Oxidative damage and response to Bacillus Calmette-Guérin in bladder cancer cells expressing sialyltransferase ST3GAL1

Abstract

Background: Treatment with Bacillus Calmette-Guérin (BCG) is the gold standard adjuvant immunotherapy of non-muscle invasive bladder cancer (NMIBC), although it fails in one third of the patients. NMIBC expresses two tumor-associated O-linked carbohydrates: the disaccharide (Galβ1,3GalNAc) Thomsen-Friedenreich (T) antigen, and its sialylated counterpart (Siaα2,3Galβ1,3GalNAc) sialyl-T (sT), synthesized by sialyltransferase ST3GAL1, whose roles in BCG response are unknown.

Methods: The human bladder cancer (BC) cell line HT1376 strongly expressing the T antigen, was retrovirally transduced with the ST3GAL1 cDNA or with an empty vector, yielding the cell lines HT1376_sT and HT1376_T, that express, respectively, either the sT or the T antigens. Cells were in vitro challenged with BCG. Whole gene expression was studied by microarray technology, cytokine secretion was measured by multiplex immune-beads assay. Human macrophages derived from blood monocytes were challenged with the secretome of BCG-challenged BC cells.

Results: The secretome from BCG-challenged HT1376_sT cells induced a stronger macrophage secretion of IL-6, IL-1β, TNFα and IL-10 than that of HT1376_T cells. Transcriptomic analysis revealed that ST3GAL1 overexpression and T/sT replacement modulated hundreds of genes. Several genes preserving genomic stability were down-regulated in HT1376_sT cells which, as a consequence, displayed increased sensitivity to oxidative damage. After BCG challenge, the transcriptome of HT1376_sT cells showed higher susceptibility to BCG modulation than that of HT1376_T cells.

Conclusions: High ST3GAL1 expression and T/sT replacement in BCG challenged-BC cancer cells induce a stronger macrophage response and alter the gene expression towards genomic instability, indicating a potential impact on BC biology and patient's response to BCG.

Keywords: Bacillus Calmette-Guérin; Glycosylation; Sialyl T antigen; Sialyltransferase; Thomsen-Friedenreich antigen.

Fig. 1

BCG-stimulation of HT1376 cells. a: Secretion of IL-6 and IL-8. HT1376_T, (white circles) and HT1376_sT (black circles) cells were challenged with BCG for 2 h, then they were washed and cultured in medium for 16 h. Cytokine concentration was determined as described in Materials and Methods section. Data of three independent experiments are reported. Statistical analysis is reported in Additional file 2. b: Time course of BCG uptake. HT1376T (white circles) and HT1376sT (black circles) cells were challenged with CMTMR-labelled BCG for 2 and 6 h, as described in the Methods section. BCG internalization was estimated as mean fluorescence intensity of the cells, measured by flow cytometry Data of three independent experiments are reported

Fig. 2

Cytokine secretion by human macrophages treated with conditioned media of BC cell lines. Cytokine secretion was measured in culture media conditioned by unstimulated macrophages (MΦ, grey circles) or stimulated with conditioned media from BCG-challenged or unchallenged BC cell lines: HT1376_T, (white circles); HT1376_sT (black circles). Cells were challenged with BCG for 2 h, then they were washed and allowed to condition the culture medium for 16 h. The conditioned culture media of BCG-challenged or unchallenged cells was used to stimulate human monocyte-derived macrophages for 24 h. MØ: cytokines released by unstimulated macrophages; MØ + unchallenged: cytokines released by macrophages stimulated with the conditioned medium of unchallenged HT1376 cells; MØ + BCG-challenged: cytokines released from macrophages stimulated with the conditioned medium of BCG-challenged cells. Data of three independent experiments are reported. Statistical analysis is reported in Additional file 2

Fig. 3

Cytotoxic effect of H₂O₂ on HT1376_T and HT1376_sT cells. a: cells were treated with 5 mM H₂O₂ or mock-treated as described in Methods and counted. Data of six experiments are reported. The HT1376_sT cells are more susceptible to the toxic effect of H₂O₂ (**p = 0.005 according to Student’s t test). b: phase contrast micrograph of representative fields of the two cell populations treated as described above, showing the stronger cytotoxic effects of H₂O₂ on HT1376_sT. Scale bar: 100 μm