Terrein induces apoptosis in HeLa human cervical carcinoma cells through p53 and ERK regulation

  • Authors:
    • Yuwarat Porameesanaporn
    • Wanlaya Uthaisang-Tanechpongtamb
    • Faongchat Jarintanan
    • Suchada Jongrungruangchok
    • Benjamas Thanomsub Wongsatayanon
  • View Affiliations

  • Published online on: February 15, 2013     https://doi.org/10.3892/or.2013.2288
  • Pages: 1600-1608
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Abstract

Terrein, a fungal metabolite derived from Aspergillus terreus, has been shown to have a variety of biological activities in human cells including inhibition of melanogenesis, as well as anti-inflammatory, antioxidant and anticancer properties. In the present study, terrein was shown to have marked anticancer activity on HeLa human cervical carcinoma cells. Terrein exhibited inhibition of proliferation within the same ranges for other cancer cell types with an IC50 at 0.29 mM. The growth inhibition that induced cell death was via apoptosis mechanisms. Chromatin condensation was observed using the Hoechst 33342 stain, a DNA-specific dye. The increase of DNA fragmentation or the sub-G0 peak was also detected by flow cytometry. The signaling used by terrein to induce apoptosis was via the death-receptor and mitochondrial pathways; the cleavage of specific fluorogenic substrates by caspase-3, -8 and -9 activities are clearly demonstrated. The mitochondria were damaged as demonstrated by the decrease of the red/green ratio of the JC-1 staining and the increase of the Bax/Bcl-2 expression ratio. Further analysis of the upstream signaling by the quantitative real-time polymerase chain reaction showed that p53, p21 and ERK were upregulated which indicates the importance of their roles on terrein signaling. This study is the first to show that terrein has an effect on the anticancer properties in cervical cancer cells by inducing apoptosis through p53 and ERK regulation. Our data may help expand the function of the terrein compound and may also aid in the discovery of new anticancer agents.

Introduction

Human cervical carcinoma is the second most common cancer among women worldwide, with about 500,000 new cases and 250,000 related deaths occurring every year, primarily in developing countries (1). Cervical cancer can be cured by radical surgery or radiotherapy for the patients diagnosed with cervical cancer in the early stages, while chemotherapy or neoadjuvant chemotherapy is the primary option for patients with advanced cervical cancer (2). However, the available chemotherapeutic agents are not completely effective in patients with advanced cervical cancer due to the lower chemosensitivity of the cervical cancer cells. Therefore, effective chemotherapeutic agents are required to improve the 5-year survival rate of these patients (3). Cancer is a disease of uncontrolled cell growth or proliferation and a lack of apoptosis; therefore, any agent that can block the cell proliferation or induce apoptosis in the cancer cells could prove to be a potent anticancer agent. To date, several anticancer drugs (such as, paclitaxel, doxorubicin, etoposide and cisplatin) already in use in the clinical setting have been proven to be apoptosis-inducing agents (47). Thus, apoptosis induction is a promising direction in the development of new anticancer agents.

Several sources from plants, marine organisms and microorganisms are used to produce anticancer agents. In microorganisms, it has been shown that both bacteria and fungi are valuable sources of bioactive compounds. However, most anticancer drugs developed from microorganisms currently used in the clinical setting are from bacteria (8). The anticancer properties of metabolites from fungi have yet to be fully elucidated. Terrein (C8H10O3) is a bioactive, fungal, secondary metabolite which was first isolated from Aspergillus terreus in 1935 (9). The chemical structure of terrein contains free hydroxyl groups at positions 4 and 5 of the cyclopentenone ring (Fig. 1) (10,11). Terrein has been reported to have several biological activities. It has been shown that terrein functions as a melanogenesis inhibitor by reducing the tyrosinase production in the spontaneously immortalized mouse melanocyte cell line of Mel-Ab (11,12). In lipopolysaccharide (LPS)-induced inflammation of human dental pulp cells, terrein has been shown to function as an anti-inflammatory agent (13). In MC3T3-E1 fibroblast cells grown on a titanium surface, biocompatible material, terrein was found to reduce the oxidative stress demonstrating anti-oxidant activity (14). Aside from the activities mentioned, terrein has also been shown to suppress the proliferation of human skin keratinocyte cells (15). Markedly, terrein has been shown to inhibit the growth of several types of cancer cells. In prostate cancer cells, terrein has been reported to work as an angiogenesis inhibitor (16). In lung cancer, terrein has been shown to function as a proteasome inhibitor that promotes cell death by apoptosis (10). Additionally, terrein has suppressive growth effects in ABCG2-expressing breast cancer cells by inducing the apoptosis mechanism (17). Thus, terrein is a promising compound, particularly for its anticancer properties; it may provide a new option in cancer therapeutics. In this study, we further examined the anticancer properties of terrein in cervical cancer cells (HeLa), as well as the signaling induced through ERK, p53 and caspase-3, -8 and -9, which have yet to be reported for terrein function.

