| Background:Breast cancer, along with lung and prostate cancers, is prone to metastasize to bone. Once bone metastases have been established, tumor cells directly or indirectly activate osteoclasts and result in bone destruction, with a clinical outcome characterized by intractable pain, nerve compression syndromes, increased risk of fractures, and hypercalcemia, and as a result, the chance of survival and the quality of life of the patient dramatically drop. Bone tissue, different from other tissues, is mainly formed by mineral substances that are relatively harder, and therefore, bone tissue shall have greater resistance to invasion of tumor cells. It is currently recognized that bone lesions in tumor bone metastases, instead of being caused by tumor cells directly, actually result from bone resorption induced by osteoclasts. Therefore, to cause destruction to bone tissue, tumo r cells must have certain ability to activate osteoclasts so as to further damage bone tissue. Extensive research has revealed that tumor cells can produce factors that induce osteoclastogenesis, namely, PTHr P and interleukins(IL)-1, IL-6 and IL-11, which act on osteoblasts to increase the production of the receptor activator of NF-κB ligand(RANKL), which is considered to be a key factor in osteoclast differentiation and plays an important role in cancer-associated bone resorption.Osteoclasts are derived from bone marrow stem cells and are effector cells of bone resorption. Tumor bone metastases are generally osteolytic in nature and rely on the ability of tumor cells to activate osteoclast bone resorption. Therefore, agents that can suppress osteoclast differentiation have therapeutic potential. Some drugs, including bisphosphonates, teriparatide and denosumab, have been used for bone treatment. However, apart from their positive effects on treatment of osteolytic bone lesions, most of these drugs have some limitations or side effects, such as osteonecrosis, thromboembolism, osteosarcoma and esophageal irritation. Therefore, there is a need to develop or discover new drugs so as to improve bone metastases therapy.Previously, we found that Quetiapine(QUE), a commonly used atypical antipsychotic drug, can promote the differentiation of neural progenitors of the oligodendrocyte lineage through the MAPK signaling pathway. Meanwhile, there are studies demonstrating that QUE can also function to regulate the NF-κB signaling pathway. The MAPK and NF-κB signaling pathways are both implicated in osteoclast differentiation. Thus, we asked whether QUE can, by suppressing the MAPK and NF-κB signaling pathways, inhibit RANKL-induced osteoclast differentiation and potentially prevent osteolytic bone lesions induced by tumor cells. This study is intended to propose novel therapeutic strategies for osteolysis as in tumor bone metastases.Objectives:1. Use models of RANKL-induced osteoclast differentiation from RAW 264.7 cells and Bone marrow-derived macrophages(BMMs) to test whether QUE can suppress osteoclastogenesis in vitro. 2. Use mouse models of tumor bone metastases to further test whether QUE can suppress activation of osteoclasts induced by tumor cells to thereby provide a bone-protecting effect. 3. Use models of RANKL-induced osteoclast differentiation from RAW 264.7 cells to investigate the molecular mechanisms by which QUE suppresses osteoclastogenesis.Methods:1. RAW 264.7 cells were cultured in culture media DMEM and α-MEM to test the effect of these culture media on proliferation and survival of RAW 264.7 cells, so as to select a suitable culture medium to be used in subsequent experiments. RAW 264.7 cells were stimulated with different concentrations of RANKL(10, 30, 50, 100, 200ng/ml), so as to select an optimum concentration of RANKL that promotes osteoclastogenesis. 2. BMMs were obtained by differential adherence sorting and gradient centrifugation, respectively, and were dosed with different concentrations of RANKL and M-CSF, so as to select an optimum method for obtaining cells and an appropriate cytokine concentration. 3. CCK-8 assay was performed to detect cell proliferation after incubation of RAW 264.7 cells with different concentrations of QUE(1μM, 10μM, 25μM, 50μM, 100μM) for 48 hours. Previously built models of RANKL-induced osteoclast differentiation were used to observe whether QUE can suppress osteoclastogenesis. Models of RANKL-induced osteoclast differentiation were treated with different concentrations of QUE(1μM, 10μM, 25μM, 50μM), and real-time quantitative PCR was performed to test effect of QUE on expression of osteoclast related genes in the process of osteoclast differentiation induced by RANKL. 4. QUE was added to osteoclast differentiation cultures beginning at day 1, day 2, day 3, and day 4, respectively, so as to test at which stage QUE functions to suppress osteoclast differentiation. 5. Immunodeficient mice were purchased to build models of bone metastases of breast cancer MDA-MB-231 cells by intramedullary injection of tumor cells. QUE(10mg/kg/d) was given through intraperitoneal injection for 6 weeks, and the mice were subjected to an X-ray system to assess the protective effect of QUE against bone lesions induced by tu mor bone metastases. H&E and TRAP staining were performed to assess tumor cell invasion and the effect of QUE in suppressing activation of osteoclasts induced by tumor bone metastases. 