Cancer Letters

Cancer Letters

Volume 434, 10 October 2018, Pages 42-55
Cancer Letters

Original Articles
2′-hydroxycinnamaldehyde inhibits cancer cell proliferation and tumor growth by targeting the pyruvate kinase M2

https://doi.org/10.1016/j.canlet.2018.07.015Get rights and content

Highlights

  • HCA inhibits proliferation of DU145 prostate cancer cells and suppresses their growth in a xenograft mouse model.

  • HCA directly binds to PKM2 in cancer cells and tumor tissues.

  • HCA increases pyruvate kinase activity by promoting the tetrameric state of PKM2.

  • HCA suppresses the release of tumor extracellular vesicles (EV) by dephosphorylating PKM2.

Abstract

It is reported that 2′-hydroxycinnamaldehyde (HCA), isolated from cinnamon, has anti-tumor effects through the modulation of multi-target molecules. In this study, we identified pyruvate kinase M2 (PKM2) as a direct target of HCA by use of biochemical methods including affinity chromatography, drug affinity responsive target stability, and cellular thermal shift assay. PKM2 is up-regulated in multiple cancer types and is considered as a potential target for cancer therapy. HCA binds directly to PKM2 and selectively decreases the phosphorylation of PKM2 at Tyr105, indicating a potential anti-proliferative effect on prostate cancer cells. As a PKM2 activator, HCA increases pyruvate kinase activity by promoting the tetrameric state of PKM2. However, HCA suppresses protein kinase activity of PKM2 by decreasing the phosphorylation at Tyr105. Moreover, this leads to a decrease of PKM2-mediated STAT3 phosphorylation at Tyr705 and a down-regulation of target genes, including MEK5 and cyclin D1. Furthermore, HCA suppresses tumor growth and the release of tumor extracellular vesicles in vivo by inhibiting the phosphorylation of PKM2. Collectively, our results suggest that HCA may be a potential anticancer agent targeting PKM2 in cancer progression.

Introduction

Cancer cells with highly proliferative phenotypes require increased glucose uptake and enhanced lactate production regardless of oxygen availability [1]. To supply the energy needed for anabolic reactions, cancer cells use aerobic glycolysis, known as the Warburg effect [2]. Compared to normal cells, cancer cells display a considerably different metabolism to support cell growth and proliferation [1,3]. Cancer cells balance the synthesis of macromolecules as well as sufficient ATP production [3]. Targeting the metabolic enzyme has gained attention as a potential therapeutics for cancer, but there are currently few molecules that target the central carbon metabolism in clinical trials [4,5]. Because rapidly proliferating cells rely on the same metabolic pathways, targeting cancer cell metabolism shows adverse effects on normal cells such as gut epithelium and bone marrow [4]. To reduce unwanted toxicity, drugs that target metabolic enzymes with metabolic differences between cancer cells and normal cells, should be explored.

Pyruvate kinase (PK) is a rate-limiting enzyme during glycolysis, and it has four isoforms (M1, M2, L, and R) with unique tissue expression patterns in mammals [6]. PKM2 is expressed in cancer cells, embryonic tissues, and adult stem cells, whereas PKM1 is expressed in many differentiated cells such as brain and muscle [7]. As a key regulator of the Warburg effect, enhanced expression of PKM2 has been reported in various cancer cell lines and samples from cancer patients [8,9]. PK and protein kinase activity of PKM2 are determined by regulating the conformational states of PKM2 [10,11]. The tetrameric state of PKM2 has high PK activity, whereas the dimeric state of PKM2 has low PK activity but high protein kinase activity [11]. While normal proliferating cells express the tetrameric form, cancer cells predominantly express the dimeric form of PKM2 [9,12]. Additionally, the conversion of tetrameric to dimeric PKM2 is regulated by oncoproteins, tyrosine kinase-mediated phosphorylation, and oxidative stress [13]. Recent studies show that the phosphorylated or acetylated PKM2 translocates into the nucleus and binds to STAT3 or β-catenin, leading to cell proliferation-related gene expression, such as MEK5, cyclin D1, and c-Myc [2,[14], [15], [16]]. Thus, inhibiting the phosphorylation of PKM2 can suppress cell proliferation and tumor growth. Several groups have reported that PKM2 inhibitors and activators suppress cancer cell proliferation and tumor growth [[17], [18], [19]]. Because therapies targeting PKM2 expression can have toxic effects towards some normal tissues, small molecules that activate PK activity of PKM2 are considered therapeutic modalities [19,20].

