Cucurbitacin IIa induces caspase-3-dependent apoptosis and enhances autophagy in lipopolysaccharide-stimulated RAW 264.7 macrophages

https://doi.org/10.1016/j.intimp.2013.03.013Get rights and content

Highlights

  • CuIIa inhibited the proliferation and migration of RAW 264.7 cells.

  • CuIIa induced a significant apoptosis in LPS-stimulated cells.

  • LPS-induced autophagy in RAW 264.7 was enhanced by CuIIa.

  • Neither MAPKs nor NF-kappaB pathways in LPS-stimulated cells were suppressed by CuIIa.

Abstract

Cucurbitacin IIa (CuIIa), a member of cucurbitacin family, is isolated from the root of Hemsleya amabilis which has been used as an ancient remedy for bacillary dysentery and gastroenteritis. The anti-inflammatory properties of CuIIa have long been recognized but the underlying mechanism is largely unknown. In this study, we investigated the anti-inflammatory effect of CuIIa on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that CuIIa inhibited the proliferation and migration of RAW 264.7 cells in a dose-dependent manner. Whereas CuIIa did not cause apoptosis in unstimulated RAW 264.7 cells, it did induce a significant apoptosis in LPS-stimulated cells, which was caspase-3-dependent and associated with downregulation of survivin. Furthermore, LPS induced autophagy in RAW 264.7 cells and this effect was further enhanced by CuIIa as evidenced by increased levels of LC3-II conjugates and formation of LC3 puncta. In addition, CuIIa disrupted actin cytoskeleton via inducing actin aggregation. However, neither the synthesis of tumor necrosis factor-α, nor the activation of the mitogen-activated protein kinases and NF-κB pathways in LPS-stimulated cells was suppressed by CuIIa treatment. Collectively, these results suggested that induction of apoptosis and enhancement of autophagy contributed to the anti-inflammatory activity of CuIIa against inflammation-related diseases.

Introduction

Cucurbitacins, a large family of triterpenoid compounds isolated from cucurbitaceous plants, possess a wide spectrum of pharmacological activities such as anti-cancer, anti-virus, anti-bacterium, and anti-inflammation activities [1]. A number of studies have revealed the anti-cancer activities of several cucurbitacins including cucurbitacin B, E, I and D [2], [3], [4], [5]. Mechanistically, these cucurbitacins have been reported to induce cell growth arrest and apoptotic cell death through inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation [4], [6], [7]. However, although the precise mechanism for damaging actin cytoskeleton is largely unknown, mounting evidence indicates that many cucurbitacins and their derivatives can disrupt actin cytoskeleton in a variety of cell types leading to cell cycle arrest at G2/M phase [2], [3], suggesting that cucurbitacins represent a group of novel therapeutic agents targeting actin cytoskeleton.

Apart from their anti-cancer activities, the anti-inflammatory properties of cucurbitacins have been revealed both in vitro and in vivo. In the in vitro studies, cucurbitacin R reduces the proliferation of phytohemagglutinin A-stimulated human T lymphocytes and decreases the production of cytokines such as interleukin (IL)-2, IL-4, IL-10, and interferon-γ [8], [9], while cucurbitacin E downregulates the level of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages [10]. In addition, 23,24-dihydrocucurbitacin D can effectively block NO production and NF-κB activation in peritoneal macrophages [11]. These anti-inflammatory activities have been confirmed in several in vivo studies. For instance, cucurbitacin E could markedly suppress carrageenan-induced paw edema in rats [10], and 23, 24-dihydrocucurbitacin B was able to decrease carrageenan-induced paw edema in mice [12]. In an adjuvant-induced arthritis model, cucurbitacin R can reduce the inflammation and bone damage in Lewis rats [8]. These observations reveal the potential application of cucurbitacins as the potent agents for anti-inflammatory therapy.