Materials and methods

Chemicals and reagents

Dulbecco’s modified Eagle’s medium (DMEM), Medium 199, fetal bovine serum (FBS), 0.25% Trypsin-EDTA, penicillin-streptomycin, TRIzol Reagent, Taq DNA Polymerase, SuperScript® VILO™ cDNA Synthesis kit and Hoechst 33342 were purchased from Gibco (Gaithersburg, MD, USA). Dimethyl sulfoxide (DMSO), RNase and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium citrate, dithiothreitol (DTT) and ethidium bromide were purchased from Sigma Chemical Co., (St. Louis, MO, USA). Material 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from USB Corp., (Cleveland, OH, USA) and agarose from Vivantis (Oceanside CA, USA). The JC-1 mitochondrial membrane potential assay kit was purchased from Biotium, Inc. (Hayward, CA, USA). The caspase colorimetric assay kit was from Calbiochem Merck KGaA (Darmstadt, Germany). Power SyBR® Green Master Mix was purchased from Applied Biosystems (Foster City, CA, USA).

Preparation of terrein

Terrein was extracted from the culture broth of fungi Aspergillus terreus CRI301. The crude extract was carried out using ethyl acetate as a solvent. The EtOAc extract was concentrated in vacuo, and then the crude extract from the broth was fractionated and purified by use of the Sephadex LH-20 (2 cm inner diameter and 125 cm long), using MeOH as an eluent. Spectroscopic analysis was used for the compound characteristics.

Cell culture and maintenance

The human cervical carcinoma cell line (HeLa) was kindly provided by Dr Mathurose Ponglikitmongkol, Department of Biochemistry, Faculty of Science, Mahidol University, Thailand. The immortalized porcine epithelial glandular (PEG) cells were kindly provided by Dr Chatsri Deachapunya, Srinakharinwirot University. The HeLa cells were maintained in DMEM supplemented with 10% FBS and with 1% penicillin-streptomycin. The PEG cells were cultured in DMEM containing 5% FBS, 1% L-glutamine, 1% non-essential amino acid, 0.1% insulin and 1% penicillin-streptomycin. Both specimens were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Cytotoxicity assay

The cytotoxicity assay was performed by the MTT method (18). The HeLa and PEG cells at 1×104 cells/100 μl/well were seeded onto a 96-well plate and incubated overnight. The cells were then treated with terrein at 5, 0.5, 0.05, 0.005, 0.0005 and 0.00005 mM for 24 h. An untreated group was combined with 1% DMSO and used as a negative control. Following 24 h of cell treatment, the MTT dye [Thiazolyl Blue Tetrazolium Bromide: (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added at 0.5 mg/ml into each well and incubated for 3 h. The formazan crystal products formed were dissolved by the addition of 100 μl of DMSO. After 15 min, the amount of purple formazan was determined by measuring the optical density (OD) using the ELISA microplate reader at 595 nm. The experiment was performed in triplicate and the percentage of cell viability was calculated as: % Viability = [OD of treated cells/OD of control cells] × 100.

Nuclear morphological observation

The effect of terrein on the nuclear morphological changes was investigated by Hoechst 33342 staining (19). Briefly, the HeLa cells at 4×105 cells/well were seeded onto a 12-well plate and treated with terrein at 0, 0.3, 0.6 and 1.5 mM for 24 h. At the end of the treatment, both the adherent and non-adherent cells were collected. Then, the cells were fixed with 3.7% (vol/vol) paraformaldehyde for 10 min at room temperature, permeated with 0.1% Triton X-100 for another 10 min at room temperature and stained with Hoechst 33342 (1 mg/ml of phosphate-buffered saline; PBS) at 37°C for 15 min. The nuclear morphology was observed with a fluorescent microscope (Olympus, Tokyo, Japan).

Analysis of apoptotic sub-G0 population

The sub-G0 population was analyzed using flow cytometry as previously described (20). The HeLa cells at 1×106 cells/well were plated on a 6-well plate and treated with terrein at 0, 0.3, 0.6 and 1.5 mM. After 24 h, the treated cells were trypsinized and washed twice with ice-cold PBS. The cell pellet was resuspended in 1 ml PBS and gently fixed (drop by drop) with 4 ml of absolute ethanol at −20°C for 5–15 min. Following centrifugation, the ethanol was discarded and 5 ml of PBS was added to the cell pellet which was then allowed to rehydrate for 15 min. Subsequently, each sample was incubated with 500 μl of 100 μg/ml RNase for 20 min at 37°C. After washing with PBS, the cell pellet was gently resuspended in 500 μl of PI solution (50 μg/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100) at 4°C in a darkened environment overnight. Each sample was measured using flow cytometry (BD FACSCanto, Becton-Dickinson, Lincoln Park, NJ, USA) using the Consort 30 program (Becton-Dickinson).