Cinnamon, a spice used daily by people all over the world, has beneficial effects when used as a treatment for cancer, allergies, bacterial/viral infections, and Alzheimer's disease [21]. Cinnamon extracts consist of active compounds, including essential oils (cinnamaldehydes), tannins, mucus, and carbohydrates [22]. One of active components, isolated from stem bark of Cinnamomum cassia named 2′-hydroxycinnamaldehyde (HCA), was screened as an anticancer drug candidate due to its inhibitory effects on farnesyl protein transferase (FPTase) [23]. HCA and its derivatives showed anti-tumor activities in various types of cancer cells [21]. To understand the molecular mechanisms of HCA, molecular targets of HCA have been reported: the proteasome subunits in colon cancer cells, the low-density lipoprotein receptor-related protein 1 (LRP1) in microglial cells and breast cancer cells, and the Pim-1 kinase in human leukemia cells [[24], [25], [26]]. Although HCA and its derivatives have various biological activities in different cell types, the molecular mechanism associated with the binding target of HCA is not completely understood.

In this study, we identified PKM2 as a direct target of HCA, and determined its anti-proliferative and anti-tumor activities in prostate cancer cells. HCA suppressed cell proliferation by inhibiting tyrosine phosphorylation of PKM2 and STAT3 as well as promoting tetramer formation of PKM2. In addition, the amount of extracellular vesicle (EV) harboring PKM2 was reduced in tumor-bearing mice treated with HCA compared to the vehicle-treated mice. Thus, HCA significantly suppressed cell proliferation and tumor growth by regulating cancer cell metabolism and oncogenic molecules.

Section snippets

Cell culture

All cell lines used in this study were originally obtained from the American Type Culture Collection (ATCC). DU145, LNCaP (human prostate cancer), HCT116, SW480, SW620 (human colon cancer), MDA-MB-231, MDA-MB-468 (human breast cancer) cells were maintained in RPMI 1640 medium (Gibco). PC3 (human prostate cancer) and HFF (human fibroblast) cells were maintained in Dulbecco's modified Eagle's medium (Gibco). MCF-10 A (human mammary epithelial) cells were maintained in DMEM-F12 (Gibco). All

Identification and validation of PKM2 as a protein target of HCA

To identify the proteins bound to HCA, we performed a pull-down assay with biotin-labeled HCA (biotin-HCA) in DU145 cell lysates (Fig. 1A). Biotin-HCA showed similar levels of anti-proliferative activities compared to the control HCA (Supplementary Figs. S1A and B). As shown in Fig. 1A, several bands were detectable from the eluate of biotin-HCA in the Coomassie blue stained gel images. Among the proteins identified by Mass spectrometry, the proteasome subunits have already been reported as a

Discussion

HCA was identified as an FPTase inhibitor [23] with a variety of biological activities [21,23]. Furthermore, preclinical evaluations of HCA and 2′-benzoyloxycinnamaldehyde (BCA) have been completed. Additionally, a phase I clinical trial is currently underway testing BCA as an antitumor drug. Although proteasome subunits, LRP1, and Pim-1 were identified as direct targets of HCA [[24], [25], [26]], the diverse mechanisms of action of HCA have not been completely defined. It is well known that

Conflicts of interest

The authors declare that there are no competing financial interests.

Acknowledgements

This work was supported by the KRIBB Research Initiative Program, the Bio-Synergy Research Project (NRF-2012M3A9C4048777), and the Bio & Medical Technology Development Program of the National Research Foundation & funded by the Korean government (NRF-2015M3A9B5030311 and NRF-2017M3A9A8032417).

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