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. 2020 Nov;9(21):8173-8185.
doi: 10.1002/cam4.3432. Epub 2020 Sep 25.

Asperolide A prevents bone metastatic breast cancer via the PI3K/AKT/mTOR/c-Fos/NFATc1 signaling pathway

Affiliations

Asperolide A prevents bone metastatic breast cancer via the PI3K/AKT/mTOR/c-Fos/NFATc1 signaling pathway

Wenli Jiang et al. Cancer Med. 2020 Nov.

Abstract

Background: Breast cancer is the leading cause of death among women with malignant tumors worldwide. Bone metastasis is the main factor affecting the prognosis of breast cancer. Therefore, both antitumor and anti-breast-cancer-induced osteolysis agents are urgently needed.

Methods: We examined the effect of Asperolide A (AA), a marine-derived agent, on osteolysis and RANKL-induced phosphoinositide 3-kinase (PI3K)/AKT/mTOR/c-FOS/nuclear factor-activated T cell 1 (NFATc1) pathway activation, F-actin ring formation, and reactive oxygen species (ROS) generation in vitro. We evaluated AA effect on breast cancer MDA-MB-231 and MDA-MB-436 cells in vitro through CCK8 assay, wound healing assay, transwell assay, Annexin V-FITC/PI staining for cell apoptosis, and cell cycle assay. Furthermore, we assessed the effect of AA in vivo using a breast cancer-induced bone osteolysis nude mouse model, followed by micro-computed tomography, tartrate-resistant acid phosphatase staining, and hematoxylin and eosin staining.

Results: Asperolide A inhibited osteoclast formation and differentiation, bone resorption, F-actin belt formation, ROS activity, and osteoclast-specific gene and protein expressions and prevented PI3K/AKT/mTOR/c-FOS/NFATc1 signaling activation in a dose-dependent manner in vitro. AA also inhibited breast cancer growth and breast cancer-induced bone osteolysis by reducing osteoclast formation and function and inactivated PI3K/AKT/mTOR signaling in vivo.

Conclusions: Our study demonstrated that AA suppressed bone metastatic breast cancer. These findings indicate AA as a potential, novel curative drug candidate for patients with bone metastatic breast cancer.

Keywords: Asperolide A; bone metastases; breast cancer; mTOR; osteoclast.

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Conflict of interest statement

