Elsevier

Biomedicine & Pharmacotherapy

Volume 103, July 2018, Pages 699-707
Biomedicine & Pharmacotherapy

Inhibition of PI3K/Akt/mTOR pathway by apigenin induces apoptosis and autophagy in hepatocellular carcinoma cells

https://doi.org/10.1016/j.biopha.2018.04.072Get rights and content

Abstract

Apigenin is a dietary flavonoid with known antioxidant and antitumor effects against several types of cancers by promoting cell death and inducing cell cycle arrest. Apigenin also regulates a variety of intracellular signal transduction pathways during apoptosis or autophagy. However, the precise mechanism underlying the anticancer effects of apigenin in liver cancer remains poorly understood. In this study, we demonstrated that apigenin has anticancer activity against hepatocellular carcinoma cells. Apigenin inhibited the cell growth and induced cell death in a dose- and time-dependent manner in HepG2 cells. We found that apigenin treatment increased the expression of LC3-II and the number of GFP-LC3 puncta. Moreover, inhibition of autophagy with 3-MA and Atg5 gene silencing strengthened apigenin-induced proliferation inhibition and apoptosis. Our data has indicated that apigenin-induced autophagy has a protective effect against cell death. Additionally, apigenin induced apoptosis and autophagy through inhibition of PI3K/Akt/mTOR pathway. Most importantly, in vivo data showed that administration of apigenin decreased tumor growth and autophagy inhibition by 3-MA significantly enhanced the anticancer effect of apigenin. Collectively, our results reveal that apigenin inhibits cell proliferation and induces autophagy via suppressing the PI3K/Akt/mTOR pathway. Our results also suggest combination of autophagy inhibitors and apigenin would be a potential chemotherapeutic strategy against hepatocellular carcinoma.

Introduction

Hepatocellular carcinoma is the sixth most common cancer and ranked as the third leading cause of cancer-related deaths worldwide [1,2]. Due to lacking specific clinical symptoms and signs, early diagnoses and treatments, hepatocellular carcinoma become almost impossible [3]. The treatment of liver cancer includes surgical resection, local ablation, transplantation, transarterial embolization and immunotherapy [4]. Although improved diagnosis and therapeutic methods, the overall 5-year survival rate of approximately 12% [5]. Therefore, novel agents and effective therapeutic strategies to treat liver cancer are urgently required.

Apigenin (4′,5,7-trihydroxyflavone), is a natural flavonoid that is widely distributed in fruits and vegetables, such as parsley, orange, tea, chamomile and seasonings [6]. In recent years, apigenin has been shown to possess significant anti-inflammatory, anti-oxidant and anti-tumor properties [7]. Previous studies have reported that apigenin could inhibit the growth and proliferation, promote apoptotic cell death, induce cell cycle arrest and mitochondrial membrane potential disruption of cancer cells in vitro and in vivo [[8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]]. However, the precise mechanism underlying the anti-tumor effects of apigenin remains poorly understood.

Autophagy, an evolutionarily conserved pathway, plays a vital role in degradation of the superfluous proteins and organelles. Autophagy is induced by a series of conditions, such as hypoxia, cellular stress and nutrient starvation [21]. Recent studies indicate that targeted autophagy may be an effective therapeutic strategy to fight cancer [22]. However, autophagy has dual roles in regulating cell survival with regard to cancer treatment [23]. Accumulating evidence suggests that in some conditions, cancer cells tend to use autophagy as a survival mechanism to avoid chemotherapeutic agents or γ-irradiation induced apoptosis [24]. Several molecular and signaling pathways play a crucial role in regulating autophagy in many types of cancers, such as the autophagy-related gene family [25] and PI3K/Akt/mTOR signaling pathway [26,27]. The mammalian target of rapamycin (mTOR) plays a critical role in regulating the balance between cell proliferation and autophagy in response to cellular stress induced by chemotherapeutics in cancer cells [28]. Previous studies have reported that apigenin could induce autophagy and apoptosis in breast cancer cells [29]. However, the relationship between apoptosis and autophagy induced by apigenin remains unknown.

In the present study, we aimed to investigate the effect of apigenin on apoptosis and autophagy in hepatocellular carcinoma cells and xenograft mouse model. Our results showed that apigenin induced apoptosis and autophagy in vitro and in vivo. We found that inhibition of autophagy led to inhibition of cell proliferation and tumor growth. Moreover, we also demonstrated that the apigenin induced autophagy and apoptosis via inhibiting PI3K/Akt/mTOR pathway.

