Neurokinin-1 receptors located in human retinoblastoma cell lines: antitumor action of its antagonist, L-732,138.
OBJECTIVE
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 microM for WERI-Rb1 and 56.78 microM 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.
Muñoz
Miguel
M
Pediatric Intensive Care Unit, Virgen del Rocío University Children's Hospital, Sevilla, Spain. mmunoz@cica.es
Rosso
Marisa
M
Coveñas
Rafael
R
Montero
Ignacio
I
González-Moles
Miguel Angel
MA
Robles
María José
MJ
D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002452:Cell Count; D049109:Cell Proliferation; D002675:Child, Preschool; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007124:Immunoenzyme Techniques; D064729:Neurokinin-1 Receptor Antagonists; D010880:Piperidines / Q000494:pharmacology; D018040:Receptors, Neurokinin-1 / Q000378:metabolism*; D019572:Retinal Neoplasms / Q000378:metabolism* / Q000473:pathology; D012175:Retinoblastoma / Q000378:metabolism* / Q000473:pathology; D013237:Stereoisomerism; D013373:Substance P / Q000378:metabolism; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D064729:Neurokinin-1 Receptor Antagonists; D010880:Piperidines; D018040:Receptors, Neurokinin-1; C086089:3,5-bis(trifluoromethyl)benzyl N-acetyltryptophan; C103788:3-((3,5-bis(trifluoromethyl)phenyl)methyloxy)-2-phenylpiperidine; D013373:Substance P; D014364:Tryptophan