Ononin Inhibits Tumor Bone Metastasis and Osteoclastogenesis By Targeting Mitogen-Activated Protein Kinase Pathway in Breast Cancer

Abstract

Breast cancer (BC) often spreads to bones, leading to bone metastasis (BM). Current targeted therapies have limited effectiveness in the treatment of this condition. Osteoclasts, which contribute to bone destruction, are crucial in supporting tumor cell growth in the bones. Breast cancer bone metastasis (BCBM) treatments have limited efficacy and can cause adverse effects. Ononin exhibits anticancer properties against various cancers. The study examined the impact of ononin on the BCBM and the signaling pathways involved. Our study utilized a variety of experimental techniques, including cell viability assays, colony formation assays, wound-healing assays, Transwell migration assays, Western blot analysis, and tartrate-resistant acid phosphatase (TRAP) staining. We examined the effects of ononin on osteoclastogenesis induced in MDA-MB-231 conditioned medium- and RANKL-treated RAW 264.7 cells. In a mouse model of BCBM, ononin reduced tumor-induced bone destruction. Ononin treatment effectively inhibited proliferation and colony formation and reduced the metastatic capabilities of MDA-MB-231 cells by suppressing cell adhesion, invasiveness, and motility and reversing epithelial-mesenchymal transition (EMT) markers. Ononin markedly suppressed osteoclast formation and osteolysis-associated factors in MDA-MB-231 cells, as well as blocked the activation of the mitogen-activated protein kinase (MAPK) pathway in RAW 264.7 cells. Ononin treatment down-regulated the phosphorylation of MAPK signaling pathways, as confirmed using MAPK agonists or inhibitors. Ononin treatment had no adverse effects on the organ function. Our findings suggest that ononin has therapeutic potential as a BCBM treatment by targeting the MAPK pathway.

Fig. 1.

Antiproliferative effects of ononin on MDA-MB-231 cell lines. (A) Chemical structure formula of ononin. (B) Ononin was administered to MDA-MB-231 cell lines at concentrations ranging from 0 to 80 μM over periods of 24 and 48 h, subsequent to which MTT assays were conducted. (C) Results from the clonogenic assays were recorded for these cells after exposure to various concentrations of ononin and DOX. (D) Colonies were quantified after exposure to ononin and DOX at the specified concentrations. (E) Wound-healing assay results following separate treatments of cells with DOX and ononin (5/20 μM). (F) The wound closure percentage was calculated. (G to I) Transwell assay results following separate treatments of cells with DOX and ononin (5/20 μM). (J and K) WB results of ERK1/2/JNK/p38 signaling pathway following separate treatments of cells with DOX and ononin (5/20 μM). ^$$$$ P < 0.0001, ****P < 0.0001, ^#### P < 0.0001.

Fig. 2.

Ability of ononin to suppress EMT evaluated by treating the cells with ononin and DOX. (A and B) WB analysis was employed to assess the expression of E-cad, N-cad, MMP-2, and MMP-9 in TNBC cells. ****P < 0.0001, ^#### P < 0.0001, ^$$$$ P < 0.0001. (C) MTT assays demonstrated that ononin (0 to 20 μM) does not cause cytotoxicity in RAW 264.7 cells after 48-h treatment. (D) Clonogenic assay of control and experimental groups of RAW 264.7 cells. (E) Ononin does not alter caspase-3 and caspase-9 in control and experimental groups of RAW 264.7 cells. (F) Quantification of the apoptosis markers. (G and H) Ononin inhibits RANKL-induced osteoclastogenesis in RAW 264.7 cells (n = 6). (I and J) The antimigratory properties of ononin were assessed using a Matrigel-coated Transwell assay on RAW 264.7 cells treated with CM.

Fig. 3.

(A and B) Ononin suppresses osteoclast differentiation induced by CM, as demonstrated by TRAP staining assay. (C) Effects of ononin on control CM [Dulbecco’s modified Eagle’s medium (DMEM)] and MDA-MB-231-induced RANKL and OPG protein expression levels. (D) The relative ratio of RANKL/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and OPG/GAPDH protein expression was calculated with a densitometer. (E) The assay of JNK/ERK/p38 pathways was performed using a WB. (F) Quantification of the ERK/JNK signaling pathways. ^$$$$ P < 0.0001, ****P < 0.0001, and ^#### P < 0.0001.