Caspase activity assay

Caspase-3, -8 and -9 activities were measured using fluorescent assay kit detection (Calbiochem Merck KGaA), according to the manufacturer’s instructions. Briefly, HeLa cells at 1×106 cells/well were placed on a 6-well plate, treated with terrein at 0, 0.3, 0.6 and 1.5 mM for 12 h. After the treatment, supernatants from cell lysates were incubated with fluorogenic substrates using DEVD-AFC (caspase-3-like), IETD-AFC (caspase-8-like) and LEHD-AFC (caspase-9-like), at 37°C for 2 h prior to monitoring with a fluorescent microplate reader with excitation set at 400 nm, and emissions at 505 nm.

Analysis of mitochondrial transmembrane potential

The changes in mitochondrial membrane potential (ΔΨm) were detected using a 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1) dye (Biotium Inc.). In healthy cells, the JC-1 accumulates in the mitochondria as JC-1 aggregates (fluorescence is red) and also in the cytoplasm as JC-1 monomers (fluorescence is green). In early apoptosis, the ΔΨm collapses, making JC-1 aggregates unable to accumulate within the mitochondria and dissipate into the JC-1 monomers leading to a loss of the red fluorescence. Therefore, collapse of the ΔΨm is exhibited by a decrease in the ratio of red to green fluorescence (21). In accordance with the terrein treatment, HeLa cells at 1×104 cells/well were placed on a 96-well plate, treated with terrein at 0, 0.3, 0.6 and 1.5 mM for 6 h. Following treatment, the cells were harvested and incubated with a JC-1 reagent solution at 37°C for 20 min, then washed twice with PBS and suspended once more in the PBS. The samples were analyzed by a fluorescence microplate reader and measured with both the red fluorescence (excitation 550 nm, emission 600 nm) and the green fluorescence (excitation 485 nm, emission 535 nm). The ratio of red fluorescence intensity vs. green fluorescence intensity was calculated and presented as the means ± SD. This experiment was performed in triplicate.

Real-time polymerase chain reaction (real-time PCR)

To analyze the effect of terrein on the expression of apoptosis-related genes (p53, Bax, Bcl-2, p21 and ERK2), real-time PCR was used. HeLa cells at 1×106 cells/well were plated on a 6-well plate, treated with terrein at 0, 0.3, 0.6 and 1.5 mM for 24 h. Following treatment, the cells were harvested and washed with 500 μl of PBS. The total RNA was extracted using a TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and quantified by use of OD measurement at 260 and 280 nm using a spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA) (all RNA samples had an A260/A280 ratio >1.8).

The isolated total RNA (2.5 μg) was reverse-transcribed to cDNA with the SuperScript VILO cDNA Synthesis kit (Invitrogen). The reaction mixture was composed of 4 μl of 5X VILO reaction mix, 2 μl of 10X SuperScript enzyme mixture, and DEPC-treated water in a total volume of 25 μl. The reaction mixture was incubated at 25°C for 10 min, at 42°C for 60 min and the reaction was terminated by heating at 85°C for 5 min. The resultant cDNA was stored at −20°C until further use. The PCR primers were obtained from BioDesign Co., Ltd., Pathumthani, Thailand. PCR primers were designed by Primer 3.0 and BLAST search to check the specificity. The primer sequences used are listed in Table I.

Table I

Oligonucleotides used in real-time PCR.

Table I

Oligonucleotides used in real-time PCR.

GeneForward primerReverse primer
p53 5′-ACTAAGCGAGCACTGCCCAA-3′ 5′-ATGGCGGGAGGTAGACTGAC-3′
p21 5′-TATGGGGCTGGGAGTAGTTG-3′ 5′-AGCCGAGAGAAAACAGTCCA-3′
Bax 5′-GCGTCCACCAAGAAGCTGAG-3′ 5′-ACCACCCTGGTCTTGGATCC-3′
Bcl-2 5′-TGTGGCCTTCTTTGAGTTCG-3′ 5′-TCACTTGTGGCCCAGATAGG-3′
ERK2 5′-GCCTGGCCCGTGTTGCAGAT-3′ 5′-CGCCCCTCCAAACGGCTCAA-3′
GAPDH 5′-GAAGGTGAAGGTCGGAGTCA-3′ 5′-GACAAGCTTCCCGTTCTCAG-3′

Real-time quantitative-PCR was performed on an ABI StepOnePlus (Applied Biosystems), using 96-well microtiter plates. The reaction was carried out in a total volume of 20 μl, containing 2.5 μl of the cDNA sample (equivalent to 75 ng), 1 μl of 0.5 μM each of the primer and 10 μl of SYBR-Green Reaction Mix (Applied Biosystems). PCR amplification was performed in duplicated wells. The cycling conditions were: 10 min polymerase activation at 95°C and 40 cycles at 95°C for 15 sec and 60°C for 60 sec. In addition, the real-time reaction of the products was examined by analyzing the melting point after each reaction. A sample without cDNA was used as a negative control and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The baseline and the threshold were set automatically by the software. The crossing point of the amplification curve with the threshold represents the cycle threshold (Ct). The fluorescence threshold Ct values were calculated, and the ΔCt values were determined using the formula ΔCt = Cttarget gene − CtGAPDH. The ΔΔCt values were then calculated based on the formula ΔΔCt = ΔCt treated − ΔCt untreated. The expression level of the target gene in the treated cells was measured relative to the level observed in the untreated cells and was quantified using the formula 2−ΔΔCT(22). The PCR products were electrophoresed on a 2% agarose gel and stained by ethidium bromide under UV light.

