*Article* **PNU-74654 Suppresses TNFR1/IKB Alpha/p65 Signaling and Induces Cell Death in Testicular Cancer**

**Wen-Jung Chen 1,2,3,† , Wen-Wei Sung 1,2,3,† , Chia-Ying Yu <sup>2</sup> , Yu-Ze Luan <sup>2</sup> , Ya-Chuan Chang <sup>2</sup> , Sung-Lang Chen 2,3,\* ,‡ and Tsung-Hsien Lee 1,4,5,\* ,‡**


**Abstract:** Testicular cancer (TC) is a rare malignancy worldwide and is the most common malignancy in males aged 15–44 years. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 interaction. We evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2, by measuring cell viability, cell cycle transition and cell death. Potential pathways were evaluated by protein arrays and Western blots. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis. Our findings demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may identify potential new treatment strategies for TC.

**Keywords:** PNU-74654; TNF receptor-1; apoptosis; testicular cancer
