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

The HT1376 cell line was established from a primary invasive transitional cell cancer of the bladder [25]. Cells were grown in DMEM (4.5 g/L glucose, Sigma), containing 10% foetal calf serum (FCS, Sigma), 2 mM L-glutamine (Sigma) and 100 μg/mL penicillin/streptomycin (Sigma). HT1376 cells expressing ST3GAL1 were generated by transduction with a retroviral vector obtained with the ViraPower Lentiviral Expression System (Invitrogen), according to manufacturer’s instructions. The cDNA of the whole coding region of human ST3GAL1 was obtained by PCR amplification of the cDNA of the colon cancer cell line HT29 with the following primer pair: forward primer: 5’-CACCATGGTGACCCTGCGGAAGAGG-3′; reverse primer: 5’-TCATCTCCCCTTGAAGATCCGG-3′. Amplification was performed for 35 cycles of the following program: denaturation 94 °C 1 min; annealing 60 °C 1 min; extension 72 °C 1 min. The PCR product was gel isolated and cloned into the pLenti6/V5 Directional TOPO cloning vector (Invitrogen) which drives the expression of inserted genes under the control of the cytomegalovirus promoter. A negative control retroviral vector was prepared with an empty plasmid. After transduction with negative control- or ST3GAL1-expressing vectors, HT1376 cells were selected with 4 μg/mL of blasticidin. The replacement of the T antigen with the sT antigen was evaluated as loss of cell reactivity with the fluorescent labeled lectin from Arachis hypogea (peanut agglutinin, PNA), conjugated with fluorescein isothiocyanate (PNA-FITC). Although selected cells were mainly negative to PNA-FITC as detected by FACS analysis, a small population of PNA-FITC positive cells was still present. To obtain a population of cells homogeneously negative for the T antigen, about 100 ST3GAL1-transduced HT1376 cells were seeded in a 10 cm Petri dish and after one month, PNA-FITC negative colonies were selected and pooled. This polyclonal cell population homogeneously negative for T antigen expression is thereafter referred to as HT1376_sT. The polyclonal cell population obtained after transduction with the negative control retroviral vector followed by blasticidin selection is referred to as HT1376_T.

Total RNA was isolated using the GenElute Mammalian Total RNA Purification kit and DNase treatment (Sigma), according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed, using the random-primers based High Capacity cDNA Archive Kit (Applied Biosystems). The expression level of ST3GAL1 (Hs00161688_m1; NM_​173344.​2 and NM_​003033.​3) was evaluated with the TaqMan assay system in a 7500 Fast Real-Time PCR System (Applied Biosystems) using the TaqMan Universal PCR Master Mix Fast, as previously described [24, 26, 27]. The efficiency of the amplification reaction for each primer-probe was above 95% (as determined by the manufacturer). Normalized mRNA expression was computed as the number of mRNA molecules of the gene of interest per 1000 mRNA molecules of the endogenous control β-actin gene, calculated using the 2^-ΔCT×1000 formula [28].

Cell pellets were homogenized in water and the protein concentration of the homogenates was determined by the Lowry method. The activity of ST3GAL1 was measured in the homogenates in the range of time and substrate concentration linearity in a 25 μL volume containing: 50 mM of 2-(N-morpholino)ethanesulphonic acid (MES) buffer pH 6.5, 0.5% Triton X-100, 23.5 μg of Galβ1,3GalNAcα1-O-benzyl (benzyl-T; Sigma) as acceptor substrate, 15 μM (640 Bq) of CMP-[¹⁴C]Sia (Amersham) and 50 μg of homogenate proteins. Reactions were incubated at 37 °C, for 2 h and the products were then isolated by hydrophobic chromatography in SepPak C18 Classic Cartridge (Waters). The columns were washed with water and eluted with 1 mL acetonitrile, which was counted in a liquid scintillation counter. The incorporation on endogenous substrates, in the absence of the acceptor substrate, was subtracted.

Commercial Connaught BCG (ImmuCyst, Sanofi Pasteur SA, France) was suspended in PBS containing 0.05% Tween 80 and stored at − 80 °C. Before each assay, BCG aggregates were discarded by centrifugation (300 × g for 5 min). To assess BCG internalization, bacteria were stained with 2 μg/mL of 5-(and-6-)(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTR, Invitrogen) for 2 h in culture medium, incubated with HT1376_T or HT1376_sT cells in a 1:10 cell/bacteria ratio for 2 h at 37 °C and analyzed by flow cytometry. To assess cytokine secretion, HT1376_T or HT1376_sT cells were challenged with unstained BCG for 2 h at 37 °C, the medium was removed and the cells were washed twice with PBS and incubated with fresh medium for 16 h. Conditioned media were used for cytokine analysis and to challenge macrophages, while cell pellets were used for RNA extraction and transcriptomic analysis.

