HSulf-1 and low-dose cadmium change cell morphology and reduce cell numbers
Preliminary data using various concentrations of sodium chlorate (5 to 200 mM) as a global inhibitor of sulfation revealed that chlorate itself induces cytotoxicity in H292 cells, and it was found that this cytotoxicity could be reduced by heparin (500 μg/ml), indicating that there is a lower limit to the amount of sulfation crucial for cell growth and proliferation (Supplementary Data 1). Matrix HSPGs are decorated with a variety of sulfate moieties, so to dissect the contribution of a single type of sulfation to cell viability we specifically targeted the 6-O-sulfate moiety with a HSulf-1 over-expression adenovirus.
Previously, we reported that HSulf-1 over-expression reduced H292 cell numbers in a dose-dependent fashion but had no effect upon hAT2 cells, and that the mechanism behind this decrease was apoptosis, likely due to the reduction in growth factor signaling caused by selective removal of 6-O-sulfates from HSPGs by HSulf-1 (Zhang et al., 2012
). In hAT2, H292, and A549 cells over-expressing HSulf-1, the sulfated glycosaminoglycan (GAG) content was indeed reduced, which confirmed the selective modification of extracellular matrix by HSulf-1 (Supplementary Data 2).
Preliminary data also indicated that exposure to even a low level of cadmium reduces viability in hAT2 cells, while H292 and A549 cells are much more resistant to cadmium's effects. Because many cancer cells exhibit increased resistance to heavy metals, such as cadmium, we wanted to determine whether HSulf-1 over-expression might sensitize H292 cells to cadmium and/or intensify the effects of cadmium on H292 cells.
H292, A549, and hAT2 cells (two to three days after isolation) were infected with adenoviruses at 10 MOI (multiplicity of infection) for transient transduced over-expression of lacZ (control) or HSulf-1, incubated for 24 hours to allow expression of the gene, and then treated with 10 μM cadmium for 48 hours. The hAT2 cells infected with lacZ adenovirus only were essentially similar to untreated cells (data not shown) and, by the fifth or sixth day after isolation, had assumed a phenotype similar to hAT1 (type I) cells, which are typically large, round, and squamous in appearance with a centrally located nucleus (). H292 cells infected with lacZ adenovirus only () appeared as small, polygonal cells with a centrally positioned nucleus, essentially similar to untreated cells (data not shown). A549 cells transduced with lacZ appeared similar to untreated cells as well (data not shown). With cadmium treatment, even at this relatively low concentration (10 μM), there was a visible decrease in hAT2 cell density and size along with assumption of more stellate or fusiform shapes (), while H292 and A549 cells were relatively unaffected by comparison (). As expected, a low MOI of HSulf-1 adenovirus did not appear to reduce the density of hAT2 cells (), but did induce some reduction in size and cell density (apparent cell numbers) of H292 cells (). In A549 cells, HSulf-1 induced increased cell refractility, but not much cell density reduction ().
Figure 1 Representative photomicrographs of hAT2, H292, and A549 cells over-expressing HSulf-1 and/or treated with cadmium. hAT2 cells (A – D), H292 cells (E – H), and A549 cells (I – L) were exposed to 10 μM cadmium for 48 hours (more ...)
Interestingly, treatment with cadmium in the presence of HSulf-1 over-expression markedly reduced the cell densities of both hAT2 () and H292 () cells and increased the refractility as well as detachment of A549 cells (). In addition to these reductions in cell density, increases in the relative numbers of stellate or fusiform cell shapes and in the numbers of floating cells and amounts of surface blebbing, as suggested by increased refractility of cell borders, were seen in all three cell types. Western blot confirmed the high basal level of HSulf-1 protein expression in hAT2 cells, the lack of HSulf-1 protein expression in non-transduced H292 and A549 cells, and its over-expression in HSulf-1-transduced cells of all three types (Supplementary Data 3).
