Human C1q Induces Apoptosis in an Ovarian Cancer Cell Line via Tumor Necrosis Factor Pathway

Abstract

Complement protein C1q is the first recognition subcomponent of the complement classical pathway that plays a vital role in the clearance of immune complexes, pathogens, and apoptotic cells. C1q also has a homeostatic role involving immune and non-immune cells; these functions not necessarily involve complement activation. Recently, C1q has been shown to be expressed locally in the microenvironment of a range of human malignant tumors, where it can promote cancer cell adhesion, migration, and proliferation, without involving complement activation. C1q has been shown to be present in the ascitic fluid formed during ovarian cancers. In this study, we have examined the effects of human C1q and its globular domain on an ovarian cancer cell line, SKOV3. We show that C1q and the recombinant globular head modules induce apoptosis in SKOV3 cells in a time-dependent manner. C1q expression was not detectable in the SKOV3 cells. Exogenous treatment with C1q and globular head modules at the concentration of 10 µg/ml induced apoptosis in approximately 55% cells, as revealed by immunofluorescence microscopy and FACS. The qPCR and caspase analysis suggested that C1q and globular head modules activated tumor necrosis factor (TNF)-α and upregulated Fas. The genes of mammalian target of rapamycin (mTOR), RICTOR, and RAPTOR survival pathways, which are often overexpressed in majority of the cancers, were significantly downregulated within few hours of the treatment of SKOV3 cells with C1q and globular head modules. In conclusion, C1q, via its globular domain, induced apoptosis in an ovarian cancer cell line SKOV3 via TNF-α induced apoptosis pathway involving upregulation of Bax and Fas. This study highlights a potentially protective role of C1q in certain cancers.

Keywords: C1q; TNF; apoptosis; complement; mTOR; ovarian cancer.

Figure 1

(A) Binding of human C1q and recombinant globular head modules, ghA, ghB, and ghC (10 µg/ml; 1 h incubation) to SKOV3 cells using immunofluorescence microscopy. Panel A shows the nucleus of the cells stained with Hoechst. Panel B shows the cells probed with anti-C1q (C1q) and anti-maltose-binding protein (MBP) (globular heads) polyclonal antibodies, followed by anti-rabbit IgG labeled with FITC; the bound proteins are visible on the cell membrane (panels C and D). (B) Flow cytometric analysis to show binding of human C1q and ghA, ghB, and ghC (10 µg/ml) to SKOV3 cells after 1 h incubation. Panel a shows the number of cells probed with anti-C1q (C1q) and anti-MBP (globular heads) antibodies followed by anti-rabbit IgG labeled with FITC, as compared to the untreated cells. Panel b shows the shift in the fluorescent intensity from untreated to treated cells.

Figure 2

SKOV3 cell viability assay via Trypan blue exclusion (A) and MTT (B) following treatment with human C1q, ghA, ghB, ghC, and maltose-binding protein (MBP) (10 μg/ml)and untreated control for 24 h (±SEM, of three independent experiments). Cell numbers were reduced by approximately 50% in the treated as compared to untreated and MBP controls. Significance was established using the unpaired one-way ANOVA test (*p < 0.05, *p < 0.01, and ***p < 0.001) (n = 6).

Figure 3

Analysis of apoptosis using immunofluorescence microscopy in SKOV3 cells treated with human C1q, ghA, ghB, and ghC (10 µg/ml) and an untreated control after 24 h. Panel A shows the nucleus stained with Hoechst. Panel B shows the cell membrane integrity marker FITC Annexin V, which binds to PS of the cell membrane of cells undergoing apoptosis. No FITC was detected in the untreated SKOV3 cells.

Figure 4

The quantitative analysis of apoptosis using FACS. SKOV3 cells, treated with C1q and globular head modules for 24 h, yielded approximately 50% (C1q, ghA, ghB, and ghC quadrant Q2-2) cells positive for both FITC and PI, significantly higher than 1.42% untreated control cells (untreated, quadrant Q2-2). Approximately 20% cells stained positive for FITC only (C1q, ghA, ghB, and ghC quadrant Q2-4).

