JAK2 regulation by licochalcone H inhibits the cell growth and induces apoptosis in oral squamous cell carcinoma

Abstract

Background: Licochalconce (LC) H is an artificial compound in the course of synthesizing LCC in 2013. So far, few studies on the effects of LCH have been found in the literature. Despite progress in treatment modalities for oral cancer, the cure from cancer has still limitations.

Purpose: The effects of LCH were investigated on human oral squamous cell carcinoma (OSCC) cells to elucidate its mechanisms.

Study design: We explored the mechanism of action of LCH by which it could have effects on JAK2/STAT3 signaling pathway.

Methods: To confirm LCH anti-cancer effect, analyzed were MTT assay, DAPI staining, soft agar, kinase assay, molecular docking simulation, flow cytometry and Western blotting analysis.

Results: According to docking and molecular dynamics simulations, the predicted pose of the complex LCH and JAK2 seems reasonable and LCH is strongly bound to active JAK2 with opened activation loop. The LCH inhibitor is surrounded by specific ATP-binding pocket in which it is stabilized by forming hydrogen bonds and hydrophobic interactions. It is shown that LCH plays as a competitive inhibitor in an active state of JAK2. LCH caused a dose-dependent decrease in phosphorylation of JAK2 and STAT3. More interestingly, LCH suppressed JAK2 kinase activity in vitro by its direct binding to the JAK2. LCH significantly inhibited the JAK2/STAT3 signaling pathway, causing the down-regulation of target genes such as Bcl-2, survivin, cyclin D1, p21 and p27. In addition, LCH inhibited cell proliferation and colony formation of OSCC cells in a dose- and time-dependent manner, as well as induction of cell apoptosis through extrinsic and intrinsic pathway. The induction of apoptosis in OSCC cells by LCH was evident in the increased production of ROS, loss of mitochondrial membrane potential, release of cyto c, variation of apoptotic proteins and activation of caspase cascade.

Conclusion: LCH not only induces apoptosis in OSCC cells through the JAK/STAT3 signaling pathway but also inhibits cell growth. It is proposed that LCH has a promising use for the chemotherapeutic agent of oral cancer.

Keywords: 7-AAD, 7-Aminoactinomycin D; Abbreviations: OSCC, Oral squamous cell carcinoma; Apaf-1, apoptotic protease activating factor-1; Apoptosis; C-PARP, cleaved Poly (ADP-Ribose) Polymerase; CHOP, CCAAT/enhancer-binding protein homologous protein; DAPI, 4′-6-diamidino-2-phenylindole; DR, Death receptor; FBS, fetal bovine serum; JAK, Janus kinase; JAK2; LC, Licochalcone; Licochalcone H; MMP, Mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; Oral cancer; PBS, phosphate-buffered saline; ROS, Reactive oxygen species; RT, Room temperature; STAT, Signal transducer and activators of transcription; STAT3; TPK, tyrosine protein kinase; cyto C, cytochrome C; tBid, truncated Bid.