IL-10, IL-10 receptor and galectin-1 are molecular targets of low dose cyclophosphamide (Cy) in a rat B cell lymphoma tumor model

MARÍA J. RICO, MARIANO F. ZACARÍAS, PABLO MATAR, NATALIA RUBINSTEIN, GABRIEL A. RABINOVICH, O. GRACIELA SCHAROVSKY

Orlando, Florida; USA

Congreso; AACR (American Association of Cancer Research); 2004

American Association of Cancer Research

IL-10, IL-10R and Galectin-1 (Gal-1) are molecular targets of low dose cyclophosphamide (Cy) in a rat B-cell lymphoma tumor model María J. Rico, Mariano F. Zacarías, Pablo Matar, Natalia Rubinstein, Marta A. Toscano, Gabriel A. Rabinovich, O Graciela Scharovsky Instituto de Genética Experimental, Facultad de Ciencias Médicas, Universidad Nacional de Rosario (MJR, MZ, PM, OGS), Rosario, Laboratorio de Inmunogenética. Hospital de Clínicas, Universidad de Buenos Aires, ARGENTINE We have already demonstrated that a single-low dose Cy has an antimetastatic effect on lymphoma (L-TACB) bearing rats by immunomodulation. Also, we have shown that IL-10 and Gal-1, a b-galactoside binding protein that induces apoptosis of T activated cells,  may represent negative regulatory pathways involved in downregulation of the immune response in those models.  Our present aim was to study the modulation, by low dose Cy, of IL-10, IL-10R and Gal-1 in the lymphoma model and the effect of Gal-1 on Cy-treated spleen cells viability. Mafosfamide (MF), a compound with the same active metabolites as those of Cy, was used for in vitro studies. Inbred e rats were s.c. challenged with L-TACB and, on day 21, primary tumors (Tu) and lymph node metastases (Me) were excised and cell suspensions prepared. Cells were incubated 96h with 0, 5 and 10 mM MF in RPMI + 10% FCS and cells and conditioned media (CM) were studied independently. Cell proliferation of Me cells was inhibited by both doses of MF, while Tu cells were not affected by them. The concentration of IL-10, measured in CM by ELISA, showed non detectable values for Tu cells, while for Me cells it was higher (p<0.001) in control cells (mean ± SE: 493±7,6 pg/ml) than those treated with 5mM (127±1,5 pg/ml) and 10 mM MF (26±3 pg/ml). The [IL-10R], determined by CELISA (cellular ELISA), for Me control cells (Optical density:1488±11,8) was higher (p<0.001) than for cells incubated with MF (236±13,9 and 76,5±3,6, respectively). No variations were detected for Tu cells. The expression of Gal-1, detected by Western blot, in Tu incubated 18 and 24h with MF was down-regulated in a non dose-dependent manner with respect to control cells, a result that was not observed in Me which showed an heterogeneous behavior. Early apoptosis, evaluated with Annexin-V, induced by 96h incubation with 4mg/ml Gal-1 of CT and control spleen cells of L-TACB-bearing rats, was lower  (p<0.01) in CT than in controls. Considering that Cy, administered in vivo, would produce the same effects than Mafosfamide in vitro, the down-regulation of IL-10 and IL-10R in metastatic cells and of Gal-1 in primary tumor cells, along with the inhibition of tumor cell proliferation and the reduction of spleen cells apoptosis, which cannot be reverted by Gal-1 addition, would enable the immune system to develop an effective antimetastatic immune response. Thus, several molecular targets of Cy on primary tumor and/or metastatic cells would be responsible for overcoming different tumor-induced immunosuppression.