Introduction
Cancer of the larynx constitutes a majority of the cancers affecting the laryngeal epithelium based on tumor location. It directly spreads to structures adjacent to the cervical lymph node, and more distantly through the blood stream via metastasis to the regional lung [1]. The overall five-year survival rate was within 50–70% over the past decade [2]. Consequently, numerous studies have been conducted to develop efficient strategies for the development of cancer chemopreventive and chemotherapeutic agents. Recently, apoptosis induced by chemotherapy has been shown to play a significant role in the elimination of cancer cells [3]. Thus, targeting the apoptotic pathway in epithelial cancer cells may effectively prevent laryngeal cancer. L-ascorbic acid (L-AA) exhibits strong antiproliferative and antioxidant effects and is known to modulate various enzymes that are involved in cancer development and progression [4]. The effects of L-AA can also be attributed to its biological role as a cofactor for a number of enzymes and most hydroxylases related to collagen synthesis, and represents a source of the signaling molecule and hydrogen peroxide. Other studies have discussed the anti-carcinogenic effect of L-AA on cancer prevention [5]. The signals associated with the molecular mechanism of cancer prevention by L-AA have been attributed to nuclear factor-κB (NF-κB), phosphatidylinositol 3’-kinase (PI3K)/Akt, and mitogen-activated protein kinase (MAPK) signaling pathways [6-8]. These signal transduction pathways are related to the regulation of cell proliferation and apoptosis, modulation of cell cycle, and activation of tumor migration [9,10]. The constitutive activation of NF-κB in several cancers, including pancreatic, lung, breast, gastric, melanoma, and head and neck cancer, is an important contributor to cancer metastasis and progression [11,12]. A recent study suggested that the level of NF-kB activity may be important in laryngeal cancer cell survival [13]. The PI3K/Akt signaling pathway might be the most central pathway in the transmission of anti-apoptotic signals in cell survival. Multiple pathways have been implicated in cell proliferation, survival, and metabolic processes. The MAPKs, including extracellular signalregulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38, are also known to play a basic role in survival, proliferation, transcription, and apoptosis. The PI3K/Akt and MAPKs signaling pathways are clearly involved in a variety of cellular functions, including cell growth, differentiation, development, and apoptosis [14]. However, the anticancer effects of L-AA in squamous carcinoma head and neck cancer cells are not clearly established.
In the present study, we explored the anti-tumor activity of L-AA in laryngeal Hep-2 cells and the mechanism underlying L-AA-induced growth inhibition and apoptosis.
Materials and Methods
1. Materials
L-AA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and all other chemicals were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies used were polyclonal goat anti-human Bcl-2, monoclonal mouse anti-human Bax, monoclonal mouse anti-human cytochrome c, polyclonal rabbit anti-human Bid, monoclonal mouse anti-human apoptosisinducing factor (AIF), polyclonal goat anti-human endonuclease G (EndoG), and polyclonal goat anti-human actin (Santa Cruz, Santa Cruz, CA, USA). Monoclonal rabbit anti-human NF-κB P65 antibodies were acquired from Cell Signaling Technology (Danvers, MA, USA).
2. Cell culture and cell viability assay
The Hep-2 cell line, delivered from a human laryngeal epidermoid carcinoma of the larynx, was purchased from a Korean Cell Line Bank (KCLB No. 10023). Hep-2 cells were cultured in Dulbecco’s modified eagle media, supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% streptomycin-penicillin. The cells were grown at 37°C in a humidified atmosphere of 5% carbon dioxide in air. Cells were incubated with or without L-AA in 96-well plates (5 × 103 cells/well) for 24 hours, and the cell viability was evaluated using MTT assay as described previously [15]. The relative cell viability was expressed as the ratio (%) of the absorbance in the experimental wells to that of the control wells (without test compound). The cytotoxic concentration for 50% cell death (CC50) was determined from the dose-response curve.
3. 4′,6-diamidino-2-phenylindole (DAPI) staining
Hep-2 cells (1 × 106/mL) were cultured in 60-mm diameter dishes in RPMI containing 1% FBS with 10 mM L-AA for 24 hours, followed by fixation of the cells in 4% paraformaldehyde for 15 minutes at room temperature before permeabilization with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes. The cells were rinsed with phosphate-buffered saline (PBS) and incubated for 15 minutes at room temperature with 0.01% DAPI stain (Sigma-Aldrich). Excess stain was removed by washing three times in PBS. Stained nuclei were visualized via fluorescence microscopy. The percentage of apoptotic cells was determined by counting at least 200 cells within the treatment group.
