Brucea javanica L Merr widely distributed in tropical and
Brucea javanica (L.) Merr, widely distributed in tropical and sub-tropical zones of Asia, is a traditional Chinese medicinal plant belong to the family of Simaroubaceae . The oil extracted from B. javanica seeds has been recorded in Pharmacopoeia of the People’s Republic of China (Guidelines for clinical use, volume 19 of Chinese herbal formula preparation), and is used as an anti-tumor drug commercially available
in China for years [21,22]. Quassinoids, such as Bruceine D (BD) etc., as the characteristic compounds isolated from the Simaroubaceae were considered to be the critical chemical ingredient exhibiting eﬃcient anti-pancreatic cancer and Hepatocellular Carcinoma activity [23–25]. However, BD's anti-cancer eﬀect on other type of cancer still needs extensive exploration. In this study, we confirmed that BD possessed anti-cancer activity against Non-small cell lung cancer (NSCLC) in vitro, and we also revealed an underlying mechanism of this function. The CCK-8 assay demonstrated a dose-dependent loss of viability in BD-treated H460, A549, H1299 and PC9 cells. Our results also showed that BD caused inhibition of colony formation in H460 and A549 cells. These results provided evidence that BD exerted anti-cancer eﬀect on NSCLC in vitro.
Since cellular apoptosis is a major pathway leading to cell growth inhibition, the present study elucidated cellular apoptosis after BD treatment. We observed significant fragmentation of genomic DNA in NSCLC 2196204-23-4 as evidenced by DAPI staining. In addition, Annexin V and PI staining reveled that BD was able to induce cellular apoptosis in
H460 and A549 cells. Moreover, in BD-treated cells PARP protein ex-pression was decreased, while Cleaved-Caspase 3 was accentuated.
JNK is a member of the mitogen activated protein kinases (MAPKs) . Previous studies have demonstrated that the JNK signaling pathway serve important roles in regulating cell survival, development and apoptosis [19,27,28]. In this study, we observed that BD increased JNK phosphorylation in H460 and A549 cells in a time-dependent manner, while the total protein level remained steady. Furthermore, treatment with a specific JNK inhibitor SP600125 eﬀectively alleviated the BD induced apoptosis in NSCLC cells. These findings demonstrated JNK activation plays a critical role in BD induced apoptosis in NSCLC H460 and A549 cells. Activated JNK phosphorylates the N terminus of the transcription factor c-Jun (a nuclear substrate), which further re-sults in an increase of AP-1 (activator protein-1) transcription activity to modulate the transcription of genes associated with apoptosis [29,30]. In our study, the mechanisms of BD promoting cell apoptosis through JNK needs to be further explored.
Furthermore, we also conducted transwell matrigel invasion assay. No significant diﬀerences were observed between the control and BD groups for A549 and H460 cells (Supplemental Fig. 4), indicating none significant eﬀect of BD on tumor invasion in NSCLC cells. In summary, BD exerts anti-tumor eﬀect by inducing apoptosis in NSCLC H460 and A549 cells in our study. The mechanisms underlying these eﬀects are corelated with activation of JNK pathway. Taken together, BD may be a potential therapeutic agent for the treatment of human NSCLC cells.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
L-NM & FW conceived of the presented idea, and together with T-BQ designed the experiment. T-BQ and H-YY carried out most of the ex-periments. L-LH, ZB, Y-LJ and WY contributed to sample preparation, data analyisis. T-BQ wrote the manuscript. All authors discussed the results, provided critical feedback, contributed to the final manuscript.
Appendix A. Supplementary data
 S.T. Lau, Z.X. Lin, P.S. Leung, Role of reactive oxygen species in brucein D-mediated p38-mitogen-activated protein kinase and nuclear factor-kappaB signalling path-ways in human pancreatic adenocarcinoma cells, Br. J. Cancer 102 (3) (2010)
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BTB and CNC homology 1 (Bach1) promotes human ovarian cancer cell T
metastasis by HMGA2-mediated epithelial-mesenchymal transition
Wenyan Hana,1, Yiqun Zhanga,1, Cong Niub,1, Jieyu Guob, Jiajia Lia, Xiangxiang Weib, Mengping Jiab, Xiuling Zhib,∗∗∗, Liangqing Yaoa,∗∗, Dan Mengb,∗ a Department of Gynecology, Obstetrics & Gynecology Hospital, Fudan University, Shanghai, 200011, China
b Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China