6. Using the models of RANKL-induced osteoclast differentiation, western blot and immunocytochemical staining were performed to assess the effect of QUE on MAPK and NF-κB signaling pathways in the process of RANKL-induced osteoclast differentiation.Results:1. Compared to the α-MEM medium, DMEM medium was more advantageous to RAW 264.7 cell proliferation, survival, and maintaining of the form. Different concentrations of RANKL were used in the process of osteoclast differentiation from RAW 264.7 cells, when the concentration was lower than 30 ng/ml, osteoclast differentiation induced by RANKL was limited, when the concentration was higher than 50 ng/ml, the effect of RANKL inducing osteoclast differentiation was obviously enhanced, and the difference from 50 ng/ml to 200 ng/ml was not obvious. RAW 264.7 cells were treated by RANKL(50 ng/ml) for 5 days, TRAP staining showed a large number of TRAP positive multinucleated osteoclast-like cell formation. 2. Compared to gradient centrifugation and magnetic bead separation methods, differential adherence method was simple and easy. BMMs isolated by the method of differential adherence could eventually differentiate into the TRAP positive multinucleated osteoclast-like cells in the presence of RANKL and M-CSF. The concentration of RANKL in 50 ng/ml and M-CSF in 25 ng/ml promoted osteoclast differentiation from BMMs. 3.Treatment of QUE in the concentration of between 1 and 50 μM showed no obvious toxic effect on the RAW 264.7 cell proliferation, which was tested by CCK-8 cell proliferation testing kit. Furthermore, under the concentration of 50 μM QUE RAW 264.7 cells were treated for 3 days and 5 days, BMMs were treated for 3, 5 and 7 days, the results showed: 50 μM QUE had no effect on the cell proliferation both in RAW 264.7 cells and BMMs. 4.Compared with the positive control group which was treated by RANKL alone, QUE in the concentrations of 1 μM and 10 μM showed no significant effect on the process of osteoclast differentiation. But when QUE was used in the concentrations of 25 μM and 50 μM, QUE could significantly suppress osteoclast differentiation from the RAW 264.7 cells induced by RANKL. Furthermore, BMMs were treated with QUE(50μM) for 7 days in the presence of RANKL and M-CSF, TRAP staining showed that: 50μM QUE could effectively suppress osteoclast differentiation from BMMs. Real-time quantitative PCR detection showed: 25μM and 50μM QUE could effectively inhibit the expression of osteoclastogenesis-related genes in RAW 264.7 cells and BMMs induced by RANKL. 5.QUE(50μM) could effectively inhibit osteoclast differentiation when QUE was added in the medium on day 1 and day 2 in the process of osteoclast differentiation from RAW 264.7 cells, but QUE showed no inhibitory effect when it was added in the medium on day 3 and day 4. 6.After treated by QUE for 6 weeks, breast cancer bone metastases mice were detected by X-ray. Results hinted QUE could significantly reduce bone destruction induced by tumor cells to thus provide bone protective effect; H&E and TRAP staining showed that there was a large scale of tumor cell invasion in both the experimental group and control group, but compared with the control group, QUE treatment significantly reduced the number of osteoclasts activated by tumor cells. 7. Effect of QUE on MAPK signaling pathway in the process of osteoclast differentiation was tested by western blot. The results showed that QUE suppressed RANKL-induced phosphorylation of p38, ERK and JNK during osteoclast differentiation. 8.In RANKL-induced osteoclast differentiation, IκBα in the NF-κB signaling pathway was degraded significantly under the effect of RANKL, while QUE could significantly suppress IκBα degradation and meanwhile suppress phospho-p65 level. Immunocytochemical staining showed: RANKL could promote nuclear translocation of p 65 between 15 and 60 minutes, while nuclear translocation of p65 was suppressed upon QUE treatment.Conclusions:1. Models of RANKL-induced osteoclast differentiation from RAW 264.7 cells and BMMs are successfully built, which provide a sound experimental basis for subsequently studying the effect of QUE in osteoclast differentiation and the mechanism thereof. 2. In osteoclast differentiation from RAW 264.7 cells induced by RANKL and osteoclast differentiation from BMMs induced by RANKL and M-CSF, QUE can significantly suppress the differentiation of osteoclast progenitor cells into osteoclasts and suppress the expression of osteoclast related genes in the process of osteoclast differentiation. 3. QUE presents suppressive effect in the early stage of osteocl ast differentiation. 4. In tumor bone metastases induced by breast cancer MDA-MB-231 cells, QUE can significantly suppress activation of osteoclasts induced by tumor bone metastases, and can thus further suppress bone lesions induced by tumor bone metastases. 5. Suppressing the activation of the MAPK and NF-κB signaling pathways during RANKL-induced osteoclast differentiation contributes to the anti-osteoclastogenic activity of QUE. |
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