Fig. 4.

(A) Diagram of the administration of ononin or DOX in the BCBM mouse model. (B) Bioluminescence imaging was conducted on days 3, 10, 17, and 25 following the implantation of cancer cells. (C) Representative radiograph images from mice administered with ononin or DOX. Arrows on the image mark the osteolytic bone lesions resulting from the injection of MDA-MB-231 cells. (D) The lesion count per mouse was determined through analysis of radiographic images from mice administered with ononin or DOX. (E) Osteolytic area (mm³) in mice treated with ononin or DOX. (F) Animals’ body weight throughout the study period of up to 40 d. (G) The survival curve was plotted based on the survival of the animal exhibiting BMs after treatment with ononin and DOX.

Fig. 5.

(A and B) H&E staining was employed to assess the impact of ononin treatment on bone tissue. (C to F) Results from TRAP staining indicated a substantial reduction in metastatic tumor load in groups treated with ononin.

Fig. 6.

CC-401 is synergistically active with ononin to exhibit inhibitory effects on TNBC cells. (A) Results of cell viability analysis following treatment with various concentrations of ononin in combination with JNKi for 24 and 48 h. (B) Results of the colony formation assay following treatment of cells with JNKi alone and in combination with ononin. (C) Number of colonies was quantified. (D) Results of the wound-healing assay following cell treatment with JNKi alone and in combination with ononin. (E to G) Results of the Transwell assay following cell treatment with JNKi alone and in combination with ononin. P < 0.0001, ****P < 0.0001. (H) The assay of JNK/ERK/p38 pathways was performed using a Western blot. (I) Quantification of the ERK/JNK signaling pathways. P < 0.0001, ^**** P < 0.0001.

Fig. 7.

(A) Cotreatment of ononin and JNKi effectively suppresses the formation of colonies in RAW 264.7 cells. (B) Cotreatment of ononin and JNKi inhibits osteoclast differentiation induced by CM, which was analyzed using a TRAP staining assay. (C) Antimigratory effects of the combination of ononin with JNKi were evaluated by Transwell assay. (D) Protein expression levels of RANKL and OPG induced by MDA-MB-231 were investigated. (E and F) The assay of ERK1/2/JNK/p38 pathways was performed using a WB. ^$$$$ P < 0.0001 and ****P < 0.0001.

Fig. 8.

Proliferation, migration, and invasion of MDA-MB-231 cells induced by PMA are inhibited by ononin (A). The MTT assay was conducted in MDA-MB-231 cells. (B and C). Clonogenic assay was performed in MDA-MB-231. (D) The wound-healing assay was utilized to measure the extent of wound closure, quantifying the migratory properties of the cells. (E to G) Non-Matrigel-coated and Matrigel-coated Transwell assay on MDA-MB-231 cells. (H and I) The assay of ERK1/2/JNK/p38 pathways was performed using a WB. ^$$$$ P < 0.0001 and ****P < 0.0001.

Fig. 9.

(A) The number of colonies formed under various concentrations of PMA with or without ononin was quantified. (B) Ononin was shown to suppress osteoclast differentiation prompted by PMA in a condition mediated by CM, as determined through TRAP staining analysis. (C) Ononin inhibits PMA-induced migratory effects that were evaluated using RAW 264.7 cells treated with CM. (D) Quantitative analysis of RANKL and OPG protein expression levels. (E and F) The assay of ERK1/2/JNK/p38 pathways was performed using a WB. ^$$$$ P < 0.0001 and ****P < 0.0001.

Fig. 10.

Mechanism of BCBM inhibition by ononin. Ononin targets the ERK1/2/JNK/p38 pathway in TNBC cells, leading to the suppression of cancer cell proliferation, migration, invasion, and ultimately metastasis. In vivo, ononin inhibits TNBC BM by targeting this pathway.