Ugonin P mitigates osteolytic bone metastasis by suppressing MDK via upregulating miR-223-3p expression

Abstract

Bone metastasis is a significant complication in advanced-stage cancers, especially breast and lung malignancies, profoundly influencing prognosis and quality of life. Osteolytic bone metastasis contains multiple interactions between cancer cells and the bone microenvironment, driving osteoclast-mediated bone resorption and deterioration while releasing growth factors that promote tumor progression. Current treatments, including surgery, radiation, and chemotherapy, often result in severe side effects, highlighting the need for effective, targeted therapies. Ugonin P, a natural compound derived from Helminthostachys zeylanica, known for its anti-inflammatory and anticancer properties. However, the effects of Ugonin P on osteolytic bone metastasis remain unclear. Our findings demonstrate that Ugonin P inhibits both RANKL-induced and lung and breast cancer-induced osteoclast formation. Bioinformatics analysis revealed that Midkine (MDK), a heparin-binding growth factor known to promote migration, is highly elevated in breast and lung cancer patients and is related with osteoclast formation. We further showed that MDK is involved in cancer-promoted osteoclastogenesis and that Ugonin P suppresses this process by upregulating miR-223-3p expression. Importantly, Ugonin P effectively blocks lung and breast cancer-facilitated osteolytic bone metastasis in vivo. These findings highlight Ugonin P as a promising therapeutic strategy for treating osteolytic bone metastasis.

Keywords: Bone metastasis; Helminthostatchys zeylanica, Ugonin P, Osteoclast.

Figure 1

Ugonin P inhibits RANKL-induced osteoclast formation. (A) Ugonin P chemical structure. (B) RAW264.7 cells were treated with Ugonin P (0, 0.1, 0.3, 1, 3 µM) for 24 hours, with cell viability assessed using an MTT assay. RAW264.7 cells were treated with RANKL (50 ng/ml) and Ugonin P (0, 0.1, 0.3, 1, 3 µM) for 5 days and stained with TRAP (C-D) (white arrow indicates osteoclasts) and F-actin ring formation (E-F). F-actin (green) and DAPI (blue). ImageJ software quantified the number of positively stained cells or mature osteoclast area. n=3 per group. *p < 0.05 compared with the control group. #p < 0.05 compared with the RANKL-treated group.

Figure 2

Ugonin P inhibits cancer-promoted osteoclast formation. (A) Steps followed to collect cancer cell-conditioned medium (CC-CM). The CC-CM was subsequently collected and applied to RAW264.7 cells, which were then incubated for 5 days. (B-D) Staining of TRAP in RAW264.7 cells treated with CC-CM (white arrow indicates osteoclasts). ImageJ software quantified the number of positively stained cells or mature osteoclast area. n=3 per group. *p < 0.05 compared with the control group. #p < 0.05 compared with the RANKL-treated group.

Figure 3

Ugonin P mitigates MDK expression in cancer cells. (A) Venn diagram showing the intersection analysis of the GEO database datasets (GSE126548, GSE191230, and GSE178196), revealing 11 potential genes. (B-C) Heatmap and KM Plot analysis of the potential genes across the datasets demonstrates MDK expression in lung cancer, breast cancer, and osteoclast differentiation. (D) Survival analysis of MDK expression in lung and breast cancer patients using the GEPIA database. (E-G) CL1-5 and MDA-MB-231 cells were treated with Ugonin P (0.1,0.3,1,3 µM) for 24 hours. MDK mRNA and protein levels were analyzed via qPCR and western blotting. n=3 per group. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group. ***p < 0.001 compared with the control group.

Figure 4

MDK is involved in Ugonin P-inhibited osteoclast formation. (A-D) CL1-5 and MDA-MB-231 cells were transfected with the MDK plasmid (Overexpress MDK- OE MDK) and treated with Ugonin P (3 µM) for 24 hours. MDK mRNA and protein levels were analyzed by qPCR, western blotting, and ELISA. (E-F). The CC-CM was collected and applied to RAW264.7 cells, which were then incubated for 5 days. Osteoclast area and number were assessed using TRAP assay. n=3 per group. *p < 0.05 compared with the CC-CM group. #p < 0.05 compared with the Ugonin P-treated group.

Figure 5

Ugonin P reduces the expression of MDK by enhancing the levels of miR-223-3p. (A) A comprehensive analysis of publicly available miRNA databases was conducted to identify miRNAs targeting MDK. (B) qPCR analysis of miRNA expression in CL1-5 and MDA-MB-231 cells treated with Ugonin P (3 µM). (C-D) Heatmap showing differentially expressed miRNAs from the GSE 135918 and GSE 57897 dataset. (E) Cells were treated with Ugonin P for 24 hours, the MDK expression was examined by qPCR. (F-G) Cells were transfected with a miR-223-3p inhibitor and subsequently treated with Ugonin P (3 µM) for 24 hours, the MDK expression was examined by qPCR and western blotting. n=3 per group. *p < 0.05 compared with the control group. **p < 0.01 compared with the control group. #p < 0.05 compared with the Ugonin P-treated group.

Figure 6

Ugonin P inhibits osteolytic bone metastasis in vivo. (A) Intra tibial injection of A549 and MDA-MB-231 cells into nude mice followed by intraperitoneally treated with Ugonin P (15 mg/kg) for alternative days for 4 weeks. (B-C) Representative IVIS images of bone metastasis at 1 and 4 weeks. (D-G) Quantification of emitted photons from each tumor by using IVIS and Mouse body weight was monitored three times a week over the four weeks. (H) After four weeks of Ugonin P treatment, all mice were humanely euthanized. The dissected legs from both the control and Ugonin P-treated groups were evaluated for tumor luminescent intensity to assess the treatment's effects. n= 4 mice per group.

Figure 7

Ugonin P reduces cancer-promoted MDK expression and osteoclast number in vivo. (A-B) Representative X-ray images of bone erosion in dissected legs. (C) Representative H&E, immunohistochemical images of MDK and TRAP-positive staining images of mouse leg bones. (D-G) The quantitative data for (C). n=3 per group. *p < 0.05 compared with the control group. **p < 0.01 compared with the control group.

Figure 8

Schematic representation illustrates the impact of Ugonin P on osteolytic bone metastasis. (The schema was generated utilizing BioRender.com). Ugonin P inhibits lung and breast cancer-promoted osteoclast formation by reducing MDK production through upregulation of miR-223-3p expression.