Lead Decreases Bone Morphogenetic Protein-7 (BMP-7) Expression and Increases Renal Cell Carcinoma Growth in a Sex-Divergent Manner

Abstract

Both tissue and blood lead levels are elevated in renal cell carcinoma (RCC) patients. These studies assessed the impact of the subchronic lead challenge on the progression of RCC in vitro and in vivo. Lead challenge of Renca cells with 0.5 μM lead acetate for 10 consecutive passages decreased E-cadherin expression and cell aggregation. Proliferation, colony formation, and wound healing were increased. When lead-challenged cells were injected into mice, tumor size at day 21 was increased; interestingly, this increase was seen in male but not female mice. When mice were challenged with 32 ppm lead in drinking water for 20 weeks prior to tumor cell injection, there was an increase in tumor size in male, but not female, mice at day 21. To investigate the mechanism underlying the sex differences, the expression of sex hormone receptors in Renca cells was examined. Control Renca cells expressed estrogen receptor (ER) alpha but not ER beta or androgen receptor (AR), as assessed by qPCR, and the expression of ERα was increased in tumors in both sexes. In tumor samples harvested from lead-challenged cells, both ERα and AR were detected by qPCR, yet there was a significant decrease in AR seen in lead-challenged tumor cells from male mice only. This was paralleled by a plate-based array demonstrating the same sex difference in BMP-7 gene expression, which was also significantly decreased in tumors harvested from male but not female mice; this finding was validated by immunohistochemistry. A similar expression pattern was seen in tumors harvested from the mice challenged with lead in the drinking water. These data suggest that lead promotes RCC progression in a sex-dependent via a mechanism that may involve sex-divergent changes in BMP-7 expression.

Keywords: BMP-7; androgen receptor; estrogen receptor α; lead; renal cell carcinoma.

Figure 1

Impact of lead on cell adhesion in Renca cells. Renca cells were challenged with 0.5 μM lead acetate for 10 passages. (A): Western blot analysis of E-cadherin in lead-challenged Renca cells, while (B) shows cell–cell aggregation in control and lead-challenged Renca cells (10×); the quantitative results are shown in (C). # indicates a significant difference from control (p < 0.0001).

Figure 2

Impact of lead on RCC progression in vitro. Renca cells were challenged with 0.5 μM lead acetate for 10 passages. (A) depicts cell proliferation. Colony formation is shown in (B, 10×); quantitative results are shown in the table (C). Wound healing in the scratch assay at 48 h is shown in (D, 10×), and the quantitative results are shown in (E). * indicates a significant difference from control (p < 0.05).

Figure 3

Impact of lead on tumor growth. Lead-Cells Tumor: Renca cells were challenged with 0.5 μM lead acetate for 10 passages prior to subcutaneous injection in the hind flank of male and female Balb/cJ mice. (A): Tumor growth at 21 days is shown; lead-challenged cells had increased tumor growth as compared to control cells as indicated # (p = 0.0127); the significant difference was confined to male mice (# p = 0.0076), but not female mice (p = 0.3858). (B): Control Renca cells were injected into male and female Balb/cJ mice—control or challenged with 32 ppm lead in drinking water for 20 weeks. There was no significant difference in tumor size when mice were examined as a group (p = 0.1125), but a significant increase was seen in male mice (# p = 0.015) but not female mice (p = 0.5972).

Figure 4

Sex hormone expression in Renca cells and tumors. (A): qPCR analysis of ERα expression in control Renca cells and tumors harvested from male and female mice injected with control cells is shown; there is an increase in expression in tumor samples as compared to cells. In (B), Western blot analysis demonstrates a similar finding of increased expression in tumor samples as compared to cells. (C) depicts qPCR analysis of AR and ERα expression in tumor samples harvested from male and female mice injected with lead-challenged Renca cells. AR expression was decreased in tumors derived from lead-challenged cells harvested from the male (# p = 0.0089) but not female mice. There were no sex-divergent effects on ERα expression; although it tended to be lower in tumors derived from lead-challenged Renca cells in males, it was not significant (p = 0.2425).

Figure 5

Sex-dependent impact on lead-induced alterations in BMP-7 expression. Renca cells were challenged with 0.5 μM lead acetate for 10 passages prior to subcutaneous injection in the hind flank of male and female Balb/cJ mice. Gene expression (A) of three samples per group using the RT² Profiler PCR Gene Array: Mouse EMT. A significant decrease (#) in BMP-7 was observed in tumors harvested from male mice, while a significant increase (#) in expression was seen in tumors harvested from female mice. (B): Immunohistochemical analysis demonstrates a decreased expression in tumors harvested from male mice (10×) (C): qPCR analysis of BMP-7 expression in tumor samples harvested from the lead drinking water expression is shown; * indicates a significant difference from the respective control.