Elsevier

Chemico-Biological Interactions

Volume 277, 1 November 2017, Pages 91-100
Chemico-Biological Interactions

Carnosic acid induces apoptosis of hepatocellular carcinoma cells via ROS-mediated mitochondrial pathway

https://doi.org/10.1016/j.cbi.2017.09.005Get rights and content

Highlights

  • Carnosic acid (CA) induces apoptosis in hepatocellular carcinoma cells.

  • CA inhibited the growth of HepG2-and SMMC-7721-xenografted tumor in mice.

  • CA-induced HCC cell apoptosis via oxidative stress-mediated mitochondrial pathway.

  • CA regulates the phosphorylation of NF-κB and mTOR involved in its pro-apoptosis.

Abstract

Carnosic acid (CA), an important bioactive phenolic diterpene mainly found in labiate plants, exerts various biological functions, including antioxidant, anti-inflammatory, antitumor, and neuroprotective activities. In the present study, we proved the deleterious effects of CA against hepatocellular carcinoma (HCC) in both in vitro and in vivo models. In vitro, CA significantly decreased cell viability, inhibited cell proliferation and migration, enhanced apoptosis, and increased caspase-3, -8, and -9 activities in HepG2 and SMMC-7721 cells. Specifically, CA led to a decreased mitochondrial membrane potential (MMP) and increases in intracellular reactive oxygen species (ROS) levels and apoptosis-related protein expression. Pre-incubation of HCC cells with N-Acetyl-l-cysteine (NAC), a ROS inhibitor, strongly suppressed CA-induced apoptotic phenomena, including reduced cell viability, excessive ROS levels, MMP decreases, and abnormal protein expression, suggesting an association of CA-induced apoptosis with oxidative stress-mediated mitochondrial pathways. In HepG2-and SMMC-7721-xenograft tumor mouse models, treatment with CA inhibited tumor growth and modulated apoptosis-related protein expression, confirming the anti-HCC effects of this chemical. Moreover, the CA-mediated anti-HCC effects associated with oxidative stress provide experimental evidence to support the potential use of CA as a drug therapy for HCC.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide and is therefore recognized as a serious disease [1]. Nearly 820,000 deaths related to HCC are occurred ever year [2]. Although an early diagnosis might lead to symptom improvement, most HCC cases do not meet the indications for surgical resection and are insensitive to radiotherapy and chemotherapy [3].

Resistance to cell death mechanisms is a characteristic of malignant tumor cells. Accordingly, apoptosis is a primary target of anti-tumor therapy [4]. The apoptotic process involves two pathways: the mitochondrial pathway and the death receptor pathway. In the former pathway, the mitochondria are considered “the storage of the suicide weapon” [5]. In response to external stimuli, mitochondrial membrane permeability increases, and apoptotic signals are released into the cytoplasm to further promote apoptosis. In particular, the Bcl-2 protein family includes pro- and anti-apoptotic mitochondrial proteins [6]. Additionally, caspase-3, an important apoptotic executor, can amplify signals produced by caspase-8 and caspase-9 [7]. Within the death receptor pathway, autocatalysis of pro-caspase-8 can decrease the mitochondrial membrane potential (MMP) [8].

Furthermore, excessive levels of reactive oxygen species (ROS) can cause an imbalance of the oxidation and antioxidant systems in the body, leading to oxidative stress that may activate the endogenous apoptosis pathway induced by mitochondrial dysfunction [9]. The mitochondria are considered the major source of intracellular ROS production. An accumulation of ROS can decrease the MMP and increase the membrane permeability, thus allowing the release of cytochrome c into the cytoplasm and the initiation of a caspase cascade reaction [10]. Notably, the plant extract shikonin (SHI) can induce oxidative stress by increasing the intracellular ROS level to potentiate arsenic trioxide (ATO)-induced DNA damage, thus inducing hepatoma cell apoptosis [11].

Recently, herbal preparations and natural compounds have been reported to possess anti-hepatocellular carcinoma effects [12], [13]. Carnosic acid (CA), a bioactive phenolic diterpene mainly found in labiate plants such as Salvia officinalis and Rosmarinus officinalis, exhibits various pharmacological effects, including antioxidant, anti-inflammatory, neuroprotective and antitumor activities [14], [15]. In a previous study, co-incubation of breast cancer cells with CA and tamoxifen led to the activation of caspase-3, downregulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL, and ultimately to apoptosis [16]. Additionally, the inhibitory effects of rosemary extract on colon cancer cell proliferation are largely attributed to CA [17]. Recent evidence suggests that CA nanoparticles can alleviate the inflammatory progression of hepatoma cells by regulating NF-κB and inducing apoptosis via Bad-regulated caspase-3 pathway [18]. However, no systematic study has evaluated CA-mediated hepatocellular carcinoma cell apoptosis via the oxidative stress-dependent mitochondrial pathway (see Fig. 1).

The present case study sought to examine the pro-apoptotic effects of CA on HCC through both in vivo and in vitro experiments. We confirmed that CA induced the apoptosis of both HepG2 and SMMC-7721 cells primarily through the oxidative stress-dependent mitochondrial apoptotic pathway. All of our data support the potential addition of CA to the list of therapeutic drugs for HCC.

Section snippets

Cell culture

The human HCC cell lines HepG2 (passage number: <10) and SMMC-7721 (passage number <10) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and China Center for Type Culture Collection (CCTCC; Beijing, China), respectively. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% streptomycin and penicillin, under a humidified atmosphere containing 95% air and 5% CO2 at 37 °C.

Cell viability assay

To assess cell viability, HepG2

CA induces apoptosis in hepatocellular carcinoma cells

Dose-dependent reductions in cell viability were observed in HepG2 and SMMC-7721 cells treated with CA. The 24-h half-maximal inhibitory concentrations of CA in HepG2 and SMMC-7721 cells were 43.7 and 74.8 μM, respectively (P < 0.001; Fig. 2A). At 50 μM, CA exerted marked inhibitory effects on HepG2 and SMMC-7721 cell colony formation, with complete inhibition at 100 μM (Fig. 2B). Additionally, the effects of CA on cell migratory abilities were evaluated using a wound healing assay. The

Discussion

The results obtained by our group and others have successfully confirmed the cytotoxic effects of CA in HCC cells, cervical carcinoma cells [22], prostate carcinoma cells [23], pancreatic cancer cells and colon cancer cells [24]. In the present study, CA exerted robust cytotoxic effects in HepG2 and SMMC-7721 cells, as evidenced by the reduction in cell viability, inhibition of cell colonization and migratory abilities, enhancement of the G2/M phase within cell cycle, and initiation of cellular

Conclusions

In summary, CA induced apoptosis in HepG2 and SMMC-7721 HCC cells inhibited xenograft tumor growth at least partially via the ROS-mediated mitochondrial apoptosis pathway. Our findings support the use of CA as a novel agent for the treatment of HCC.

Conflict of interest

The authors have declared that there is no conflict of interest.

Ethical approval

The experimental animal protocol was approved by the Animal Ethics Committee of Jilin University (20,160,504).

Acknowledgements

This work was supported by the Natural Science Foundation of China (Grant No. 81402955), Science and Technology Key Project in Jilin Province of China (Grant No. 20150203002NY and 20160204029YY) and Special Projects of the Cooperation between Jilin University and Jilin Province (SP2017-1-1-(8)).

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