Kadsurenone is a useful and promising treatment strategy for breast cancer bone metastases by blocking the PAF/PTAFR signaling pathway

Abstract

Breast cancer (BC) is characterized by high incidences of bone metastases. Current treatment strategies for BC bone metastases primarily focused on breaking the 'vicious osteolytic cycle'. Platelet-activating factor (PAF) is a potent phospholipid mediator, which has previously reported biological activities in BC progression and osteoclast differentiation by activating its receptor PAF receptor (PTAFR). However, the role of PAF in the mediation of BC bone metastases remains elusive. In the present study, it was revealed that the upregulation of PTAFR was associated with an increased incidence of bone metastases. It was also revealed that PAF significantly enhanced the processes of BC cell migration and BC mediated osteoclastogenesis. These results suggest that PAF serves a promotion role in BC bone metastases. It was further demonstrated that the natural PAF antagonist Kadsurenone may effectively attenuate each process by partially blocking the PAF/PTAFR signaling pathway. Therefore, targeting PAF/PTAFR by Kadsurenone may be a promising treatment strategy for BC bone metastases.

Keywords: Kadsurenone; PAF; bone metastases; breast cancer; osteoclastogenesis; vicious osteolytic cycle.

Figure 1.

Bioinformatics analysis of PTAFR expression in BC tissues and OG cell lines. (A) Expression fold changes of PTAFR in patients with BS vs. normal breast tissues. The data were obtained from the Oncomine database. (B) Survival analysis using the PTAFR mRNA expression level for incidences of bone metastasis in Minn's dataset, P-values were calculated on the basis of a log-rank test. (C) Comparison of PTAFR mRNA relative expression in OG and non-OG cell lines following Rankl stimulation. Data were obtained from the GSE43811 dataset and are presented as the mean ± standard error of the mean (n=3). P-values were calculated using a Student's t-test. **P<0.01 and ***P<0.001. OG, osteoclastogenic; PTAFR, platelet-activating factor receptor; BC, breast cancer.

Figure 2.

Kadsurenone inhibits PAF induced BC cell migration. (A) Chemical structure of Kadsurenone. (B) Effect of Kadsurenone on the viability of BC cell line MDA-MB-231. MDA-MB-231 cells were treated with the indicated concentrations of Kadsurenone. (C) Transwell assay of MDA-MB-231 cells induced by PAF (200 nM) with the presence or absence of the indicated concentrations of Kadsurenone. (D) Graphs represent the mean fold changes of migrated cells from three independent experiments. (E) Relative NF-κB luciferase activity of MDA-MB-231 cells induced by PAF (200 nM) with the presence or absence of indicated concentrations of Kadsurenone. Data are presented as the mean ± standard error of the mean (n=3). Scale bar, 100 µm. P-values were calculated using one-way analysis of variance followed by Tukey's post hoc test. **P<0.01 and ***P<0.001. PAF, platelet-activating factor; BC, breast cancer; NF, nuclear factor.

Figure 3.

Kadsurenone inhibits breast cancer cells induced osteoclastogenesis. (A) Representative images of Trap⁺ osteoclasts. MDA-MB-231 and RAW264.7 cells were co-cultured and treated with the indicated concentrations of Kadsurenone and stained on day 7. The (B) number and (C) area of Trap⁺ osteoclasts per field are reported. Scale bar, 100 µm. Data are presented as the mean ± standard error of the mean (n=3). P-values were calculated using one-way analysis of variance followed by Tukey's post hoc test. *P<0.05 and ***P<0.001 vs. the control. Trap, tartrate-resistant acid phosphatase.

Figure 4.

Kadsurenone directly inhibits RANKL induced osteoclastogenesis. (A) Effect of Kadsurenone on the viability of mouse BMMs. Mouse BMMs were isolated and treated with the indicated concentrations of Kadsurenone. (B) Representative images of Trap⁺ osteoclasts. Mouse BMMs were isolated and induced for osteoclastogenesis with RANKL (50 ng/ml). Cells were treated with thye indicated concentrations of Kadsurenone and stained on day 7. The (C) number and (D) area of Trap⁺ osteoclasts per field are indicated (E) Representative images of actin ring staining. Mouse BMMs were isolated and induced for osteoclastogenesis with RANKL (50 ng/ml). Cells were treated with the indicated concentrations of Kadsurenone and stained on day 7. (F) The bar chart represents the mean number of actin rings for three independent experiments. Scale bar, 100 µm. Data are presented as the mean ± standard error of the mean (n=3). P-values were calculated using one-way analysis of variance followed by Tukey's post hoc test. **P<0.01 and ***P<0.001 vs. the control. BMMs, bone marrow monocytes; RANKL, receptor activator for NF-κB ligand; Trap, tartrate-resistant acid phosphatase.

Figure 5.

Kadsurenone inhibits RANKL induce OC marker gene expression by inhibiting the NF-κB signaling pathway. (A) RT-qPCR analysis of the OC marker gene Ctsk and Trap expression. Mouse BMMs were isolated and induced for osteoclastogenesis with Rank l (50 ng/ml). Cells were treated with or without 1 µM Kadsurenone. mRNAs were harvested on the indicated days. (B) RT-qPCR analysis of the OC marker genes Trap, Nfatc1 and Ctsk expression. Mouse BMMs were isolated and induced for osteoclastogenesis with RANKL (50 ng/ml). Cells were treated with the indicated concentrations of Kadsurenone. mRNAs were harvested on day 3. (C) Relative NF-κB (left) and Nfatc1 (right) luciferase activity of mouse BMMs induced by RANKL (50 ng/ml) with the presence or absence of the indicated concentrations of Kadsurenone. Data are presented as the mean ± standard error of the mean (n=3). P-values were calculated using one-way analysis of variance followed by Tukey's post hoc test. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. BMMs, bone marrow monocytess; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OC, osteoclast; Ctsk, cathepsin K; Nfatc1, nuclear factor of activated T cells 1; Trap, tartrate-resistant acid phosphatase; RANKL, receptor activator for NF-κB ligand; NF, nuclear factor.