EGFR family

Normal cell growth is tightly regulated through activation of cellular signal transduction pathways. Growth factors and their receptors play a fundamental role in the communication between outside the cell surface and the inside compartments. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, also known as the HER or ErbB family, include four known members: EGFR (HER1/

ErbB1), HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4 [4]. The EGFR is composed of an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain [5]. The ErbB tyrosine kinases and the EGF-like peptides form a complex system [6]. Binding with ligands, such as epidermal growth factor (EGF) or transforming growth factor (TGF)-α, EGFR will be activated by dimerization and autophosphorylation of tyrosine kinase domain with subsequent initiation of downstream signaling molecules [7]. EGFR activates two major downstream intracellular signaling pathways: (1) Ras-Raf-mitogen-activated protein kinase kinase (MEK)-mitogen-activated protein kinase (MAPK) and (2) the phosphoinositide 3-kinase (PI3K)-Akt/protein 3-kinase B-mammalian target of rapamycin (mTOR) cascades [8; 9]. The Ras-Raf-MEK-MAPK pathway modulates several cellular processes including gene transcription, G1/S cell-cycle progression, and cellular proliferation. The PI3K pathway regulates anti-apoptotic and prosurvival signal cascades [10].

1.3 EGFR targeting cancer therapies

Tumor cells are able to synthesize and to respond to a number of different peptide growth factors [11]. Dysregulation of EGFR pathways by overexpression or constitutive activation can promote tumor processes including angiogenesis and metastasis [12].

This is associated with poor prognosis in many human malignancies [13]. Two major classes of EGFR-targeted therapies have been developed: the anti-ErbB monoclonal antibodies (MAb) and ErbB-specific tyrosine kinase inhibitors (TKIs) [14]. Monoclonal antibodies and TKIs clearly differ in their mode of action at target level. Anti-EGFR MAb such as cetuximab and panitumumab bind to the extracellular domain of EGFR on the surface of tumor cells, thus preventing EGFR ligands from interacting and activating the receptor, as well as receptor-ligand internalization [15]. By contrast, TKIs such as gefitinib and erlotinib block the binding of adenosine triphosphate to the intracellular

signaling [16].

1.4 Gefitinib

A quinazoline-derived agents that are developing by specific ATP competitors of EGFR tyrosine kinase, one representative of which is gefitinib [17]. Gefitinib was the first commercially available EGFR TKI. It has now been approved for the treatment of advanced colorectal cancer, squamous cell carcinoma of the head and neck, advanced non-small-cell lung cancer, as well as pancreatic and breast cancer [18]. The recommended dose for use was established at 250 mg/day [19]. Gefitinib shows antiproliferative activity in various human cancer cell types. It inhibits the production of pro-angiogenic factors and induces apoptosis in numerous in vitro and xenograft models [20]. This sensitivity to gefitinib is associated with dependence on both Akt and ERK1⁄

2 pathways [21; 22]. Moreover, tumor growth inhibition by gefitinib is potentiated by combination with a variety of cytotoxic anticancer agents [23].

Anti-EGFR therapy has shown significant efficacy in some cancers; however, no therapeutic response was seen in the majority of cancer patients [24]. In addition, patients initially responsive to anti-EGFR therapy develop resistance over time of treatment. Potential mechanisms of resistance to EGFR-targeted therapy might rely on activation of alternative RTKs which bypass the EGFR pathway or constitutive activation of signaling pathways downstream EGFR, as well as EGFR gene amplification and receptor mutations [25].