Statistical analysis

The results of each experiment were expressed as the means ± standard deviation (SD, for each group n=3). The data were processed with the GraphPad Prism 5 software. Statistical significance was assessed by one-way ANOVA analysis of variance to evaluate the significance of differences between the groups.

Results

Effect of terrein on cell viability

Terrein was tested for its cytotoxicity against human cervical cancer cells (HeLa) and normal cells (PEG) by the MTT assay. As shown in Fig. 2, terrein significantly inhibited the growth of PEG and HeLa cells in relation to the concentration used. The IC50 values were at 0.53 mM for PEG and 0.29 mM for HeLa. It is noteworthy that the percentage of cell viability comparing the cancer and the normal cells differed significantly when using terrein at a concentration of 0.5 mM which was approximately 18 and 60%, respectively. The results indicate a considerable potential of the cytotoxicity effect on human cervical cancer HeLa cells with lower toxicity on normal PEG cells.

Terrein induces apoptosis in HeLa cells

To evaluate the mode of cell death induced by terrein in HeLa cells, the experiment was carried out by staining cells with the DNA specific dye, Hoechst 33342. The cell samples were compared between the terrein-treated cells and the untreated control HeLa cells. The concentrations of terrein were 0, 0.3, 0.6 and 1.5 mM and were treated for 24 h. As depicted in Fig. 3, the untreated control cells displayed normal, round nuclei (Fig. 3a), while the cells treated with terrein exhibited characteristics of apoptosis, such as cell shrinkage, nuclear condensation and fragmentation in a dose-dependent manner (Fig. 3b–d).

As the apoptotic cells with fragmented nuclei appear as cells with hypodiploid DNA content and could be detected at sub-G0 peak with flow cytometry, the numbers of the apoptotic sub-G0 population were quantified. The result demonstrated that the terrein-treated HeLa samples had significantly increased in the sub-G0 phase as compared to the untreated sample (Fig. 4Aa–d). The statistics of each phase of the cell cycle showed that the sub-G0 populations increased as the doses of terrein increased from 11.90 to 26.37 and 84.93%. (Fig. 4B). These results suggest that apoptosis is the mode of cell death used by terrein against HeLa cells.

Induction of apoptotic signaling is triggered by the death receptor and the mitochondrial pathway

Apoptosis is triggered by sequential activation of caspases, a group of cysteine proteases, and proceeds primarily through two pathways. The extrinsic or death receptor pathway involves activation of caspase-8 and is initiated by ligand interaction with death receptors. Second, the intrinsic or mitochondrial pathway is activated by an imbalance between pro-apoptotic and anti-apoptotic proteins from the Bcl-2 family at the mitochondria and cytosol, resulting in the release of cytochrome c from the mitochondria, which in turn activates caspase-9. Both caspase-8 and caspase-9 activate caspase-3 which acts as a common downstream part of the two major apoptosis pathways resulting in apoptosis (23). To address the apoptotic pathway in the terrein-treated HeLa cells, measuring of the fluorogenic substrate cleavage was performed. The result of the fluorescence intensity showed that terrein significantly activated caspase-8, caspase-9 and caspase-3 function after 12 h of treatment. Caspase activity increased significantly when compared to the control group of untreated cells in a concentration-dependent manner (Fig. 5). In addition, the activity of each caspase was inhibited by their specific inhibitor provided by the kit (data not shown). These results suggest that terrein activates the signaling of both the death receptor and mitochondrial pathways.

To confirm the cascade, the damage to the mitochondria was analyzed using a specific dye for mitochondrial, JC-1, staining. Upon quantification by flow cytometry, the HeLa cells treated with terrein at 6 h presented with decreasing ΔΨm as compared to the control group of untreated cells in a dose-dependent manner (Fig. 6). Then, we investigated the expression of Bcl-2 family proteins and whether they were involved with the damage to the mitochondria. The expression of Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) was selected for investigation. As a result, terrein increased the expression of Bax (Fig. 7a) and decreased the expression of Bcl-2 (Fig. 7b) in a dose-dependent manner by real-time PCR. An increase in the Bax/Bcl-2 ratio (Fig. 7c) indicates that upregulation of these Bcl-2 family proteins are upstream events causing damage to the mitochondria.

