The effect of substance P (SP) on adhesion of Jurkat leukemia cells and squamous carcinoma cells (SCC) to vascular endothelial cells and role in metastasis

Metastasis is the leading cause of fatality in 90% of cancers, and approximately 60% of all cancer cases will have either regional or distant metastasis at initial diagnosis. Global data shows that survival rates drop significantly with the advance of disease stage and metastatic activity; therefore, patients with localised disease have a far better prognosis than those with disseminated tumours. In this aspect, Leukaemia and oral squamous-cell carcinoma (OSCC) are invasive neoplasms. Both cancers are considered the worst prognosis cancers with an overall survival rate of 50% or slightly higher. The accumulating research suggests that inflammation is more likely to act in favour of tumour initiation and metastasis. The striking similarity which exists between the out flux of tumour mass in the circulation and that of inflammatory exudates in wound healing points to the possibility that the same mediators might be utilised for both purposes. Substance P (SP) is a primary regulator of neurogenic inflammation mainly acting as a trigger for vasodilatation, plasma protein extravasation and leukocyte adhesion to vascular endothelial cells. SP has been proposed as a model that can explain the inflammation-tumour relationship. Therefore, the link between the role of SP and the extravasation of tumour cells into the circulation represents an attractive opportunity for unravelling the metastatic mechanisms. We carried a systematic review which identified nine inflammatory mediators associated with metastasis. The 16 articles reported lymph node metastasis and one article reported both lymph node and distant metastasis. The inflammatory mediators identified were CXCR4 (six studies), CXCL12 (SDF-1), CCR7, IL-6 (two studies each), IL-18, CCL20 (MIP-3), CXCL1 (GRO-1), CCL3, CXCR2 (one study each). This review systematically summarises the evidence of the prognostic role of inflammatory mediators in predicting metastasis/metastatic stages in OSCC. Additionally, it compares the available evidence from clinical and experimental animal settings. Our experimental results showed that SP increased the adhesion of Jurkat Leukaemia cell lines in a time-course treatment with a peak adhesion increase at three-four hours. SP increased the adhesion of H157, CAL27, and DOK cells to HUVEC endothelial cells (P < 0.001), significantly, in a time-dependent treatment with peak adhesion at three hours. It has been demonstrated that SP is expressed by several tumours and several roles have been proposed for its action in tumour growth and progression. Our study describes a new role for SP in stimulating an early onset adhesion of tumour-endothelial adhesion. We found that treating endothelial cells with either stimulating factor or inhibitor produces more potent levels of adhesion which may 1) explain the organ-specific metastasis of cancers; and 2) highlight the active interaction of tumour-endothelial cells during adhesion. Moreover, our data suggests that inhibition of adhesion levels – achieved using cycloheximide, which blocks translation of messenger RNA on the cytosolic 80S ribosomes but does not inhibit the organelle protein synthesis – did not achieve higher levels such as the one inflicted with monoclonal antibodies. This might suggest that tumour cells highly express adhesion molecules as well as inflammatory receptors. Our data also suggests that in Jurkat cells, CAL27 and BICR22, SP 1mcg/ml treatment has increased both CD 11 (not significant) and CD 49 (P < 0.05), but not CD15s expression in 1-48 hour time-scale treatment, as indicated by the FACS analysis. Anti-human monoclonal antibodies to VCAM or ICAM significantly inhibited adhesion levels to below the untreated baseline levels. The NK-1R antagonist was only effective in inhibiting adhesion molecule expression in Jurkat and CAL27. No other effect was noted in the other OSCC cell lines. We hypothesised that adhesion molecules were the main requirement of the adhesion process, and adhesion molecule expression followed the pattern predicted which increased from no expression in the normal oral keratinocytes to the lymph node positive cell line H157 and declined in the metastatic cells BICR22, for the three adhesion molecules. The pre-cancer cell line DOK had an elevated expression profile which agrees with previous studies, suggesting an early invasive/metastatic phenotype in the tumour cycle. Our results did not prove any significant effect for SP on the release of MMP-2 in either Jurkat cells or OSCC cell lines. This was against the predicted pattern in which we hypothesised that SP would trigger up-regulation of MMP enzymes to facilitate the transmigration of tumour cells through the endothelium. The resulting model represents a novel approach in cancer treatment where the main target is prevention of metastasis. This paradigm shift has a powerful potential to develop effective anti-metastatic therapies through interfering with the metastatic cycle. The data resulting from the systematic review as well as the experimental findings can be integrated for future implementation in two main categories. In conclusion, the study identifies a new role for Substance P in mediating an early onset adhesion of cancer cells to endothelial cells. The study also highlights the role of the NK-1R antagonist as a novel therapy in inhibiting this adhesion in those cell lines. A combined therapy of the NK-1R antagonist and monoclonal anti-adhesion molecule would be powerful in preventing the onset of metastasis. These primary data warrant further research through animal models to confirm this role for Substance P

Transforming growth factor-β1 treatment of oral cancer induces epithelial-mesenchymal transition and promotes bone invasion via enhanced activity of osteoclasts

