J Bone Metab.  2019 Aug;26(3):179-191. 10.11005/jbm.2019.26.3.179.

Mechanical and Mechanosensing Properties of Tumor Affected Bone Cells Were Inhibited via PI3K/Akt Pathway

• Lee T

• KMID: 2457437
• DOI: http://doi.org/10.11005/jbm.2019.26.3.179

Abstract

BACKGROUND
Osteolytic metastasis is a common destructive form of metastasis, in which there is an increased bone resorption but impaired bone formation. It is hypothesized that the changed mechanical properties of tumor affected bone cells could inhibit its mechanosensing, thus contributing to differences in bone remodeling.
METHODS
Here, atomic force microscopy indentation on primary bone cells exposed to 50% conditioned medium from Walker 256 (W) carcinoma cell line or its adaptive tumor (T) cells was carried out. Nitric oxide levels of bone cells were monitored in response to low-magnitude, high-frequency (LMHF) vibrations.
RESULTS
A stronger sustained inhibitive effect on bone cell viability and differentiation by T cells as compared to that of its cell line was demonstrated. This could be attributed to the higher levels of transforming growth factor-β1 (TGF-β1) in the T-conditioned medium as compared to W-conditioned medium. Bone cell elastic moduli in W and T-groups were found to decrease significantly by 61.0% and 69.6%, respectively compared to control and corresponded to filamentous actin changes. Nitric oxide responses were significantly inhibited in T-conditioned group but not in W-conditioned group.
CONCLUSIONS
It implied that a change in cell mechanical properties is not sufficient as an indicator of change in mechanosensing ability. Moreover, inhibition of phosphoinositide 3-kinase/Akt downstream signaling pathway of TGF-β1 alleviated the inhibition effects on mechanosensing in T-conditioned cells, further suggesting that growth factors such as TGF-β could be good therapeutic targets for osteoblast treatment.

Keyword

Bone metastasis; Bone neoplasms; Elastic modulus; Mechanosensing; Nitric oxide

MeSH Terms

Actins
Bone Neoplasms
Bone Remodeling
Bone Resorption
Cell Line
Cell Survival
Culture Media, Conditioned
Elastic Modulus
Intercellular Signaling Peptides and Proteins
Microscopy, Atomic Force
Neoplasm Metastasis
Nitric Oxide
Osteoblasts
Osteogenesis
Sensitivity Training Groups
T-Lymphocytes
Vibration
Walkers
Actins
Culture Media, Conditioned
Intercellular Signaling Peptides and Proteins
Nitric Oxide

Figure

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  Fig. 1 (A) Radiograph of aggressive malignant lesion (white arrow) induced with elevated periosteal reaction (arrowheads) and a soft tissue mass with aneurysmal features at 50 days. (B) Histological image of tumor burdened bone, (C) colony formation of Walker 256 cells and (D) adaptive tumor cells at 14 days, ×10 magnification.

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  Fig. 2 Vimentin and cytokeratin expression of adaptive tumor cells and Walker 256 cells, at ×60 magnification, scale bar: 30 µm.

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  Fig. 3 (A) Growth inhibition of bone cells and (B) alkaline phosphatase activity inhibition of bone cells by Walker 256 (W) and adaptive tumor (T) conditioned medium for 3, 5, and 7 days of exposure. *Indicated P<0.05 and **indicated P<0.001 for one-way analysis of variance analysis, with Tukey's honestly significant difference post-hoc test.

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  Fig. 4 (A) Von Kossa staining of control well, (B) Walker 256 (W)-conditioned well and (C) adaptive tumor (T)-conditioned well at ×4 magnification, scale bar: 200 µm. (D) Collagen I staining of control well, (E) W-conditioned well and (F) T-conditioned well at ×20 magnification, small scale bar: 17 µm, large scale bar: 96 µm.

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  Fig. 5 (A) F-actin staining of control well, (B) Walker 256 (W)-conditioned well and (C) adaptive tumor (T)-conditioned well at ×40 magnification, scale bar: 50 µm. Cell nucleus was counterstained with 4′,6-diamidino-2-phenylindole. (D) Plot of indentation force vs. indentation depth, (E) Bar chart plot of elastic moduli for control, W-conditioned and T-conditioned groups. **Indicated P<0.001.

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  Fig. 6 (A) Bar chart plot of normalized nitric oxide levels across vibrated control, Walker 256 (W)-conditioned and adaptive tumor (T)-conditioned groups with treatment and without treatment of LY294002, *indicated P<0.05. (B) F-actin staining of vibrated control, W- and T-conditioned wells, with or without treatment of LY294002 at ×60 magnification, scale bar: 30 µm.

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