investigate the effect of persistent high-dose PTHrP on breast cancer bone metastasis and bone metabolism by X-ray, pathological section and testing biochemical markers of bone turnover, biomechanical response, as well as to observe tumor growth. Lay a foundation for the development of anti-PTHrP antibody to inhibit tumor bone metastasis.Methods In preliminary test, establishing mice model of breast cancer by injecting tumor cells suspension and by injecting tumor tissue mass suspension respectively to select a better method of establishing mice model, and selected one mouse randomly in each group to do pathological sections of the tumor to confirm, the conclusion was that the later is the better method. Fourty female health BALB/C mice about 4-6 weeks old were randomly divided into 3 groups, including control group (number12), model group (number14)and experimental group(number14), The last two groups were designed for establishing mouse model of breast cancer by injecting tumor tissue mass suspension, 10 days later, take mice whose tumor diameter greater than 4mm long as successful mice model. finally, there were 12 mice selected in each group. Then model group was to be injected with normal saline intraperitoneally every day, while experimental group with PTHrP 400μg/kg. after 40 days, take whole body bone X-ray, select the suspected bone lesions to make decalcification paraffin section, HE staining, to observe pathology. measure whole body bone mineral density (BMD), the biomechanical parameters and the bone metabolic related indices, such as serum Ca, P,ALP,β-CTX, BGP and BSP. Divest tumor to comparing tumor size and weight. Values are expressed as mean±SD. Statistical significance was evaluated by Independent–Samples T test for comparison. Inter-group comparison uses one-way ANOVA, using four table data Exact Test to compare the rate. All statistical tests were performed on raw data by SPSS 13.0 process.Results The method of establishing mouse model of breast cancer by injecting tumor tissue mass suspension is better than by injecting tumor cells suspension. X-ray showed that there were obvious osteolytic bone destruction in experimental group (including 1 fracture), the same as visual study, and decalcified bone paraffin section confirmed it was a metastasis. Compared with control group and model group, BMD and biomechanical parameters of the experimental group (including peak load, elastic load, cross sectional moment of inertia, elastic modulus of femur and peak load of lumbar decreased rapidly by 12.37%, 11.72%, 19.4%, 26.16%, 34.71% and by 13.12%, 15.61%, 17.24%, 15.47%, 32% respectively) had significant differences (P < 0.01). Serum calcium level of different groups showed no statistical differences ( P > 0.05). compared with the other two group, P, BSP,β-CTX levels of experimental group were significantly higher(increased by 31.5%, 31.58%, 81.25% and 21.92%, 14.93%, 58.82% respectively), while ALP and BGP levels were significantly lower(decreased by 19.57%, 17.43% and 12.48%, 11.54% respectively()P<0.01 or P<0.05), but there was no significant difference between control group and model group (P > 0.05). The tumor size and weight of experimental group were significantly greater than the model group[1.123±0.192(g)vs 0.859±0.176(g); 824.18±189.79(cm3)vs 297.07±106.70(cm3)].Conclusion X-ray ,pathological section ,biochemical markers of bone turnover and biomechanical response can be taken as diagnostic standard of tumor bone metastasis .PTHrP1-34 can promote breast cancer osteolytic bone metastases, reduce bone mineral density and change the bone biomechanical properties. It is a major candidate factor responsible for the osteoclastic bone resorption present at sites of breast carcinoma metastatic to bone. In addition, some cell factors released during bone destruction may promote tumor growth.