This study investigates relationships between EMT and bone invasion by OSCC. Three OSCC cell lines, SCC25, HN5, and Tca8113 were artificially induced to display EMT by adding 5 ng/mL of TGF-β1 to culture media for 1–3 days. Cell morphology and phenotypic changes was examined by immunocytochemical staining of CK and VIM. EMT markers, cell-invasion factors, and osteoclast-related molecules were analysed at mRNA, gelatine and protein levels by real-time PCR, gelatine zymography and Western blotting respectively. Mature osteoclasts differentiated from Raw264.7 cells were treated by conditioned medium (CM) of OSCC cells with/without TGF-β1. Immunohistochemistry was performed to validate proteins of CK, VIM, E-cad and Snail1 in OSCC tissue samples with bone invasion. Results showed minimal staining of VIM was found in SCC25 and HN5, while Tca8113 cells stained strongly. EMT markers Twist1 and N-cad were up-regulated; Snail1 and E-cad down-regulated in all cells. Of factors associated with invasion, MMP-2 was unchanged and MMP-9 increased in SCC25 and Tca8113, while MMP-2 was increased and MMP-9 unchanged in HN5. For osteoclast-related molecules, both MT1-MMP and RANKL were up-regulated, while OPG was down-regulated in all cells. CM of OSCC cells pre-treated with TGF-β1 showed to prolong survival of osteoclasts up to 4 days. All target molecules were validated in OSCC samples of bone invasion. These findings suggest that TGF-β1 not only induces EMT to increase the capacity of OSCC for invasion, but also promotes factors which prolong osteoclast survival. TGF-β1 may enhance the ability of MMP2/9 in resorbing bone and favouring invasion of cancer cells

Substance P (SP) increases the adhesion of oral squamous cell carcinoma (OSCC) to human umbilical vein endothelial cells (HUVEC) uhrough upregulating adhesion molecules; implication for metastasis

Objectives: Interaction of cancer cells with endothelial cells plays an important role in metastasis. Our study hypothesised that SP might play a role in the early onset adhesion of OSCC to HUVEC cells lines via upregulating the adhesion molecule expression. Methods: CAL 27, SCC25, DOK, H157, BICR22 (OSCC) and HUVEC cell lines were used. Adhesion molecules’ expression was measured by flowcytometry. The ECM645 adhesion assay was used for quantification of tumour-endothelial adhesion. Matrix metalloprotienease-2 (MMP-2) expression in OSCC cells was measured by total ELISA kit. Results: Treatment with SP increased the adhesion of H157, CAL27 and DOK cells in a time-dependent manner, which was inhibited SP receptor antagonist. Monoclonal anti-adhesion antibodies inhibited the adhesion between OSCC and HUVEC. SP treatment increased (very late antigen-4) VLA-4 adhesion molecule in CAL27 and SCC25 cells and (lymphocyte function associated antigen-1) LFA-1 in BICR22. SP treatment increased MMP-2 release but it was not significant. Conclusion: The study showed that SP could promote early onset oral cancer–endothelial cell adhesion. The study provided insights into the regulatory mechanism of this adhesion that it can occur in acute phase and in pre-cancer cells. These findings may provide potential new therapeutic targets for metastasis prevention

Identification of inflammatory mediators associated with metastasis of oral squamous cell carcinoma in experimental and clinical studies: systematic review

Metastasis, whether regional or distant, remains the main cause of morbidity and recurrence in oral cancer. The accumulating evidence suggests that inflammatory mediators are strong drivers for cancer progression and spread. However, the precise role of these inflammatory mediators in mediating specific metastatic stage is poorly understood due to lack of integration/validation of experimental research data and the clinical trials, i.e., the data produced from research is not translated to clinical therapeutic targets. This, in turn, results in the lack of developing reliable biomarker that can be used for accurate diagnosis/prognosis of the tumour spread. We have performed a systematic review to assess the role of inflammatory mediators as potential markers for diagnosis/prognosis of oral squamous cell carcinoma (OSCC) metastasis. We carried out a systematic search the PubMed, Web of Science, Embase and Scopus databases under the guidelines for Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Australian National Health and Medical Research Council (NHMRC). Articles were divided into two groups; experimental (in-vivo) and clinical studies. The REporting recommendations for tumour MARKer prognostic studies Scale (REMARK) was used to assess the quality of the studies for the clinical search while Animal research: Reporting In-vivo experiments (ARRIVE) guidelines were used to assess the quality of the animal studies. Sixteen articles in the clinical group and four articles in the experimental group were included in the final review. We identified nine inflammatory mediators; CXCR4, CXCL12 (SDF-1), CCR7, IL-6, IL-18, CCL20 (MIP-3), CXCL1 (GRO-1), CCL3, CXCR2. This panel of inflammatory mediators can provide a framework for hypothesis testing of the potential value of these mediators in metastatic prognosis. We recommend carrying a large cohort study with data pooling for adequate assessment and testing of the inflammatory panel of mediators