Efficacy of HDAC Inhibitors Belinostat and Panobinostat against Cisplatin-Sensitive and Cisplatin-Resistant Testicular Germ Cell Tumors

Abstract

Novel treatment options are needed for testicular germ cell tumor (TGCT) patients, particularly important for those showing or developing cisplatin resistance, the major cause of cancer-related deaths. As TGCTs pathobiology is highly related to epigenetic (de)regulation, epidrugs are potentially effective therapies. Hence, we sought to explore, for the first time, the effect of the two most recently FDA-approved HDAC inhibitors (HDACis), belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. In silico results were validated in 261 patient samples and differential expression of HDACs was also observed across cell lines. Belinostat and panobinostat reduced cell viability in both cisplatin-sensitive cells (NCCIT-P, 2102Ep-P, and NT2-P) and, importantly, also in matched cisplatin-resistant subclones (NCCIT-R, 2102Ep-R, and NT2-R), with IC50s in the low nanomolar range for all cell lines. Treatment of NCCIT-R with both drugs increased acetylation, induced cell cycle arrest, reduced proliferation, decreased Ki67 index, and increased p21, while increasing cell death by apoptosis, with upregulation of cleaved caspase 3. These findings support the effectiveness of HDACis for treating TGCT patients in general, including those developing cisplatin resistance. Future studies should explore them as single or combination agents.

Differential gene expression in cisplatin-resistant and -sensitive testicular germ cell tumor cell lines

“…Of course, we are aware of the fact that our study has some limitations. These result mainly from the combination of cell lines used, as only one pair of TGCT cell lines originates from the same cell clone and resistant derivative was not really made CDDPresistant, for instance by cultivation with sub-lethal doses of this drug, as used in recent works in the field [96][97][98]. Instead, the parental CDDP-sensitive cell line was in vitro differentiated using differentiation-inducing medium.…”

Targeting Germ Cell Tumors with the Newly Synthesized Flavanone-Derived Compound MLo1302 Efficiently Reduces Tumor Cell Viability and Induces Apoptosis and Cell Cycle Arrest

“…Additionally, a cisplatin resistant clone of NCCIT (NCCIT-R) (kindly provided by Prof. Daniel Nettersheim and generated by Dr. Christoph Oing and Prof. Friedemann Honecker) was included. Cisplatin resistance was validated and this clone was obtained through prolonged culturing under sublethal doses of cisplatin (fully described in [ 15 ]).…”

“…Cell viability was assessed at 24 h, 48 h, and 72 h of treatment with DAC and MLo1302, as described in [ 15 ]. Cells were plated at density of 8000, 6000, and 4000 cells/well (for NCCIT, 2102EP, and NTERA-2, respectively), incubated with Resazurin (Canvax Biotech, Córdoba, Spain) and quantification was performed in a microplate reader (560 nm) (Fluostar Omega, BMG Labtech, Ortenberg, Germany).…”

“…Optical density (OD) values were corrected to Resazurin-only wells and normalized for day zero. IC 50 values were computed as described [ 15 ].…”

“…Apoptosis assay was performed at 72 h of treatment with MLo1302 according to the protocol detailed in [ 15 ], using the Cell-APOPercentageTM apoptosis assay kit (Biocolor, Carrickfergus, UK). For this assay, cells were seeded in 24-well plates at a density of 40,000, 30,000, and 35,000 cells/well (for NCCIT, 2102EP, and NTERA-2, respectively).…”