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Abstract

Furano-1,2-naphthoquinone (FNQ), a biologically active component of Avicennia marina, has been demonstrated to display anticancer activity. FNQ exerted cytotoxicity with the G₂/M cell cycle arrest and apoptosis in Ca9-22 cells. FNQ-induced G₂/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (CDK) 1 and 2 with concomitant induction of p27. FNQ-induced apoptosis was accompanied by Bax and Bad upregulation, and the downregulation of Bcl-2, Bcl-X_L, Mcl-1, and X-linked inhibitor of apoptosis (XIAP), resulting in cytochrome C release and sequential activation of caspase-9 and caspase-3. Mechanistic studies showed that FNQ suppressed Src phosphorylation, PI3K, and Akt activation in Ca9-22 cells. Moreover, the Src inhibitor PP2 reduced the phosphorylation of Src and activation of PI3K/Akt, which was comparable with FNQ treatment. The combined treatment of FNQ with PP2 enhanced the cell cycle arrest and apoptosis and also led to the downregulation of Bcl-X_L, Mcl-1, XIAP, cyclin A, cyclin B, CDK1, and CDK2 and upregulation of p27, Bax, and Bad. These findings suggest that FNQ-mediated cytotoxicity of Ca9-22 cells is related with the G₂/M cell cycle arrest and apoptosis via inactivation of Src and PI3K/Akt–mediated signaling pathways.

Keywords

apoptosis Akt Ca9-22 cells FNQ PI3K Src

Introduction

The survival rate for patients with squamous cell carcinoma (SCC) of the oral cavity (OSCC) remains low, despite advances in diagnosis and treatment.¹ OSCC usually develops in areas of the epithelium exposed to carcinogens and likely results from accumulation of cellular and genetic alterations, leading to the aberrant expression of many proteins involved in cell growth regulation.^1,2

Many cytotoxic and DNA damaging agents arrest the cell cycle at the G₁, S, or G₂/M phase and induce apoptotic cell death.³ This process is regulated by the coordinated action of cyclin-dependent kinases (CDKs) in association with their specific regulatory cyclin proteins.⁴ Activation of the complex of CDK1 and CDK2 with cyclin A and cyclin B is required for transition from G₂ to the M phase of the cell cycle.⁵ In addition, caspase activation is often regulated by various cellular proteins including members of the inhibitor of apoptosis (IAP) or Bcl-2 families.⁶ Previous reports have demonstrated that some Bcl-2 family members (such as Bax, Bcl-X_L, Mcl-1, Bcl-2, and Bad), located on the mitochondrial membrane, can alter the permeability of the mitochondrial membrane and trigger the activation of caspases, leading to apoptotic cell death.⁷

The c-Src nonreceptor tyrosine kinase is overexpressed and activated in OSCC and has been implicated in the development, growth, progression, and metastasis.⁸ Src mediates its biological effect through upregulation of the antiapoptotic genes such as Bcl-X_L, Mcl-1, and downstream signaling cascades, including the phosphatidylinositol-3-kinase (PI3K) and Akt proteins.⁹ PI3K is a dimeric enzyme composed of an inhibitory/regulatory (p85) subunit and a catalytic (p110) subunit. The p85 subunit anchors to ErbB receptor docking sites, and the p110 subunit is responsible for the phosphorylation and activation of the protein serine/threonine kinase Akt,¹⁰ which plays a major role in cell survival, apoptosis, and oncogenesis of tumors.^11,12

Previous studies have shown that 1,2- and 1,4-naphthoquinones fused with furan or pyran ring are important groups for cytotoxicity to cancer cell lines, with 1,2-naphthoquinones having better activity.^13-16 Naphtho[1,2-b]furan-4,5-dione (FNQ; Figure 1A), a 1, 2-furanonaphthoquinone, was originally isolated from Avicennia marina belonging to the family Avicennaceae,¹⁷ and can be synthesized by a chemical process.¹⁸ FNQ was found to show potent cytotoxicity against KB (human epidermoid carcinoma), HeLa (human cervical carcinoma), and HepG2 (human hepatocellular carcinoma) cell lines.¹⁵ Nevertheless, the cellular signaling pathways involved in these effects of FNQ remain unknown. Oral cancer is one of the most common malignant and devastating of human tumors. Ca9-22 cells were more sensitive to FNQ treatment than 2 oral cancer cell lines (CAL27 and SAS). The aim of this study is to evaluate effects of FNQ on the induction of apoptosis and cell cycle arrest and to examine the molecular mechanisms involved, especially activation of Src and PI3K/Akt-mediated downstream signaling in Ca9-22 cells.