Apoptotic signaling is mediated by p53 and ERK activation

The tumor suppressor gene, p53, is known to be responsible for the inhibition of cell growth and/or the commitment to apoptosis. Meanwhile, p53 protein regulates the expression of the downstream effector p21, a potent inhibitor of cell cycle kinases, in which both are in response for DNA damage. In addition, p53 regulates apoptosis via upregulation of the expression of Bax and blocks the function of Bcl-2. Thus, it is possible that a substantial increase in the Bax/Bcl-2 ratio may have resulted from p53 function. The level of the expression of p53 and p21 were then examined. As shown in Fig. 8a and b, the expression of mRNA from both p53 and p21 was upregulated suggesting that the upstream signaling of the mitochondrial pathway was induced by terrein. To investigate this signaling triggered by terrein, further upstream mediators of p53 were evaluated. As is well known, several protein kinases may function to activate p53 and ERK2 may be the kinase that is responsible for the DNA damage. Therefore, we selected ERK2 to study the level of expression in response to the terrein treatment. As depicted in Fig. 8c, the mRNA expression of ERK2 increased following terrein treatment in a dose-dependent manner indicating the involvement of ERK signaling.

Discussion

The present study is the first to demonstrate that terrein, a fungal metabolite, induces apoptosis in cervical cancer cells via p53 and ERK signaling. As previously shown, terrein has a variety of effects including anti-inflammatory (13), anti-oxidant (14), anti-proliferative (15) and skin-whitening properties (1112). The effects of terrein on cancer cells have also been reported. In androgen-dependent prostate cancer cells (LNCaP-CR), terrein demonstrates angiogenesis inhibition by blocking the secretion of angiogenin with an IC50 of 13 μM (16). In human lung tumoral cell lines (NCI-H292), terrein acts as proteasome inhibitor by suppressing the chymotrypsin- and trypsin-like activities with the IC50 of 0.3 mM. Also, in these lung tumor cells, terrein was able to induce apoptotic cell death at concentrations of 0.15 mM and 0.3 mM (10). In breast cancer cells (MCF-7), terrein markedly inhibited cell proliferation in IC50 of 1.1 nM (17). Meanwhile, for normal cells, it has been shown that terrein has of non-cytotoxic effects in human keratinocyte at the concentration of 1–50 μM (15). Comparing these data, our study found that the IC50 for cervical cancer cells was at 0.29 mM, while in normal porcine epithelial glandular (PEG) cells it was at 0.53 mM (Fig. 2). These data suggest that the dose of terrein to induce cancer cell death is cell type-dependent. The concentration appears to be high (mM range) but this effective dose has almost the same value exhibited in lung tumor cells. The inhibition concentration at 50% of terrein treatment in HeLa cells did not differ significantly from normal PEG cells. This indicates that terrein also exhibits cytotoxic action on normal cells. However, at approximately 0.5 mM of terrein, the percentage of cell viability of HeLa cells was approximately 18%, while in the normal PEG cells it was approximately 60% which was represented by the difference in the sensitivity.

The evaluation of the mechanism used by terrein to trigger cervical cancer cell death is implicated via apoptosis. As shown in Figs. 25, chromatin condensation, DNA fragmentation and caspase activation were clearly demonstrated. These are distinct characteristics of the apoptosis mechanism (24). To develop an anticancer agent, apoptosis is the preferable mechanism as it does not trigger the inflammation process observed in necrosis, another form of cell death. As previously shown, several anticancer drugs use apoptosis as their target mechanism, therefore, terrein is another promising compound for development as an anticancer agent (47).

The pathway to induce apoptosis is initiated by two major pathways, the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. The extrinsic pathway integrates extracellular signals through the binding of external ligands to death receptors located at the plasma membrane such as the Fas/FasL interaction. Engagement of these receptors by their specific ligand induces their trimerization and leads to the assemblage of the death-inducing signaling complex (DISC). In this complex, procaspase-8 is activated and in turn cleaves and activates executioner caspases including caspase-3, caspase-6 or caspase-7 (25,26). The intrinsic pathway is triggered by the activation of the pro-apoptotic Bcl-2 family proteins known as Bax or Bak. These proteins have shown the ability to form pores in the mitochondrial outer membranes, thereby allowing permeabilization of cytochrome c release to cytosol. Cytochrome c binds to the adaptor apoptotic protease activating factor-1 (Apaf-1) forming a large multi-protein structure known as the apoptosome. The apoptosome then recruits and activates procaspase-9 into the active form which further activates the downstream effector caspases for the death receptor pathway, finally resulting in cell death (27,28).

However, several reports have shown that the cascade of the extrinsic and intrinsic pathway is not fully separated in some cases. Caspase-8 can initiate death via caspase-3 directly or it can trigger the mitochondrial pathway via Bid cleavage (25). Cells that perform directly to the cascade from caspase-8 to caspase-3 are called type I cells, while the cells relative to the cascade initiated from caspase-8 and that have death enhancement via the mitochondrial pathway are called type II cells (29). As shown in this study, terrein activates both caspase-8 and caspase-9 (Fig. 5). Also, the changes in the ratio of Bax/Bcl-2 expression and the dissipation of the ΔΨm were detected (Figs. 6 and 7). These data suggested that terrein-induced apoptosis in cervical cancer cells may display as type II signaling, which is consistent with the reports that HeLa cells triggered by apoptosis-inducing agents usually perform as type II cells (30,31).

