Cardamonin, a Novel Blocker of STAT3 and Nuclear Factor-κB Activation, Suppresses Proliferation, Arrests Cell Cycle, and Potentiates Apoptosis In Human Multiple Myeloma Cells
Authors: Qin You , Sun Chunyan , Guo Tao , Wang Yadan , Zhang Lu , Chen Lei , She Xiaomei , Ai Lisha , Yan Han , Gregg Nicholas Miller , Fang Jun , Hu Yu 
Published: October 2019

Abstract
Abstract 1820

Background and Objective:
Although there have been introductions of novel and more potent therapeutic regimens, multiple myeloma (MM) remains an incurable hematological malignant disorder. Current goals in MM treatment are focused on finding new therapies that target the deregulated signaling cascades which promote MM cell survival and proliferation, such as STAT3 and nuclear factor-κB (NF-κB) (Bharti AC et al, Blood 2004). Cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) is isolated from Alpinia katsumadai (Zingiberaceae), a plant widely used in traditional Chinese medicine. Previous reports have demonstrated that cardamonin possesses diverse pharmacologic actions, such as anti-inflammation (Hatziieremia S et al, Br J Pharmacol 2006), anti-melanogenesis (Cho M et al, Biochem Biophys Res Commun 2009) and anti-platelet properties (Jantan I et al, Phytomedicine 2008). However, very little is known about anti-myeloma activity of cardamonin. Thus, in the present report we investigated the therapeutic potential of cardamonin against MM, specifically studying the capacity of cardamonin to inhibit STAT3 and NF-κB pathways.

Methods and Results:
Cardamonin directly inhibited the growth of MM cell lines in a dose-dependent (from 0 μM to 100 μM) and time-dependent (from 0 h to 72 h) manner by using a cell counting kit-8 assay kit. Growth inhibition of MM cell lines, including RPMI 8226, U266 and ARH-77 cells, was demonstrated with an IC50 of less than 50 μM cardamonin at 24 h. Cardamonin also induced chemosensitivity to vincristine, doxorubicin, and dexamethasone in MM cells. Flow cytometric analysis showed that cardamonin caused accumulation of MM cells in G2 phase (from 14.6% to 81.9% for RPMI 8226, 24 h at 10 μM). A more precise evaluation of cell cycle by Hochest 33342 and 5-ethynyl-2′-deoxyuridine (EdU) fluorescence staining indicated that the cells in S phase were reduced in cardamonin-treated cells. Indeed, cardamonin strongly induced cell apoptosis as shown by annexin V-fluoroisothyocyanate analysis (from 5.3% to 49.4% for RPMI 8226, 48 h at 25 μM), which was also proved by a combination of acridine orange and ethidium bromide staining assay. Furthermore, cardamonin significantly enhanced the apoptotic effects of bortezomib from 23.1% to 74.3% and of thalidomide from 20.1% to 65.8%. Because of the pivotal role of STAT3 and NF-κB in MM cell survival and proliferation, we explored whether the above effects of cardamonin were mediated by interfering with STAT3 and NF-κB pathways by western blot analysis and immunofluorescence. We found that cardamonin blocked IL-6-inducible STAT3 phosphorylation and sequent STAT3 nuclear translocation. The constitutive phosphorylation of STAT3 found in certain cells was also abrogated by treatment with cardamonin in a dose- and time-dependent manner. In addition, we discovered that NF-κB, constitutively active in all human MM cell lines examined, was downregulated by cardamonin through suppression of phosphorylation of NF-κB p65 as evaluated by western blot. This correlated with reduction of the nuclear retention of p65. Moreover, cardamonin suppressed phosphorylation of IκBα, an inhibitor of NF-κB, and phosphorylation of Akt, which has been shown to phosphorylate p65. Additionally, immunoblotting analysis indicated that the expression of STAT3 and NF-κB-regulated gene products associated with proliferation (cyclin D1, TF and COX2), antiapoptosis (Bcl-2, Bcl-xL, Survivin, XIAP and Bfl-1/A1), invasion (ICAM-1), and angiogenesis (vascular endothelial growth factor) were down-regulated by cardamonin. ELISA assay showed that IL-6 release was also suppressed by cardamonin in certain MM cells.

Conclusions:
Taken together, our results suggest that cardamonin is a potent in vitro inhibitor of STAT3 and NF-κB pathways, which provides the molecular basis for its anti-myeloma activities, including suppression of proliferation, arrest of cell cycle, and induction of apoptosis.

Disclosures:
No relevant conflicts of interest to declare.