HSulf-1 significantly reduces H292 and A549 cell viability with cadmium treatment
Since HSulf-1 over-expression worsened the effects of low concentrations of cadmium on hAT2, H292, and A549 cells, further experiments using a commercial MTT assay were performed to determine the optimal dose of cadmium to use on each cell type in order to trigger some adverse or signaling effects without inducing significant and rapid cell death from toxicity. The survival rates of lacZ over-expressing hAT2 cells treated with cadmium concentrations ranging from 1 to 30 μM decreased from 100 to 27% of untreated controls after 24 hours (). Similarly, the transduction of HSulf-1 into hAT2 cells subsequently treated with the same range of cadmium concentrations did not significantly change the survival rates at 24 hours, which decreased from 100% to 25% of untreated, nontransduced control cells (). At 48 hours, in lacZ over-expressing hAT2 cells, the survival rates ranged from 100% of untreated controls at 1 μM to 14% at 30 μM with a steep decline at 20 μM, suggesting toxicity at that concentration. Similarly, HSulf-1 over-expression along with increasing levels of cadmium further decreased hAT2 survival percentage from 100% down to 8%, a decrease that was not significantly different from that of lacZ-transduced cells over most of the dose range and with the same steep decline at 20 μM ().
Figure 2 Dose response to cadmium of hAT2, H292, and A549 cells over-expressing lacZ or HSulf-1. Cells transduced at 10 MOI for lacZ or HSulf-1 over-expression were exposed to increasing concentrations of cadmium for 24 or 48 hours. Cells were cultured in 1 mg/ml (more ...)
In H292 cells, by comparison, the survival rates of lacZ over-expressing cells at 24 hours gradually decreased from 98% down to 55% of untreated controls over cadmium concentrations ranging from 5 to 60 μM (), with only a slightly greater decrease over most of the range at 48 hours, from 92% to 37% of untreated controls (), confirming some resistance to cadmium's effects in these cancer cells. However, H292 cells over-expressing HSulf-1 and treated in culture with cadmium chloride at the same range of concentrations had viability rates that gradually decreased from 98% to 33% of untreated controls after 24 hours, significantly less than control at the higher doses (), and from 95% down to 18% of untreated controls at 48 hours, with significance over most of the dose range ().
Similarly, in A549 cells, the survival rates of lacZ over-expressing cells at 24 hours gradually decreased from 96% down to 60% of untreated controls over cadmium concentrations ranging from 5 to 60 μM (), with only a slightly greater decrease over most of the range at 48 hours, from 99% to 41% of untreated controls (). However, A549 cells over-expressing HSulf-1 and treated in culture with cadmium chloride at the same range of concentrations had viability rates that gradually decreased from 96% to 42% of untreated controls after 24 hours, significantly less than control at the higher doses (), and from 99% down to 33% of untreated controls at 48 hours, with significance over most of the dose range ().
These results confirm that H292 and A549 cells are more resistant to cadmium than are hAT2 cells but indicate that in these transformed cell lines, HSulf-1 over-expression can specifically exacerbate injury from high concentrations of cadmium chloride with short exposure times and from a range of concentrations after long exposures.
Because all three cell types exhibited some phenotypic response with lowest toxicity to 10 μM cadmium, this dose was used for further experiments to explore the mechanism behind the observed reduction in cell numbers in cadmium-treated HSulf-1-expressing cells.
Apoptosis is exacerbated by HSulf-1 over-expression in cadmium-treated H292 and A549 cells
To determine whether the observed reduced viability of HSulf-1-expressing cells treated with low-dose cadmium is caused by apoptosis, hAT2, H292, and A549 cells were transduced at 10 MOI with lacZ or HSulf-1 over-expression adenoviruses, incubated overnight, and then treated with 10 μM cadmium chloride as above. After 48 hours of cadmium treatment, cells were detached; TUNEL assay was performed and analyzed by light microscopy. Results, confirmed in representative photographed fields (), indicated that HSulf-1 transduction at 10 MOI did not induce apoptotic cell death in hAT2 cells, with only rare or no co-localization of yellow-green BrdU-labeled foci (DNA fragments) with blue (PI) nuclei (), but did induce apoptotic cell death in both H292 and A549 cells ( and ). However, cadmium at 10 μM did induce an increased number of FITC-labeled foci indicative of apoptosis in hAT2 cells () but not in H292 or A549 cells ( and ). The combination of HSulf-1 and cadmium induced apoptosis in all three cell types (, , and ).
Figure 3 TUNEL assay in hAT2 cells treated with HSulf-1 and/or cadmium. hAT2 cells were adenovirally-transduced (10 MOI) for lacZ or HSulf-1 over-expression and treated with or without 10 μM cadmium for 48 hours. TUNEL assay was performed on harvested (more ...)
Figure 4 TUNEL assay in H292 cells treated with HSulf-1 and/or cadmium. H292 cells were adenovirally-transduced (10 MOI) for lacZ or HSulf-1 over-expression and treated with or without 10 μM cadmium for 48 hours. TUNEL assay was performed on harvested (more ...)