4. Flow cytometry
An annexin V FLUOS staining kit (Sigma-Aldrich) was used to measure the level of annexin V binding according to the manufacturer’s instructions. Briefly, the cells were trypsinized in PBS, collected by centrifugation, and resuspended in 100 μL of binding buffer containing annexin V. The cells were then incubated at room temperature for 15 minutes in the dark, and analyzed by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA). Several controls were used to optimize the instrument settings and determine the gating for the Windowsbased platform.
5. Caspase activity and inhibition of apoptosis with a caspase inhibitor
The activities of caspase-3, -8, and -9 in L-AA-treated cells were measured colorimetrically using a commercial assay kit (Calbiochem, San Diego, CA, USA) according to the manufacturer’s instructions. To determine the inhibition of apoptosis, a general caspase inhibitor z-VAD-fmk (50 μM), was added to the culture medium 1 hour before L-AA treatment. The cytotoxic effect of L-AA was measured using MTT in the presence or absence of caspase inhibitors.
6. Protein extraction and Western blot analysis
Cell lysates were prepared in NP-40 lysis buffer (30 mM Tris-Cl, pH 7.5, 1 mM ethylenediaminetetraacetic acid [EDTA], 150 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride [PMSF], and protease inhibitor mixture containing 1 μg/ mL aprotinin and leupeptin), and the total protein content was quantified using the BCA protein assay (Pierce, Rockford, IL, USA). Equal amounts of total protein (100 μg) were electrophoretically separated via 12% sodium dodecyl sulphatepolyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was blocked with 5% nonfat dry milk and incubated with primary antibodies for 1 hour at room temperature. The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich) for 2 hours. The blots were then washed and reacted with enhanced chemiluminescence (ECL) solutions (Amersham Pharmacia Biotech) and exposed to ECL Hyperfilm ((American BioSciences, Blauvelt, NY, USA)). The relative intensity of each band was determined using a computerized software program.
7. Extraction of nuclear protein
Hep-2 cells reaching 80% to 90% confluence were incubated in a medium containing 1% FBS in 0–10 mM of L-AA for 24 hours. The cells were resuspended in 500 μL of cold buffer A [50 mM Tris (pH 7.4), 150 mM NaCl, 0.2 mM EDTA, 3% (v/ v) glycerol, and 1.5 mM MgCl2]. The cells were allowed to swell for 5 minutes on ice and then lysed with 500 μL of buffer B (buffer A containing 0.05% Nonidet P-40; Sigma-Aldrich). The cell lysates were gently layered onto an equal volume of buffer C [10 mM Tris (pH 7.4), 25% (v/v) glycerol, and 1.5 mM MgCl2] and centrifuged for 5 minutes at 200 × g. The white nuclear pellet was resuspended in 75 μL of a cold high-salt lysis buffer [20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM PMSF]. This suspension was agitated for 30 minutes at 4℃ and microcentrifuged for 15 minutes at 4℃. The resulting supernatant was stored in aliquots at –80℃. The protein was quantified spectrophotometrically using the BCA assay (Pierce) with bovine serum albumin as the standard.
8. Electrophoretic mobility shift assay (EMSA)
EMSA was performed using a gel shift assay (Promega, Madison, WI, USA). Briefly, the oligonucleotide with the consensus sequence for NF-κB (5′-AGTTGAGGGGA CTTTCCCAGG-3′) was end-labeled with [g-32P]-adenosine triphosphate (3 Ci/mmol; Amersham Pharmacia Biotech) using T4 polynucleotide kinase. The labeled oligonucleotide was then purified in a Microspin G-25 column (Sigma-Aldrich) and used as an EMSA probe. The nuclear extract proteins (15 μg) were pre-incubated with the binding buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM dithiothreitol, 4% (v/v) glycerol, and 0.05 mg/mL poly(deoxyinosine-deoxycytidine)] for 5 minutes, and then incubated with the labeled probe for 15 minutes at 37℃. Each sample underwent electrophoresis in a 5% non-denaturing polyacrylamide gel with a 0.5X Tris-borate-EDTA buffer (pH 7.4) at 150 V for 4 hours. The gel was dried and subjected to autoradiography. In the competition study, a 50-fold excess of the unlabeled oligonucleotide was incubated with the radiolabeled probe in the reaction mixture.
