Effects Of Sinomenine On Breast Cancer Cell-induced Osteolysis And The Molecular Mechanisms

Background and objectives:Bone is a common metastatic site of advanced malignancies,about 80%of which is caused by breast cancer,prostate cancer and lung cancer.Cancer bone metastases induces osteoclast activation by releasing a series of factor,resulting in osteolytic lesions,followed by a series of skeletal related events including pathological fractures,hypercalcemia,unbearable bone pain and spinal cord compression syndrome,etc.Osteoclasts are derived from bone marrow mononuclear macrophage lineage cells,which are the unique bone-resorbing cell in the body.In physiological condition,the bone resorption of osteoclasts and the bone formation of osteoblasts can maintain a dynamic balance.In the presence of stimulating factors,excessive activation of osteoclasts,will cause a series of bone destruction related diseases.It is reported that osteoclasts were over-activated in some malignancies.Therefore,development of drugs targeting osteoclasts is considered to be the most effective strategy for alleviating the bone destruction caused by tumor cells,and improving the quality of life and prognosis of patients.Currently,bisphosphonate zoledronic acid and the monoclonal antibody denosumab,as representative drugs,are used for the clinical prevention and treatment of skeletal-related events(SREs)caused by bone metastases,all of which are targeting osteoclasts.However,in recent years,studies have shown that long-term use of bisphosphonates can inhibit the natural regeneration of bone tissue,resulting in non-spinal fractures patients with delayed fracture healing,causing femoral shaft fractures.Denosumab the nuclear factor-KB receptor activator Ligand(RANKL)inhibitor,is a fully human monoclonal antibody,which works by preventing the development of osteoclasts which are cells that destroy bone.It is produced by Amgen Corporation and approved by the U.S.FDA for the prevention of bone-related events in patients with solid tumor bone metastases in November 2010.However,the drug has serious complications such as mandibular necrosis and it is also relatively expensive and requires subcutaneous injection,which causes great inconvenience to patients.So it is urgent to develop the convenient,safe and effective drugs for prevention and treatment of(SREs)caused by tumor bone metastasis.Sinomenine is a pure compound extracted from the traditional Chinese medicinal plant Sinomenium acutum,with significant analgesic and sedative properties and anti-inflammatory effects.Sinomenine has been widely used clinically in the treatment of rheumatism and arthritis.In recent years,scholars at home and abroad pay more and more attention to its role in anticancer activities.Studies have found that sinomenine has a strong inhibitory effect on breast cancer,prostate cancer,lung cancer and liver cancer and other tumors.Compared with the current clinical drugs zoledronic acid and denosumab,sinomenine has shown good analgesic efficacy.In addition,the most common adverse reactions during zoledronic acid therapy included influenza-like symptoms such as fever,joint pain,myalgia and transient bone pain.Studies have found that Celecoxib,a selective cyclooxygenase-2(COX-2)inhibitor can alleviate the above adverse symptoms.Sinomenine,as a selective COX-2 inhibitor,can also be used to prevent the influenza-like symptoms adverse reactions caused by administration of zoledronic acid.In addition,our previous findings have suggested that sinomenine can inhibit RANKL and LPS-induced osteoclastogenesis.These datas suggest that sinomenine might suppress tumor-induced osteoclast formation,which has not been reported.Therefore,the objective of our study is to investigate the effect of sinomenine on breast cancer cell MDA-MB-231-induced osteolytic damage and elucidate the molecular immune mechanism.Methods:1.To value the effects of sinomenie on breast cancer cell MDA-MB-231 induced osteolyis in vivo.The human MDA-MB-231 breast cancer cells were injected directly into the tibias of female nude mice.Four weeks later,mice were sacrified and tibias were excised and processed for Micro-CT analysis and histologic examination.Micro-CT analysis was used to detect the tibial bone parameters(BMD,BV/TV,Tb.N and Tb.Sp)and HE staining was used to evaluated the effect of sinomenine on the trabecula bone structures in nude mice.2.To observe the effect of sinomenine on the breast cancer cell MDA-MB-231 and conditioned media of cultured breast cancer cell MDA-MB-231(CM)-induced