Figure 5 TUNEL assay in A549 cells treated with HSulf-1 and/or cadmium. A549 cells were adenovirally-transduced (10 MOI) for lacZ or HSulf-1 over-expression and treated with or without 10 μM cadmium for 48 hours. TUNEL assay was performed on harvested (more ...)
Apoptotic cell numbers were normalized to total cell numbers to obtain apoptotic ratios. In hAT2 cells, over-expression of HSulf-1 alone induced apoptosis in only 4% of cells, which was not significantly different from the lacZ over-expressing control. However, cadmium, in the presence of lacZ or HSulf-1 over-expression, induced apoptosis in 35% or 32% of cells, respectively, at a significantly higher level than apoptosis in untreated cells but not significantly different due to the transduced gene ().
In contrast to hAT2 cells, cadmium treatment of H292 or A549 cells alone was not a significant factor in induction of apoptosis, while HSulf-1 over-expression alone induced apoptosis in 15% or 12% of transduced cells, respectively ( and ). Further, the combination of HSulf-1 and cadmium induced apoptosis in up to 28% or 20% of total H292 or A549 cells, respectively, which was significantly greater than for HSulf-1 over-expression alone ( and ). Thus, it appears that the reductions in H292 and A549 cell numbers caused both by HSulf-1 alone and by the combination of cadmium and HSulf-1 over-expression are due primarily to an increase in apoptosis.
Cadmium induces activation of apoptosis pathway genes in hAT2 cells but not in H292 or A549 cells
Apoptosis array data demonstrated that in hAT2 cells over-expressing lacZ, expression of fifteen pro-apoptotic genes (APAF1, CARD8, CASP10, DAPK1, FAS, NOD1, TNFRSF21, TNFSF10, BCL2L11, BIK, BNIP3, CIDEA, HRK, LTA, and TNFRSF9), three anti-apoptotic genes (BAG3, BIRC3, and BNIP1), and two genes with functions unrelated to apoptosis (CD27, TNFSF8) was altered by cadmium treatment (). Of the fifteen proapoptotic genes, eight (APAF1, NOD1, CARD8, CASP10, DAPK1, FAS, TNFRSF21, and TNFSF10) were down-regulated and seven (BCL2L11, BIK, BNIP3, CIDEA, HRK, LTA, and TNFRSF9) were up-regulated by cadmium, while all of the three anti-apoptotic genes were up-regulated. However, three of the genes (HRK, LTA, and BCL2L11), all pro-apoptotic, were up-regulated with much higher fold changes than any of the other genes (). These results are further illustrated by scatter plot analysis (), in which the distance outside the 2-fold difference from identity with no-cadmium control (lacZ only) for these three genes (labeled) is clearly evident and which, on balance, indicates apoptosis pathway activation.
Genes that were up- or down-regulated in hAT2 cells by HSulf-1 or cadmium
Figure 6 Scatter plot analysis of Apoptosis PCR array results for (A) hAT2, (B) H292, and (C) A549 cells exposed to cadmium. hAT2, H292, and A549 cells over-expressing lacZ were exposed to cadmium for 48 hours. Total RNA was isolated and reverse-transcribed, and (more ...)
In contrast, in H292 cells over-expressing lacZ, only three apoptotic genes were altered by cadmium alone. Of them, two pro-apoptotic genes (BIK and CIDEA) were slightly down-regulated and one pro-apoptotic gene (DFFA) was up-regulated to a small extent, which suggests a lack of significant pathway activation in H292 cells by cadmium (). This is illustrated by scatter plot analysis, which shows a clustering of relevant genes at identity with the no-cadmium control (lacZ only), and only a small fold change for those outside the non-significant range ().
Genes that were up- or down-regulated in H292 cells by HSulf-1 or cadmium
Similarly, in A549 cells, cadmium treatment of lacZ over-expressing cells only activated seven genes, including four anti-apoptotic genes and one apoptotic gene. Of the four anti-apoptotic genes, two were down-regulated (BCL2A1 and NFkB1) and two were up-regulated (BCL2 and NOL3). The only pro-apoptotic gene, TP53, was down-regulated (). This relative lack of apoptosis pathway activation by cadmium is illustrated by scatter plot analysis ().