The concentration of cytokines IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IFN-γ and TNF-α was measured in a 96-well strip plate from a commercial MIBA kit (Bio-Rad), as recommended by manufacturer’s instructions. Fluorescence was read in a Luminex 100 Bio-Plex Liquid Array Multiplexing System reader (Bio-Rad) and the data analyzed with the Bio-Plex Manager v5 software (Bio-Rad).

Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation (GE Healthcare) from the peripheral blood of healthy blood-donors. For this use, no study approval was necessary. The only authorization required was that obtained from the Blood Collection Service of the Pizzardi Hospital in Bologna, Italy, which keeps the rights on donors’ blood samples. Macrophages were obtained by differentiation of monocytes by culture in RPMI 1640 (Sigma) medium supplemented with 20% FCS, 2 mM L-glutamine and 100 μg/mL penicillin/streptomycin. After 7 days, monocyte-derived macrophages were detached with a cell scraper and dispensed in 24 well plates at a cell density corresponding approximately to 50% of confluence. One day later, macrophages were incubated with standard unconditioned culture medium or with the media conditioned by HT1376_T or HT1376_sT cells either BCG-challenged (as described above) or mock challenged. After 2 h, the conditioned media were replaced by fresh medium, which was collected 24 h later and stored at − 80 °C for the detection of cytokines secreted by macrophages.

Cells in exponential growth phase were incubated in serum-free medium containing 5 mM H₂O₂ for 1 h. The medium was then replaced with fresh complete medium and the cells were harvested and analyzed 24 h later. Mock-treated cells were incubated as above without H₂O₂. The cytotoxic effect of H₂O₂ was determined by counting the number of cells in six replicas seeded in 6-wells plates. Representative fields were photographed with an inverted phase contrast microscope.

Total RNA was isolated by the guanidinium thiocyanate-method [29] and converted to labelled single strand cDNA (ssDNA) by the commercial Whole Transcript Expression kit (Ambion), according to the manufacturer’s instructions. Labelled ssDNA fragments were hybridized in a Human Transcriptome Array 2.0 overnight. After staining with phycoerythrin-streptavidin, fluorescence was read in a GeneChip Scanner 3000 7G (Affymetrix). After statistical analysis (see below), array data were functionally analyzed by the ArrayStar v2.0 software (DNASTAR) and through a literature search of the biological roles of modulated genes. Gene nomenclature followed the HUGO Gene Nomenclature Committee rules (https://​www.​genenames.​org/​) in italic uppercase letters. With exception of cytokines, proteins had the same name as the gene, represented in regular uppercase.

Microarray raw data were background-subtracted, normalized and summarized with the robust multi-array average (RMA) algorithm implemented in the Affy package of Bioconductor (www.​bioconductor.​org), which utilizes R software. Differentially expressed genes between query and control assay were selected by application of the two tail ANOVA, followed by the Benjamini-Hochberg false discovery rate test with a p or q ≤ 0.05 cut-off and by the log₂ expression ratio, considering only variations ≥0.5. MIBA data were analyzed by ANOVA, followed by Tukey multiple comparison test. H₂O₂ toxicity data were analyzed by the Student’s t test. The software used was Graphpad Prism, version 7.0.

The mock-transduced cell line HT1376_T, like the wild type HT1376, expressed high and homogeneous T expression, while the ST3GAL1-transduced cell line HT1376_sT displayed an homogeneous low PNA reactivity which could be reverted to high reactivity after sialidase treatment (Additional file 1B). Both the ST3GAL1 activity (Additional file 1C) and the ST3GAL1 mRNA measured by real time PCR (Additional file 1D) were very high in HT1376_sT although almost undetectable in HT1376_T. The level of ST3GAL1 activity and PNA reactivity reached by HT1376_sT cells was similar to that displayed by the wild type BC cell line 5637 strongly expressing ST3GAL1 (data not shown [24]). This is consistent with the notion that, in BC cells, ST3GAL1-mediated α2,3 sialylation of galactose (Additional file 1A) masks the T antigen [24].

