The in vitro and in vivo effect of a chemokine inhibitor CTCE-9908 in combination with Kynurenine metabolites on tumour adhesion

In this study, the effect of CTCE-9908 (a CXCR4 chemokine receptor inhibitor) and combination treatment with three kynurenine metabolites (quinolinic acid, L-kynurenine, and kynurenic acid) was investigated on cell viability and adhesion in melanoma and endothelioma cell lines since they are highly metastatic cancers. L-kynurenine (L-kyn) was identified as a promising antiproliferative kynurenine metabolite based on the in vitro experiments conducted on endothelioma cells; therefore, L-kyn was used in combination treatment with CTCE-9908 to investigate tumour adhesion by quantifying adhesion proteins, namely E-cadherin, paxillin, and focal adhesion kinase (FAK) in vitro in melanoma cells and in vivo in murine models.

The study aimed to explore the in vitro effects of L-kyn, quinolinic acid (Quin), and kynurenic acid (KA) on endothelioma sEnd-2 cells and on endothelial EA.hy926 cells (control cell line). The in vitro effect at 24 h, 48 h, and 72 h exposure to a range of 1 mM to 4 mM of the respective kynurenine metabolites on the two cell lines in terms of cell morphology, cell cycle progression, and induction of apoptosis was assessed. The half inhibitory concentration (IC₅₀), as determined using nonlinear regression, for L-kyn, Quin, and KA was 9.17 mM, 15.56 mM and 535.40 mM, respectively. Optical transmitted light differential interference contrast and hematoxylin and eosin staining revealed cells blocked in metaphase, formation of apoptotic bodies, and compromised cell density in L-kyn-treated cells. A statistically significant increase in the number of cells present in the sub-G₁ phase was observed in the L-kyn-treated sample. It can be concluded that L-kyn exerts an antiproliferative effect on the endothelioma sEnd-2 cell line by decreasing cell growth and proliferation as well as a metaphase block. These hallmarks suggest cell death via apoptosis.

In addition, the effect of kynurenine metabolites and CTCE-9908 on adhesion in melanoma B16-F10 cells and control cell lines, namely the human immortalised human keratinocyte cell line (HaCaT) and endothelial EA.hy926 cells, was investigated using the crystal violet assay, in which 96-well plates were coated with fibronectin and collagen IV. Results showed a statistically significant decrease in the percentage cell number in L-kyn- and CTCE-9908-treated sEnd-2 cells when compared to the vehicle control, suggesting the inhibitory effect of the compounds on adhesion. In the non-cancerous cell lines HaCaT and EA. hy926, there was a decrease in percentage cell number after treatment with L-kyn, phorbol 12-myristate 13-acetate (PMA), a positive control for adhesion, and CTCE-9908, suggesting a decrease in cell adhesion in the non-cancerous cell lines.

The in vivo research revealed that the intravenous injections of a combination treatment of chemokine inhibitor CTCE-9908 and L-kyn downregulates the metastatic characteristic of melanoma in C57BL/6 mice inoculated with B16-F10 cells. A decrease in tumour volume was observed in the combination treatment when compared to the treatments alone. Cell morphology was assessed by means of immunohistochemistry and it showed an increase in adhesion protein staining, namely E-cadherin in mice treated with CTCE-9908 when compared to control mice. This was confirmed by an increase in E-cadherin in mice serum by enzyme-linked immunosorbent assay (ELISA). These findings suggest that CTCE-9908 and L-kyn may possess anti-metastatic properties against melanoma. Data from this study provides novel insights into the mechanism of action of CTCE-9908 and kynurenine metabolites and suggests that CTCE-9908 and L-kyn represent promising adjunct chemotherapeutic agents against melanoma and endothelioma.