Figure 1.

Effect of FNQ on the cell viability of Ca9-22 cells. (A) Chemical structure of FNQ. (B) Effect of FNQ on the viability of HGF, SAS, CAL27, and Ca9-22 cells. Cells were incubated with the indicated concentrations of FNQ for 24 hours. (C) Effect of FNQ on the viability of Ca9-22 cells. Cells were incubated with 0, 1, 2, and 4 µM of FNQ for 6, 12, 24, and 48 hours. Cell viability assays were performed using the MTT assay as described in materials and methods. The graph displays the mean ± standard deviation of 3 independent experiments.

Materials and Methods

Chemicals

Dulbecco’s modified eagle medium: nutrient mixture F-12 (DMEM/F12) medium, fetal bovine serum (FBS), trypan blue, penicillin G, and streptomycin were obtained from Gibco BRL (Gaithersburg, MD). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), ribonuclease (RNase), rhodamine123, and propidium iodide (PI) were purchased from Sigma Chemical (St Louis, MD). The following antibodies were used for Western blotting: anti-Akt, Src, cytochrome C, XIAP, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Bax, Bcl-2, Bcl-X_L, Mcl-1, cyclin A, cyclin B, CDK1, CDK2, p21, and p27 antibodies (BD Biosciences, Franklin Lakes, NJ); anti-phospho-Akt, PI3K (p110), and PI3K (p85) antibodies (Upstate, Temecula, CA); and anti-phospho-Src antibody (Cell Signaling Technology, Beverly, MA). The colorigenic synthetic peptide substrates, Ac-DEVD-pNA and Ac-LEHD-pNA, were purchased from BioVision (Mountain View, CA). The Src-specific inhibitor PP2 and PI3K inhibitor Wortmannin were purchased from Calbiochem (San Diego, CA). Annexin V and PI double staining kits were a product of Upstate. Antimouse and antirabbit IgG peroxidase-conjugated secondary antibodies were purchased from Pierce (Rockford, IL). Hybond ECL transfer membrane and ECL Western blotting detection kit were obtained from Amersham Life Sciences (Buckinghamshire, UK).

Cell Culture

Human OSCC cell lines, Ca9-22, SAS, and CAL27, were obtained from the Health Science Research Resources Bank (Osaka, Japan). Human gingival fibroblasts (HGF) were kindly provided by Dr Chien-Chih Chiu, Department of Biotechnology of Kaohsiung Medical University. Cells were maintained in DMEM/F12 medium supplemented with 10% FBS, 2 mM glutamine, and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin) at 37°C in a humidified atmosphere of 5% CO₂.

Preparation of FNQ Solution

FNQ was synthesized from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient 1-pot reaction, according to a previously reported procedure.¹⁸ FNQ dissolved in DMSO was administered to cells, with a final concentration of DMSO at 0.1% in the medium.

Cell Viability Assay

The viability of cells was determined by the MTT assay, and the trypan blue dye exclusion assay was performed to confirm and verify cell viability. Cells were seeded at a density of 1 × 10⁵ cells/3 mL cell culture medium into 12-well plates. After 24 hours of incubation, the cells were treated with vehicle (DMSO, 0.1% in media) or the increasing concentrations of FNQ in medium for the indicated times. Then, the MTT solution (1.2 mg/mL) was added to each well and cells were incubated for a further 4 hours. MTT is reduced by the mitochondrial dehydrogenases of viable cells to a purple formazan product. The MTT-formazan product dissolved in DMSO was estimated by measuring absorbance at 570 nm in an ELISA plate reader. For the trypan blue dye exclusion assay, cells were seeded at a density of 1 × 10⁵ cells/well in 12-well plates for 24 hours; then FNQ was added to the medium at various indicated concentrations and times. After incubation, cells were exposed to 0.2% trypan blue and were then counted in a hemocytometer.

