Kinetics of expression of galectin-1 and its effects on the viability of spleen cells in a model of rat B cell lymphoma treated with cyclophosphamide

MARÍA J. RICO, MARIANO ZACARIAS, NATALIA RUBINSTEIN, JUAN M. ILARREGUI, MARTA A. TOSCANO, GABRIEL A. RABINOVICH, GRACIELA SCHAROVSKY

Toronto, Canadá

Congreso; American Association of Cancer Research AACR; 2005

American Association of Cancer Research

Kinetics of Galectin-1 (Gal-1) expression and its effect on spleen cells viability  of B-cell lymphoma bearing rats treated with a single-low dose Cyclophosphamide (Cy)    Mariano F. Zacarías*; María J. Rico*; Juan M. Ilarregui; Marta A. Toscano; Natalia Rubinstein; Gabriel A. Rabinovich; O. Graciela Scharovsky   Instituto de Genética Experimental, Facultad de Ciencias Médicas, Universidad Nacional de Rosario (MFZ, MJR, OGS), Rosario, Laboratorio de Inmunogenética. Hospital de Clínicas, Universidad de Buenos Aires (NR, JMI, MAT, GAR), ARGENTINE. *Contributed equally to this work   We have already demonstrated that a single-low dose of Cy has an antimetastatic effect on lymphoma (L-TACB) bearing rats by immunomodulation. We also showed that Gal-1, a beta-galactoside binding protein with immunoregulatory properties, plays a key role in tumor escape from the immune response in this model. The aim of the present study is to explore the kinetics of expression of Gal-1 in spleen, primary tumor and metastasis of Cy-treated L-TACB bearing rats and its effect on  spleen cells viability. Inbred e rats were s.c. challenged with L-TACB (day 0), were distributed in two groups: I) Control, without further treatment and II) Treated on day 14 with Cy i.p. (10 mg/kg). The tumor size was measured twice a week and group I) rats were sacrificed on days 0, 7, 10, 14 and 19 and group II) rats on day 19. Primary tumors, spleens and lymph node metastases were excised (n=4/day), tissues homogenates prepared and Gal-1 expression determined by Western Blot. In primary tumors, the expression of Gal-1 on day 19 was higher than on day 7 (p<0.05). On the contrary, in spleen, Gal-1 expression on day 19, was lower than on day 0 (basal levels) (p<0.05). In primary tumor from treated rats on day 19 (II-19), Gal-1 expression was lower than in I-19 (p<0.05). In spleen II-19, Gal-1 expression was higher than in I-19 (p<0.05). Gal-1 expression in lymph node metastases was lower than in primary tumor on day 19 (p<0.001). The correlation between Gal-1 expression and tumor volume was positive and significant (Pearson Correlation; r2: 0.35; p=0.0122).  Spleen cells suspensions (day 19) from L-TACB bearing rats treated on day 14 with 10mg/kg Cy (group III) or without treatment (group IV), were incubated for 96h with 4 (A) or 10 mg/ml (B) of Gal-1. Spleen cells viability was determined by MTS/PMS colorimetric assay. Results showed that cell viability was not different between groups III-A and III-B; lower in group IV-A than in IV-B (p<0.05);.higher in III-A than in IV-A (p<0.01); higher in III-B than in IV-B (p<0.05). The increased Gal-1 expression during tumor progression is positively correlated with tumor volume. Cyclophosphamide restores the basal levels of Gal-1 in spleen and primary tumor. The decrease of Gal-1 expression in metastasis would suggest a competition for the same expression pathways between Gal-1 and molecules involved in the metastatic phenotype. Gal-1 was not able to revert the increased viability of spleen cells induced by Cy. Therefore, Cy would modulate molecule/s involved in the cell death pathways utilized by Gal-1 when inducing activated T lymphocytes apoptosis.