The Effects And Mechanisms Study Of Iguratimod Alleviating Bone Destruction Induced By Breast Cancer Bone Metastasis

Background and objective:Bone is one of the most common sites of metastasis in patients with cancer. The occurrence of bone metastasis may lead to decreased quality of life and poor prognosis. With the deeper understanding of the mechanisms underlying bone metastasis and the impact of "vicious circle" in bone microenvironment, the treatments of bone metastasis were provided important new insights. Recently, it is believed that tumor cells which invaded into bone microenvironment produced active factors including PTHrP, TNF-?, IL-1?,IL-6 and RANKL. These factors may directly impact the activity of osteoclasts or indirectly act on osteoblasts which further provide pro-osteolysis factors including RANKL. RANKL recognized the RANK on osteoclast precursors and activated downstream NF-?B and MAPK pathways, leading to the activation of osteoclasts. It is believed that the over-activation of osteoclasts induced by tumor cells is the main mechanism for cancer induced bone destruction and cytokines are involved in mediating this activation process. Iguratimod, known as a novel disease-modifying anti-rheumatic drug, has shown its anti-rheumatic effects by suppressing the production of cytokines (TNF-?, IL-1?, IL-6, IL-8 and IL-17) and inhibiting the activation of NF-?B. Given the role of NF-?B in osteoclastogenesis and the impact of cytokines on "vicious circle", we tested the effects of iguratimod on cancer induced bone destruction and explored the mechanisms.Methods:1) Rats were inoculated with Walker 256 in left tibia and treated with iguratimod for 7 days. X-ray test and H&E staining were used to detect bone destruction levels in rats' tibias. The activation of osteoclasts in rats'tibias was tested by TRAP staining. Mechanical paw withdrawal thresholds and expression levels of p-ERK and c-Fos in spinal cord were tested to detect the changes of bone pain.2) Bone marrow macrophages were isolated and cultured in vitro. Iguratimod was added into culture medium to detect the effects of iguratimod on RANKL-induced osteoclastogenesis. TRAP staining was used to investigate the formation of osteoclasts. The activation levels of NF-?B and MAPK pathways downstream RANK receptor and the expression of c-Fos and NFATcl were measured by western blot. Expressions of several target genes of NFATc1 were detected by real-time PCR.3) Real-time PCR was used to examine the influences of iguratimod on mRNA levels of cytokines produced by MDA-MB-231 cells. Phosphorylation of NF-?B p65 in iguratimod-treated MDA-MB-231 cells was detected by immunoblotting. The effects of iguratimod on the proliferation of MDA-MB-231 cells and MCF-7 cells were detected by CCK-8 assay and flow cytometry. The effects on migration and invasion of cancer cells were determined by wound-healing and transwell assay.Results:1) X-ray test and H&E staining showed that the bone destruction severity was decreased in rats treated with iguratimod. And the numbers of activated osteoclasts were also reduced in iguratimod-treated rats. Meanwhile, mechanical paw withdrawal thresholds and expression levels of p-ERK and c-Fos were declined in dose-dependent manner in rats with iguratimod 2) TRAP staining and CCK-8 assay showed that iguratimod significantly inhibited osteoclastogenesis induced by RANKL without impacting the cell viability. Furthermore, immunoblotting indicated that the activation of NF-?B and MAPK pathway induced by RANKL-RANK combination were suppressed by iguratimod, which resulted in the down-regulation of c-Fos and NFATc1. The expressions of several osteoclast-specific genes were found declined in the test of real-time PCR.3) Iguratimod decreased the transcription levels of TNF-?, IL-1? and IL-6 in MDA-MB-231 in a concentration-dependent manner and partially impaired NF-?B signaling by suppressing the phosphotylation of NF-?B p65 subunit. In addition, high dose (30 ?g/ml) iguratimod slightly suppressed the proliferation of cancer cells but failed to inhibit their migration and invasion activity.Conclusions:1) Iguratimod alleviated the bone destruction induced by breast cancer bone metastasis in a rat model.2) Iguratimod suppressed the activation of NF-?B and MAPK pathways induced by RANKL and subsequently down-regulated the expressions of osteoclast-specific genes, so that the formation and activation of osteoclasts were repressed. 3) Iguratimod may alleviate bone destruction by partially decreasing the expressions of TNF-?, IL-1? and IL-6 in an NF-?B dependent manner, while had little effect on the tumor proliferation and invasion.