Flow Cytometry Analysis

Controlled and treated cells were harvested, washed in cold phosphate-buffered saline (PBS), fixed in 70% ethanol, and stored at 4°C. DNA was treated with RNase A solution (500 units/mL) at 37°C for 15 minutes and stained by PI (50 µg/mL) in 1.12% sodium citrate at room temperature before analysis. Flow cytometric determination of DNA content was done by Coulter EPICS XL Flow Cytometer (Coulter Corp, Miami, FL). The fractions of the cells in G₀/G₁, S, and G₂/M phases were analyzed using Multicycle (Phoenix Flow System, San Diego, CA) cell cycle analysis software.

Assessment of Apoptosis

The accumulation of sub-G1 population in Ca9-22 cells was determined by flow cytometry. Cells were seeded onto 6-cm dishes and treated with or without the indicated FNQ for 24 hours. Cells were then washed twice with ice-cold PBS and collected by centrifugation at 200g for 5 minutes at 4°C. Cells were fixed in 70% (vol/vol) ethanol at 4°C for 30 minutes. After fixation, cells were treated with 0.2 mL of DNA extraction buffer (0.2 M Na₂HPO₄ and 0.1 M citric acid buffer, pH 7.8) for 30 minutes, centrifuged, and resuspended in 1 mL of PI staining buffer (0.1% Triton X-100, 100 µg/mL RNase A, 500 µg/mL PI in PBS) at 37°C for 30 minutes. Cytometric analyses were performed using a flow cytometer (FACS Calibur, BD Biosciences) and CellQuest software. Approximately 10 000 cells were counted for each determination.

Apoptotic cells were quantified by annexin V and PI double staining by using a staining kit purchased from Upstate (Temecula, CA). In brief, 10⁶ cells were grown in 35-mm diameter plates and were labeled with annexin V-flourescein isothiocyanate (FITC; 10 µg/mL) and PI (20 µg/mL) before harvesting. After labeling, all plates were washed with a binding buffer and harvested by scraping. Cells were resuspended in the binding buffer in a concentration of 2 × 10⁵ cells/mL before analysis by flow cytometry.

Protein Extraction and Western Blotting

Cells were harvested and extracted with lysis buffer (50 mM Tris–HCl, pH 7.5, 137 mM sodium chloride, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 0.1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, and 2 µg/mL aprotinin). The lysates were centrifuged at 20 000g for 30 minutes, and the protein concentration in the supernatant was determined using a bicinchoninic acid protein assay kit (Pierce). Equal amounts of protein were separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and then electrotransferred to a polyvinylidene difluoride membrane. Membranes were blocked with a solution containing 5% nonfat dry milk Tris-buffered saline Tween-20 (TBST) buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) for 1 hour and then washed with TBST buffer. Cells were then indicated with the primary antibodies, washed, and monitored by immunoblotting with specific antibodies. The blot was developed by enhanced chemiluminescence.

Preparation of Cytosolic Fraction for Assessment of Cytochrome C

The mitochondrial and cytosolic fractions were prepared by resuspending cells in ice-cold buffer A (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 17 µg/mL phenylmethylsulfonylfluoride, 8 µg/mL aprotinin, and 2 µg/mL leupeptin, pH 7.4). The cells were broken by passing 10 times through a 22-gauge needle. Unlysed cells and nuclei were pelleted by centrifugation for 10 minutes at 750g. The supernatant was then centrifuged at 100 000g for 15 minutes. This pellet, representing the mitochondrial fraction, was resuspended in buffer A. The supernatant was again centrifuged at 100 000g for 1 hour. The supernatant from this final centrifugation step represents the cytosolic fraction.