To assess the effect of ST3GAL1 overexpression and the consequent replacement of the T with the sT antigen on cytokine production following BCG stimulation, we challenged the two HT1376 cell lines with BCG. Among the several cytokines tested (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IFN-γ and TNF-α), only IL-8 was detectable in media conditioned by unchallenged HT1376_T or HT1376_sT cells. After BCG challenge, IL-6 became detectable in both HT1376_sT and HT1376_T cells, while the IL-8 secretion showed a clear tendency to up-regulation after BCG challenge, especially in HT1376_T cells (Fig. 1a). The p values are reported in Additional file 2.

To establish whether the different IL-8 production by HT1376_sT after BCG challenge was due to a higher BCG uptake by these cells, we measured BCG internalization by the two HT1376 cell populations by flow cytometry. As shown in Fig. 1b, there were no significant differences in the uptake of fluorescent BCG by HT1376_T and HT1376_sT cells, ruling out the possibility that differences in cytokine production by BCG-stimulated HT1376_T and HT1376_sT was dependent on different BCG internalization rate.

To investigate the role played by ST3GAL1 expression and the consequent T/sT replacement on the stimulation of innate immunity by BCG-challenged BC cells, the secretion of cytokines by human macrophages stimulated with the secretome of BCG-challenged BC cells was measured. Unstimulated macrophages secreted high levels of IL-8 and very low levels of TNF-α, IL-6, IL-1β and IL-10. This pattern of secretion was not significantly changed by stimulation with the secretome of BCG-unchallenged HT1376_T or HT1376_sT cells (Fig. 2). In contrast, the secretome from both BCG-challenged cell lines, in particular that from HT1376_sT, significantly increased the secretion of IL-6, IL-1β, TNF-α and IL-10 whereas IL-8 secretion was hardly affected by the secretome of BCG-challenged cells, regardless of their ST3GAL1 expression (Fig. 2). It should be noted that the level of secretion of IL-8 and IL-6 by macrophages was about 10 and 3-fold higher, respectively, than the level of either in HT1376 cells (Fig. 1), ruling out the possibility of a significant contamination of cytokines secreted by macrophages with those secreted by HT1376 cells. The cytokines IL-2, IL-4, IL-12 and IL-17 were not secreted by macrophages in any of the tested conditions (data not shown). The p and q values are reported in Additional file 2.

The impact of ST3GAL1 overexpression and of T replacement by sT on the global transcriptional activity of HT1376 cells was evaluated by expression microarray technology. Of the 254 genes, which displayed ST3GAL1-dependent modulation by at least a log₂ expression ratio ≥ 1.0 (meaning a fold change of at least 2) with p or q values ≤0.05, 106 were up-regulated and 148 were down-regulated. The complete list of genes showing significant modulation (changes equal or higher than 2-fold) in HT1376_sT vs. HT1376_T is reported in Additional file 3. At least 29 genes putatively regulating malignancy because of their involvement in the control of apoptosis, cell growth, angiogenesis, inflammation or proteolysis, were differentially expressed in HT1376_sT cells, compared with HT1376_T cells (Additional file 4). A search in the literature revealed that 18 genes were modulated towards increased malignancy and 14 towards decreased malignancy (Additional file 4). Remarkably, among the 18 genes modulated towards increased malignancy in HT1376_sT cells, 11 down-regulated genes can be collectively referred to as “caretaker genes” because of their involvement in DNA repair and mitotic fidelity (Additional file 4). Moreover, if threshold was lowered to include genes down-regulated above 1.3-fold, the number of down-regulated caretaker genes rose to 34 (Table 1). As a functional validation of gene expression data, cells were exposed to the genotoxic effects of the oxidizing agent H₂O₂. As expected for a cell line with impaired DNA repair mechanisms, H₂O₂ -treatment resulted in a reduced viability in HT1376_sT than in HT1376_T cells (Fig. 3a and b).

To investigate the effect of a differential T/sT expression on BCG challenging, the global transcriptional activity was also compared in BCG-challenged HT1376_T and HT1376_sT cell lines. While in HT1376_sT cells a group of 38 genes showed significant modulation by BCG (p and q values ≤0.05) with a log₂ expression ratio ≥ 0.5 (fold change at least 1.4) (Additional information 5), the tendency to modulation showed by a few genes in HT1376_T cells never reached statistical significance (data not shown). Modulated genes in HT1376_sT cells fell into several broad functional categories (Additional information 5). In particular, a set of genes related to “aminoacid and protein biosynthesis” were prevalently down-regulated in BCG-challenged HT1376_sT cells (Additional file 5). This functional category includes genes involved in the biosynthesis and transport of aminoacids, attachment of aminoacids to tRNAs and regulation of translation. Other categories of modulated genes include “cell growth” and “inflammatory and immune response” (Additional file 5).