BMP-2 Promotes Oral Squamous Carcinoma Cell Invasion by Inducing CCL5 Release

<div><p>Bone morphogenetic protein-2 (BMP-2)-containing bone grafts are useful regenerative materials for oral and maxillofacial surgery; however, several <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> studies previously reported cancer progression-related adverse effects caused by BMP-2. In this study, by quantifying the rhBMP-2 content released from bone grafts, the rhBMP-2 concentration that did not show cytotoxicity in each cell line was determined and applied to the <i>in</i><i>vitro</i> monoculture or coculture model in the invasion assay. Our results showed that 1 ng/ml rhBMP-2, while not affecting cancer cell viability, significantly increased the invasion ability of the cancer cells cocultured with fibroblasts. Cocultured medium with rhBMP-2 also contained increased levels of matrix metalloproteinases. rhBMP-2-treated cocultured fibroblasts did not show a prominent difference in mRNA expression profile. Some cytokines, however, were detected in the conditioned medium by a human cytokine antibody array. Among them, the cancer invasion-related factor CCL5 was quantified by ELISA. Interestingly, CCL5 neutralizing antibodies significantly reduced the invasion of oral cancer cells. In conclusion, our results suggest that 1 ng/ml rhBMP-2 may induce invasion of oral squamous cell carcinoma (OSCC) cells by CCL5 release in coculture models. Therefore, we propose that a careful clinical examination before the use of rhBMP-2-containing biomaterials is indispensable for using rhBMP-2 treatment to prevent cancer progression.</p></div

Human CCL5 ELISA of monocultured or cocultured medium with or without 1 ng/ml rhBMP-2.

<p>Data shown are means of three independent repeats, and error bars indicate standard deviation. *P<0.05 compared to the control.</p

MMP and cytokine antibody array for cocultured YD-38 and fibroblasts.

<p>(a–c) MMP array for cocultured YD-38 cells either untreated (a) or treated with rhBMP-2 (b). (c) Quantification of the levels of various MMPs. (d–f) Cytokine array of cocultured YD-38 cells either untreated (d) or treated with rhBMP-2 (e). (f) Quantification of the levels of various cytokines. Red boxes in (d) and (e) show the changes of signal intensities of CCL5 before and after 1 ng/ml rhBMP-2 treatment, respectively, which were quantified and compared in the red box of (f).</p

Invasion assay of coculture models treated with rhBMP-2 and CCL5 neutralizing antibodies.

<p>(a–f) Representative invasion results of YD-10B (a and b), YD-38 (c and d), and HSC-2 (e and f) cocultured cells treated with either 1 ng/ml rhBMP-2 only (a, c, and e) or both rhBMP-2 and 0.05 µg/ml CCL5 neutralizing antibodies (b, d, and f). (g) Quantification of the invasion ability of OSCC cell lines. Data shown are means of three independent repeats, and error bars indicate standard deviation. *P<0.05 compared to the control.</p

Invasion assay in monoculture or coculture models.

<p>(a) Schematic diagram of the invasion assay and cellular changes. Carcinoma cells on the transwell penetrate the collagen membrane when cocultured with fibroblasts and then finally attach to the lower surface of the transwell. (b–g) Cocultured YD-10B (b and c), YD-38 (d and e), and HSC-2 (f and g) cells either untreated (b, d, and f) or treated with 1 ng/ml rhBMP-2 (c, e, and g). (h) Quantification of the invasion ability of OSCC cells. Data shown are means of three independent repeats, and error bars indicate standard deviation. *P<0.05 compared to the control.</p