Flow Cytometric Detection of ΔΨm

To assess changes in mitochondrial membrane potential (ΔΨm), FNQ-treated cells were incubated with 40 nM rhodamine 123 for 15 minutes at 37°C. The rhodamine 123 data were recorded using an FL1 photomultiplier. Sample data (10 000 cells) were used to prepare histograms with the CellQuest data analysis program (BD Biosciences). Dead cells and debris were excluded from the analysis by electronic grating of forward-scatter and side-scatter measurements.

Assays of Caspase Activity

Cells (1 × 10⁶/dish) were seeded in 10-cm dishes. After 24 hours of incubation, cells were treated with 1, 2, and 4 µM of FNQ for 24 hours, respectively. After treatment, cells were collected and washed 3 times with PBS and resuspended in 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, and 10 mM EGTA. Cell lysates were clarified by centrifugation at 18 000g for 3 minutes, and clear lysates containing 50 µg of protein were incubated with 100 µM of enzyme-specific colorigenic substrates at 37°C for 2 hours. The alternative activities of caspase-3 and caspase-9 were determined as the cleavage of the colorimetric substrate by measuring the absorbance at 405 nm.

Statistical Analysis

The values given are means ± standard deviation. Statistical analysis between 2 samples was performed using Student’s t test. Statistical comparisons of more than 2 groups were performed using 1-way analysis of variance with Bonferroni’s post hoc test. In all cases, P < .05 was considered as significant.

Results

Effect of FNQ on the Cell-Cycle Arrest and Apoptosis

To verify the effects of FNQ on cell viability, CAL27, SAS, and Ca9-22 cells were treated with increasing concentrations of FNQ for 24 hours, and cell viability was assessed using the MTT assay. FNQ treatment caused cell mortality of CAL27, SAS, and Ca9-22 cells with an estimated 50% cell death (IC₅₀) values of 4.4, 3.2, and 2.2 µM, respectively. Ca9-22 cells were the most sensitive to FNQ treatment (Figure 1B). Meanwhile, a parallel treatment of FNQ to noncancerous cells, HGF, revealed these cells to be markedly less sensitive to the effects of FNQ than Ca9-22 cells (Figure 1B). Therefore, Ca9-22 cells were selected for the following experiments. As shown in Figure 1C, FNQ resulted in time-dependent and dose-dependent decreases in cell number relative to control cultures. In Ca9-22 cells, till 6 hour exposure of FNQ, no obvious effect could be found regardless of the dose of FNQ. However, a trend of decrease of cell viability as the dose of FNQ increased was observed after 12 hour exposure, a significant decrease in cell viability was observed in a dose-dependent manner following 24 hour exposure (Figure 1C). Therefore, 1, 2, and 4 µM FNQ at 24 hours were chosen for further experiments to investigate the mechanism related to the FNQ induced cytotoxicity.

To determine whether the cytotoxicity of FNQ was attributed to cell cycle arrest or apoptosis in Ca9-22 cells, the effects of FNQ treatment on cell cycle arrest and apoptosis were examined. As shown in Figure 2A, treatment of cells with 0, 1, 2, and 4 µM of FNQ for 18 hours resulted in G₂/M phase accumulation of cells corresponding to 18.9%, 34.1%, 31.6%, and 26.0%, respectively. FNQ treatment induced a significant accumulation of cells in the G₂/M phase with concomitant decreases of cells at the G₀/G₁and S phases.

Figure 2.

(A) Effect of FNQ on cell cycle arrest and apoptosis in Ca9-22 cells. Cells were treated with 0, 1, 2, and 4 µM of FNQ or treatment with PP2 (a Src-specific inhibitor; 10 µM) for 24 hours, and then the cells were washed, fixed and stained with PI, and analyzed for DNA content by flow cytometry as described in “Materials and Methods.” G0/G1, S, and G2/M indicate cell phase, and sub-G1 DNA content refers to apoptotic cells. Data shown are representative of 3 independent experiments. *P < .05, compared with control population. (B) Flow cytometric analysis of phosphatidylserine externalization (Annexin V binding) and cell membrane integrity (PI staining). Cells also were treated with 0, 1, 2, and 4 µM of FNQ or with PP2 for 24 hours, and then analyzed for apoptosis by flow cytometry for Annexin V and PI staining. Data shown are representative of 3 independent experiments. *P < .05, compared with control Annexin V positive population; *P < .05, compared with both Annexin V and PI positive population.

