Dimethyl sulfoxide induces chemotherapeutic resistance in the treatment of testicular embryonal carcinomas

Abstract

Dimethyl sulfoxide (DMSO) is an amphipathic molecule that is used as a solvent in biological studies and as a vehicle for drug therapy. The present study was designed to evaluate the potential effects of DMSO as a solvent in the treatment of testicular embryonal carcinomas (ECs). DMSO was applied to two human EC cell lines (NEC8 and NEC14), with the treated cells evaluated in relation to cisplatin (CDDP) resistance, differentiation (using Vimentin, Fibronectin, TRA-1-60, and SSEA-1 and -3 as markers) and stemness (denoted by expression of SOX2 and OCT3/4). Furthermore, DNA methyltransferase (DNMT-1, -3A and -3L) expression and methylation status were analyzed. DMSO induced resistance to CDDP, aberrant differentiation and reduction of stemness-related markers in each of the EC cell lines. The expression levels of DNMT-3L and -3A were reduced in response to DMSO, while this treatment also affected DNA methylation. The data demonstrated that DMSO perturbed differentiation, reduced stemness and induced resistance to CDDP in human EC cells. Therefore, DMSO could reduce drug efficacy against EC cells and its use should be carefully managed in the clinical application of chemotherapy.

Keywords: cisplatin; differentiation; dimethyl sulfoxide; embryonal carcinoma; methylation.

Figure 1.

DMSO enhances resistance to CDDP without induction of MDR-1 or MRP-1 in human EC cells. (A) Viability of human EC cells after 48 h of CDDP exposure together with DMSO treatment. Two EC cell lines, NEC8 and NEC14, were treated with DMSO at the indicated concentrations for 72 h, and various doses of CDDP were additionally applied for 48 h. The effects on cell growth were determined by use of the WST-8 assay (n=3; vertical bars, standard deviation). IC₅₀ values were indicated by each colored-arrow and value. DMSO decreased the cytotoxicity of CDDP in a dose-dependent manner; 0.8% (v/v) DMSO induced ~3-fold more resistance to CDDP than 0% (v/v) DMSO in each cell line. (B) Micrographs of NEC8 and NEC14 cells cultured in medium containing 15 mM CDDP with/without 0.8% (v/v) DMSO. The NEC8 and NEC14 cells retained their morphology at high doses of CDDP with DMSO, with exposure to CDDP alone leading to a significant reduction in cell numbers. Scale bar, 100 µm. (C) Western blot analysis of the major drug efflux pumps, MDR-1 and MRP-1. No induction of either pump was observed in response to DMSO treatment in the NEC8 and NEC14 cells. Cell lysate (*) of U-2OS/DOXO35 was used as the positive control for MDR-1. DMSO, dimethyl sulfoxide; CDDP, cisplatin; EC, embryonal carcinoma; IC₅₀, half maximal inhibitory concentration.

Figure 2.

DMSO perturbs differentiation and reduces the stemness characteristics of human embryonic carcinoma cells. (A) Western blotting. Vimentin, Fibronectin and TRA-1-60 were used as markers of differentiation. SOX2 and OCT3/4 were used as stemness-related markers. DMSO dose-dependently induced the aberrant expression of Vimentin and TRA-1-60 in the NEC8 and NEC14 cells. DMSO also reduced SOX2 expression. α-tubulin was used as a loading control. (B) Immunocytochemical evaluation of TRA-1-60, SSEA-1 and SSEA-3 in the NEC8 cells. DMSO significantly induced TRA-1-60 expression, but did not affect SSEA-1 and SSEA-3 expression. Scale bar, 100 µm. DMSO, dimethyl sulfoxide; CDDP, cisplatin.

Figure 3.

DMSO reduces DNMT-3L and -3A expression, and increases DNA methylation levels in human embryonic carcinoma cells. (A) Western blot analysis for the DNMT family. DNMT-3A and -3L expression was dose-dependently reduced by DMSO in the NEC8 and NEC14 cells. The expression of DNMT1 showed little change between the control and DMSO-treated cells. (B) Immunocytochemical evaluation of DNMT3L in the NEC8 cells. DNMT3L was reduced and delocalized from the cell nuclei by DMSO exposure. Scale bar, 100 µm. (C) Methylation status of LINE1 DNA repetitive elements in the NEC8 and NEC14 cells. This COBRA assay indicated five possible digestion products of 285, 247, 166, 126 and 38 bp, signifying methylated repetitive elements. The upper undigested bands represent unmethylated repetitive elements or repetitive elements in which the restriction site has been mutated. The size of the undigested and digested PCR products is shown on the left. The reference mark (*) indicates a male PBL sample that was a highly methylated LINE1 control. The band intensities were quantitated through densitometric analysis, with the extent of methylation shown below each lane. DMSO, dimethyl sulfoxide; CDDP, cisplatin.