Table 1.
Studies with epidrugs in testicular germ cell tumours
| Drug | Category | Phase of study | Cell lines/Patients | Dosage | Assays/Endpoints | Main Results | Notes | Ref. |
|---|---|---|---|---|---|---|---|---|
| 5-aza-2ʹdeoxycytadine | DNMT inhibitor | Pre-clinical | TERA1, TERA2 and NCCIT | 10 µM | MSP and bisulphite sequencing (methylation) | Can reverse aberrant DNA methylation, mainly in SCGB3A promoter | [29] | |
| 5-aza-2ʹdeoxycytidine | DNMT inhibitor | Pre-clinical | NT2/D1, 833 K and their cisplatin-resistant clones, TERA-1, 577 M and 2102EP | 10 nM | Cell-TiterGlo (viability) | Pre-treatment with low doses of this compound restores cisplatin cytotoxic response by inducing p53 and/or re-expressing genes (MGMT, RASSF1A, and HOXA-9) | The effect of 5-aza-2´deoxytidine depends on DNMT3B expression, but cisplatin sensitivity not | [40] |
| 5-aza-2ʹdeoxycytadine | DNMT inhibitor | Pre-clinical | 2102EP, TERA1, NTERA2 and H7 | 10 nM | Flow cytometry (apoptosis) qRT-PCR (transcript levels) Western Blot (protein levels) |
There is an induction of apoptosis and differentiation | First study that shows that DNMT3B can act as oncogene | [42] |
| 5-azacytidine | DNMT inhibitor | Pre-clinical | TCam-2 | 10 µM | qRT-PCR (transcript levels) MTT (viability) MSP and bisulphite sequencing (methylation) |
Restores sensitivity to cisplatin, causing changes in methylation pattern and downregulation of pluripotency genes (NANOG and OCT3/4) | [28] | |
| 5-aza | DNMT inhibitor | Pre-clinical | NT2/D1 and its cisplatin-resistant clone | 10 nM | Cleavage of PARP1 and Western Blot (apoptosis) MSP and bisulphite sequencing (methylation) |
Low doses of 5-aza lead to DNA damage and apoptosis by activation of p53 targets, global hypomethylation and downregulation of pluripotency genes | First study that shows that induction of p53 is not associated with increased p53 mRNA, but with p53 stability | [43] |
| Guadecitadine (SGI-110) |
DNMT inhibitor | Pre-clinical | NT2/D1 and its cisplatin-resistant clone | 5 nM | Cell-TiterGlo (viability) | Exposure to low concentration results in a decrease of tumour cells growth, induces p53 target genes, re-expresses RASSF1A and SOX15 and, consequently, restores cisplatin sensitivity | Using in vivo (mouse) models, combination of SGI-110 with cisplatin causes complete tumour regression | [44] |
| 5-aza | DNMT inhibitor | Pre-clinical | 2102EP and NCCIT and their cisplatin-resistant clones | 10–20 nM | Trypan Blue (viability) Cleavage of Caspase 3 and PARP1 Western Blot Flow cytometry (apoptosis) |
Anti-tumour activity as a single agent at low concentration, but more effectively when combined with cisplatin | Demonstrated that the effect of 5-aza is independent of TP53 mutational status | [17] |
| 5-aza alone or in combination with TSA | DNMT inhibitor and HDAC inhibitor | Pre-clinical | JEG-3 and primary choriocarcinoma stem-like cells | 75 µM (5-aza) 100 nM (TSA) |
qRT-PCR (transcript levels) Western Blot (protein levels) |
5-aza as a single agent leads to decreased DNMT1 and DNMT3B. In combination with TSA it also reduces the expression of pluripotency genes (NANOG, OCT3/4, SOX2, and ABCG2) | This study introduces a natural compound (curcumol) for treatment of choriocarcinoma cells with satisfactory results | [36] |
| 5-azacytidine | DNMT inhibitor | Clinical trial, phase II | 17 patients with advanced germ cell tumours | 150 mg/m2/day | Response to treatment Disease-free survival |
All patients progressed; 16/17 died | [45] | |
| 5-azacytidine | DNMT inhibitor | Clinical trial, phase II | 4 patients with testicular cancer | 150–225 mg/m2 | Response to treatment | Two of four patients presented partial responses | [46] | |
| Hydralazine in combination with valproate | DNMT inhibitor and HDAC inhibitor | Clinical trial, phase II | One patient with non-seminoma | 83–182 mg (hydralazine) 700 mg (valproate) |
Response to treatment Disease-free survival Overall survival |
Patient with stable disease | [47] | |
| TSA | HDAC inhibitor | Pre-clinical | P19 | 10–100 ng/mL | TUNEL and flow cytometry (apoptosis) | TSA inhibits cell progression but alone does not induce differentiation (only in combination with retinoic acid) | [61] | |
| CBB | KDM inhibitor | Pre-clinical | F9, NCCIT, NTERA2, HELA, 293, NIH3TS | 5,27–11,16 µM | Spectrometry | CBB blocks demethylation activity of LSD1 on mono- and- di-methylated H3K4, inducing differentiation | To mimic in vivo models, authors treated mouse F9 embryonic stem cells and obtained similar results in in vitro analysis | [62] |
| CBB3001 | KDM inhibitor | Pre-clinical | F9 | 21,25 µM | Spectrometry | CBB3001 inhibits specifically LSD1, reducing cell growth and downregulates SOX2 and OCT3/4 | [63] | |
| TSA and vorinostat as a single agent | HDAC inhibitor | Pre-clinical | GH | Various concentrations | qRT-PCR (transcript levels) Cleavage of PARP-1 and caspase 3 and Western Blot (apoptosis) |
Both drugs restore GTAp63 and induce apoptosis. Combination with cisplatin causes complete cell death | First study that reports the induction of GTAp63 as relevant for treatment | [66] |
| Romidepsin | HDAC inhibitor | Pre-clinical | TCam-2, 2102EP-R, NCCIT-R, NT2/D1-R, JAR and JEG-3 | 1–10 nM | XTT (viability) Cleavage of PARP-1 and Western Blot and flow cytometry (apoptosis) qRT-PCR (transcript levels) |
Romidepsin is highly toxic at low concentration, inducing stress, apoptosis and cell cycle arrest | Used in vivo models (mouse). Romidepsin kills SurePath cells by inducing apoptosis | [67] |
| JQ1 | Bromodomain inhibitor | Pre-clinical | NCCIT, NT2/D1, 2102EP and their cisplatin-resistant clones, TCam-2, FS1 and MPAF | 100–500 nM | XTT (viability) Western Blot (protein levels) Flow cytometry (apoptosis) qRT-PCR (transcript levels) |
JQ1 not only increases G1 arrest and apoptosis, but also differentiation, and inhibits angiogenesis | First study that used bromodomain inhibitor in TGCTs | [58] |
| Animacroxam | HDAC inhibitor and cellular cytoskeletal dynamics inhibitor | Pre-clinical | 2102EP and resistant clone, NCCIT | 0,1–3,2 µM | ELISA iCELLigence AC-DEVD-AMC Flow cytometry |
Animacroxam has an anti-proliferative effect and inhibits cell migration | Authors used CAM as in vivo model, observing reduction of tumour growth | [69] |
DNMT: DNA methyltransferase; MSP: Methylation-specific PCR; qRT-PCR: Real-Time Quantitative Polymerase Chain Reaction; TSA: trichostatin A; HDAC: Histone deacetylase; KDM: Histone demethylases; ELISA: Enzyme-Linked Immunosorbent Assay; CAM: Chick Chorioallantoic Membrane