Section snippets

Reagents and antibodies

Apigenin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for cleaved-caspase-3(1:1500), cleaved-caspase-9(1:1000), cleaved-PARP(1:1500), Bcl-2(1:1000), Bax(1:1000), PI3K(1:1500), p-PI3K(1:1000), Akt(1:1000), p-Akt(1:800), mTOR(1:1000), p-mTOR(1:800), Atg5(1:1000), Beclin1(1:1000) and SQSTM1(1:1500) were obtained from Cell Signal Technology (Boston, USA). Antibodies for LC3B(1:1000) and actin(1:5000) were purchased from Sigma (St. Louis, MO, USA). Atg5 siRNA, GFP-LC3B plasmid,

Apigenin inhibited cell growth and induced apoptosis in hepatocellular carcinoma cells

To investigate the effect of apigenin on the viability of hepatocellular carcinoma cells, we treated HepG2 cells with apigenin We found that apigenin inhibited cell viability in a time- and dose-dependent manner (Fig. 1A and B). Moreover, Annexin V and PI staining assay showed that apigenin induced cell death in a dose-dependent manner (Fig. 1C and D). To further explore the mechanism by apigenin induced cell death in HepG2 cells, we detected the anti-apoptotic and pro-apoptotic protein

Discussion

Apigenin, one of the common natural flavonoids, has been demonstrated to inhibit tumour cell proliferation, angiogenesis and induce apoptosis in various types of tumor cells [31]. Our previous study has been demonstrated that apigenin inhibits hepatoma cell growth both in vitro and in vivo, which is associated with induction of cell cycle arrest and apoptosis [32]. However, the special mechanism underlying the anti-tumor effects of apigenin in hepatoma cell is still not well understood. In the

Conflict of interest

The authors state that they have no conflict of interests.

References (47)

  • V. Papadimitrakopoulou

    Development of PI3K/AKT/mTOR pathway inhibitors and their application in personalized therapy for non–small-cell lung cancer

    J. Thorac. Oncol.

    (2012)
  • C. Xu et al.

    Effect of evodiagenine mediates photocytotoxicity on human breast cancer cells MDA-MB-231 through inhibition of PI3K/AKT/mTOR and activation of p38 pathways

    Fitoterapia

    (2014)
  • X. Zhang et al.

    A novel protoapigenone analog RY10-4 induces breast cancer MCF-7 cell death through autophagy via the Akt/mTOR pathway

    Toxicol. Appl. Pharma

    (2013)
  • H.B. El-Serag

    Hepatocellular carcinoma

    N Engl. J. Med. New. Engl. J. Med.

    (2011)
  • Q. Du et al.

    Synergistic anticancer effects of curcumin and resveratrol in Hepa1-6 hepatocellular carcinoma cells

    Oncol. Rep.

    (2013)
  • D. Patel et al.

    Apigenin and cancer chemoprevention: progress, potential and promise (review)

    Int. J. Oncol.

    (2007)
  • H. Zhu et al.

    Apigenin induced apoptosis in esophageal carcinoma cells by destruction membrane structures

    Scanning

    (2015)
  • X.Y. Hu et al.

    5-Fluorouracil combined with apigenin enhances anticancer activity through mitochondrial membrane potential (ΔΨm)-mediated apoptosis in hepatocellular carcinoma

    Clin. Exp. Pharmacol. Physiol.

    (2015)
  • Y. Liao et al.

    Apigenin induces the apoptosis and regulates MAPK signaling pathways in mouse macrophage ANA-1 cells

    Plos One

    (2014)
  • M.D. Shi et al.

    Apigenin, a dietary flavonoid, inhibits proliferation of human bladder cancer T-24 cells via blocking cell cycle progression and inducing apoptosis

    Cancer Cell Int.

    (2015)
  • S. Shukla et al.

    Apigenin inhibits prostate cancer progression in TRAMP mice via targeting PI3K/Akt/FoxO pathway

    Carcinogenesis

    (2014)
  • S. Park et al.

    Apigenin induces ROS dependent apoptosis and ER stress in human endometriosis cells

    J. Cell Physiol.

    (2018)
  • J. Fang et al.

    Apigenin inhibits tumor angiogenesis through decreasing HIF-1α and VEGF expression

    Carcinogenesis

    (2007)
  • Cited by (271)

    • Effects of apigenin on gastric cancer cells

      2024, Biomedicine and Pharmacotherapy
    View all citing articles on Scopus
    View full text