Figure 5

Relative quantification comparisons of TNF-α (A) and NF-κB (B,C) mRNA expression in SKOV3 cells treated with C1q, ghA, ghB, and ghC. The transcriptional expressions of both TNF-α and NF-κB were upregulated (log₁₀ 0.5- to 1-fold) after 12 h treatment and NF-κB at 24 h was downregulated. Significance was ascertained using the unpaired one-way ANOVA test (*p < 0.05, *p < 0.01, and ***p < 0.001) (n = 3).

Figure 6

Relative quantification comparisons of Fas (A) and Bax (B,C) mRNA expression in SKOV3 cells treated with C1q, ghA, ghB, and ghC (10 µg/ml). Fas expression was upregulated (~log₁₀ 0.5-fold) at 12 h (C1q only) and 24 h (both for C1q and globular head modules). Bax expression was upregulated (~log₁₀ 0.5-fold) at 24 h. Significance was determined using the unpaired one-way ANOVA test (*p < 0.05, *p < 0.01, and ***p < 0.001) (n = 3).

Figure 7

Caspase activation in SKOV3 cells following treatment with C1q and globular head modules. (A) Western blot analysis of full-length/total caspase 3 at 32 kDa after 12 and 24 h of the treatment with C1q, ghA, ghB, ghC, and untreated. (B) The cleaved caspase 3 was observed at 17 kDa after 24 h of treatment with C1q, ghA, ghB, ghC, and untreated (C) Western blot analysis for β-actin as a loading control for 12 h and 24 h at 45 kDa. (D) The activation of caspase 3 was also shown by immunofluorescence microscopy at 24 h in parallel with apoptosis staining for Annexin V-FITC, where activated caspase 3 was clearly visible in the cytoplasm probed with CY3 at 24 h.

Figure 8

(A) Relative quantification comparisons of mammalian target of rapamycin (mTOR) (a), RAPTOR (b), and RICTOR (c) mRNA expression in SKOV3 cells treated with C1q, ghA, ghB, and ghC (10 µg/ml) at 6 h. Significance was obtained using the unpaired one-way ANOVA test (*p < 0.05, *p < 0.01, and ***p < 0.001) (n = 3). (B) Immunofluorescence microscopy to show the presence of mTOR detected abundantly in the cytoplasm (green) of the untreated cells, compared to SKOV3 cells treated with C1q and globular heads at 15 h time point.

Figure 9

Illustration of the likely apoptosis pathway in action following C1q binding to SKOV3 cells. Exogenous treatment of SKOV3 cells with C1q and individual globular head modules can induce upregulation of TNF-α and Fas. TNF-α stimulation triggers two parallel, but contrasting, pathways: the pro-apoptotic caspase (15) and the anti-apoptotic NFκB-IκB inhibitor of apoptosis proteins (IAP) pathway (16). The cell survival or death, however, depends upon interactions among the components of these two pathways as well as other factors such as mitochondrial dysfunction (15, 16). TNF-α can bind to TNF type I receptor (TNFR1), which is then internalized to first form a complex with TNFR1-associated DEATH domain, receptor-interacting protein 1 (RIP1), TNF receptor-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis protein-1 (c-IAP1) (complex I), triggering the upregulation of nuclear factor-κB (NF-κB). A second complex (complex II) is later formed when complex I bind to Fas-associated protein with death domain (FADD), which is recruited upon activation of Fas (15, 17). Complex II subsequently causes downstream activation of apoptosis signal by activating caspase cascade, resulting in the cleavage of initiator caspase 8 and effector caspase 3 leading to apoptosis (, –19). However, NF-κB upregulation may intersect apoptotic pathway as it can promote the induction of various cellular apoptosis inhibitors such as TRAF1, TRAF2, cIAP-1, c-IAP-2, and X-linked inhibitor of apoptosis protein (XIAP) (15, 20).