Inhibition of lung cancer cell proliferation by CD40 signaling through tumor necrosis factor I.

Background and Objective: CD40 signaling induces growth inhibition in some tumor cells in vitro, but the precise molecular mechanism remains unclear. This study was to investigate the biological effects and mechanisms of CD40 stimulation on proliferation of lung cancer cell lines NCI-H460 and A549, changes in tumor necrosis factor receptors (TNFRs) and membrane tumor necrosis factor alpha (mTNF-α). Methods: The expression of CD40 on the cell surface, and changes in TNFR and mTNF-αexpression after CD40 stimulation were detected by the immunofluorescence technique and flow cytometry. Changes in protein contents of TNFR as well as mTNF-α expression after CD40 stimulation were measured by western blot. The cell proliferation rate was determined by MTT assay. The content of soluble TNF-α(sTNF-α) in the supernatant of lung cancer cells was measured by ELISA assay. Results: The expression rates of CD40 in NCI-H460 and A549 were (89.0 ± 3.2)% and (62.2 ± 4.5)%, respectively. After 48 h of CD40 stimulation, the expression rates of TNFRI in NCI-H460 and A549 became significantly higher [(36.2 ± 4.6)% and (38.5 ± 5.9)%] than those in the corresponding control cells [(15.2 ± 3.1)% and (7.2 ± 1.9)%] (p < 0.05); while the expression rates of TNFRII were significantly lower than those in the control cells [(18.0 ± 1.6)% and (5.8 ± 1.2)% vs. (58.1 ± 3.6)% and (38.8 ± 4.3)%] (p < 0.05); the expression rates of mTNF-αwere decreased in the two cell lines [(8.7 ± 1.1)% and (7.0 ± 0.9)%] as compared to those in control cells [(15.0 ± 2.1)% and (26.5 ± 3.2)%] (p < 0.05). The level of TNFRI protein was elevated with the downregulation of TNFRII protein in NCI-H460 and A549. The level of mTNFαprotein remained unchanged in the two cell lines. TNF-αwas not detectable in the supernatant of lung cancer cells. Moreover, cell proliferation of NCI-H460 and A549 were inhibited after CD40 stimulation (p < 0.05), but the inhibition effect disappeared after blocking TNFRI. Blocking of TNF-α inhibited cell proliferation of the two cell lines (p < 0.05), but a synergistic effect was not observed after simultaneous stimulation with CD40. Conclusion: CD40 signaling inhibits the proliferation of CD40-positive lung cancer cells through mTNF-α/TNFRI in vitro.