1.5 Apoptosis

The maintenance of cellular homeostasis is fundamental for tissue integrity in multicellular organisms. Programmed cell death (apoptosis) is a highly conserved mechanism that has evolved to maintain cell numbers and cellular positioning within tissues comprised of different cell compartments [26]. It is activated in response to diverse signals during normal tissue development, homeostasis, and disease pathogenesis [27; 28]. Apoptosis was first described in 1972 by Currie and colleagues [29]. Mammals have two distinct, but ultimately converging, apoptosis signaling

pathways: the extrinsic (also called ‘death receptor’) pathway, which is activated by death receptors; and the intrinsic (also called ‘mitochondrial’ or ‘Bcl-2-regulated’) pathway [30]. Apoptosis requires controlled degradation of cellular macromolecules by hydrolytic enzymes, and therefore proteases play a central role in this process [31; 32;

33; 34]. These proteases were collectively referred to as caspases (cysteine aspartic acid specific proteases) [35]. Since activated caspases are demonstrated in apoptotic cells, these proteases could presumably cleave their target proteins anywhere in the cell and at different times to complete the execution phase of apoptosis [36]. The activation of caspase-3 is a final parameter of caspase family on apoptosis. Poly(ADP-ribose) polymerase (PARP), a nuclear protein implicated in DNA repair, is one of the earliest proteins targeted for a specific cleavage to the signature 89-kDa fragment during apoptosis [37]. PARP is one of the prime target proteins for caspase-3 [38]. Failure of this apoptosis regulation results in pathological conditions such as developmental defects, autoimmune diseases, neurodegeneration or cancer [39]. On the other hand, apoptosis-regulating proteins also provide targets for drug discovery and new approaches to the treatment of cancer [40].

Mitochondria functions can control cellular life and death [41]. Mitochondria play important roles in cell death through the release of pro-apoptotic factors such as cytochrome c and apoptosis-inducing factor (AIF), which activate caspase-dependent and caspase-independent cell death [42]. On apoptosis, cytochrome c and AIF release to the cytosol, mitochondrial fragmentation as well as mitochondrial hyperpolarisation followed by an oxidative burst, and breakdown of mitochondrial membrane potential [43].

1.6 Securin and cancer

Pituitary tumor-transforming gene (PTTG) was isolated from rat pituitary tumor cells in 1997 [44]. It consists of a homologous family of proteins expressed in different species that includes Cut2 in fission yeast, Pds1 in budding yeast, Pimples in Drosophila, and securin in human [45; 46; 47; 48]. Securin acts as an anaphase inhibitory protein that plays an important role in preventing abnormal chromosome segregation during mitosis [47]. The activity of separase is inhibited by securin in

metaphase [49]. At the metaphase-anaphase transition, securin is degraded with subsequent release of separase to mediate the separation of sister chromatids by cleavage of the chromosomal cohesin [50]. In normal condition, securin also maintains genomic stability [51]. Recently, it has been shown that securin regulates DNA repair following UV and X-ray damages [52].

Securin overexpression has been reported in a variety of endocrine-related tumors, especially pituitary, thyroid, breast, ovarian, and uterine tumors, as well as nonendocrine-related cancers involving the central nervous system, pulmonary system, and gastrointestinal system [53; 54; 55; 56; 57]. It has been shown that securin can promote the cell proliferation and tumorigenesis [58; 59]. Securin levels correlate with tumor invasiveness, and it has been identified as a key signature gene associated with tumor metastasis [60].

1.7 ATF3 and cancer

Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP response element-binding (CREB) family of transcription factors [61; 62]. The common feature that these proteins share is the basic-region leucine zipper (bZIP) element [63].

This domain is responsible for specific DNA binding, while the leucine zipper region is responsible for forming homodimers or heterodimers with other bZIP-containing proteins [64]. ATF/CREB proteins were initially identified for their binding to the cyclic AMP response element (CRE) in various promoters, which has the consensus sequence TGACGTCA [65].

ATF3 is an adaptive-response gene that participates in cellular processes to adapt to extra- and/or intracellular changes, where it transduces signals from various receptors to activate or repress gene expression [66; 67]. Emerging evidence suggests that ATF3 may play another critical function in host defence by regulating the delicate balance between proliferative and apoptotic signals that contribute to the development of cancer [68]. Interestingly, ATF3 has been demonstrated to play differing roles in cancer development depending on the cell type and context [69]. There are many studies that support an oncogenic role of ATF3 [70]. ATF3 expression contributes to the successful propagation of human cancer [71]. In contrast to the studies above, some evidence

suggests that ATF3 may be able to inhibit tumourigenesis [72]. The loss of ATF3 function results in loss of tumour suppression [73].

