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Neurokinin-1 Receptors Located in Human Retinoblastoma Cell Lines: Antitumor Action of Its Antagonist, L-732,138

¹From the Pediatric Intensive Care Unit, Virgen del Rocío University Children’s Hospital, Sevilla, Spain; ²Laboratory of Neuroanatomy of the Peptidergic Systems, Institute of Neurosciences of Castilla y León, Salamanca, Spain; and ³University of Granada, School of Dentistry, Department of Oral Medicine, Granada, Spain.

PURPOSE. The authors have recently demonstrated that substance P and L-733,060 induce cell proliferation and cell inhibition, respectively, in human retinoblastoma cell lines. However, the presence of neurokinin-1 receptors has not been demonstrated in such cell lines, nor is it known whether other neurokinin-1 receptor antagonists exert antitumoral action against retinoblastoma cell lines. The purpose of this study was to demonstrate the presence of neurokinin-1 receptors in the human retinoblastoma cell lines WERI-Rb-1 and Y-79 and to study the growth inhibitory capacity of the neurokinin-1 receptor antagonist L-732,138 against those cell lines. The authors also sought to demonstrate that the administration of L-732,138 or L-733,060 induces apoptosis in retinoblastoma cells and that neurokinin-1 receptors and substance P are present in primary retinoblastoma.

METHODS. Immunoblot analysis was used to determine neurokinin-1 receptors, and a Coulter counter was used to determine viable cell numbers; this was followed by application of the tetrazolium compound WST-8, a colorimetric method, to evaluate cell viability. DAPI stain was applied to assess chromatin condensation, characteristic of apoptosis, and immunoperoxidase was used to demonstrate neurokinin-1 receptors and substance P in eyes with primary retinoblastoma.

RESULTS. Neurokinin-1 receptors were present in both retinoblastoma cell lines studied. Three identical bands (isoforms of approximately 33, 58, and 75 kDa) were observed in both cell lines. Moreover, L-732,138 inhibited the growth of both cell lines studied, with and without previous administration of substance P. This inhibition occurred in a dose-dependent manner, with the IC₅₀ values of 60.47 µM for WERI-Rb1 and 56.78 µM for Y-79. Moreover, apoptosis was observed in both cell lines after the administration of L-732,138 or L-733,060. In fixed eyes with primary retinoblastoma, a high density of neurokinin-1 receptors was observed in tumor cells, whereas a very low number of such cells contained substance P.

CONCLUSIONS. This study showed that the same isoforms of the neurokinin-1 receptor are present in human retinoblastoma cell lines WERI-Rb-1 and Y-79. Both L-732,138 and L-733,060 can induce apoptosis in these cell lines and therefore can act as antitumoral agents. Primary retinoblastoma specimens display neurokinin-1 receptor immunolabeling. These results suggest that the neurokinin-1 receptor may be a promising new target for the treatment of retinoblastoma.