Ethyl Acetate Fraction of Hedyotis diffusa Willd Induces Apoptosis via JNK/Nur77 Pathway in Hepatocellular Carcinoma Cells

Weimin Ning; Nishan Xu; Chunhong Zhou; Lifang Zou; Jingyu Quan; Hua Yang; Zinbin Lu; Huihui Cao; Junshan Liu

doi:10.1155/2022/1932777

Ethyl Acetate Fraction of Hedyotis diffusa Willd Induces Apoptosis via JNK/Nur77 Pathway in Hepatocellular Carcinoma Cells

Evid Based Complement Alternat Med. 2022 Aug 24:2022:1932777. doi: 10.1155/2022/1932777. eCollection 2022.

Authors

Weimin Ning¹, Nishan Xu², Chunhong Zhou², Lifang Zou², Jingyu Quan², Hua Yang², Zinbin Lu², Huihui Cao², Junshan Liu^{2

3}

Affiliations

¹ Dongguan Hospital of Chinese Medicine affiliated to Guangzhou University of Chinese Medicine, Dongguan 523005, China.
² Traditional Chinese Pharmacological Laboratory, Third Level Research Laboratory of State Administration of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China.
³ Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou 510515, China.

Abstract

Background: Hepatocellular carcinoma (HCC) is characterized by poor diagnosis and high mortality. Novel and efficient therapeutic agents are urgently needed for the treatment. Hedyotis diffusa Willd (HDW) is used to treat cancers, especially HCC in China.

Purpose: The study aimed to identify the main anti-HCC extract in HDW and to explore the mechanism of the active extract.

Materials and methods: The high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was used for the simultaneous determination of main compounds in the ethyl acetate fraction of HDW (EHDW). The toxicity test of different HDW fractions was carried out on larvae at 2 day-post-fertilization (dpf) for 72 h. The in vivo anti-HCC effect of different HDW fractions was evaluated on a zebrafish tumor model by immersion administration. The antiproliferative effect of HDW fractions was determined with MTT assay, as well as hematoxylin and eosin (HE) staining assay. Hoechst 33258 staining was used to observe changes in nucleus morphology. Flow cytometry analysis was used to investigate apoptosis induction. Western blot analysis was used to examine apoptosis-related proteins, and key proteins in JNK/Nur77 signaling pathway. SP600125 was served to validate the apoptotic mechanism.

Results: EHDW showed the strongest tumor cell growth inhibitory effect on zebrafish tumor model. Further study revealed that EHDW induced apoptosis in zebrafish tumor model and in cultured Hep3B cells. Meanwhile, it has been shown that the levels of BCL2-associated X (Bax), cytochrome c (cyto c), cleaved-caspase 3, and poly-ADP-ribose polymerase (PARP) cells were upregulated. In contrast, the level of antiapoptotic B cell lymphoma-2 (Bcl-2) was downregulated in Hep3B cells. Additionally, EHDW activated JNK/Nur77 pathway by increasing the levels of p-JNK^{(Thr183/Tyr185)} and p-Nur77^(Ser351). Further study showed that blockage of JNK by SP600125 reversed EHDW-induced JNK/Nur77 pathway and the downstream apoptotic proteins.

Conclusion: In conclusion, EHDW exerted the anti-HCC effect, which may be attributed to the activation of JNK/Nur77 pathway. This study supported the rationale of HDW as an HCC therapeutic agent.