The entire
Hypericum japonicum herb, named “Tianjihuang,” is widely used for the treatment of infectious hepatitis, acute and chronic hepatitis, and tumour in China [
1]. An 85% ethanol-treated water extract is documented in the Chinese Pharmacopoeia as an injection for the treatment of viral hepatitis [
2,
3]. Moreover,
H. japonicum is used as an animal feed in China because of its widespread growth. These records demonstrate the clinical safety of
H. japonicum. However, the molecular mechanisms of its effects are unclear. To better understand the mechanisms of
H. japonicum, its chemical composition was systematically isolated and analysed in our previous study. In this study, we identified jacarelhyperol A (Jac-A), a characteristic constituent of
H. japonicum, as a potent inhibitor of Bcl-2 proteins
via high throughput screening of an in-house natural product library (NPL).
The Bcl-2 family of proteins play an important role in apoptosis through the balance of antiapoptotic proteins (e.g., Bcl-2, Bcl-x
L, Mcl-1) and proapoptotic proteins (e.g., Bak, Bax, Bad, Bid) [
4]. The ability of antiapoptotic proteins to form heterodimers with a number of proapoptotic proteins is believed to play a crucial role in their antiapoptotic function [
5]. Antiapoptotic Bcl-2 proteins are overexpressed in a variety of tumours, which can protect cancer cells from apoptosis [
6,
7]. Owing to their important functions in regulating cell death, the pharmacological inhibition of Bcl-2 proteins is a promising strategy for apoptosis induction or sensitisation to chemotherapy [
8]. Protein sequence analysis and structure-function studies revealed that the BH3 domain of proapoptotic proteins is the fundamental motif for the dimerisation with antiapoptotic proteins [
9]. The three-dimensional structure of a complex of Bcl-x
L and the Bak BH3 domain peptide showed that the Bak peptide is an amphipathic α-helix that binds to a hydrophobic groove on the surface of Bcl-x
L[
10]. Based on these studies, screening new ligands that bind to the same pocket became an anti-cancer drug discovery strategy to search for antiapoptotic protein inhibitors [
11]. To screen for Bcl-2 protein inhibitors, we used fluorescence polarisation (FP), whose basic principle is that a fluorescent peptide tracer (Flu-Bid-BH3) and a nonfluorescent small molecule inhibitor compete for binding to the Bid BH3 domain of Bcl-2 proteins. Jac-A was chosen as the candidate compound for further research because of its high affinity with Bcl-2 proteins and favorable binding mode with Bcl-x
L. Then, we tested its anti-cancer activity
in vitro and
in vivo. Jac-A possesses a broad antitumour effect for all tested cancer cells and remarkably inhibited the proliferation of leukaemia cells. Moreover, Jac-A not only induced K562 cell apoptosis
in vitro, but also inhibited human K562 cell growth in a mouse xenograph tumour model, which provided evidence for using
H. japonicum as an anti-cancer herbal medicine. We also proved that Jac-A’s effect is partly caspase-dependent and it can disrupt the heterodimerization between anti-apoptotic Bcl-2 family members with pro-apoptotic Bcl-2 family members.