Park Moon Nyeo, Park Hyunmin, Rahman Md Ataur, Kim Jeong Woo, Park Se Sun, Cho Yongmin, Choi Jinwon, Son So-Ri, Jang Dae Sik, Shim Bum-Sang, Kim Sung-Hoon, Ko Seong-Gyu, Cheon Chunhoo, Kim Bonglee
Department of Pathology, College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea.
Korean Medicine-Based Drug Repositioning Cancer Research Center, College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea.
Front Oncol. 2022 Mar 7;12:791365. doi: 10.3389/fonc.2022.791365. eCollection 2022.
BK002 consists of Nakai (AJN) and Rohrbach (MFR) that have been used as herbal medicines in China and Korea. AJN and MFR have been reported to have anti-inflammatory, anti-oxidative, and anti-cancer activities, although the synergistic targeting multiple anti-cancer mechanism in castration-resistant prostate cancer (CRPC) has not been well reported. However, the drug resistance and transition to the androgen-independent state of prostate cancer contributing to CRPC is not well studied. Here, we reported that BK002 exerted cytotoxicity and apoptosis in CRPC PC3 cell lines and prostate cancer DU145 cell lines examined by cytotoxicity, western blot, a LIVE/DEAD cell imaging assay, reactive oxygen species (ROS) detection, quantitative real-time polymerase chain reaction (RT-PCR), and transfection assays. The results from our investigation found that BK002 showed more cellular cytotoxicity than AJN and MFR alone, suggesting that BK002 exhibited potential cytotoxic properties. Consistently, BK002 increased DNA damage, and activated p-γH2A.X and depletion of survivin-activated ubiquitination of pro-PARP, caspase9, and caspase3. Notably, live cell imaging using confocal microscopy found that BK002 effectively increased DNA-binding red fluorescent intensity in PC3 and DU145 cells. Also, BK002 increased the anti-proliferative effect with activation of the C/EBP homologous protein (CHOP) and significantly attenuated PI3K/AKT expression. Notably, BK002-treated cells increased ROS generation and co-treatment of N-Acetyl-L-cysteine (NAC), an ROS inhibitor, significantly preventing ROS production and cellular cytotoxicity, suggesting that ROS production is essential for initiating apoptosis in PC3 and DU145 cells. In addition, we found that BK002 significantly enhanced miR-192-5p expression, and co-treatment with BK002 and miR-192-5p inhibitor significantly reduced miR-192-5p expression and cellular viability in PC3 and DU145 cells, indicating modulation of miR-192-5p mediated apoptosis. Finally, we found that BK002-mediated CHOP upregulation and PI3K downregulation were significantly reduced and restrained by miR-192-5p inhibitor respectively, suggesting that the anti-cancer effect of BK002 is associated with the miR-192-5p/PI3K/CHOP pathway. Therefore, our study reveals that a combination of AJN and MFR might be more effective than single treatment against apoptotic activities of both CRPC cells and prostate cancer cells.
BK002由中井（AJN）和罗尔巴赫（MFR）组成，在中国和韩国一直被用作草药。据报道，AJN和MFR具有抗炎、抗氧化和抗癌活性，尽管在去势抵抗性前列腺癌（CRPC）中协同靶向多种抗癌机制的研究尚未充分报道。然而，导致CRPC的前列腺癌的耐药性以及向雄激素非依赖状态的转变尚未得到充分研究。在此，我们报告BK002在CRPC的PC3细胞系和前列腺癌DU145细胞系中表现出细胞毒性和诱导凋亡作用，这通过细胞毒性检测、蛋白质免疫印迹法、活/死细胞成像分析、活性氧（ROS）检测、定量实时聚合酶链反应（RT-PCR）和转染实验得以验证。我们的研究结果发现，BK002比单独的AJN和MFR表现出更强的细胞毒性，表明BK002具有潜在的细胞毒性特性。一致地，BK002增加了DNA损伤，激活了p-γH2A.X，并使survivin激活的pro-PARP、caspase9和caspase3的泛素化减少。值得注意的是，使用共聚焦显微镜的活细胞成像发现，BK002有效地增加了PC3和DU145细胞中DNA结合红色荧光强度。此外，BK002通过激活C/EBP同源蛋白（CHOP）增强了抗增殖作用，并显著减弱了PI3K/AKT的表达。值得注意的是，经BK002处理的细胞增加了ROS的产生，而ROS抑制剂N-乙酰-L-半胱氨酸（NAC）的共同处理显著抑制了ROS的产生和细胞毒性，这表明ROS的产生对于启动PC3和DU145细胞中的凋亡至关重要。此外，我们发现BK002显著增强了miR-192-5p的表达，并且BK002与miR-192-5p抑制剂的共同处理显著降低了PC3和DU145细胞中miR-192-5p的表达和细胞活力，表明miR-192-5p介导的凋亡受到调控。最后，我们发现BK002介导的CHOP上调和PI3K下调分别被miR-192-5p抑制剂显著降低和抑制，这表明BK002的抗癌作用与miR-192-5p/PI3K/CHOP途径相关。因此，我们的研究表明，AJN和MFR的组合可能比单一治疗对CRPC细胞和前列腺癌细胞的凋亡活性更有效。
