There is limited understanding of the effects of the anaphylatoxin C5a in attenuating tumour growth in murine breast cancer. C5a exerts a pro-inflammatory role that induces regression of tumour growth (Gunn and others 2010) and this action is favourable against tumour immunity (Patel and others 1993). In the current study, C5aR were found to be express abundantly on the cell membrane of EMT6 murine mammary cancer cells (see Fig. 1). The expression of the C5aR was reported to be extensively on both immune and non-immune cells, leukocytes (e.g., neutrophils, basophils, monocytes, mast cells) (26, 27, 28) and several tissues (17), supporting similar reports of C5aR expression in lung (19) and cervical cancer cells from syngenic mice (16).
The current study has also shown, for the first time, that a C5aR agonist directly inhibited the growth and viability of mammary tumour cells. Conversely, treatment with a C5aR antagonist significantly enhanced tumour cell proliferation. This was an unexpected finding and contrasted to a study by Markiewski and others (2008) that showed that C5aR activation led to progression of cervical cancer in mice (28).
Several factors may account for the differences between the current study and that of Markiewski and others, 2008 (28). A synthetic C5a synthetic agonist was used in the current study since the C5a molecule itself is unstable (20, 30, 31). EP54 reduced the number and viability of the EMT6 mammary tumour cells, while PMX205 enhanced the growth of these cells. It could be speculated that autogenous production of C5a may limit but not inhibit the growth of EMT6 mammary tumour cells. A further factor to consider is that EP54 is a dual C5a/C3a receptor agonist (32) and activation of the C3a receptor (C3aR) may contribute to its anti-tumour activity, although PMX205 is a specific C5aR antagonist. Finally, the Markiewski and others, 2008 study investigated the effects of C5a on cervical tumour cells and there are reports to suggest that C5a may have varying effects in different cancer cell types (28).
The initial hypothesis for the current study was that C5a released during the inflammatory response, particularly chronic inflammation may initiate and propagate neoplastic changes in cells, based on the findings of Markiewski and others, 2008 (28). However, the current study actually supported other studies that proposed that C5a is a molecular adjuvant inducing anti-tumour immune responses in mouse models of mammary cancer and melanoma (20, 33). The immune properties of C5a potentially derived from the migration of myeloid cells into the mammary tumours with the subsequent release of inflammatory mediators contributing towards tumour cell death (20). However, the current study also showed that C5a might have a direct effect on tumour cell growth and viability, particularly the in vitro results, which removed the confounding effects of immune cells or their products. This study needs to be repeated to see if the effects are consistent with other neoplastic cell lines. Importantly, administration of EP54 also reduced the growth of the EMT6 tumours in vivo. The mechanism by which EP54 may inhibit growth in tumour cells is unclear, but certain peptides will kill cells and delay cell division (34) and EP54 induced significant increases in the signaling proteins for TNF-α, caspase-3 and C5a, compared to C5aR antagonist-treated group.
Further evidence to support the anti-tumour activity of C5a can be found in the response of some neoplastic cells able to express soluble or membrane-associated regulators of complement (e.g.: CD55) (35, 36) that protect against complement-dependent cytolysis (37, 38). Our initial observation indicates that C5a magnitude was higher in the C5a agonist treatment, which might leads to the activation of apoptosis. The increase in TNF-α following EP54 treatment was also interesting since TNF-α is used to treat local or metastatic melanomas and other unresectable tumours, particularly in conjunction with other cytostatic drugs (39). The response of TNF-α to EP54 treatment was lower in vitro than in vivo, which agrees with other reports (40, 41) and may reflect synergies with other mediators in vivo.
In addition to TNF-α, we found that EP54 increased the concentration of caspase-3, in vitro and in vivo. This contrasted with an earlier study in which a C5aR agonist had a protective effect on cultured rat granule neurons by inhibiting the activation of caspase-3 (42). In contrast, other studies have shown that an increased concentration of caspase-3 induced apoptosis in tumour cells (43), including in response to other chemotherapeutic drugs (26). However, it should also be noted that caspase can be present as an active and/or inactive form and apoptosis will depend on the relative ratio of the two forms (44) Furthermore, human breast cancer carcinoma cells (MCF-7) have been reported to resist the apoptotic stimuli by deletion of 47 base pairs in exon 3 of CASP3 gene, which is necessary for caspase-3 expression (42, 45).
Finally, antagonism of the C5aR by PMX205 induced a significant increase in the signaling protein for VEGF-α, with subsequent vascular growth known to be supportive of tumourigenesis (46). Increased expression of VEGF-α was also found to promote tumour growth and angiogenesis in vivo in a nude mouse model (47, 48). In the current study, measurement of VEGF-α activity could only be undertaken in vivo since active blood vessels are required. Similarly, expression of VEGF-α has been shown to increase tumour growth, angiogenesis and metastases in vivo in a nude mouse model, but had no effect on the growth of MCF cells in vitro (12, 47, 49).