NK-1 receptor antagonists induce apoptosis and counteract substance P-related mitogenesis in human laryngeal cancer cell line HEp-2

It has been demonstrated that substance P (SP) induces cell proliferation and neurokinin-1 (NK-1) receptor antagonists inhibit growth in several human cancer cell lines, but it is currently unknown whether such actions are exerted on human laryngeal carcinoma cell line HEp-2. In addition, the presence of NK-1 receptor has not been demonstrated in this cell line. We carried out an in vitro study of the growth inhibitory capacity of the NK-1 receptor antagonists L-733,060 and L-732,138 against human laryngeal carcinoma cell line HEp-2. Coulter counter was used to determine viable cell numbers followed by application of the tetrazolium compound MTS. Furthermore, an immunoblot analysis was used to determine the NK-1 receptor, and the 4',6-diamidino-2-phenylindole (DAPI) method was applied to demonstrate apoptosis of the laryngeal carcinoma cells. We observed the presence of several NK-1 receptors isoforms (34, 46, 58 and 75 kDa). Nanomolar concentrations of SP increased the growth rate of the cell line and micromolar concentrations of L-733,060 and L-732,138 inhibited the growth of the HEp-2 cells in a dose-dependent manner, with and without previous administration of SP. The 50% inhibition concentration values were 21.34 mu M and 37.97 (48 h) respectively for HEp-2. NK-1 receptor presence on HEp-2 cells was confirmed by western blotting. DAPI staining revealed the presence of apoptosis following NK-1 receptor antagonists treatment. We demonstrated that NK-1 receptors were present in this laryngeal cancer cell line; these findings demonstrate that SP acts as a mitogen on the human laryngeal carcinoma cell line HEp-2 through the NK-1 receptor, and also indicate that both NK-1 receptors antagonists induced apoptosis of the tumour cells. This new action, reported here for the first time, suggests that the NK-1 receptor is a new and promising target in the treatment of human laryngeal carcinoma.