Genes that were up- or down-regulated in A549 cells by HSulf-1 or cadmium
HSulf-1 over-expression with cadmium treatment activates apoptosis pathway genes in H292 and A549 cells
Initially, normalizing the “HSulf-1 + cadmium” array data to the “lacZ only” data gave scatter plots () and gene tables (, , and ) that confirmed that apoptosis was triggered by the combination of HSulf-1 and cadmium in all three cell types. However, this analysis obscured the separate contributions of the HSulf-1 over-expression and cadmium to apoptosis caused by the combination in the cells. Normalizing the “HSulf-1 + cadmium” data to that of “lacZ + cadmium” revealed that in hAT2 cells, expression of only two pro-apoptotic genes (TNFRSF9 and HRK), one anti-apoptotic gene (BAG3), and a pro-proliferation gene (CD70) was specifically altered by the HSulf-1 contribution to cadmium's effects (). One of the pro-apoptotic genes was up-regulated and the other was down-regulated, the anti-apoptotic gene was down-regulated, and the pro-proliferation gene was up-regulated. This relative lack of significant pathway activation is illustrated by scatter plot analysis, which shows a clustering of relevant genes with no significant change and small fold change for those outside the range ().
Figure 7 Scatter plot analysis of Apoptosis PCR array results for (A) hAT2, (B) H292, and (C) A549 cells exposed to the combination of HSulf-1 over-expression and cadmium treatment. hAT2, H292, and A549 cells were exposed to cadmium for 48 hours after adenovirally-transduced (more ...)
Genes that were up- or down-regulated in hAT2 cells by the HSulf-1/cadmium combination, normalized to lacZ/cadmium results
Figure 8 Scatter plot analysis of the contribution of HSulf-1 to the apoptotic effects of cadmium. (A) hAT2, (B) H292, and (C) A549 cells were exposed to cadmium for 48 hours after adenovirally-transduced over-expression of lacZ or HSulf-1. Genes significantly (more ...)
The contribution of HSulf-1 to cadmium-induced injury was much more striking in the H292 and A549 cells. In H292 cells, eight pro-apoptosis genes (BAX, BIK, BNIP3, CASP7, CIDEA, FAS, TNF, and TNFRSF9) were significantly up-regulated, several by nearly 10-fold or better. Two pro-apoptosis genes (CASP9 and TNFSF10) were moderately down-regulated and two anti-apoptosis genes (AKT1 and BCL2A1) were moderately up-regulated. Two pro-proliferation genes (CD70 and TNFSF8) and a gene thought to be involved with apoptosis but with an unidentified function (CD27) were up-regulated (). Furthermore, in A549 cells, fourteen of the array genes showing activation due to HSulf-1's contribution were pro-apoptotic. Twelve pro-apoptosis genes (BAK1, BAX, CASP3, CASP6, CD40LG, LTA, MCL1, NOD1, RIPK2, TNFRSF9, TNFRSF25, and TNFSF10) were up-regulated while only two pro-apoptosis genes were down-regulated (BCL2L11 and LTBR). Two anti-apoptosis genes (CFLAR and TNFRSF1B) were up-regulated and one anti-apoptosis gene (NFkB1) was down-regulated (). Scatter plot analysis illustrates these results ().
Genes that were up- or down-regulated by HSulf1/cadmium combination vs cadmium in H292 cells
Genes that were up- or down-regulated by HSulf1/cadmium combination vs cadmium in A549 cells
Importantly, whereas cadmium itself strongly induced activation of several pro-apoptotic genes in hAT2 cells, almost no apoptotic pathway activation in H292 or A549 cells was triggered by cadmium alone. In contrast, the HSulf-1 contribution to cadmium's effects up-regulated many more pro-apoptosis genes in both H292 and A549 cells than it did in hAT2 cells. Data points representing those genes in H292 and A549 cells () showed increased and significant activation compared to genes in hAT2 cells (). These results indicate that HSulf-1 exacerbates apoptosis pathways activated by cadmium in H292 and A549 cells but not in hAT2 cells.
