Inhibition Of Proliferation Of Lung Cancer Cells By CD40 Signaling Through Tumor Necrosis Factor I

Objective: CD40 signaling induces growth inhibition in some tumor cells in vitro, but the precise molecular mechanism is still unclear. This study was to investigate the biological effects and mechanisms of CD40 stimulation on proliferation of lung cancer cell lines and the changes of tumor necrosis factor receptors(TNFR), as well as membrane tumor necrosis factor alpha(mTNF-α) after CD40 stimulation, and its possible mechanism.Methods: Human lung cancer cell lines NCI-H460, A549 and SPC-A-1 were chosen as target cells and CD40 signal was stimulated by agonistic CD40 monoclonal antibody(5C11). Immunofluorescence technique and flow cytometry were used to detect the expression of CD40 on cell surface and the changes of TNFR and mTNF-αexpression after CD40 stimulation. Western blot was used to measure the changes of protein contents of TNFR as well as TNF-αexpression after CD40 stimulation. MTT assay was used to determine the effect of CD40 stimulation after blocking TNFRI as well as TNF-α. ELISA assay was used to measure the change of the content of TNF-αin the supernatant fluid of lung cancer cells.Results: (1) The expression of CD40 on cell line varied from (2.3±0.4)% to (89±3.2)%. (2) There was an up-regulation of TNFRI together with down-regulations of TNFRII and mTNF expression on cell surface of NCI-H460 and A549 after CD40 stimulation(P<0.05). (3)There was an up-regulation of TNFRI protein together with a down-regulation of TNFRII protein expression on NCI-H460 and A549 after CD40 stimulation. There was no detectable change of mTNF protein expression on the two cell lines after CD40 stimulation. (4) There was no detectable change of TNFR,mTNF-αexpression on the cell line SPC-A-1 after CD40 stimulation. (5) There was no detectable TNF-αin the supernatant of lung cancer cells. (6) There was an inhibition of cell proliferation on cell lines NCI-H460 and A549 after CD40 stimulation(P<0.05)while no inhibition was observed on cell line SPC-A-1. (6) The effect of inhibition of cell proliferation induced by CD40 stimulation was not observed after blocking TNF signaling.CONCLUSION: CD40 stimulation could induce inhibition of cell proliferation of CD40 positive cell lines NCI-H460 and A549 and cause changes of TNFR and mTNF-αexpression. The effect of inhibition of cell proliferation induced by CD40 stimulation was not observed after blocking TNFRI. Theses changes indicate that mTNF-α/TNFRI pathway was involved in the inhibition of the proliferation of lung cancer cells induced by CD40 signaling in vitro.