osteoclastogensis in pre-osteoclastic RAW264.7 cells in vitro.MTT assay was used to determine the cytotoxicity of sinomenine on RAW264.7 cells.The effect of sinomenine on MDA-MB-231 and CM-induced the differentiation of RAW264.7 cells into osteoclasts by TRAP staining and the regulation of sinomenine on CM-induced osteoclast resorption activity by bone resorption pits assay.The effect of sinomenine on CM-induced osteoclast-related gene expression was detected by qRT-PCR analysis.3.To investigate the mechanism of sinomenine on CM-induced osteoclastogensis in pre-osteoclastic RAW264.7 cells.The effect of sinomenine on the expression of NF-?B transcriptional activity by NF-?cB luciferase reporter gene assay.Western bloting analysis was used to examine the protein expression of NF-?B,MAPKs(p38,JNK and ERK 1/2),c-Fos,and NFATc1 and qRT-PCR analysis was used to measure the gene expression level of c-Fos and NFATc1 in RAW264.7 cells.4.To investigate the effect of sinomenine on the cytokines secreated by MDA-MB-231 cells that can induce osteoclastogensis.We evaluated the effects of sinomenine on the gene expression of TNF-a,IL-1,TGF-?,PTH-rP,M-CSF,IL-6 and IL-8 were detected by qRT-PCR analysis in MDA-MB-231.And then,we further examined the effect of sinomenine on IL-8 protein secretion by MDA-MB-231 cells using ELISA.Finally,in order to confirm the inhibiting function of sinomenine on the IL-8 signaling pathway in MDA-MB-231-induced osteoclast formation,we used qRT-PCR analysis to detect the effect of sinomenine on the expression of IL-8 receptor CXCR1 in RAW264.7 cells.Results:1.Sinomenine can inhibit MDA-MB-231 cells-induced bone destruction in vivo.Micro-CT analysis and HE staining showed that compared with the model group,150 mg/kg sinomenine could effectively inhibit the level of bone erosion induced by breast cancer cells.2.Sinomenine can inhibit MDA-MB-231 cells and the conditioned medium(CM)-induced osteoclast precursor cells differentiation into osteoclasts in vitro.MTT assay revealed that there was no obvious toxic effect on RAW264.7 cells when the dosage of sinomenine was less than 1mM.TRAP staining showed that sinomenine could significantly inhibit the formation of osteoclasts induced by MDA-MB-231 cells and the conditioned medium compared with the control group.The bone resorption pit assay indicated that compared with the control group,0.5mM sinomenine can reduce the bone resorption area.The qRT-PCR analysis indicated that compared with the control group,sinomenine could significantly reduce the gene expression of TRAP and OSCAR,which were important genes in the process of osteoclast differentiation.3.Sinomenine can suppress osteoclast formation by directly decreasing the expression of downstream protein c-Fos and transcription factors NFATcl induced by conditioned medium in RAW264.7 cells,not by NF-?B and MAPKs signaling pathways.NF-?B luciferase reporter gene results showed that 0.25-1 mM sinomenine could not inhibit the increase of CM-induced NF-?B luciferase reporter gene expression in RAW264.7 cells.The results of Western bloting demonstrated that compared with the control group,0.25-1 mM sinomenine did not affect the phosphorylation of NF-?B p65 and MAPKs(JNK,ERK,p38)in RAW264.7 cells.However,sinomenine could significantly inhibit the protein expression of c-Fos and NFATcl in RAW264.7 cells.The qRT-PCR analysis was futher confirmed that sinomenine regulated the expression of c-Fos and NFATcl in RAW264.7 cells.4.In addition to the downstream signal of osteoclast formation,sinomenine can also significantly reduced the secretion of IL-8 from breast cancer cell MDA-MB-231 and the expression of CXCR1 in the osteoclast precursor cell RAW264.7.The qRT-PCR analysis declared that sinomenine significantly inhibited the gene expression levels of IL-8 in MDA-MB-231 cells.Furthermore,it was found that sinomenine could suppress the protein expression levels of IL-8 in MDA-MB-231 cells,and that of IL-8 in breast cancer cells and osteoclast precursor cells co-culture system by ELISA.The results of qRT-PCR analysis showed that sinomenine could significantly inhibit the expression of IL-8 receptors CXCR1 in osteoclast precursor cells.Conclusions:1.In vivo,sinomenine can inhibit MDA-MB-231cells-induced bone destruction.2.In vitro,sinomenine can inhibit MDA-MB-231 cells and the conditioned medium-induced osteoclast precursor cells differentiation into osteoclasts.3.Sinomenine can inhibit MDA-MB-231 cell-induced osteoclast formation and osteolysis through the IL-8-CXCR1 and c-Fos-NFATcl signaling pathways.