p53 plays an important role in several cellular processes. It controls the cell cycle, cell senescence and cell apoptosis. To regulate the apoptosis mechanism, p53 mediates the expression of several proteins that are involved in the release of cytochrome c from the mitochondria, and Bax, Noxa, Puma, AIP1 and APAF1 are also included (32). We also demonstrated that Bax is upregulated upon treatment with terrein, and this may be due to the function of the transcriptional activation by p53. As shown by our results, the level of p53 expression increased upon treatment with terrein (Fig. 8a). In addition, the level of p21, the cyclin-dependent kinase 2 inhibitor that is transcriptionally activated by p53, was also upregulated (Fig. 8b). These data support the critical role of p53 in terrein-mediated cervical cancer cell death which correlates with previous studies of bioactive agents, such as capsaicin (33), eurycomanone (34), flavonoid quercetin (35), kaempferol-7-O-β-D-glucoside (36) and cisplatin (37).

Our study also analyzed the role of extracellular signal-regulated kinase (ERK) signaling and whether or not it is involved in terrein-induced apoptotic cell death. As previously described, ERK2 is involved in cell death by interaction with phosphorylated p53 (38). Thus, we determined the level of ERK2 expression in response to terrein treatment. As depicted in Fig. 8c, the level of the expression of ERK2 increased in a dose-dependent manner. These data suggest that ERK may act upstream of p53 and that consequently leads to cell death by apoptosis. In addition, it has been reported in HeLa cells that ERK activation is associated with the upregulation of p53 expression upon treatment with shikonin (39) and H2O2(40). Otherwise, it is assumed that ERK may act upon the activation of caspase-8. As it has been shown, the prolonged activation of ERK1/2 induces FADD-independent caspase-8 activation and cell death (41,42). As is demonstrated by our study, the upregulation of ERK2 is possibly an important mediator that activates p53, caspase-8 and caspase-9, leading to the destruction of the cancer cells.

In conclusion, our study demonstrated that terrein is a potential candidate as an anticancer agent as it was shown to induce cytotoxicity and apoptosis in cervical cancer cells. The apoptosis pathway may be type II signaling which mediates through ERK signaling. ERK acts as a mediator to regulate the activation of both caspase-8 and p53. The downstream effect of the p53, particularly Bax, was upregulated and significantly leads to the dissipation of the ΔΨm. Consequently, caspase-9 and caspase-3 are activated finally initiating the cleavage of all cellular substrates and genetic materials.

Acknowledgements

This study was supported by the Commission on Higher Education, Ministry of Education and Faculty of Medicine, Srinakharinwirot University, Thailand.

References

1 

Jit M, Demarteau N, Elbasha E, Ginsberg G, Kim J, Praditsitthikorn N, Sinanovic E and Hutubessy R: Human papillomavirus vaccine introduction in low-income and middle-income countries: guidance on the use of cost-effectiveness models. BMC Med. 54:2–9. 2011.PubMed/NCBI

2 

Thomas GM: Improved treatment for cervical cancer-concurrent chemotherapy and radiotherapy. N Engl J Med. 340:1198–1200. 1999. View Article : Google Scholar : PubMed/NCBI

3 

Ren G, Zhao YP, Yang L and Fu CX: Anti-proliferative effect of clitocine from the mushroom Leucopaxillus giganteus on human cervical cancer HeLa cells by inducing apoptosis. Cancer Lett. 262:190–200. 2008. View Article : Google Scholar : PubMed/NCBI

4 

Park SJ, Wu CH, Gordon JD, Zhong X, Emami A and Safa AR: Taxol induces caspase-10-dependent apoptosis. J Biol Chem. 279:51057–51067. 2004. View Article : Google Scholar : PubMed/NCBI

5 

Wang S, Konorev EA, Kotamraju S, Joseph J, Kalivendi S and Kalyanaraman B: Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms. J Biol Chem. 279:25535–25543. 2004. View Article : Google Scholar : PubMed/NCBI

6 

Day TW, Wu CH and Safa AR: Etoposide induces protein kinase Cδ- and caspase-3 dependent apoptosis in neuroblastoma cancer cells. Mol Pharmacol. 76:632–640. 2009.

7 

Tanida S, Mizoshita T, Ozeki K, Tsukamoto H, Kamiya T, Kataoka H, Sakamuro D and Joh T: Mechanisms of cisplatin-induced apoptosis and of cisplatin sensitivity: potential of BIN1 to act as a potent predictor of cisplatin sensitivity in gastric cancer treatment. Int J Surg Oncol. 2012:8628792012.PubMed/NCBI

8 

Newman DJ and Cragg GM: Microbial antitumor drugs: natural products of microbial origin as anticancer agents. Curr Opin Investig Drugs. 10:1280–1296. 2009.PubMed/NCBI

9 

Raistrick H and Smith G: Studies in the biochemistry of micro-organisms: the metabolic products of Aspergillus terreus Thom. A new mould metabolic product-terrein. Biochem J. 29:606–611. 1935.PubMed/NCBI