9. Statistical analyses
The data are expressed as means ± standard error of mean. Statistical analyses were conducted using Student’s t-test and Turkey’s tests for the determination of post hoc differences between group means. Statistical significance was determined at p ＜ 0.05.
Results
1. L-AA induced apoptotic cell death in human laryngeal Hep-2 cancer cells
The effect of L-AA on the viability of Hep-2 cells was evaluated via MTT assay with different concentrations of L-AA. L-AA decreased the viability of Hep-2 cells in a time- and dose-dependent manner. As shown in Fig. 1A, the concentration required for 50–60% inhibition over a period of 24 hours and 48 hours was 5 mM and 1 mM, respectively. The cells undergoing apoptosis showed profound structural changes, including nuclear disintegration and condensation. After a 24-hours treatment with 5 or 10 mM L-AA, the nuclei were stained with DAPI to assess the changes in nuclear morphology. The nuclei of the L-AA-treated cells contained clearly condensed chromatin, showing apoptotic changes (Fig. 1B). In addition, the L-AA-induced apoptosis of epithelial cells was further analyzed for the presence of annexin V-conjugated cells via fluorescence analysis. As shown in Fig. 2, incubation with 5 to 10 mM L-AA induced a dose-dependent increment in the number of annexin V-positive apoptotic cells.
2. L-AA induced the release of cytochrome c from mitochondria into the cytosol and upregulated AIF and EndoG levels
Apoptosis and cell death in most cancer cells are activated by p53 during the initial stages of apoptosis. The Bcl-2 family also plays a major role in apoptosis, controlling the release of mitochondrial cytochrome c into the cytosol [16,17]. The expression of p53 and the Bcl-2 family was analyzed in L-AAinduced apoptosis of Hep-2 cells. As shown in Fig. 3A, after L-AA exposure, the expression of the p53 and Bax proteins was significantly higher, while the expression of the Bcl-2 protein was significantly lower than that of the control group.
The release of cytochrome c, AIF, and EndoG from mitochondria is essential for programmed cell death. To elucidate the mitochondrial role in Hep-2 cells undergoing L-AA-induced apoptosis, the release of cytochrome c, AIF, and EndoG from mitochondria into the cytosol was determined by Western blot. The levels of the mitochondrial and cytosolic fractions isolated from Hep-2 cells exposed to different doses of 5 or 10 mM L-AA for 24 hours were measured. Compared to the control, the contents of cytochrome c, AIF, and EndoG were significantly increased after exposure to L-AA (Fig. 3B).
3. L-AA-induced apoptosis was caspase dependent in Hep-2 cells
Caspases play a crucial role during the apoptosis of various cancer cells. The activities of caspase-8, -9 and -3 were examined in L-AA-treated Hep-2 cells. After exposure to different concentrations of L-AA for 24 hours, the activities of the three caspases were significantly increased (Fig. 4A). To confirm the role of caspases in L-AA-induced cell death, the cells were pretreated with the general caspase inhibitor z-VAD-fmk before exposure to L-AA and cell viability was subsequently measured. Treatment with 50 or 100 mM z-VAD-fmk inhibited the cell death induced by L-AA in Hep-2 cells (Fig. 4B). These results indicated that L-AA-induced apoptosis in Hep-2 cells was mediated via caspase.
4. L-AA-induced apoptosis is mediated via NF-κB, MAPK, and Akt signal pathways
The PI3K/Akt and MAPKs signaling pathways play a crucial role in apoptosis of many cells [14]. The PI3K/Akt and MAPKs signaling pathways were investigated to determine the cellular apoptotic pathways associated with L-AA-induced Hep- 2 cell death. Fig. 5A showed that the phosphorylation of JNK, p38 MAPK and Akt was observed from 30 minutes to 36 hours after treatment with 10 mM L-AA, and peaked at 24 hours after treatment, whereas that of ERK1/2 decreased at 36 hours. Many studies proposed that the regulation of NF-κB activity is important in cancer cell survival [18]. The known target genes of NF-κB include a number of anti-apoptotic genes, such as the Bcl-2 family. L-AA-induced apoptosis of Hep-2 cells inhibited constitutive NF-κB activation. EMSA results showed that L-AA treatment dose-dependently decreased the DNA- binding activity of NF-κB in the Hep-2 cells. In addition, the protein level of the p65 subunit of NF-κB in the nuclear lysates of Hep-2 cells was also decreased by L-AA-treatment (Fig. 5B).