Cucurbitacin IIa (also known as 25-O-acetyl-23,24-dihydrocucurbitacin F, Fig. 1A), a member of cucurbitacin family, is purified from the root of medicinal plant Hemsleya amabilis (Cucurbitaceae), which has been used as an ancient remedy for bacillary dysentery and gastroenteritis [13]. Like other cucurbitacins, cucurbitacin IIa (CuIIa) has been recently shown to inhibit cancer cell growth both in vitro and in xenografted tumor model via disrupting actin cytoskeleton but independent of STAT3 activity [14], [15]. However, the anti-inflammatory effect of CuIIa and the underlying mechanism have not been well characterized. In this study, we explored the anti-inflammatory effect of CuIIa in LPS-stimulated RAW 264.7 macrophage cell line. Our data revealed that CuIIa induced apoptosis and enhanced autophagy in LPS-stimulated RAW 264.7 cells, but failed to suppress the mitogen-activated protein kinases (MAPKs) and NF-κB activation and tumor necrosis factor (TNF)-α expression. These data suggest a novel mechanism for the anti-inflammation action of CuIIa.

Section snippets

Reagents and antibodies

Cucurbitacin IIa (CuIIa; purity ≥ 98%) was obtained from the National Institutes for Food and Drug Control (Beijing, China). Propidium iodide (PI), dimethyl sulfoxide (DMSO) and lipopolysaccharide (Escherichia coli O55:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CuIIa was dissolved in DMSO at 20 mM, and stored at − 20 °C. Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, RNase A, L-glutamine and fetal bovine serum (FBS) were purchased from Gibco/Invitrogen

CuIIa inhibited cell proliferation and migration of RAW 264.7 cells

We initially explored the effects of CuIIa on the proliferation and migration of RAW 264.7 cells. Cell proliferation assays showed that CuIIa dose-dependently inhibited the proliferation of RAW264.7 cells as compared with untreated (control) cells (Fig. 1B). The IC50 values were 122.32 μM and 6.42 μM for 24 h and 48 h, respectively. A transwell system was used to evaluate the migration ability of RAW 264.7 cells in the presence or absence of CuIIa. As shown in Fig. 1C and D, CuIIa markedly

Discussion

As an active component of H. amabilis which has been used as an ancient remedy for bacillary dysentery and gastroenteritis, CuIIa has long been identified as an anti-inflammatory agent, although the underlying mechanism of action is unclear. In this study, we investigated the anti-inflammatory effect of CuIIa in LPS-activated RAW 264.7 cells. Our results indicate that CuIIa had little influence on the MAPK and NF-κB activation and TNF-α expression in RAW 264.7 cells in response to LPS

Acknowledgments

This work is supported by grants from the National Natural Science Foundation of China (No. 81173604) and the Fundamental Research Funds for the Central Universities (No. 21612411 and No. 21609403).

References (40)

  • Y.C. Lu et al.

    LPS/TLR4 signal transduction pathway

    Cytokine

    (2008)
  • S. Shelly et al.

    Autophagy is an essential component of Drosophila immunity against vesicular stomatitis virus

    Immunity

    (2009)
  • M. Miro

    Cucurbitacins and their pharmacological effects

    Phytother Res

    (1995)
  • Y. Zhang et al.

    Cucurbitacin B induces rapid depletion of the G-actin pool through reactive oxygen species-dependent actin aggregation in melanoma cells

    Acta Biochim Biophys Sin (Shanghai)

    (2011)
  • W.W. Huang et al.

    Cucurbitacin E induces G(2)/M phase arrest through STAT3/p53/p21 signaling and provokes apoptosis via Fas/CD95 and mitochondria-dependent pathways in human bladder cancer T24 cells

    Evid Based Complement Altern Med

    (2012)
  • J. Sun et al.

    Cucurbitacin Q: a selective STAT3 activation inhibitor with potent antitumor activity

    Oncogene

    (2005)
  • M.A. Blaskovich et al.

    Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice

    Cancer Res

    (2003)
  • J.M. Escandell et al.

