Korean J Physiol Pharmacol.  2018 Sep;22(5):503-511. 10.4196/kjpp.2018.22.5.503.

Lysophosphatidic acid enhances breast cancer cells-mediated osteoclastogenesis

• Nam JS
• Sharma AR
• Nguyen LT
• Jagga S
• Lee YH
• Sharma G
• Lee SS

• KMID: 2419000
• DOI: http://doi.org/10.4196/kjpp.2018.22.5.503

Abstract

Lysophosphatidic acid (LPA) is known to play a critical role in breast cancer metastasis to bone. In this study, we tried to investigate any role of LPA in the regulation of osteoclastogenic cytokines from breast cancer cells and the possibility of these secretory factors in affecting osteoclastogenesis. Effect of secreted cytokines on osteoclastogenesis was analyzed by treating conditioned media from LPA-stimulated breast cancer cells to differentiating osteoclasts. Result demonstrated that IL-8 and IL-11 expression were upregulated in LPA-treated MDA-MB-231 cells. IL-8 was induced in both MDA-MB-231 and MDA-MB-468, however, IL-11 was induced only in MDA-MB-231, suggesting differential LPARs participation in the expression of these cytokines. Expression of IL-8 but not IL-11 was suppressed by inhibitors of PI3K, NFkB, ROCK and PKC pathways. In the case of PKC activation, it was observed that PKCδ and PKCμ might regulate LPA-induced expression of IL-11 and IL-8, respectively, by using specific PKC subtype inhibitors. Finally, conditioned Medium from LPA-stimulated breast cancer cells induced osteoclastogenesis. In conclusion, LPA induced the expression of osteolytic cytokines (IL-8 and IL-11) in breast cancer cells by involving different LPA receptors. Enhanced expression of IL-8 by LPA may be via ROCK, PKCu, PI3K, and NFkB signaling pathways, while enhanced expression of IL-11 might involve PKCδ signaling pathway. LPA has the ability to enhance breast cancer cells-mediated osteoclastogenesis by inducing the secretion of cytokines such as IL-8 and IL-11.

Keyword

Breast cancer; Interleukin-8; Interleukin-11; Lysophosphatidic acid; Osteoclastogenesis

MeSH Terms

Breast Neoplasms*
Breast*
Culture Media, Conditioned
Cytokines
Interleukin-11
Interleukin-8
Neoplasm Metastasis
Osteoclasts
Receptors, Lysophosphatidic Acid
Culture Media, Conditioned
Cytokines
Interleukin-11
Interleukin-8
Receptors, Lysophosphatidic Acid

Figure

• 

  Fig. 1 Stimulation of IL-8 and IL-11 expression in MDA-MB-231 cells by LPA.MDA-MB-231 cells were treated with 10 μM LPA for indicated times (1h, 3 h and 6 h), quantitative expression of IL-8 (A) and IL-11 (B) mRNA were determined by RT-PCR in normalization with GAPDH. *p<0.05, **p<0.01, significant difference compared to control at 1 h.

• 

  Fig. 2 Stimulation of IL-8 and IL-11 expression by LPA in MDA-MB-231 and MDA-MB-468 cells.MDA-MB-231 and MDA-MB-468 cells were treated with 10 μM LPA for 3 h, quantitative expression of IL-8 (A) and IL-11 (B) mRNA was determined by RT-PCR in normalization with GAPDH. After 12 h of stimulation of LPA, the concentrations of IL-8 (C) and IL-11 (D) were measured by ELISA. *p<0.05, **p<0.01.

• 

  Fig. 3 Effects of PI3K, NFKB, ROCK and PKC inhibitors on induction of IL-8 and IL-11 by LPA in MDA-MB-231 cells.MDA-MB-231 cells were pretreated with specific inhibitors for 30 min, prior to LPA (10 μM) treatment for 3 h. Quantitative expression of IL-8 (A) and IL-11 (B) mRNA were evaluated by RT-PCR in normalization with GAPDH. *p<0.05, **p<0.01.

• 

  Fig. 4 LPA stimulated IL-8 and IL-11 mRNA expression through regulation of different PKC subtypes.(A) MDA-MB-231 cells were treated with LPA (10 μM). Protein levels of p-PKC were detected by western blot at 0–4 h. β-actin was used as loading control, (B) MDA-MB-231 cells were pretreated with specific inhibitors for 30 min, prior to LPA (10 μM) treatment for 3 h. Quantitative expression of IL-8 and IL-11 mRNA were evaluated by RT-qPCR in normalization with GAPDH. *p<0.05, **p<0.01.

• 

  Fig. 5 LPA-stimulated breast cancer cells conditioned media induced osteoclast formation.RAW264.7 cells after stimulation with 30 ng/ml RANKL for 2.5 days were exposed to CM from breast cancer cells (MDA-MB-231 or MDA-MB-468) treated with or without LPA for 2.5 days (see Materials and Methods). (A) RT real-time PCR analysis for expression of TRAP mRNA. (B) Cell viability was assessed by MTT. (C) Osteoclast formation was assessed by TRAP staining and osteoclast number was counted. (D) Representative microcopy picture of cells stained with TRAP.

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