To examine whether FNQ decreases cell viability by inducing apoptosis, a proportion of sub-G₁ DNA was determined using flow cytometry. FNQ treatment of Ca9-22 cells resulted in a dose-dependent increase in the accumulation of cells in the sub-G₁ phase (Figure 2A). Phos-phatidylserine translocation to the cell surface is an indicator of apoptotic cells. To confirm whether the growth inhibition of FNQ was caused by apoptosis in vitro, Annexin V-FITC/PI double staining assay was used. In Figure 2B, the percentage of Annexin V–positive cells, indicative of early apoptosis, increased from 0.6% in the control cells to 0.9%, 19.1%, and 30.1% in cells treated with 1, 2, and 4 µM of FNQ for 18 hours, respectively. Moreover, FNQ treatment increased the percentage of cells positive for both Annexin V and PI (hallmarks of late apoptosis) from 2.9% in the controls to 3.5%, 15.6%, and 34.2%.¹⁹ These results suggest that FNQ inhibited the cell viability of Ca9-22 cells through the G₂/M phase arrest and apoptosis.

Effect of FNQ on the Expression of Cell Cycle-Regulating Proteins

The cell cycle is tightly regulated through a complex network of positive and negative regulatory molecules, including cyclin dependent kinase (CDKs), cyclin and Cdk inhibitor (CDKI). To elucidate the role of the cell cycle–regulating proteins in the G₂/M cell cycle arrest induced by FNQ, the protein extracts of FNQ-treated cells were prepared. As shown in Figure 3, FNQ treatment dose-dependently caused a marked decrease in cyclin A, cyclin B, CDK1, and CDK2 expression. In addition, the expression level of p27 increased in FNQ-treated cells in comparison with the controls. These results suggested that FNQ induced G₂/M arrest, possibly by altering the G₂/M cell cycle-related proteins in Ca9-22 cells.

Figure 3.

Western blot analysis of FNQ and PP2 (a Src-specific inhibitor) on the expression levels of Bcl-2 family proteins and XIAP. Cells were treated with 0, 1, 2, and 4 µM of FNQ or with 10 µM PP2 for 24 hours. Whole cell extracts were prepared and 60 µg proteins of these extracts were resolved by SDS-PAGE, and then probed with specific antibodies. The amount of β-actin was measured as an internal control. Densitometry shows data from 3 experiments. *P < .05 versus control.

Effect of FNQ on the Expression Levels of Bcl-2 and IAPs Family Proteins and Apoptosis by Mitochondrial Pathway

Figure 4 showed that FNQ treatment resulted in downregulation of Bcl-2, Bcl-X_L, and Mcl-1, while FNQ notably upregulated Bax and Bad expression. In addition to Bcl-2 family proteins, the IAP family proteins regulate apoptotic signaling cascades by blocking caspase activities.²⁰ FNQ treatment resulted in reduced protein expression of XIAP (Figure 4).

Figure 4.

Accumulating evidence suggests that mitochondria play a critical role in apoptosis by modulating Bcl-2 family members, such as Bcl-2 and Bax, and releasing apoptogenic factors, such as cytochrome C.²¹ Consistent with these results, cytochrome C release was noted after FNQ treatment (Figure 5A). Since cytochrome C release is linked to the loss of ΔΨm, the effect FNQ on ΔΨm was examined using the mitochondria-specific dye rhodamine 123. As indicated in Figure 5B, compared with untreated control cells (9.8%), approximately 68.2% of cells shifted toward the left following treatment with 4 µM FNQ, thereby indicating that the FNQ treatment induced a dissipation of ΔΨm. To verify caspase-3 and caspase-9 activity, we performed an in vitro assay based on the proteolytic cleavage of the chromophore p-nitroanilide (pNA) from the specific colorigenic peptide substrates (Ac-DEVD-pNA and Ac-LEHD-pNA for caspase-3 and caspase-9, respectively). A marked increase in the activity of both caspase-3 and caspase-9 was observed in FNQ-treated cells (Figure 5C). These results suggest that FNQ-induced apoptosis is mediated through a mitochondria-dependent pathway.

