Transient receptor potential Vanilloid 1 (TRPV1) in haematological malignancies

Transient receptor potential vanilloid-1 (TRPV1) is a member of the TRP family of channels that are responsible for nociceptive, thermal and mechanical sensations. It is primarily associated with neuronal cells, but has been detected in different non-neuronal cells, including leukocytes. Capsaicin (CAP), the active ingredient of hot chilli peppers, is one of a number of related endogenous and plant-derived compounds (broadly termed ‘vanilloid-like agents’) that have been shown to induce apoptosis and inhibit cell proliferation in some cancer cells, through both TRPV1-dependent and -independent mechanisms. The expression and function of TRPV1 in haematological malignancies however, has not been extensively investigated. Specific targeting by vanilloid-like agents toward TRPV1 on cancerous cells in patients with haematological malignancies may represent a novel therapeutic approach to treating these diseases. This thesis investigated the expression and function of TRPV1 in haematological malignancies, using both blood cancer cell lines and blood samples obtained from patients with different blood cancers. The specific aims were to; 1) study the effect of TRPV1 agonists and antagonists on the viability of THP-1, U266B1 and U937 haematological malignant cell lines, 2) validate and optimise Western blotting and flow cytometry protocols to detect TRPV1 expression in leukocytes, 3) investigate TRPV1 expression in THP-1, U266B1 and U937 cells, and 4) compare TRPV1 expression in leukocytes obtained from patients with blood cancers to normal subjects. The thesis begins with a comprehensive review and discussion on TRPV1 structure and function, as well as its expression and role in health and disease. In particular, there is a focus on the role of TRPV1 in cancer, including haematological malignancies (Chapter 1). In Chapter 2, the effect of CAP on the metabolic activity of three malignant haematological cell lines, THP-1, U266B1 and U937, was investigated. Metabolic activity assays were performed using the alamarBlue® method. CAP induced cytotoxicity in all three cell lines in a concentration-dependent manner. A biphasic effect on metabolic activity was observed on THP-1 cells [EC50, IC50 (95% CI) = 32.9 (19.9-54.3), 219 (144-246) μM]. U266B1 cells were more resistant to CAP-induced death than THP-1 and U937 cells. TRPV1 and CB1 antagonists (SB452533 and AM251, respectively) suppressed the CAP-induced increase in THP-1 cell metabolic activity (P<0.001). These experiments suggest that CAP inhibits the metabolic activity of malignant haematological cells through a non-TRPV1-dependent mechanism. Chapters 3 and 4 represent the experimental work and trouble-shooting conducted to develop, validate and optimise methods for the detection of TRPV1 expression in human cells. Western blotting (Chapter 3) and flow cytometric (Chapter 4) methods have been previously published, however few have documented the use of appropriate controls for the detection of TRPV1, suggesting that data in the literature may not necessarily be valid. A problem identified in the current study was the correct application of negative controls, particularly to assess the specificity and therefore suitability of the primary antibody used in these methods. These optimised protocols were then used to investigate the expression of TRPV1 in human malignant haematological cell lines (Chapter 5) and leukocytes obtained from patients with blood cancers (Chapter 6). Increased expression of TRPV1 protein was observed in THP-1, U266B1 and U937 cells compared to normal leukocytes. Furthermore, a TRPV1 dimer was detected in U266B1 cells. Interestingly, TRPV1 was detected in non-haematological cell lines that have previously been used as TRPV1-negative cells for Western blotting, including untransfected- and TRPV1-transfected (without tetracycline to switch TRPV1 transcription off) HEK293 and RAW264.7 cells. This latter finding highlights the need for appropriate negative (and positive) controls in both flow cytometric and Western blotting studies of TRPV1. Expression of TRPV1 in leukocytes obtained from patients with a range of haematological malignancies, including multiple myeloma (MM) and B-cell non-Hodgkin’s Lymphoma (B-NHL), was then investigated (Chapter 6). TRPV1 expression was detected in all patients and controls using flow cytometry, but not Western blotting. Using flow cytometry, a sub-group of patients (4/49=8.2%, MM=2, B-NHL=2) demonstrated increased expression of TRPV1 relative to the remainder of the cohort. TRPV1 was found to be similar to the control group for 91.8% of all patients. There were no significant differences in TRPV1 expression (assessed using flow cytometry) between patients with MM and B-NHL, or between de novo patients and those undergoing treatment. Using Western blotting, TRPV1 (~95kDa) was detected in one MM and four B-NHL patients, although interestingly, a 240kDa band was also detected in both a B-NHL and a MM patient. In addition, although C-reactive protein was elevated (≥5 mg/L) in 25% of all patients, it was not associated with higher TRPV1 expression. These results indicate that TRPV1 expression in leukocytes is relatively increased in a small subset of patients with blood cancers, and is not associated with inflammation. Furthermore, some patients may have a unique isoform of TRPV1 that warrants further investigation. In summary, this study has generated new data and knowledge on the role of TRPV1 in haematological cells, including those from patients with blood cancers. A number of novel findings have been reported. Firstly, the inhibition of cell metabolic activity by the TRPV1 agonist, CAP, was found to be independent of TRPV1 activation in malignant haematological cell lines. Secondly, optimised Western blotting and flow cytometric methods for the detection of TRPV1 expression were developed and successfully validated. Thirdly, increased TRPV1 expression was demonstrated in the THP-1, U266B1 and U937 malignant haematological cell lines. Finally, increased TRPV1 expression was observed in some patients with MM and B-NHL, but was not associated with inflammation. The results presented in this thesis can be used as a basis for future studies of TRPV1 function in other human cells and cancers.