Diterpenoid Pseudolaric Acid B Induces M-Phase Arrest And Apoptosis Primarily Through Jnk Stress Signaling In Lncap Prostate Cancer Cells

Pseudolaric acid B (PAB) is a major diterpenoid component isolated from the root bark of the traditional Chinese medicinal plant Pseudolarix kaempferi Gordon (Pinaceae). PAB has shown potential as an anticancer agent via inducing cell cycle arrest, apoptosis and DNA damage in several cancer models. To explore potential use of PAB for prostate cancer chemoprevention, we used human LNCaP prostate cancer cells that harbor wild-type p53, functional androgen receptor (AR), and defective PTEN that are characteristic of early stage prostate adenocarcinomas to study cell fates and signaling mechanisms upon PAB exposure in cell culture. Our results show that PAB inhibited LNCaP cell proliferation in a dose- and time-dependent manner (IC 50 ~5.4 µM at 24 h and ~ 0.2 µM at 72 h). PAB induced M phase cell cycle arrest as confirmed by increased phosphorylation of histone H3 (p-H3 Ser10). PAB also induced apoptosis evidenced by Annexin V staining and cleavage of the canonical caspase substrate PARP. PAB at lethal exposure concentration decreased AR protein and its best-known target prostate-specific antigen (PSA). Timewise, PAB treatment increased p-H3 (mitosis marker) and the expression of another mitosis/prosurvival protein survivin several hours ahead of PARP cleavage. PAB induced p-H2A.X, which would mark DNA double-strand breaks, and the best-known DNA damage response protein P53 and its target P21Cip1 preceding cPARP. PAB stimulated phosphorylative activation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 decreased PAB-induced p-H2A.X, P53/P21Cip1 and cleavage of PARP as well as M phase arrest. Furthermore, PAB significantly increased 29,79-dichlorofluorescin diacetate (DCF)-reactive oxygen species (DCF-ROS) in LNCaP cells and addition of antioxidant N-acetyl-L-cysteine (NAC) attenuated P53/P21Cip1 cascade and cPARP/apoptosis, but did not affect M phase arrest. Knocking-down of P53 or P21Cip1 by siRNA attenuated cPARP and apoptosis without affecting p-H2A.X. Taken together, these results suggest a rapid induction of M phase arrest by PAB that was primarily mediated by JNK stress signaling, either parallel to or upstream of ROS-DNA damage-driven P53-P21Cip1 apoptosis cascade in LNCaP cells. Citation Format: Kartick C. Pramanik, Nehal Gupta, Min Ye, Junxuan Lu, Cheng Jiang. Diterpenoid pseudolaric acid B induces M-phase arrest and apoptosis primarily through JNK stress signaling in LNCaP prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 271.