Cabazitaxel overcomes cisplatin resistance in germ cell tumour cells

Abstract

Background: Cisplatin-based chemotherapy is highly effective in metastasized germ cell tumours (GCT). However, 10-30 % of patients develop resistance to cisplatin, requiring salvage therapy. We investigated the in vitro activity of paclitaxel and the novel taxane cabazitaxel in cisplatin-sensitive and -resistant GCT cell lines.

Methods: In vitro activity of paclitaxel and cabazitaxel was determined by proliferation assays, and mode of action of cabazitaxel was assessed by western blotting and two screening approaches, i.e. whole proteome analysis and a human apoptosis array.

Results: Activity of paclitaxel and cabazitaxel was not affected by cisplatin resistance, suggesting that there is no cross-resistance between these agents in vitro. Cabazitaxel treatment showed a strong inhibitory effect on colony formation capacity. Cabazitaxel induced classical apoptosis in all cell lines, reflected by cleavage of PARP and caspase 3, without inducing specific changes in the cell cycle distribution. Using the proteomic and human apoptosis array screening approaches, differential regulation of several proteins, including members of the bcl-2 family, was found, giving first insights into the mode of action of cabazitaxel in GCT.

Conclusion: Cabazitaxel shows promising in vitro activity in GCT cells, independent of levels of cisplatin resistance.

Keywords: Apoptosis; Cabazitaxel; Cisplatin resistance; Germ cell tumour.

Fig. 1

IC₅₀ values for cisplatin, cabazitaxel, and paclitaxel in cisplatin-sensitive and -resistant germ cell tumour cell lines Half maximal inhibitory concentration (IC₅₀), determined by trypan blue staining, for the different germ cell tumour cell lines and their cisplatin-resistant counterparts. All analyses were performed in triplicates, and repeated at least three times (*p < 0.05). a Cisplatin treatment; b cabazitaxel treatment; c paclitaxel treatment

Fig. 2

Colony formation assay Cells were cultivated in drug-free medium for 10 days after 48 h treatment with the different substances (controls: solvent treated cells). Cells were treated with the respective IC₅₀ of cisplatin and cabazitaxel in the different cell lines (*p < 0.05). a NTera2; b NTera2-R; c 2102Ep; d 2102Ep-R; e NCCIT; f NCCIT-R

Fig. 3

Quantification of apoptosis Cells were treated with the respective IC₅₀ of cisplatin or cabazitaxel for 48 h. Anisomycin was used as a positive control for induction of apoptosis. The experiment was performed in triplicate. Using an annexin V-FLUOS/PI-double staining, the amount of cells in apoptosis was determined (*p < 0.05). a NTera2; b NTera2-R; c 2102Ep; d 2102Ep-R; e NCCIT; f NCCIT-R

Fig. 4

Cell cycle distribution of the different cell lines after cytotoxic treatment Cells were treated with the solvent (control) or with different concentrations of cisplatin or cabazitaxel for 48 h. Cisplatin treatment at the respective IC₅₀ was used as a positive control for induction of a G₂/M arrest. Cabazitaxel was used at three different dose levels (IC₁₀, IC₅₀ and IC₇₅). All experiments were performed in triplicate. a NTera2; b NTera2-R; c 2102Ep; d 2102Ep-R; e NCCIT; f NCCIT-R

Fig. 5

Detection of apoptosis in GCT cells treated with cisplatin and cabazitaxel Western blotting analysis of markers of classical apoptosis: Cells were treated with the solvent (control) or with the IC₅₀ of cisplatin or cabazitaxel for 48 h. a NTera2 and NTera2-R cells; b 2102Ep and 2102Ep-R cells; c NCCIT and NCCIT-R cells

Fig. 6

Analysis of protein markers of apoptosis, regulated under cytotoxic treatment. a Densitometric analysis and graphical presentation of the fold change of several pro-and anti-apoptotic proteins in NTera2 cells under treatment with the IC₅₀ of cabazitaxel for 48 h, detected using the human apoptosis array (RayBiotech). Bars show fold changes relative to untreated cell. b Analysis of selected pro- and anti-apoptotic proteins under treatment with cisplatin and cabazitaxel in NTera2 and NTera2-R cells, using Western blotting and specific antibodies

Fig. 7

Differentially expressed proteins of solvent-treated versus cabazitaxel-treated NTera2 cells detected by 2D-PAGE gel analysis. Spot identification of proteins altered in expression after treatment with cabazitaxel, and validation analyses using Mini 2D Western blotting analysis are shown. a NTera2 cells; down-regulated spots; b NTera2 cells, up-regulated spots; c NTera2-R cells, down-regulated spots; d NTera2-R cells, up-regulated spots; e glutathione S-transferase P (GSTP1) (control and after cabazitaxel treatment); f α-enolase (control and after cabazitaxel treatment)