α-Tocopheryl Succinate Inhibits Osteolytic Bone Metastasis of Breast Cancer by Suppressing Migration of Cancer Cells and Receptor Activator of Nuclear Factor-κB Ligand Expression of Osteoblasts

Abstract

Background: Breast cancer is one of the most common cancers affecting women and has a high incidence of bone metastasis, causing osteolytic lesions. The elevated expression of receptor activator of nuclear factor-κB ligand (RANKL) in cancer activates osteoclasts, leading to bone destruction. We previously reported that α-tocopheryl succinate (αTP-suc) inhibited interleukin-1-induced RANKL expression in osteoblasts. Here, we examined the effect of αTP-suc on osteolytic bone metastasis in breast cancer.

Methods: To examine the effect of αTP-suc on the metastatic capacity of breast cancer, MDA-MB-231-FL cells were injected into the left cardiac ventricle of BALB/c nude mice along with intraperitoneal injection of αTP-suc. The mice were then analyzed by bioluminescence imaging. To investigate the effect of αTP-suc on osteolysis, 4T1 cells were directly injected into the femur of BALB/c mice along with intraperitoneal injection of αTP-suc. Microcomputed tomography analysis and histomorphometric analysis of the femora were performed.

Results: αTP-suc inhibited cell migration and cell growth of 4T1 cells. In line with these results, bone metastasis of MDA-MB-231-FL cells was reduced in mice injected with αTP-suc. In addition, αTP-suc decreased osteoclastogenesis by inhibiting 4T1-induced RANKL expression in osteoblasts. Consistent with these results, 4T1-induced bone destruction was ameliorated by αTP-suc, with in vivo analysis showing reduced tumor burden and osteoclast numbers.

Conclusions: Our findings suggest that αTP-suc may be efficiently utilized to prevent and treat osteolytic bone metastasis of breast cancer with dual effects.

Keywords: Alpha-tocopheryl succinate; Breast neoplasms; Osteolysis.

Fig. 1. Effect of α-tocopheryl succinate (αTP-suc) on the cell migration and cell viability of 4T1 cells. (A, B) Cell migration of serum-starved 4T1 cells in response to 10% fetal bovine serum in the presence of dimethyl sulfoxide (DMSO) or different concentrations of αTP-suc (1–10 µM) was assessed in transwell chambers for 12 hr. (A) Representative images of hematoxylin and eosin staining, ×100 magnification; and scale bar, 200 µm. (B) The number of migrated 4T1 cells per field (n=6 per each well). (C) The 4T1 cells were cultured with DMSO or different concentrations of αTP-suc (1–10 µM) for 2 days and then cell viability was measured. The results shown are representative of three independent experiments (n=3), and the values are expressed as mean±standard deviation. ^***P<0.001 vs. vehicle treatment by unpaired Student's t-test. FBS, fetal bovine serum.

Fig. 2. Effect of α-tocopheryl succinate (αTP-suc) on metastasis by intracardiac injection of MDA-MB-231-FL (MDA-FL) cells. MDA-MB-231-FL cells or phosphate-buffered saline was injected into the left cardiac ventricle and metastasis of cancer was analyzed on day 7 after injection of cancer. Starting 1 day before cancer injection, αTP-suc (0.1 mg/head) or dimethyl sulfoxide (DMSO) was injected intraperitoneally every 2 days (n =5 each). (A) Scheme of the intracardiac injection mouse model. (B) Representative bioluminescence images. (C) Bioluminescence imaging analysis of tumor burden in the whole body. The values are expressed as mean±standard error of the mean. ^†P<0.05 vs. DMSO-injected mice with injection of MDA-MB-231-FL cells by one-way repeated-measures analysis of variance followed by Dunnett's test. i.p., intraperitoneally.

Fig. 3. Effect of α-tocopheryl succinate (αTP-suc) on osteoclast differentiation in triple co-culture and on 4T1 cell-conditioned medium (4T1-CM)-induced receptor activator of nuclear factor-κB ligand (RANKL) expression in osteoblasts (OBs). (A, B) Primary bone marrow cells and OBs were co-cultured with or without 4T1 cells in the presence of dimethyl sulfoxide (DMSO) or αTP-suc (10 µM) for 8 days. (A) Representative images of tartrate-resistant acid phosphatase staining, ×100 magnification; and scale bar, 200 µm. (B) The number of osteoclasts. ^***P<0.001 vs. DMSO-treated control by unpaired Student's t-test. (C, D) OBs were cultured with or without 4T1-CM (30%) in the presence of DMSO or different concentrations of αTP-suc (1–10 µM) for 24 hr. (C) Levels of RANKL messenger RNA (mRNA) (left panel) and osteoprotegerin (OPG) mRNA (right panel) were measured by real-time polymerase chain reaction analysis. (D) Levels of RANKL protein (left panel) and OPG protein (right panel) in the culture media were measured by enzyme-linked immunosorbent assay. The results shown are representative of three independent experiments (n=3), and the values are expressed as mean±standard deviation. ^***P<0.001 vs. untreated control and ^†††P<0.001; ^††P<0.01; ^†P<0.05 vs. DMSO-treated cells in response to 4T1-CM by unpaired Student's t-test. BMC, bone marrow cell.

Fig. 4. Effect of α-tocopheryl succinate (αTP-suc) on the bone destruction by 4T1 cells in the mouse model of intrafemoral injection. The 4T1 cells were directly injected into the left femur. Starting 1 day before cancer injection, αTP-suc (0.1 mg/head) or dimethyl sulfoxide (DMSO) was injected intraperitoneally every 2 days (n=5 each). After 7 days of 4T1 injection, left femora were collected and analyzed by microcomputed tomography (micro-CT) scanning. (A) Scheme of the intrafemoral injection mouse model. (B) Representative reconstructed images by micro-CT (scale bar, 0.7 mm). (C) Quantification of trabecular bone parameters: bone volume per total volume (BV/TV, left upper panel), trabecular thickness (Tb.Th, right upper panel), trabecular number (Tb.N, left bottom panel), and trabecular separation (Tb.Sp, right bottom panel). The values are expressed as mean±standard error of the mean. ^***P<0.001; ^*P<0.05 vs. phosphate-buffered saline-injected mice and ^†P<0.05 vs. DMSO-injected mice with injection of 4T1 cells by one-way repeated-measures analysis of variance followed by Dunnett's test. n.s., not significant; i.p., intraperitoneally.

Fig. 5. Effect of α-tocopheryl succinate on tumor burden and osteoclast number by 4T1 cells. Histological analysis of bone metastasis from the experimental groups in Figure 4 (n=5). (A) Representative images of hematoxylin and eosin (H & E)-stained femur sections, ×100 magnification; and scale bar, 500 µm. (B) Histomorphometric quantification of tumor burden from H & E sections. (C) Representative images of tartrate-resistant acid phosphatase (TRAP)-stained femur sections, ×400 magnification; and scale bar, 200 µm. (D) Histomorphometric quantification of osteoclast numbers from TRAP-stained sections. ^†P<0.05 vs. dimethyl sulfoxide (DMSO)-injected mice with injection of 4T1 cells by one-way repeated-measures analysis of variance followed by Dunnett's test. T, tumor; BM, bone marrow; B, bone; αTP-suc, α-tocopheryl succinate.