Alpha-Pinene As a Novel Therapeutic Agent for T-Cell Tumors
Authors: Abe Masaya , Asada Noboru , Kimura Maiko , Fukui Chie , Yamada Daisuke , Wang Ziyi , Miyake Masayuki , Takarada Takeshi , Ono Mitsuaki , Aoe Michinori , Kitamura Wataru , Matsuda Masayuki , Moriyama Takashi , Matsumura Akifumi , Maeda Yoshinobu 
Published: December 2023

T-cell acute leukemia and lymphoma have poor prognoses, necessitating the development of novel therapeutic agents. In this study, we investigated the antitumor activity of α-pinene, a monoterpene compound, against T-cell tumors. We evaluated the effects of α-pinene on cell growth inhibition in vitro using the murine T-cell tumor cell line EL-4 and the human-derived T-cell tumor cell line Molt-4. In addition, we assessed the effects of limonene, another monoterpene with reported tumor-suppressive effects, as a comparative agent.
Both α-pinene and limonene demonstrated concentration- and time-dependent inhibition of cell growth in both the EL-4 and Molt-4 cell lines (Figure 1). Moreover, α-pinene exhibited greater potency than limonene in inhibiting the growth of various hematologic malignancies. Importantly, α-pinene and limonene had minimal effects on the growth of normal murine spleen T cells. Further investigation of the mechanisms of action of α-pinene revealed its ability to induce apoptosis and cell cycle arrest in EL-4 and Molt-4 cells. Transcriptomic profiling of α-pinene-treated EL-4 cells using RNA-seq identified differentially expressed genes associated with cellular damage and mitochondrial dysfunction. Increased intracellular ROS levels and mitochondrial impairment were observed in the T-cell tumors treated with α-pinene. The activation of intrinsic apoptotic pathways involving EGR1, p53, BCL-2, and BAX was found to contribute to α-pinene-induced apoptosis in T-cell tumors. Moreover, α-pinene inhibited the NF-κB signaling pathway, resulting in the reduced nuclear translocation of NF-κB p65 and decreased total intracellular NF-κB p65 levels in EL-4 cells. Additionally, we discovered that α-pinene induced ferroptosis, a newly identified programmed cell death process, in EL-4 cells through lipid peroxidation and iron accumulation. Activation of the system x c−/GSH/GPX4 axis and upregulation of iron transporters, such as Slc39a8 and TfR1, were observed in α-pinene-induced ferroptosis. Treatment with the ferroptosis inhibitor Fer-1 partially reversed the tumor-inhibiting effect of α-pinene in EL-4 cells. Finally, we administrated α-pinene to mice subcutaneously injected with luciferase-expressing EL-4 to evaluate the efficacy of α-pinene in vivo. The tumor growth was significantly inhibited by α-pinene treatment (vehicle: 1055 ± 101 mm 3; α-pinene: 701 ± 82 mm 3, p &lt; 0.01, Figure 2) without adverse effects on body weight or behavior. Immunohistochemistry analyses revealed a significant increase of CD8 + T-cells (vehicle: 8.1 ± 2.7 cells / field of view; α-pinene: 43.1 ± 12.2 cells / field of view , p &lt; 0.05) and NK1.1 + cells (vehicle: 5.1 ± 1.0 cells / field of view ; α-pinene: 41.1 ± 9.8 cells / field of view , p &lt; 0.01) in the tumors, suggesting that α-pinene may have an indirect antitumor effect by recruiting immune cells into tumors.
Overall, our findings demonstrate the antitumor activity of α-pinene against T-cell tumors through the induction of apoptosis, cell cycle arrest, and ferroptosis. α-Pinene shows promise as a potential therapeutic agent for T-cell tumors and warrants further investigation for its clinical application.