The role of NF-κB, Akt, and MAPK signaling pathways in LAA- induced apoptosis was analyzed by pretreating the cells with 10 mM BAY11-7082 (NF-κB inhibitor), 20 mM LY294002 (PI3K/Akt inhibitor), 10 mM SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) for 1 hour before L-AA addition. After the inhibitor treatment, flow cytometry analysis of the apoptosis of L-AA-induced Hep-2 cells showed a decrease in the level of annexin-V-positive cells compared with the L-AAtreated group (Fig. 5C and 5D). These results strongly indicated that NF-κB, Akt, and MAPK signal pathways were involved in the L-AA-induced apoptosis of Hep-2 cells.
Discussion
Several years of investigation into the effect of L-AA on vital cellular functions and the role of key physiological antioxidants revealed that many signaling intermediates play an anticancer role. However, other studies have demonstrated that L-AA may occasionally act as a pro-oxidant inducing the destruction of tumors. Kang et al. [19] demonstrated that L-AA induced apoptosis of cancer cells by inhibiting the iron uptake. Apoptosis is important in controlling the cell number and proliferation as part of normal development. It is also essential for the elimination of cancer cells [20]. The present study showed that the decreased cell viability following L-AA treatment of Hep- 2 cells was the result of apoptosis induction. Morphological analyses of the L-AA-induced Hep-2 cells revealed apoptotic characteristics, such as the appearance of apoptotic bodies and chromatin condensation, when compared to control cells via DAPI staining. Moreover, flow cytometry with annexin-V staining confirmed that L-AA dose-dependently increased the cellular the ratio of apoptotic death.
The apoptotic process is mainly intrinsic, whereas the extrinsic pathway is mediated by cysteine proteases known as caspases. The extrinsic pathway is triggered by the death receptor, which induces the activation of the initiator caspase-8 and -10, followed by the activation of caspase-3. Active caspase-8 also cleaves cleaves BID to truncated BID (tBID), which induces the release of cytochrome c by the mitochondrial membrane in the intrinsic pathway.
The intrinsic pathway of apoptosis is triggered by cellular stress. The mitochondrial stress is induced by factors such as DNA damage. One of the stress signal regulators of the mitochondria- mediated apoptosis is the Bcl-2 family of proteins. The Bcl-2 family includes pro-apoptotic molecules such as Bax, Bak, and Bad, which are directly involved in pore formation at the mitochondrial membrane. Bax promotes mitochondrial integrity, dissipates mitochondrial membrane potential, and facilitates the release of mitochondrial proteins such as cytochrome c, AIF, and EndoG. Anti-apoptotic molecules such as Bcl-2 and Bcl-xL act as repressors of apoptosis by preventing mitochondrial pore formation and binding with pro-apoptotic protein. In the present study, L-AA treatment not only reduced the expression of Bcl-2, but also increased the expression of Bax. Furthermore, L-AA treatment dose-dependently increased p53 protein in Hep-2 cells. Many studies have shown that p53 regulates the transcription genes that play an important role in cell cycle and apoptosis.
Apoptosis is induced by p53 via several pathways, including the Bcl-2 family. Other reports showed that L-AA induced apoptosis via caspase activation in various cells [21,22]. The present results showed that L-AA exposure dose-dependently activated the expression of caspase-8, -9, and -3. Interestingly, the general caspase inhibitor z-VAD-fmk completely inhibited the L-AA-induced cell death, indicating that L-AA activated the classical caspase-dependent signaling pathway. Thus, we assumed that L-AA regulated the expression of Bcl- 2 family of proteins and increased the release of cytochrome c, AIF, and EndoG, leading to the activation of caspase-9, -8, and -3 and thereby induced apoptosis in Hep-2 cells.