Figure 5.

Effects of FNQ on cytochrome C release (A), mitochondrial membrane potential (ΔΨm; B), and caspase activation (C). (A) Accumulation of cytosolic cytochrome C. Cells were treated with 0, 1, 2, and 4 µM of FNQ for 24 hours. For cytochrome C analysis, the cytosolic fractions were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane. Western blot analysis was performed using a cytochrome C antibody. Densitometry shows data from 3 experiments. *P < .05 versus control. (B) Effects of FNQ on mitochondrial membrane potential. Cells were exposed to 4 µM FNQ for 24 hours, then stained with rhodamine 123 and analyzed immediately by flow cytometry as described in “Materials and Methods.” The number indicates the percentage of cells with a reduced ΔΨm level. (C) Activation of caspase-3 and caspase-9 by FNQ. cells were treated with 0, 1, 2, and 4 µM of FNQ for 24 hours. Cell lysates were prepared and the enzymatic activities of the caspase-3 and caspase-9 like protease were determined by incubation of 50 µg/mL of total protein with colorigenic substrates for 2 hours at 37°C. Release of chromophore pNA was monitored spectrophotometrically. Data are presented as mean and standard deviation with n = 3. *P <.05 versus control.

Effect of FNQ on Phosphorylation of Src

FNQ significantly inhibited phosphorylation of Src in Ca9-22 cells in a dose-dependent and time-dependent manner (Figure 6A and B). The results clearly indicated that FNQ had a potentially inhibitory effect on Src phosphorylation without altering the total Src protein level. Moreover, PP2, a specific pharmacological inhibitor of Src,²² was used as a positive control; a decrease in Src phosphorylation following treatment with PP2 was also found (Figure 6B).

Figure 6.

(A) Effect of FNQ on inhibition of the phospho (p)-Src, PI3K (p110 and p85), and phospho (p)-Akt in a time-dependent manner. (B) Effect of FNQ, PP2, or wortmannin (Wort) on inhibition of the phospho (p)-Src, PI3K (p110 and p85), and phospho (p)-Akt in a dose-dependent manner. Cells were treated with 0, 1, 2, and 4 µM of FNQ or with PP2 (10 µM) and wortmannin (10 µM) for 24 hours, after which whole cell extracts were prepared, and 60 µg proteins of these extracts were resolved by SDS-PAGE, and then probed with specific antibodies. The amount of β-actin was measured as an internal control. Densitometry shows data from 3 experiments. *P < .05 versus control.

Effect of FNQ on Activation of PI3K/Akt, Downstream Targets of Src Signal Pathway

The PI3K/Akt pathway promotes cell survival and is a well-established downstream effector of Src signaling.⁹ FNQ treatment resulted in decreased expression of the p110 and p85 subunits of PI3K and p-Akt in a time-dependent (Figure 6A) and dose-dependent manner (Figure 6B). It was next determined whether FNQ-induced phosphorylation/activation of cellular signal transducing elements occurs downstream of the events followed by Src activation. PP2 reduced the phosphorylation of Src and activation of PI3K/Akt, which was comparable with FNQ treatment. However, PI3K inhibitor wortmannin²³ abolished PI3K and Akt activation, but wortmannin did not affect Src activation (Figure 6B). These results show that FNQ inhibits Src kinase activity and downstream PI3K/Akt signaling in Ca9-22 cells.

