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

Abstract

Less toxic treatment strategies for testicular germ cell tumor (TGCT) patients are needed, as overtreatment is a concern due to the long-term side effects of platin-based chemotherapy. Although clinical benefit from classical hypomethylating agents has to date been limited, TGCTs show an abnormal DNA methylome indicating the potential of treating TGCTs with hypomethylating drugs. We tested, for the first time in TGCT cell lines, a new synthetic flavonoid compound (MLo1302) from the 3-nitroflavanone family of DNA methyltransferase (DNMT) inhibitors. We show that MLo1302 reduces cell viability (including of cisplatin resistant cell line NCCIT-R), with IC50s (inhibitory concentration 50) within the nanomolar range for NCCIT and NTERA-2 cells, and proved its cytotoxic effect. Exposure to MLo1302 reduced DNMT protein expression, similar to decitabine, and showed a partial effect in cell differentiation, reducing protein expression of pluripotency markers. RT2 profiler expression array indicated several dysregulated targets, related to activation of apoptosis, differentiation, and cell cycle arrest. We validated these data by showing increased apoptosis, increased protein expression of cleaved caspase 8 and activated caspase 2, and reduced proliferation (BrdU assay), with increase in CDKN1A and decrease in MIB-1 expression. Therefore, synthetic drugs designed to target DNA methylation in cells may uncover effective treatments for TGCT patients.

The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors

“…3H). This differential sensitivity to cisplatin was not attributed to cell differentiation, as no significant changes in expression of NANOG, OCT3/4 or SOX2 between knockdown and scramble cells were found, nor morphological changes, as documented in [24] (Supplementary Fig. 4).…”

“…As mentioned, m 6 A is overall strongly implicated in cell fate decisions and regulation of pluripotency [18][19][20][21]. Since our hypothesis was that m 6 A would also be relevant in tumors' degree of differentiation, we assessed the impact of ATRA-induced differentiation (a classical feature of ATRA treatment in these cell lines, as described in [24,31]) on the total amount of m 6 A and on the expression levels of its key effectors, comparing with the parental non-differentiated matched cells. We found a statistically significant downregulation of METTL3, WTAP, VIRMA, METTL14, METTL4 and YTHDF3 in ATRA-treated NCCIT cells compared to the vehicle (Supplementary Fig.…”

“…The (T)GCT cell lines TCam-2, NCCIT, 2102Ep and NT2 were kindly provided by Prof. Leendert Looijenga, and cultured as described [23]. Cell lines have been previously authenticated [details reported in [24]]. The cisplatin-resistant and cisplatin-sensitive clones of NCCI T were kindly provided by Prof. Daniel Nettersheim, and established by Dr. Christoph Oing and Prof. Friedemann Honecker.…”

“…Briefly, cells were seeded on T25 culture flasks, left to adhere for 24 h and then treated with 10 μM of the drug for 10 days, with drug renewal every 2 days. Importantly, cell morphology analysis in NT2 cell line, as well as assessment of pluripotency/differentiation markers, were performed to document neuronal-like differentiation of the cells, as previously reported by our team [24].…”

Cancer Mechanisms and Emerging Therapies

“…Lobo et al [ 1 ] present the effects of a synthetic flavonoid, MLo1302, in the context of a testicular germ cell tumor. This compound is designed to target the DNA methyltransferase enzyme responsible for the abnormal methylome in malignant pathologies including testicular germ cell cancer and the subsequent repression of the gene expression.…”