Low level exposure to monomethyl arsonous acid-induced the over-production of inflammation-related cytokines and the activation of cell signals associated with tumor progression in a urothelial cell model

Abstract

Human bladder cancer has been associated with chronic exposure to arsenic. Chronic exposure of an immortalized non-tumorigenic urothelial cell line (UROtsa cells) to arsenicals has transformed these cells to a malignant phenotype, but the involved mechanisms are not fully understood. Chronic inflammation has been linked with cancer development mainly because many pro-inflammatory cytokines, growth factors as well as angiogenic chemokines have been found in tumors. In this study the chronology of inflammatory cytokines production was profiled in UROtsa cells chronically exposed to the toxic arsenic metabolite, monomethylarsonous acid [50 nM MMA(III)] to know the role of inflammation in cell transformation. Acute 50 nM MMA(III) exposure induced over-production of many pro-inflammatory cytokines as soon as 12 h after acute exposure. The same cytokines remain over-regulated after chronic exposure to 50 nM MMA(III), especially after 3 mo exposure. At 3 mo exposure the sustained production of cytokines like IL-1, IL-6, IL-8 and TNF is coincident with the appearance of characteristics associated with cell transformation seen in other arsenic-UROtsa studies. The sustained and increased activation of NFkappaB and c-Jun is also present along the transformation process and the phosphorylated proteins p38 MAPK and ERK 1/2 are increased also through the time line. Taken together these results support the notion that chronic inflammation is associated within MMA(III)-induced cell transformation and may act as a promoting factor in UROtsa cell transformation.

Figure 1

Acute exposure to 50 nM MMA(III) leads to over-production of inflammatory cytokines in UROtsa cells. Cytokine production profile in UROtsa cells after 12 (A) and 36 (B) hr of exposure to MMA(III). Cells were exposed to 50 nM MMA(III) for 12 and 36 hr and the supernatant were recovered and analyzed for inflammatory cytokines by 16-plex ELISA array. Graph represents the mean of three independent experiments ± SD of the exposed cells. Asterisks indicate a significant difference [(*), p<0.05, (***) p<0.001] compared with none exposed cells as indicated by Bontferroni ‘s post two way ANOVA test.

Figure 2

Chronic exposure to 50 nM of MMA(III) leads to a sustained over-production of inflammatory cytokines associated with cell proliferation and survival. The figure shows the cytokine production profile in UROtsa cells exposed to 50 nM MMA(III) for 1, 3, 5 or 12 mo. The bars represented the mean of three independent experiments ± SD. Asterisk indicate a significant difference [(***) P< 0.001, (*) P< 0.05 ] compared with non exposed cells as indicated by Bontferroni ‘s post two way ANOVA test.

Figure 3

MIF is over-produced after chronic exposure to MMA(III) in a time dependent fashion. (A) Representative Western Blot of 12 kDa monomeric MIF band (N≥3). HL-60 cells whole lysate was used as positive control. (B) Histogram of MIF densitometric analysis (mean ± SD). Asterisks indicate a significant difference [(***) P<0.001 compared to non treated cells) as indicated by one way analysis of variance and Dunnett's Multiple Comparison as post-Test analysis.

Figure 4

NFKb is constitutively translocated into the nucleus in 50 nM MMA(III) chronically exposed UROtsa cells. Chronically exposed UROtsa cells were cultured in a 96 well plate in a density of 5 × 10⁵ cells/well. After 24 hr, the cells were fixed and permeabilized, specific antibodies against the p65 subunit of NFКβ were added to the plate. The secondary antibody labeled with Daylight-549 (Thermo Scientific) leads the cell to fluoresce in red. Hoechst was used to stain the nuclei; when NFКβ (red) is translocated into the nucleus (blue) the nucleus color looks pink-purple. The images were obtained using a Delta Vision deconvolution inverted microscope.

Figure 5

NFКβ activation in chronically exposed cells to MMA(III). The NFКβ activation assessed by ELISA was two times higher in UROtsa cells exposed for 1, 3, 5, or 12 mo to 50 nM MMA(III). The chronically exposed cells were cultured in 100 mm flasks at 90% confluence, the nuclear proteins were recovered from the cells, quantified and adjusted to assay NFКβ activation using an ELISA based system specific for the phosphorylated p65 subunit. Asterisks indicate a significant difference [(***) P<0.001 or 0.05 (*)] compared to non treated cells as indicated by one way analysis of variance and Dunnett's Multiple Comparison as post-Test analysis.

Figure 6

UROtsa cells exposed for 3, 5, or 12 mo showed a higher c-Jun phosphorylation than non-exposed cells. MMA(III) chronically exposed UROtsa cells were allowed to reach 80% confluence and then were serum starved for 24h. Cell proteins were recovered, quantified and adjusted to assay c-Jun activation trough the use of specific antibodies against phosho-c/Jun by Western Blot. A) Representative blot shows higher c-Jun phosphorylation in chronically exposed cells (N≥3). NIH/3T3 cell whole lysate was used as positive control. B) Histogram of densitometric analysis using imageJ software. Asterisks indicate a significant difference [(***) P<0.001 compared to non treated cells) as indicated by one way analysis of variance and Dunnett's Multiple Comparison as post-Test analysis.

Figure 7

Chronic exposure to 50 nM MMA(III) induces the sustained c-Jun translocation into the nucleus in UROtsa cells. Chronically exposed UROtsa cells were cultured in a 96 well plate in a density of 5 × 10³ cells/well. After 24 hr the cells were serum starved for additional 24 h, then fixed and permeabilized. Specific antibodies against phospho c-Jun were added to the plate. The secondary antibody labeled with Daylight-488 leads the cells nuclei to fluoresce in green-yellow when reached the target. Daylight-549 antibodies against total NFКβ were used for contrast in color images. The images were obtained using a deconvolution inverted microscope.

Figure 8

Inflammatory cell signaling is over activated in UROtsa cells chronically exposed to 50 nM MMA(III). The proteins from chronically exposed cells were recovered when the cells reached 90% confluence and then adjusted to assay an inflammation 4-plex signaling array (Quansys) A) The left image depicts the target localization in each well of the 96 well plate and the right image are representative images showing the correspondent results after chemiluminescent reaction (N≥3) . B) Histograms of densitometric analysis. Cells exposed to 1, 3, or 12 mo show and over activation in AKT p38 and p44/42 (Erk 1/2) MAPKs, while 12 mo exposed cells also showed an increase in total AKT and phospho STAT3. Cells exposed for 5 mo show a decrease in the activation of P38 MAPK and STAT3.

Figure 9

Erk 2 is over expressed in chronically exposed cells to MMA(III). A) Representative western blot show a higher phosphorylation in Erk 2 in cells exposed chronically to 50 nM MMA(III) (N≥3). AKLL myocites treated with insulin whole cell lysate was used as positive control. B) Histogram of densitometric analysis. Asterisks indicate a significant difference [(***) P<0.001) compared to non treated cells as indicated by one way analysis of variance and Dunnett's Multiple Comparison as post-Test analysis.

Figure 10

Chronic exposure of UROtsa cells to MMA(III) over-expresses p-P38 MAPK. A) Representative blot shows higher phosphorylation of p-38 MAPK in UROtsa cells exposed chronically for 3, 5 and 12 mo to 50 nM MMA(III). (N≥3). HeLa plus heat shock whole cell lysate was used as positive control . B) Histogram of densitometric analysis. Asterisks indicate a significant difference [(***) P<0.001) compared to non treated cells as indicated by one way analysis of variance and Dunnett's Multiple Comparison as post-Test analysis.