Effect of Blockade of Src Activation on G₂/M Phase Arrest and Apoptosis in Ca9-22 Cells

To further demonstrate that FNQ-induced apoptosis and G₂/M cell cycle arrest is mediated through the Src signaling pathway, cells were treated with Src inhibitor PP2 (10 µM) or in combination with FNQ for 18 hours. As shown in Figure 2A, inhibition of Src activation by PP2, similar to the effects of FNQ, also led to apoptosis and G₂/M arrest. Cotreatment of FNQ with PP2 significantly increased apoptosis and G₂/M arrest in Ca9-22 cells. The effect of FNQ on reducing the protein levels of cyclin A, cyclin B, CDK1, and CDK2 and increasing the protein level of p27 in Ca9-22 cells was also enhanced with PP2 treatment (Figure 3). These results suggest the Src signaling pathway was involved in FNQ-induced G₂/M cell cycle arrest.

To assess whether FNQ-mediated upregulation of Bax and Bad and downregulation of Bcl-2, Bcl-2, Mcl-1, and XIAP in Ca9-22 cells occur by inhibition of Src-mediated pathway, PP2 was used. The results showed that a single treatment with PP2 led to upregulation of the expression levels of Bax and Bad and downregulation of the levels of Bcl-2, Bcl-X_L, Mcl-1, and XIAP, in a way similar to that of FNQ (Figure 4). In addition, a combined treatment of PP2 with FNQ could further enhance the above effects. These data again support that the cytotoxic effects of FNQ are mediated through the Src-dependent signaling pathway.

Discussion

The goal of this study was to determine whether FNQ exerted its anticancer effects through the abrogation of the Src signaling pathway in Ca9-22 cells. We found that FNQ suppressed Src activation in parallel with the inhibition of PI3K/Akt activation. FNQ also significantly upregulated the expression levels of Bax, Bad, and p27 and downregulated Mcl-1, Bcl-X_L, Bcl-2, XIAP, cyclin A, cyclin B, CDK1, and CDK2 expression in a dose-dependent manner.

A number of CDKs regulate the cell cycle event in mammalian cells.²⁴ Among the CDKs that regulate cell cycle progression, CDK1 and CDK2 are activated primarily in association with cyclins A and B in the G₂/M phase progression.²⁴ In this study, cell cycle analysis revealed a prominent G₂/M arrest of Ca9-22 cells on FNQ treatment (Figure 2A), and CDK1, CDK2, cyclin A, and cyclin B proteins were decreased by FNQ treatment in a dose-dependent manner (Figure 3). These data suggest that cell cycle arrest is mediated by limiting the supply of CDK1, CDK2, cyclin A, and cyclin B, which are essential executors in regulating passage into mitosis. Moreover, the tumor suppressor genes p53, p21, and p27 play critical roles in regulating cell cycle.²⁵ Our results indicate that FNQ induced cell cycle arrest in Ca9-22 cells with a concomitant increase in the level of p27 (Figure 3). Thus, the G₂/M-phase cell cycle arrest in FNQ-treated cells seems to be mediated by downregulation of CDK1, CDK2, cyclin A, and cyclin B in association with induction of p27.