There is no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Compounds from the marine algae‐derived endophytic fungus Aspergillus wentii EN‐48 inhibited receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast formation with low or no cytotoxicity in vitro. The chemical structure and CAS (Chemical Abstracts Service) number of each compound (A). Macrophage‐colony stimulating factor (M‐CSF)‐dependent bone marrow macrophages (BMMs) were treated with 50 ng/mL RANKL with or without compounds for 5 d. Tartrate‐resistant acid phosphatase (TRAcP) staining was performed until osteoclasts formed (n = 3 per group) (B). The number of TRAcP‐positive osteoclasts (nuclei ≥ 3) was quantified (percentage of control) (C). The area/size of TRAcP‐positive osteoclasts (nuclei ≥ 3) was quantified (percentage of control) (D). The cytotoxic effect of each compound on M‐CSF‐dependent BMMs was measured by CCK8 assay. BMMs were cultured with 50 ng/mL M‐CSF with compounds (0.5 μmol/L) for 48 h (n = 3 per group) (E). Cell viability is expressed as a percentage of control (CTRL). Data are shown as mean ± SD; n = 3 per group; **P < .01 and ***P < .005, compared with untreated groups.
FIGURE 2
FIGURE 2
Asperolide A (AA) attenuates receptor activator of NF‐κB ligand (RANKL)‐induced osteoclastogenesis, hydroxyapatite absorption, ROS production, and F‐actin ring formation without cytotoxicity. Effect of AA on macrophage‐colony stimulating factor (M‐CSF)‐dependent bone marrow macrophages (BMM) cell viability by cell counting kit‐8 (CCK8) assay. BMMs were cultured with 50 ng/mL M‐CSF without or with AA (0.25, 0.5, 1, and 10 μmol/L) for 48 and 96 h (n = 3 per group) (A). CCK8 assay was performed. Cell viability is expressed as percentage of the nontreatment group (0 μmol/L). M‐CSF‐dependent BMMs were treated with 50 ng/mL RANKL and then treated without or with AA (0.5 and 1 μmol/L) for 5 days. Cells were fixed and tartrate‐resistant acid phosphatase (TRAcP) staining was performed until osteoclasts formed (n = 3 per group). The number of TRAcP‐positive cells (nuclei > 3) was quantified (n = 3 per group). The area of TRAcP‐positive cells (nuclei > 3) was quantified (n = 3 per group) (B). M‐CSF‐dependent BMMs were treated with AA (0.5 and 1 μmol/L) on bone‐mimicking hydroxyapatite‐coated OsteoAssay culture plates until bone resorption pits were observed. Bone resorption pits were observed using an inverted light microscope. Quantification of resorption area (C). F‐actin ring images from mature osteoclasts were observed using a fluorescence microscope. Quantification of the number of F‐actin rings (D). Intracellular ROS levels of nontreatment and AA‐treated BMMs were detected using the DCFDA fluorescence probe. Integral optic density value reflected the ROS levels in different groups of cells (E). Data are shown as mean ± SD; n = 3 per group; *P < .05, compared with the untreated group cells. ROS, reactive oxygen species. DCFDA, 2ʹ,7ʹ‐dichlorofluorescin diacetate
FIGURE 3
FIGURE 3
Asperolide A (AA) suppresses osteoclastic‐specific genes and inhibits receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast formation via the PI3K/AKT/mTOR/c‐Fos/NFATc1 signaling pathway. The expressions of cathepsin k (CTSK), c‐FOS, NFATc1, and TRAP genes were examined by quantitative real time polymerase chain reaction (A). The expressions of c‐FOS and NFATc1 proteins were examined by western blot (B). Macrophage‐colony stimulating factor (M‐CSF)‐dependent bone marrow macrophages were plated in 6‐well plates in starvation culture medium for 2 h, pretreated without or with 1 μmol/L AA for 60 min, and then treated without or with 50 ng/mL RANKL for 5, 10, 20, 30, and 60 min. Western blot was performed to evaluate phosphorylated mTOR (P‐mTOR), total mTOR, phosphorylated PI3K (P‐PI3K), total PI3K, phosphorylated AKT (P‐AKT), total AKT, and β‐actin (C). MDA‐MB‐231 and MDA‐MB‐436 cells were plated in 6‐well plates in starvation culture medium for 2 h and pretreated without or with AA and BEZ235 for 60 min. Western blot was performed to detect phosphorylated mTOR (P‐mTOR), total mTOR, phosphorylated PI3K (P‐PI3K), total PI3K, phosphorylated AKT (P‐AKT), total AKT (D). Data are shown as mean ± SD; n = 3 per group; *P < .05
FIGURE 4
FIGURE 4
Asperolide A (AA) inhibited the proliferation, migration, and invasion and induced cell cycle arrest and apoptosis of MDA‐MB‐231 and MDA‐MB‐436 cells. Cell viability (%) of nontreatment or AA treatment groups was calculated (A, B). AA inhibited cell migration as determined by wound healing assays (C). Crystal violet‐stained cells were captured and the number of invasive cells was counted (D). AA induced cell cycle arrest in S + G2/M phase and quantification of cells in S + G2/M (%) are shown (E). Apoptosis was determined using Annexin V‐FITC + PI staining and apoptosis rate (%) in each group (F). Data are shown as mean ± SD; n = 3 per group; *P < .05 compared with the untreated group cells
FIGURE 5
FIGURE 5
Asperolide A (AA) treatment inhibited breast cancer development and prevented breast cancer‐induced bone metastasis and osteolysis in vivo. Establishment of breast cancer‐induced bone metastasis nude mouse model and evaluation of AA effects (A). MDA‐MB‐231 cells were injected directly into the tibial plateau. AA inhibited tumor growth in the tibia (B). Body weight of mice (C). Tissue length of tibial tumor section (D). Tissue width of tibial tumor section (E). Tissue volume of tibial tumor section (F). Representative micro‐CT images indicated that AA prevented osteolysis (G). Quantitative analyses of bone structure parameters, including BS/BV (%), BV/TV (%), Tb.N, Tb.Sp (n = 6 per group) (H‐K). Representative tartrate‐resistant acid phosphatase (TRAcP) staining images showed that AA inhibited osteoclast formation (L). Quantitative analyses of TRAcP‐positive cell number (OC.N/BS) and area (OC.S/BS) (M, N). Representative immunohistochemistry images showed that AA inhibited the expression of P‐PI3K (L). Quantitative analyses of P‐PI3K positive area (%) (O). Data are shown as mean ± SD; n = 6 per group; *P < .05, ***P < .001, and ****P < .005, compared with the sham or CTRL groups
FIGURE 6
FIGURE 6
A potential working model for the suppression of Asperolide A in bone metastatic breast cancer

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