Osteorin attenuates osteolysis during bone metastasis of cancers through inhibiting osteoclastogenesis

doi:10.3877/cma.j.issn.1674-0807.2023.06.002

Abstract

Abstract:

Objective

To investigate the effect of osteocrin on osteoclastogenesis and osteolysis during bone metastasis of breast cancer (BC) or colorectal cancer (CRC).

Methods

In vivo experiments: 4T1 BC cells or MC-38 CRC cells were injected into medullary space of tibial of Balb/c female mice or C57BL/6 male mice with treatment of osteocrin (osteocrin group) or vehicle (control group), respectively. The samples were harvested at 14 or 21 days after injection. Safranin O-Fast green staining was used to detect the trabecular area and TRAP staining was utilized to detect the number of osteoclasts. Furthermore, the severity of bone loss was detected by micro-CT. In vitro experiments: osteoclastogenesis was induced under the stimulation of conditional medium collected from 4T1 cells or MC-38 cells in the presence of receptor activator of nuclear factor-kappa B ligand (RANKL)and macrophage colony-stimulating factor (M-CSF) and treated by osteocrin or vehicle. The TRAP staining and ELISA were used to detect the number of osteoclasts and TRAP protein level. The real-time PCR were performed to detect the level of osteoclastogenic biomarkers. Western blot was performed to analyze the effect of osteocrin on the phosphorylation of AKT. The number of osteoclasts, bone mineral density or bone volume markers were compared between two groups using t test, among multiple groups using one-way analysis of variance.

Results

The results of in vitro experiments showed that the number of osteoclasts in the culture environment of 4T1 and MC-38 cell conditioning medium was significantly lower in the osteocrin group than that in the control group [4T1: 4.60±1.14 vs 10.60±1.67, t=6.626, P<0.001; MC-38: 4.00±1.00 vs 10.20±1.92, t=6.395, P<0.001). The results of in vivo experiments showed that in the breast cancer bone metastasis model of mice, the proportion of osteoclasts area to trabecular bone area was significantly lower in the osteocrin group than in the control group [14 d: (5.55±1.56)% vs (12.79±1.08)%, t=7.640, P<0.001; 21 d: (7.21±1.41)% vs 16.96±2.00)%, t=7.966, P=0.001); in the colorectal cancer bone metastasis model of mice, the proportion of osteoclasts area to trabecular bone area was significantly lower in the osteocrin group than in the control group [14 d: (5.57±0.87)% vs (10.69±0.64)%, t=9.500, P<0.001; 21 d: (8.47±0.86)% vs (15.53±2.81)%, t=4.805, P=0.003). Real-time PCR showed that the mRNA expression levels of two osteoclast differentiation markers OCSTAMP and NFATC1 in the osteocrin group were significantly lower than those in the control group (OCSTAMP: 4T1, t=10.018, P=0.001; MC-38, t=24.537, P<0.001; NFATC1: 4T1, t=3.356, P=0.028; MC-38, t=3.377, P=0.028). Compared with the control group, the phosphorylation level of AKT protein in the osteocrin group was significantly decreased (4T1: t=3.289, P=0.030; MC-38: t=5.363, P=0.006). Treatment with SC79 (AKT pathway activator) reversed the ability of osteocrin to inhibit osteoclast formation (4T1: F=8.598, P=0.005; MC-38: F=9.125, P=0.007). Micro-CT scanning showed that bone marrow density was significantly improved after treatment of osteocrin in bone metastasis models of BC or CRC, respectively.

Conclusions

Osteocrin can prevent osteolytic lesions during bone metastasis of BC and CRC through directly inhibiting osteoclast formation via downregulation of AKT pathway.

Key words: Breast neoplasms, Colorectal neoplasms, Metastasis, Osteoclasts

Xia Kang, Hao Tian, Jin Qian, Yuan Gao, Hongming Miao, Xiaowei Qi. Osteorin attenuates osteolysis during bone metastasis of cancers through inhibiting osteoclastogenesis[J]. Chinese Journal of Breast Disease(Electronic Edition), 2023, 17(06): 329-339.

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URL: https://zhrxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-0807.2023.06.002

https://zhrxbzz.cma-cmc.com.cn/EN/Y2023/V17/I06/329

Figures/Tables 21

References 31

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基因名称	上游引物序列(5′-3′)	下游引物序列(5′-3′)
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
OCSTAMP	GTCTTCCCGCTGGCCATGAACTAC	GGAAGCGTGTGAGGCGGTAGTAGG
NFATC1	AGTACTGCCATCCGAGAGA	GAATGCCAGTGAGAGCAGAG

基因名称	上游引物序列(5′-3′)	下游引物序列(5′-3′)
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
OCSTAMP	GTCTTCCCGCTGGCCATGAACTAC	GGAAGCGTGTGAGGCGGTAGTAGG
NFATC1	AGTACTGCCATCCGAGAGA	GAATGCCAGTGAGAGCAGAG

条件培养基	样本数	对照组	骨织素组	t值	P值
4T1	3	41.66±1.38	35.24±0.32	7.843	0.001
MC38	3	44.58±1.54	33.71±0.69	11.142	<0.001

条件培养基	样本数	对照组	骨织素组	t值	P值
4T1	3	41.66±1.38	35.24±0.32	7.843	0.001
MC38	3	44.58±1.54	33.71±0.69	11.142	<0.001

标志物	条件培养基	样本数	对照组	骨织素组	t值	P值
OCSTAMP	4T1	3	1.00±0.15	0.08±0.04	10.018	0.001
	MC-38	3	1.00±0.07	0.06±0.01	24.537	<0.001
NFATC1	4T1	3	1.00±0.46	0.10±0.03	3.356	0.028
	MC-38	3	1.00±0.18	0.53±0.16	3.377	0.028

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