In addition, the results of the present study indicate that FNQ treatment induces apoptotic death in Ca9-22 cells by the intrinsic pathway involving the modulation of Bcl-2 family proteins, cytochrome C release, and then activation of caspase-9 and caspase-3. In most cancer cells, apoptosis depends on the mitochondrial “intrinsic” pathway, in which both caspase activation and activity are tightly controlled.⁶ This process is regulated by pro- and anti-apoptotic proteins, such as members of the Bcl-2 family, regulating the mitochondrial pathway prior to caspase activation. For example, caspase-9 activation is inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-X_L and facilitated by pro-apoptotic Bcl-2 family proteins such as Bax and Bad.⁶ This agrees with the finding of a previous study showing a concomitant increase in Bax and Bad expression and a decrease in Bcl-2 and Bcl-X_L expression following exposing breast cancer cells to dehydrocostuslactone.²⁶ In various cancer cells, AG1478 and gefitinib (Iressa, an epidermal growth factor receptor tyrosine kinase inhibitor) have been shown to induce the expression of pro-apoptotic Bcl-2 family members such as Bax and Bad and to inhibit anti-apoptotic members such as Bcl-2 and Bcl-X_L.²⁷ Recent studies suggest that Mcl-1 cooperates with Bcl-X_L to tether the proapoptotic proteins such as Bax, a multidomain and BH-3-exclusive domain protein, and further prevent their activation.²⁸ IAPs have been reported to inhibit apoptosis because they functionally inhibit activation of caspases such as caspase-3 and caspase-7. IAPs are also able to abrogate cytochrome C–induced activation of caspase-9.²⁰ Our data reveal that the protein expression of XIAP was decreased by FNQ treatment (Figure 4). The present findings that upregulation of Bax and Bad and downregulation of Bcl-2, Bcl-X_L, and Mcl-1 were observed with FNQ-treated cells are consistent with these suggestions. Finally, the disruption of mitochondrial membrane potential, release of cytochrome C, and then activation of caspase-9 and caspase-3 were detected in FNQ-treated cells.

Src is expressed at high levels in most cancers and is as-sociated with lower rates of survival.⁸ The data presented here reveal that FNQ treatment resulted in a dose-dependent and time-dependent decrease in the level of p-Src in Ca9-22 cells (Figure 6A and B). Decreased protein expression of p-Src following treatment with the Src inhibitor PP2 was also detected. However, the total Src protein levels did not change in cells treated with FNQ or PP2. This indicates that FNQ suppresses the activation of Src. The signaling pathways induced by activated Src include the PI3K/Akt, which play a significant role in mitogenesis and cell survival.⁹ Akt/protein kinase B (PKB) belongs to the downstream molecules of PI3K and previous studies have demonstrated that the protein kinase Akt plays a major role in the survival of cells under various conditions.²⁹ FNQ was found to induce inactivation of PI3K/Akt in a time-dependent and dose-dependent manner (Figure 6A and B). Since the Src and PI3K/Akt signaling pathways are involved in FNQ-induced cell death, the possible connections between these pathways were examined. Inhibition of Src with PP2 affected Src and PI3K/Akt activation as assessed by phosphorylation. In contrast, PI3K inhibitor wortmannin did not affect Src activation (Figure 6A and B). This indicates that Src is an upstream activator of PI3K/Akt and the results reflect that the major pathway of FNQ-mediated signals is initiated by abolishing Src activation.

Additionally, recent studies suggest that Src also regulates the cell cycle and apoptosis.³⁰ To address the importance of Src in the process, the combined treatment of FNQ with PP2 also led to upregulation of Bad and Bax and downregulation of Bcl-2, Bcl-X_L, Mcl-1, and XIAP in Ca9-22 cells (Figures 4 and 5A), which was comparable with FNQ treatment. Consistent with these results, release of cytochrome C and then activation of caspase-9 and caspase-3 were detected in FNQ-treated cells (Figure 5C). Perturbing cell cycle progression by alterations in cell cycle–related protein expression plays a vital role in the proliferation of cancer cells. As seen in Figure 3, cotreatment of FNQ with PP2 further reducing the protein levels of cyclin A, cyclin B, CDK1, and CDK2 and increasing the level of p27 was similar with those of FNQ treatment. Collectively, these data indicate that the cytotoxic effect of FNQ is closely mediated through the Src pathway.

Conclusion

The growth inhibition of FNQ is highly related to G₂/M phase cell cycle arrest and apoptosis in Ca9-22 cells. FNQ-induced G₂/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B and their activating partners CDK 1and 2 with concomitant induction of p27. FNQ-induced apoptosis was accompanied by Bax and Bad upregulation and the downregulation of Bcl-2, Bcl-X_L, Mcl-1, and XIAP, resulting in cytochrome C release and sequential activation of caspase-9 and caspase-3. Moreover, the modulation of Src and PI3K/Akt–mediated signaling pathway is involved in FNQ-induced G₂/M cell cycle arrest and apoptosis in Ca9-22 cells.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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