The MAP kinases, including ERK, p38, and JNK, are activated in response to a variety of stimuli and mediate signals important for the generation of biological responses [23]. In general, ERK plays a vital role in cell growth and division, and mediates survival. However, Park et al. [24] reported the roles of ERK and p38 in L-AA-induced apoptosis of acute myeloid leukemia cells. In addition, JNK and p38 MAPKs are activated by diverse stimuli such as oxidative stress and UV [25,26]. However, the functions of these MAPK signals depend on the type of cell system. Many studies have shown that the MAPK signaling pathway plays an important role in the pharmacological action of chemotherapeutic drugs [27,28].
The Akt cellular pathway is particularly relevant to proliferation and cell survival in various cancer cells. Activation of this pathway via phosphorylation and functional inactivation of several pro-apoptotic targets, including the Bcl-2 family member BAD and the protease caspase-9, in vitro prevented apoptosis of malignant cells [29]. In addition, Akt exerts its anti-apoptotic effects by maintaining mitochondrial interactions and preventing both Bax conformational change and its mitochondrial translocation, which appears to be crucial to Akt-mediated inhibition of cytochrome c release and apoptosis [30]. In the present study, L-AA increased the levels of phosphorylated Akt in Hep-2 cells, demonstrating that the downregulation of cytochrome c release may be mediated via Akt signaling. These results suggest that the Akt pathway may be involved in L-AA-induced apoptosis.
NF-κB exists in a latent form in the cytoplasm of unstimulated cells. The NF-κB family in mammals includes p50, p65, c- Rel, p52, and RelB, which constitute the p50/p65 dimer. The transcription factor NF-κB plays a pivotal role in the carcinogenesis of various cancers, serving as one of the key elements in the apoptotic pathway, via regulation of its target gene products including Bcl-2 and Bcl-xl. Thus, it is reasonable to identify NF-κB as a target of L-AA for induction of apoptosis in Hep-2 cells. Our data showed that L-AA treatment significantly downregulated the expression of p65 and inhibited the nuclear translocation of NF-κB, suggesting that NF-κB mediates the induction of apoptosis in Hep-2 cells by L-AA. The activation of ERK1/2, JNK, and p38 MAPKs and of Akt and NF-κB by L-AA in various cancer cells precedes the cleavages of caspase-9 and -3, indicating that the MAPK, Akt and NF-κB signaling pathways are upstream of the caspase cascades, as reported in the human placenta, leukemia, anemia, and carcinoma cell lines [8,31,32]. L-AA-induced activation of MAPK, Akt, and NF-κB was sustained for 24 hours, suggesting that the signaling may have played a role in L-AA-induced apoptosis [6,24,33]. In our experiments investigating the functional role of these cascades, we found that inhibition of this pathway using p38- and JNK-specific inhibitors SB203580 and SP600125, Akt-specific inhibitor LY, and NF-κB specificinhibitor BAY11-7082 suppressed the L-AA dependent induction of apoptosis. Thus, it appears that important signals regulating the L-AA-dependent responses are mediated by MAPK, Akt, and NF-κB. All these events appear to contribute to the induction of apoptosis, and the upstream regulatory mechanisms of such pro-apoptotic signals are analyzed in the present study. Our data clearly establish that L-AA treatment induces phosphorylation of MAPK and Akt and downregulation of NF-κB in Hep-2 cells.
In conclusion, L-AA induced apoptosis via intrinsic and extrinsic pathways by targeting the Bcl-2 family, resulting in the mitochondrial release of cytochrome c, followed by the nuclear translocation of AIF and EndoG in Hep-2 cells. The target signal of L-AA-induced apoptosis may be mediated via MAPK, PI3K, and NF-κB signaling pathways. Our finding on the mechanism of L-AA-induced apoptosis in Hep-2 cells may contribute to the process of developing molecular signaltargeted therapy for laryngeal epithelial cancer Hep-2 cells.
In this study, we explored the anti-tumor activity of L-AA in squamous carcinoma head and neck cancer cells and demonstrated that L-AA efficiently inhibited growth and induced apoptosis of laryngeal Hep-2 cells. These anti-tumor activities of L-AA were mediated via activation of the NF-κB/Akt/MAPK pathway. Thus, we propose L-AA as a promising chemotherapeutic agent for the treatment of human squamous carcinoma head and neck cancer via regulation of the NF-κB/Akt/MAPK signaling pathway.
