Tumor-Suppressive Cross-Linking of Anti- T. cruzi Antibodies in Acute Lymphoblastic Leukemia

Abstract

Parasites have been associated with possible anticancer activity, including Trypanosoma cruzi, which has been linked to inhibiting the growth of solid tumors. To better understand this antitumor effect, we investigated the association of anti-T. cruzi antibodies with B cells of the acute lymphoblastic leukemia (ALL) SUPB15 cell line. The antibodies were generated in rabbits. IgGs were purified by affinity chromatography. Two procedures (flow cytometry (CF) and Western blot(WB)) were employed to recognize anti-T. cruzi antibodies on SUPB15 cells. We also used CF to determine whether the anti-T. cruzi antibodies could suppress SUPB15 cells. The anti-T. cruzi antibodies recognized 35.5% of the surface antigens of SUPB15. The complement-dependent cytotoxicity (CDC) results demonstrate the cross-suppression of anti-T. cruzi antibodies on up to 8.4% of SUPB15 cells. For the WB analysis, a band at 100 kDa with high intensity was sequenced using mass spectrometry, identifying the protein as nucleolin. This protein may play a role in the antitumor effect on T. cruzi. The anti-T. cruzi antibodies represent promising polyclonal antibodies that have the effect of tumor-suppressive cross-linking on cancer cells, which should be further investigated.

Keywords: Trypanosoma cruzi; acute lymphoblastic leukemia; anti-T. cruzi antibodies; tumor-suppressive cross-linking.

Figure A1

SUPB15 proteins of the antigenic site recognized by anti T. cruzi antibodies. The 28 proteins identified from the protein peptides present a 100 kDa antigenic site by mass spectrometry, are shown by red stars.

Figure 1

Life cycle of Trypanosoma cruzi.

Figure 2

Recognition of membrane proteins of SUPB15 with anti-T. cruzi antibodies by FC. The quadrant Q1-LR shows the percentage of cells that recognize anti-T. cruzi antibodies. (a) SUPB15 and Alexa 488 (with no Ab), (b) SUPB15 with preimmunized antibody and Alexa 488, and (c) SUPB15 with antibodies anti-T. cruzi and Alexa 488 antibodies.

Figure 3

Determination of the CDC with anti-T. cruzi antibodies; the quadrant Q8-LR shows the percentage of cytotoxic. (a) SUPB15 autofluorescence, (b) SUPB15 and complement (with-out Ab), (c) SUPB15 with preimmunized antibody and complement, and (d) SUPB15 with antibodies anti-T. cruzi and complement. Orange CMRA and Blue live/dead staining.

Figure 4

Protein profile and recognition of SUPB15 with anti-T. cruzi antibodies by WB. Shown: (1) Molecular weight. (2) Western blot, protein extract of SUPB15 (1000 μg); preparation was separated by SDS_PAGE 12% and electro-transferred to nitrocellulose membranes, reactivity was carried out with the anti-T. cruzi antibodies 1:100, and anti-rabbit IgG coupled to peroxidase 1:10,000 was used for immunostaining and rebelled with 4-chloro-naphthol. (3) Electrophoretic separation: protein extract of SUP15 (1000 μg) separated by SDS-PAGE 12% for 2 h, Coomassie stains.

Figure 5

Determination of nucleolin in SUPB15; the quadrant Q4-LR shows the recognition percentage. (a) SUPB15 and Alexa 647 antibodies (autofluorescence); (b) SUPB15 with anti-nucleolin and Alexa 647 antibodies.

Figure 6

Determination of the CDC by anti-nucleolin monoclonal antibody. The quadrant Q2-LR shows the percentage of cytotoxic cells. (a) SUPB15 and complement (with no Ab) and (b) SUPB15 with antibodies against nucleolin (clone: NCL/902, NeoBiotechnologies) and complement. Orange CMRA and Blue live/dead staining.