The promoting effect of neutrophil-derived BAFF molecule on the proliferation and life span of CAL-27 oral squamous carcinoma cells.

Tumor-associated neutrophils (TANs) are the major cellular component of the tumor microenvironment and have been shown to release of different bioactive molecules such as B-cell activating factor (BAFF). The data on the interactions between OSCC cells and neutrophils are limited and do not explain the actual role of the BAFF in the development of the OSCC. In the present study we examined the direct effect of neutrophils-derived BAFF on the OSCC cell line CAL-27 proliferation and apoptosis. PMNs of OSCC patients and healthy control were isolated from whole blood and separated by magnetic selection with monoclonal anti-human CD16 antibodies. CD-16 - positive neutrophils were incubated in the presence of TGF-β and/or LPS as well as flavonoids (luteolin and quercetin). CAL-27 cells were co-incubated with supernatants of neutrophils. BAFF expression in neutrophils, BAFF-R expression on CAL-27 cells and apoptosis of CAL-27 cells were assessed by flow cytometry. To determine the CAL-27 cells proliferation, the MTT test was used. Expression of select mitochondrial proteins in CAL-27 cells were measured by Western blot. Neutrophils from OSCC patients showed significantly higher expression of BAFF than those from the healthy controls. The results obtained revealed upregulation of the proliferation and downregulation of the apoptosis of the CAL-27 cells in the presence of the supernatants of TGF-β-treated neutrophils. Flavonoids reduced BAFF expression in neutrophils of patients with OSCC and control group. Lower intensity of apoptosis in CAL-27 cells was associated with the increased expression of anti-apoptotic Bcl-2, Mcl-1 and activated form of PI3K kinase (pPI3K) and simultaneously reduced expression of pro-apoptotic Bax protein in the presence of rhBAFF, as well as of supernatants of neutrophils derived from OSCC patients. In conclusion, the data presented confirm the previously suggested role of neutrophil-derived BAFF in OSCC development. The favorable effects of examined flavonoids on tumor-promoting BAFF expression in neutrophils suggest that they might be promising candidates as chemo-preventive agents in the therapy of patients with OSCC.