10 

Demasi M, Felicio AL, Pacheco AO, Leite HG, Lima C and Andrade LH: Studies on terrein as a new class of proteasome inhibitors. J Braz Chem Soc. 21:299–305. 2010. View Article : Google Scholar

11 

Park SH, Kim DS, Kim WG, Ryoo IJ, Lee DH, Huh CH, Youn SW, Yoo ID and Park KC: Terrein: a new melanogenesis inhibitor and its mechanism. Cell Mol Life Sci. 61:2878–2885. 2004. View Article : Google Scholar : PubMed/NCBI

12 

Kim DS, Lee S, Lee HK, Park SH, Ryoo IJ, Yoo ID, Kwon SB, Baek KJ, Na JI and Park KC: The hypopigmentary action of KI-063 (a new tyrosinase inhibitor) combined with terrein. J Pharm Pharmacol. 60:343–348. 2008. View Article : Google Scholar : PubMed/NCBI

13 

Lee JC, Yu MK, Lee R, Lee YH, Jeon JG, Lee MH, Jhee EC, Yoo ID and Yi HK: Terrein reduces pulpal inflammation in human dental pulp cells. J Endod. 34:433–437. 2008. View Article : Google Scholar : PubMed/NCBI

14 

Lee YH, Lee NH, Bhattarai G, Oh YT, Yu MK, Yoo ID, Jhee EC and Yi HK: Enhancement of osteoblast biocompatibility on titanium surface with Terrein treatment. Cell Biochem Funct. 28:678–685. 2010. View Article : Google Scholar : PubMed/NCBI

15 

Kim DS, Lee HK, Park SH, Lee S, Ryoo IJ, Kim WG, Yoo ID, Na JI, Kwon SB and Park KC: Terrein inhibits keratinocyte proliferation via ERK inactivation and G2/M cell cycle arrest. Exp Dermatol. 17:312–317. 2007. View Article : Google Scholar : PubMed/NCBI

16 

Arakawa M, Someno T, Kawada M and Ikeda D: A new terrein glucoside, a novel inhibitor of angiogenin secretion in tumor angiogenesis. J Antibiot. 61:442–448. 2008. View Article : Google Scholar : PubMed/NCBI

17 

Liao WY, Shen CN, Lin LH, Yang YL, Han HY, Chen JW, Kuo SC, Wu SH and Liaw CC: Asperjinone, a nor-neolignan, and terrein, a suppressor of ABCG2-expressing breast cancer cells, from thermophilic Aspergillus terreus. J Nat Prod. 75:630–635. 2012. View Article : Google Scholar : PubMed/NCBI

18 

Uthaisang W, Reutrakul V, Krachangchaeng C, Wilairat P and Fadeel B: VR-3848, a novel peptide derived from Euphobiaceae, induces mitochondria-dependent apoptosis in human leukemia cells. Cancer Lett. 208:171–178. 2004. View Article : Google Scholar : PubMed/NCBI

19 

Oubrahim H, Stadtman ER and Chock PB: Mitochondria play no roles in Mn(II)-induced apoptosis in HeLa cells. Proc Natl Acad Sci USA. 98:9505–9510. 2001. View Article : Google Scholar : PubMed/NCBI

20 

Feng Q, Cao HL, Xu W, Li XR, Ren YQ and Du LF: Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G2/M arrest. Acta Pharmacol Sin. 31:519–527. 2011. View Article : Google Scholar : PubMed/NCBI

21 

Cossarizza A, Baccarani CM, Kalashnikova G and Franceschi C: A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun. 197:40–45. 1993.PubMed/NCBI

22 

Gomez-Lazaro M, Galindo MF, Concannon CG, Segura MF, Fernandez-Gomez FJ, Llecha N, Comella JX, Prehn JH and Jordan J: 6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA. J Neurochem. 104:1599–1612. 2008. View Article : Google Scholar : PubMed/NCBI

23 

Puerto HLD, Martins AS, Milsted A, Souza-Fagundes EM, Braz GF, Hissa B, Andrade LO, Alves F, Rajão DS, Leite RC and Vasconcelos AC: Canine distemper virus induces apoptosis in cervical tumor derived cell lines. Virol J. 334:2–7. 2011.PubMed/NCBI

24 

Elmore S: Apoptosis: a review of programmed cell death. Toxicol Pathol. 35:495–516. 2007. View Article : Google Scholar : PubMed/NCBI

25 

Ashkenazi A: Targeting the extrinsic apoptosis pathway in cancer. Cytokine Growth Factor Rev. 19:325–331. 2008. View Article : Google Scholar : PubMed/NCBI

26 

Gu Q, Wang JD, Xia HHX, Lin MC, He H, Zou B, Tu SP, Yang Y, Liu XG, Lam SK, Wong WM, Chan AO, Yuen MF, Kung HF and Wong BC: Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer. Carcinogenesis. 26:541–546. 2005. View Article : Google Scholar : PubMed/NCBI

27 

Budihardjo I, Oliver H, Lutter M, Luo X and Wang X: Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol. 15:269–290. 1999. View Article : Google Scholar : PubMed/NCBI

