Simin Liu,1,* Zhiyuan Tian,2 Taiming Zhang,3 Jirong Zhang,3 Yanlei Huo,1,* Chao Ma1 

1Department of Nuclear Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China; 2Department of Nuclear Medicine, Zibo Ninth People’s Hospital, Zibo, Shandong, People’s Republic of China; 3Department of Medical Imaging, People’s Hospital of Shuangjiang Autonomous Country, Lincang, Yunnan, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Chao Ma, Department of Nuclear Medicine, Shanghai Tenth People’s Hospital, Tongji University, Yanchangzhong Road 301, Shanghai, 200072, People’s Republic of China, Tel +86 21-66302075, Email mc_7419@hotmail.com

Objective: To explore analgesic effect and bone repair mechanism of non-radioactive technetium-99 conjugated with methylene diphosphonate (99Tc-MDP, brand name, Yunke) on bone metastases (BM).
Procedures: In vivo experiment, mouse BM models of prostate cancer RM-1 cell were constructed and divided into Control, Yunke, 99Tc+SnCl2 and MDP groups based on medicine composition. Tumor specimens were inspected for size, X-ray, microCT and histopathology. In vitro experiment, with Cell Counting Kit-8 (CCK8), scratch, clone, apoptosis, Polymerase Chain Reaction (PCR) and Western Blot experiments, effects of Yunke on RM-1 cells and osteoclast-related cells were observed.
Results: In vivo experiment, there was no difference in tumor size between Yunke and control group. Contrasted with control group, in Yunke group, trabecular spacing (Tb.Sp) of tumor bone was lower, bone volume/total volume (BV/TV) on marrow cavity and bone cortex were higher. Tunnel staining showed that positive rate of apoptosis in Yunke group was higher than that in control group. Ki67 staining showed that Yunke could not inhibit proliferation of tumor cells. In vitro experiment, CCK8 and scratch experiments showed that Yunke neither can inhibit proliferation nor can inhibit migration of RM-1 cells. High concentration of Yunke promoted late apoptosis of RM-1 cells. Yunke could inhibit BMM cell proliferation, differentiation of osteoclasts, and osteoclast-related transcription factors. Yunke displayed different degrees of inhibitory effects on MAPKs signaling pathway during osteoclast differentiation. It had obvious inhibitory effects on osteoclast-related transcription factors, such as cFOS, NFATC1, ACP-5, CTSK, D2 and MMP-9, the strongest inhibitory effects were observed with ACP-5, CTSK and D2. Yunke also displayed different degrees of inhibitory effects on protein activities of JNK, pERK, ERK and pP38.
Conclusion: Yunke cannot inhibit the proliferation and migration of RM-1 cells, so we think it is not recommended for the treatment of primary tumors and prevention of occurrence of tumors metastatic to bones. The mechanism of therapeutic effect of Yunke on BM by inhibiting proliferation of BMM, inhibiting MAPKs signal transduction and activation of transcription factors during differentiation process of BMM-derived osteoclasts, inhibiting number and size of osteoclasts, inhibiting bone resorption and protecting bone destruction through enhancing bone hardness and bone mass. Thereby, we believe that Yunke is more suitable for promoting the repair induced by BMs, delaying its progression and reducing the occurrence of SREs.

Keywords: 99Tc-MDP, bone metastases, molecular mechanisms, RM-1, prostate cancer