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Malignant cells are often resistant to complement activation through the enhanced expression of complement inhibitors. In this work, we examined the protective role of factor H, CD46, CD55, and CD59 in two non-small cell lung cancer cell lines, H1264 and A549, upon activation of the classical pathway of complement. Complement was activated with polyclonal Abs raised against each cell line. After blocking factor H activity with a neutralizing Ab, C3 deposition and C5a release were more efficient. Besides, a combined inhibition of factor H and CD59 significantly increased complement-mediated lysis. CD46 and CD55 did not show any effect in the control of complement activation. Factor H expression was knockdown on A549 cells using small interfering RNA. In vivo growth of factor H-deficient cells in athymic mice was significantly reduced. C3 immunocytochemistry on explanted xenografts showed an enhanced activation of complement in these cells. Besides, when mice were depleted of complement with cobra venom factor, growth was recovered, providing further evidence that complement was important in the reduction of in vivo growth. In conclusion, we show that expression of the complement inhibitor factor H by lung cancer cells can prevent complement activation and improve tumor development in vivo. This may have important consequences in the efficiency of complement-mediated immunotherapies.

Previously, we showed that magnolol induces cell-cycle arrest in cultured colon and liver cancer cells through an upregulation of the p21 protein. The aim of this study was to delineate the molecular mechanism underlying this magnolol-induced increase of p21 protein. Thus our RT-PCR analysis demonstrated that the mRNA levels of p21 were increased at 1 h after magnolol treatment and sustained for at least 24 h. The p21 promoter activity was also increased by magnolol treatment. Western blot analysis demonstrated that treatment of COLO-205 cells with magnolol increased the levels of phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment of the cells with PD98059 abolished the magnolol-induced upregulation of p21 protein, suggesting the involvement of an ERK pathway in the magnolol-induced upregulation of p21 in COLO-205 cells. Ras inhibitor peptide abolished the magnolol-induced increase of phosphorylated ERK protein levels, increase of p21 protein, and decrease of thymidine incorporation. Moreover, treatment of COLO-205 with magnolol increased the phosphorylated Raf-1 protein (the Ras target molecule). Pretreatment of the cells with Raf-1 inhibitor reversed the magnolol-induced decrease in thymidine incorporation. Treatment of the cells with CaM kinase inhibitor, but not protein kinase A (PKA) inhibitor or phosphatidylinosital 3-kinase (PI3K) inhibitor, abolished the magnolol-induced activation of ERK and decrease of thymidine incorporation. Taken together, our results suggest that magnolol activates ERK phosphorylation through a Ras/Raf-1-mediated pathway. Subsequently, p21 expression is increased, and finally thymidine incorporation is decreased.

The complement system is important for protection from invading pathogens, removal of waste products and guidance of the immune response. Furthermore, complement can be also targeted to cancer cells. However, membrane-bound inhibitors over-expressed by certain types of tumor cells restrict the cytotoxic activity of complement. Herein we report that non-small cell lung cancer (NSCLC) cells produce soluble complement inhibitors factor I (FI) and C4b-binding protein (C4BP). FI is a serine protease capable of degrading the activated complement components C3b and C4b, whilst C4BP acts as its cofactor. Furthermore, NSCLC cells express membrane-bound regulators and shed membrane cofactor protein (MCP), which shares cofactor function with C4BP. Secretion of FI from NSCLC cells was higher than previously reported for any non-hepatic source and FI produced by these cells could efficiently support cleavage of C3b and C4b. In vitro functional assays revealed that additional FI significantly decreased C3 deposition and complement-dependent lysis, particularly when cofactors were added. Our results demonstrate that soluble inhibitors produced by NSCLC cells may provide further protection from complement beyond the level ensured by membrane-bound inhibitors and, as such, contribute to the aggressive phenotype of these lung cancer cells.

Background Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), increases survival in patients with advanced EGFR-positive non-small cell lung cancer when administrated in combination with chemotherapy. In this study, we investigated the role of complement activation in the antitumor mechanism of this therapeutic drug. Results EGFR-expressing lung cancer cell lines were able to bind cetuximab and initiate complement activation by the classical pathway, irrespective of the mutational status of EGFR. This activation led to deposition of complement components and increase in complement-mediated cell death. The influence of complement activation on the activity of cetuximab in vivo was evaluated in xenografts of A549 lung cancer cells on nude mice. A549 cells express wild-type EGFR and have a KRAS mutation. Cetuximab activity against A549 xenografts was highly dependent on complement activation, since complement depletion completely abrogated the antitumor efficacy of cetuximab. Moreover, cetuximab activity was significantly higher on A549 cells in which a complement inhibitor, factor H, was genetically downregulated. Conclusions We demonstrate for the first time that the in vivo antitumor activity of cetuximab can be associated with a complement-mediated immune response. These results may have important implications for the development of new cetuximab-based therapeutic strategies and for the identification of markers that predict clinical response.