Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer

Abstract

This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 hr. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 hr. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

Bromate-induced Changes in p21 DNA Methylation and Histone Acetylation in Renal Cells

“…This hypothesis is supported by our previous studies showing that treatment of HEK293 and NRK cells with the HDAC inhibitor TSA upregulated p21 protein expression (Scholpa et al, 2014). We also demonstrated that TSA-induced p21 expression correlated with the increased acetylation of lysine 9 and 14 on histone H3 (H3K9/14 Ac) in the p21 promoter region (Scholpa et al, 2016). However, we do not know the effect of short-and long-term exposure of BrO 3 À on these cells, nor do we know the persistence of these changes.…”

“…The first set of samples was collected from passage 1 (P1) of the subchronic regimen, which was continued for 18 days (P6), ie, the cells were treated for 18 days and passaged every 3 days for sample collection. The rationale for this regimen is explained in our previous studies (Scholpa et al, 2014(Scholpa et al, , 2016Zhang et al, 2010Zhang et al, , 2011. In short, we designed a sub-chronic in vitro regimen to obtain the environmentally relevant concentrations and exposures of bromate.…”

“…TGBS does not rely on fragmentation and because each amplicon is its own read, the library preparation and data analysis are comparatively simplified. We recently demonstrated the ability of TGBS to assess p21 DNA methylation in renal cells in response to 5-Aza (Scholpa et al, 2016).…”

“…For example, treatment of human colon cancer cells with HDAC inhibitors TSA or sodium butyrate increased the transcription of p21 by inducing the acetylation of histones H4 and H3 (Fang et al, 2004). We recently showed that TSA increases p21 protein expression in renal cells (Scholpa et al, 2014) by increasing the acetylation of lysine 9 and 14 on histone H3 (H3K9/14 Ac) (Scholpa et al, 2016). However, the effects of BrO 3 À on H3K9/14 Ac levels at the p21 promoter region are not known.…”

Targeted Gene Bisulfite Sequencing Identifies Differential Methylation in p21

“…The positive results with 5-Aza suggest that the SIE-1 is an important region for epigenetic regulation of p21 expression. This assertion is strengthened by the fact that we have already shown that 5-Aza alone induced p21 protein expression in these cells 43 . To our knowledge this is the first report demonstrating the methylation of specific DNA bases within p21 promoter that are altered by 5-Aza exposure.…”