28 

Ghobrial IM, Witzig TE and Adjei AA: Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin. 55:178–194. 2005. View Article : Google Scholar : PubMed/NCBI

29 

Jin Z, Li Y, Pitti R, Lawrence D, Pham VC, Lill JR and Ashkenazi A: Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling. Cell. 137:721–735. 2009. View Article : Google Scholar : PubMed/NCBI

30 

Kim SH, Kim SH, Lee SC and Song YS: Involvement of both extrinsic and intrinsic apoptotic pathways in apoptosis induced by genistein in human cervical cancer cells. Ann NY Acad Sci. 1171:196–201. 2009. View Article : Google Scholar : PubMed/NCBI

31 

Hougardy BM, van der Zee AG, van den Heuvel FA, Timmer T, de Vries EG and de Jong S: Sensitivity to Fas-mediated apoptosis in high-risk HPV-positive human cervical cancer cells: relationship with Fas, caspase-8, and Bid. Gynecol Oncol. 97:353–364. 2005. View Article : Google Scholar : PubMed/NCBI

32 

Gross A, Jockel J, Wei MC and Korsmeyer SJ: Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17:3878–3885. 1998. View Article : Google Scholar : PubMed/NCBI

33 

Yang W, Gong X, Zhao X, An W, Wang X and Wang M: Capsaicin induces apoptosis in HeLa cells via Bax/Bcl-2 and caspase-3 pathways. Asian J Traditional Med. 1:3–4. 2006.

34 

Mahfudh N and Pihie AHL: Eurycomanone induces apoptosis through the up-regulation of p53 in human cervical carcinoma cells. J Cancer Mol. 4:109–115. 2008.

35 

Priyadarsini RV, Murugan RS, Maitreyi S, Ramalingam K, Karunagaran D and Nagini S: The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-κB inhibition. Eur J Pharmacol. 649:84–91. 2010.PubMed/NCBI

36 

Xu W, Liu J, Li C, Wu HZ and Liu YW: Kaempferol-7-O-β-D-glucoside (KG) isolated from Smilax china L. rhizome induces G2/M phase arrest and apoptosis on HeLa cells in a p53-independent manner. Cancer Lett. 264:229–240. 2008.

37 

Wang X, Martindale JL and Holbrook NJ: Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem. 275:39435–39443. 2000. View Article : Google Scholar

38 

Yeh PY, Chuang SE, Yeh KH, Song YC, Chang LLY and Cheng AL: Phosphorylation of p53 on Thr55 by ERK2 is necessary for doxorubicin-induced p53 activation and cell death. Oncogene. 23:3580–3588. 2004. View Article : Google Scholar : PubMed/NCBI

39 

Wu Z, Wu LJ, Tashiro S, Onodera S and Ikejima T: Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis. Chin Med J. 118:671–677. 2005.PubMed/NCBI

40 

Singh M, Sharma H and Singh N: Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway. Mitochondrion. 7:367–373. 2007. View Article : Google Scholar : PubMed/NCBI

41 

Zhuang S and Schnellmann RG: A death-promoting role for extracellular signal-regulated kinase. J Pharmacol Exp Ther. 319:991–997. 2006. View Article : Google Scholar : PubMed/NCBI

42 

Cagnol S and Chambard JC: ERK and cell death: mechanisms of ERK-induced cell death-apoptosis, autophagy and senescence. FEBS J. 277:2–21. 2010. View Article : Google Scholar : PubMed/NCBI

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April 2013
Volume 29 Issue 4

Print ISSN: 1021-335X
Online ISSN:1791-2431

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Spandidos Publications style
Porameesanaporn Y, Uthaisang-Tanechpongtamb W, Jarintanan F, Jongrungruangchok S and Thanomsub Wongsatayanon B: Terrein induces apoptosis in HeLa human cervical carcinoma cells through p53 and ERK regulation. Oncol Rep 29: 1600-1608, 2013.
APA
Porameesanaporn, Y., Uthaisang-Tanechpongtamb, W., Jarintanan, F., Jongrungruangchok, S., & Thanomsub Wongsatayanon, B. (2013). Terrein induces apoptosis in HeLa human cervical carcinoma cells through p53 and ERK regulation. Oncology Reports, 29, 1600-1608. https://doi.org/10.3892/or.2013.2288
MLA
Porameesanaporn, Y., Uthaisang-Tanechpongtamb, W., Jarintanan, F., Jongrungruangchok, S., Thanomsub Wongsatayanon, B."Terrein induces apoptosis in HeLa human cervical carcinoma cells through p53 and ERK regulation". Oncology Reports 29.4 (2013): 1600-1608.
Chicago
Porameesanaporn, Y., Uthaisang-Tanechpongtamb, W., Jarintanan, F., Jongrungruangchok, S., Thanomsub Wongsatayanon, B."Terrein induces apoptosis in HeLa human cervical carcinoma cells through p53 and ERK regulation". Oncology Reports 29, no. 4 (2013): 1600-1608. https://doi.org/10.3892/or.2013.2288