Presentation Authors: Toshiki Etani*, Taku Naiki, Satoshi Nozaki, Takashi Nagai, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Satoru Takahashi, Nagoya, Japan, Takayoshi Suzuki, Kyoto, Japan, Takahiro Yasui, Nagoya, Japan
Introduction: Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be discovered, is a novel tesuticular tumor therapy target. NCL1 and NCD38, novel selective cell-active inhibitors of LSD1, were discovered at our university. We analyzed the efficacy of LSD1 inhibitors in tesuticular tumor in vitro and in vivo.
Methods: Various tests were used to confirm the pathways responsible for germ cell tumor growth, and to characterize the anticancer effects of NCL1 and NCD38. Cell viability was assessed using a WST-8 assay with standard vehicle or NCL1 and NCD38 in NTERA2 and Tera-1 cells (human testicular embryonal carcinoma cell lines). Flow cytometry and western blotting were performed to assess the status of apoptosis and cell cycle in NCL1- and NCD38-treated NTERA2 cells. Mice injected with subcutaneous NTERA2 cell-derived tumors were injected intraperitoneally with vehicle or NCL1 and NCD38 twice a week, and tumor growth was monitored. Finally, tissue arrays were prepared from formalin-fixed, paraffin-embedded tissue specimens of 35 seminoma patients who underwent orchiectomy.
Results: The WST assay revealed a reduction in the number of viable cells after NCL1 and NCD38 treatment in a dose-dependent manner in both cell lines. Western blotting confirmed that NCL1 and NCD38 treatment induced caspase-dependent apoptosis, and that Oct4 and SOX2 expression decreased. Flow cytometry confirmed that NCL1 and NCD38 significantly induced apoptosis in a dose-dependent manner. Subcutaneous tumor volume was significantly lower in mice treated with NCL1 and NCD38 than in controls. TUNEL analysis showed that NCL1 and NCD38 treatment induced apoptosis in NTERA2 subcutaneous tumors. No adverse effect was observed with regard to body weight, blood toxicity, or the gross morphology of several organs in mice. Tissue array analysis showed that LSD1 expression in human seminoma specimens was significantly higher than that in noncancerous specimens.
Conclusions: Germ cell tumor growth was effectively suppressed with NCL1 and NCD38 both in vitro and in vivo in the absence of adverse events. The drugs act via induction of apoptosis and the suppression of SOX2 and OCT4, indicating the potential of these inhibitors as therapeutic agents for germ cell tumor.