    Cucurbitacin R reduces the inflammation and bone damage associated with adjuvant arthritis in Lewis rats by suppression of tumor necrosis factor-alpha in T lymphocytes and macrophages

    J Pharmacol Exp Ther

    (2007)
  • C.S. Park et al.

    Inhibition of nitric oxide generation by 23,24-dihydrocucurbitacin D in mouse peritoneal macrophages

    J Pharmacol Exp Ther

    (2004)
  • R.R. Peters et al.

    Nitric oxide and cyclooxygenase may participate in the analgesic and anti-inflammatory effect of the cucurbitacins fraction from Wilbrandia ebracteata

    Life Sci

    (2003)
  • Cited by (28)

    • The surface morphology of Platycodon grandiflorus polysaccharide and its anti-apoptotic effect by targeting autophagy

      2022, Phytomedicine
      Citation Excerpt :

      Luteolin can attenuate METH-induced neurotoxicity by inhibiting the PI3K/Akt pathway, reducing p53 accumulation, as well as p53-regulated apoptosis and autophagy (Tan et al., 2020). Cucurbitacin IIa can activate Caspase-3-dependent apoptosis and promote autophagy in LPS-induced RAW 264.7 macrophages and destroy the actin cytoskeleton, which may at least partially explain its anti-inflammatory effects (He et al., 2013). Polygala polysaccharide (PTP) can induce FAS/FAS-L-mediated apoptosis and autophagy in SPC-A-1 cells.

    • Co-assemblies based on natural Hemslecin A and β-sitosterol as a new sight for synergistic anti-gastric cancer efficacy in TCM

      2022, Colloids and Interface Science Communications
      Citation Excerpt :

      Hemsleya penxianensis (Cucurbitaceae), as a commonly folk medicine used for the treatment of tuberculosis, diabetes, bronchitis, coronary heart disease mainly distributed in Sichuan, Yunnan, Guizhou, Zhejiang province, was first recorded in “Tianbao Materia Medica” [1]. Previous phytochemical studies have made clear that the pharmacodynamic substance basis of H. penxianensis are cucurbitane type triterpenoids as well as steroids like β-sitosterol [2], among which hemslecin A [3] is the dominant ingredient of this plant, exhibiting various activities including anti-tumor, anti-bacteria and anti-inflammation, etc. [4–6], In the process of exploring the mechanism of natural small molecules acting as pharmacodynamics, a class of natural small molecules (NSMs) have been found to self-assemble into natural product gels (NPGs) without modification in different media in recent years [7–9].

    • Cucurbitacins: Bioactivities and synergistic effect with small-molecule drugs

      2020, Journal of Functional Foods
      Citation Excerpt :

      Cucurbitacin IIa not only inhibited the proliferation and migration of RAW 264.7 cells in a dose-dependent manner, but also enhanced LPS induced autophagy. In addition, Cucurbitacin IIa disrupted actin cytoskeleton via inducing actin aggregation (He et al., 2013). Cucurbitacin IIb exhibited anti-inflammatory activity by regulating a variety of cellular behaviors, such as time-dependent and dose-dependent inhibition of lymphocyte proliferation, and multiple signaling pathways, such as blocking the nuclear translocation of NF-κB p65 (Wang et al., 2014).

    • Cucurbitacin IIa interferes with EGFR-MAPK signaling pathway leads to proliferation inhibition in A549 cells

      2019, Food and Chemical Toxicology
      Citation Excerpt :

      CuIIa induced apoptosis by inhibiting survivin independent of JAK2/STAT3 phosphorylation (Boykin et al., 2011). In other work (He et al., 2013), CuIIa was found to induce caspase-3-dependent apoptosis via increasing caspase-3 cleavage and survivin degradation, thus enhancing autophagy in lipopolysaccharide-stimulated RAW 264.7 macrophages. Surprisingly, to date, although EGFR pathway is a hot topic in drug exploration for cancer therapy, few reports are available on impact of CuIIa with this pathway.

    View all citing articles on Scopus
    1

    These two authors contributed equally to this work.

    View full text