Interactions between HSulf-1 over-expression and cadmium treatment
In hAT2 cells, synergistic/antagonistic effects of cadmium and HSulf-1 were seen by the Association Test on three genes (HRK, TNFRSF9, and CD70), which accounted for only 13% of total activated genes (). In the interaction graph for each of these genes, the X-axis indicates the lacZ control cells at the left with the HSulf-1 over-expressing cells at the right, while the Y-axis represents the regulatory fold-changes revealed in the PCR array. The dashed line (NA) represents “No Cadmium treatment” and the solid line (Cd) represents cadmium chloride (10 μM) treatment. Horizontal lines indicate no change, positively-sloped lines indicate up-regulation, and negatively-sloped lines indicate down-regulation. Thus, for example, the solid line in each graph indicates the change in expression of the examined gene from that seen in the lacZ over-expression control to that seen with HSulf-1 over-expression, both when in the presence of cadmium. Solid and dashed lines in parallel indicate that there are no synergistic or antagonistic effects. A solid line with a more positive slope than the dashed line indicates a synergistic effect between HSulf-1 and cadmium on that gene, while a solid line with a more negative slope than the dashed line indicates there is an antagonistic effect. Accordingly, in hAT2 cells, the HSulf-1 and cadmium combination showed synergistic effects on TNFRSF9 (pro-apoptotic) and CD70 (pro-proliferation) (). Interestingly, the combination exhibited significant and large antagonistic effects on HRK (pro-apoptotic) in hAT2 cells (), suggesting that HSulf-1 may actually counteract the up-regulation of HRK by cadmium in these normal cells.
Figure 9 Gene expression interaction analysis in hAT2 cells. Genes up- or down-regulated in hAT2 cells by HSulf-1/Cadmium were tested for synergistic or antagonistic interactions by the Association Test. Dashed line, No Cadmium; Solid line, Cadmium treatment at (more ...)
In H292 cells, of the 25 genes whose expression was either up- or down-regulated by cadmium after over-expression of HSulf-1, compared to the “No Cadmium” lacZ control, synergistic/antagonistic effects were seen on eleven genes (BAX, BIK, BNIP3, CASP4, CASP7, FAS, GADD45A, TNFSF10, TP53, TNF, and TNFRSF9), which accounted for 44% of the activated genes (). Based on the slopes of the lines, the HSulf-1 and cadmium combination showed significant synergistic effects on six pro-apoptotic genes (BAX, BIK, BNIP3, CASP4, CASP7, and FAS) () and on an anti-proliferation gene (GADD45A) (). Small but significant antagonistic effects were seen on two additional pro-apoptotic genes (TNFSF10 and TP53) (). Although the cadmium and HSulf-1 combination did not show any significant synergistic effects on two pro-apoptotic genes (TNF and TNFRSF9) (), HSulf-1 does up-regulate the expression of these two genes compared to cadmium treatment alone.
Figure 10 Gene expression interaction analysis in H292 cells. Genes up- or down-regulated in H292 cells by HSulf-1/Cadmium were tested for synergistic or antagonistic interactions by the Association Test. Dashed line, No Cadmium; Solid line, Cadmium treatment at (more ...)
Of the 21 genes in A549 cells that were activated by the combination of HSulf-1 and cadmium, synergistic/antagonistic effects or associations were seen on 6 genes (BCL2L11, LTA, TNFRSF9, NOL3, CD40LG, and TP73), which accounted for 29% of activated genes (). Based on the slopes of the lines, antagonistic effects were seen on one pro-apoptotic gene (BCL2L11) () while synergistic effects were seen on three pro-apoptotic genes (LTA, TNFRSF9, and CD40LG) (). In the anti-apoptotic gene NOL3 (), there is an association effect between the HSulf-1 over-expression and cadmium treatment, but there is no synergistic/antagonistic effect. In TP73, although no significant synergistic effects were seen, HSulf-1 does up-regulate its expression compared to cadmium treatment alone ().
Figure 11 Gene expression interaction analysis in A549 cells. Genes up- or down-regulated in A549 cells by HSulf-1/Cadmium were tested for synergistic or antagonistic interactions by the Association Test. Dashed line, No Cadmium; Solid line, Cadmium treatment at (more ...)
The results of the Association Test indicate that pathway activation in both H292 and A549 cells treated with cadmium and over-expressing HSulf-1 exacerbated apoptosis in these transformed lung cells, while the combination, although lethal, did not add to cadmium's effects on normal human alveolar epithelial cells.
Western analysis confirmation of selected PCR array results
Western analysis was performed on H292 and A549 cells which were treated with cadmium after transduction of HSulf-1. In H292 cells, BAX was up-regulated by HSulf-1 alone and in combination with cadmium, and the increase in BAX protein was confirmed by Western analysis. In A549 cells, CASP3, which was up-regulated by HSulf-1 alone and in combination with cadmium, showed increased protein expression in Western analysis as well ().
Figure 12 Western blot confirmation of selected PCR array results. Apoptosis array analysis found that BAX in H292 cells and CASP3 in A549 cells were up-regulated by HSulf-1 alone and in combination with cadmium. Western analysis confirms that the up-regulation (more ...)