Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

Abstract

Engineering malignant cells to express a heterologous α-gal antigen can induce heterograft hyperacute rejection, resulting in complement-dependent cytolysis (CDC) of tumor cells, which has been considered as a novel strategy for antitumor therapy. A549 cells engineered to express Galα1-3Galβ1-4GlcNAc-R (α-gal) epitope exhibited strong resistance to CDC treated by normal human serum (NHS) in a previous study. We hypothesized that the expression of membrane-bound complement regulatory proteins (mCRPs) decay accelerating factor (CD55) and protectin (CD59) influenced the efficacy of the α-gal/NHS-mediated antitumor effect to tumor cells in vitro. The present study confirmed that A549 cells expressed high levels of CD55 and CD59, whereas Lovo cells expressed relatively low levels of these proteins. A549 and Lovo cells transfected with plasmids containing or lacking the α-gal epitope were evaluated for their susceptibility to CDC by NHS and detected using a trypan blue exclusion assay. α-gal-expressing Lovo (Lovo-GT) cells were almost completely killed by α-gal-mediated CDC following incubation with 50% NHS, whereas no cytolysis was observed in α-gal expressing A549 (A549-GT) cells. Abrogating CD55 and CD59 function from A549-GT cells by various concentrations of phosphatidylinositol-specific phospholipase C (PI-PLC) or blocking antibodies increased the susceptibility of cells to CDC, and the survival rate decreased significantly comparing to the controls (P<0.05). The findings of the present study indicated that using the α-gal/NHS system to eliminate tumor cells via inducing the complement cascade reaction might represent a feasible approach for the treatment of cancer. However, high levels of mCRP expression may limit the efficacy of this approach. Therefore, an improved efficacy of cancer cell killing may be achieved by combining strategies of heterologous α-gal expression and mCRP downregulation.

Keywords: complement-dependent cytolysis; membrane-bound complement regulatory proteins; α-gal epitope.

Figure 1.

Expression of CD55 and CD59 in tumor cells. (A) Cell lines were incubated with fluorescein isothiocyanate-conjugated anti-human CD55 and CD59 monoclonal antibodies, and analyzed by flow cytometry. Error bars depict standard deviations. (B) The protein level of CD55 and CD59 in A549 and Lovo cells were detected by western blotting. β-actin protein levels served as the loading control. CD55, decay accelerating factor; CD59, protectin.

Figure 2.

Establishing stable transfected α-gal-expressing cell lines. (A) α-1,3GT mRNA expression in A549, A549-V, A549-GT, Lovo, Lovo-V and Lovo-GT cells were detected by reverse transcription-polymerase chain reaction. The amplified product of α-1,3GT was detected by agarose gel electrophoresis in lane 2, 4 and 6 of the two gels. GAPDH was used as loading control in lane 1, 3 and 5 of the two gels. (B) Expression of α-gal epitope in each group of cells were detected by direct immunofluorescence (magnification, ×200). FITC-conjugated BS-IB4 lectin staining was performed to probe α-gal epitope. (B-a) A549-GT, (B-b) A549, (B-c) A549-V, (B-d) Lovo-GT, (B-e) Lovo, (B-f) Lovo-V and (B-g) positive control PIEC cells. (C) Expression of α-gal epitope in each group of cells were stained with FITC-BS-IB4 lectin, then analyzed by flow cytometry. Error bars depict standard deviations. FITC, fluorescein isothiocyanate; α-gal, Galα1-3Galβ1-4GlcNAc-R; α-1,3GT, α1,3-galactosyltransferase; PIEC, pig iliac arterial endothelial cells; A549-GT, α-gal expressing A549; A549-V, control.

Figure 3.

Expression of CD55 and CD59 on α-gal-expressing cells influences their sensitivity to CDC. (A) A549, A549-V, A549-GT, Lovo, Lovo-V, Lovo-GT and positive control PIEC cells were incubated with various dilutions of NHS (0, 15, 30, 50%) and survival rates were analyzed by trypan blue staining. Error bars showed standard deviations (*P<0.05 vs. the control). (B) A549, A549-V, A549-GT Cells were pre-treated with various concentrations of PI-PLC (0.001, 0.01, 0.05, 0.1, 0.2, or 0.5 U/ml), incubated with 50% NHS, and survival rates were analyzed by trypan blue staining. Error bars showed standard deviations. *P<0.05, vs. the control. CD55, decay accelerating factor; CD59, protectin; NHS, normal human serum; PI-PLC, phosphatidylinositol-specific phospholipase C; A549-GT, α-gal expressing A549; A549-V, control.

Figure 4.

Effects of PI-PLC treatment on CD55 and CD59 protein level in A549-GT cells. (A) Following 0.1 U/ml PI-PLC treatment, CD55 and CD59 were tested by western blot in A549, A549-V, A549-GT, Lovo, Lovo-V and Lovo-GT cells, compared with that prior to PI-PLC treatment. (B) After 0.1 U/ml PI-PLC treatment, A549-GT cells was incubated with fluorescein isothiocyanate-conjugated anti-human monoclonal antibodies. CD55 and CD59 were analyzed by flow cytometry, compared with that prior to PI-PLC treatment. Error bars showed standard deviations. *P<0.05 vs. the control. CD55, decay accelerating factor; CD59, protectin; PI-PLC, phosphatidylinositol-specific phospholipase C; A549-GT, α-gal expressing A549; A549-V, control.

Figure 5.

Effect of anti-CD55 and anti-CD59 on CDC in α-gal-expressing cells. The A549, A549-V and A549-GT cells were pre-incubated with each antibodies (anti-CD55, anti-CD59 and anti-CD55 with anti-CD59) (10 µg/ml), then 50% normal human serum was added and the survival rates were calculated. Error bars showed standard deviations. *P<0.05 vs. the control. CD55, decay accelerating factor; CD59, protectin; α-gal, Galα1-3Galβ1-4GlcNAc-R; A549-GT, α-gal expressing A549; A549-V, control.