1.8 The purpose of the study

The induction of apoptosis has been considered to be the major mechanism for these gefitinib mediated anticancer effects. However, the precise downstream signaling molecules of EGFR-independent have not yet been elucidated. In this study, we investigated the effects of gefitinib on the EGFR-independent cell death signaling pathways in human cancer cells. We also studied the role and regulation between securin and ATF3 on the cell viability following gefitinib treatment.

2. Materials and methods

2.1 Chemicals and reagents

Gefitinib was purchased from proteinkinase.de (Biaffin GmbH & Co KG, Kassel, Germany) and dissolved in DMSO. Gefitinib analogues were kindly provided by Dr. C.

Chen (National Dong-Hwa University, Hualien, Taiwan) and dissolved in DMSO. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI), the Cy3-labeled mouse anti-β-tubulin (c-4585) and Hoechst 33258 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3,3’-dihexiloxadicarbocyanine (DiOC6) was purchased from Calbiochem (San Diego, CA, USA). BODIPY FL phallacidin (B-607) was purchased from Invitrogen (Carlsbad, CA, USA). A431 cell lysate (12-301) were purchased from MILLIPORE (Temecula, CA, USA).

2.2 Antibodies

Anti-phospho-EGFR (05-1128) and anti-EGFR (05-104) antibodies were purchased from MILLIPORE (Temecula, CA, USA). Anti-caspase-3 (3004-100) was purchased from BioVision Research Products (Mountain View, CA, USA). Anti-PARP (#9542) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-securin (ab-3305) was purchased from Abcam (Cambridgeshire, UK).

Anti-ATF3 (sc-188) and the FITC (fluorescein isothiocyanate)-labeled goat anti-mouse IgG (sc-2010) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The Cy3-labeled goat anti-rabbit IgG was purchased from Amersham Pharmacia Biotech (Little Chalfont Buckinghamshire, UK). Anti-actin (MAB1501) antibodies were purchased from CHEMICON International, Inc. (Temecula, CA, USA).

2.3 Cell lines and cell culture

RKO was a colorectal carcinoma cell line that expressed the wild type p53 proteins.

The A549 cell line was derived from lung carcinoma that contained the wild type p53.

BFTC905 cells were derived from bladder carcinomas of Chinese patients. The MCF7 cell line was derived from breast adenocarcinoma of 69 years adult female. A375 cells were derived from skin carcinomas of malignant melanoma. The securinwild type and -null HCT116 colorectal carcinoma cell lines were kindly provided by Dr. B. Vogelstein of Johns Hopkins University (Baltimore, MD). RKO and A375 cells were maintained in DMEM medium (Gibco, Life Technologies, Grand Island, NY). A549, BFTC905 and MCF7 cells were cultured in RPMI-1640 medium (Gibco, Life Technologies, Grand Island, NY, USA). The securin-wild type and -null HCT116 cells were cultured in McCoy’s 5A medium (Sigma Chemical). The complete medium was supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin and sodium bicarbonate. These cells were maintained at 37°C and 5% CO2 in a humidified incubator (310/Thermo, Forma Scientific, Inc., Marietta, OH).

2.4 Cytotoxicity assay

The cells were plated in 96-well plates at a density of 1 × 10⁴ cells/well for overnight. Following gefitinib of its analogues treatment for 24 h, the cells were washed with phosphate-buffered saline (PBS) and were replaced fresh medium for cultured 2 days. Thereafter, the medium was replaced and the cells were incubated with 0.5 mg/ml of MTT in complete medium for 4 h. The surviving cells converted MTT to formazan that generates a blue-purple color when dissolved in dimethyl sulfoxide. The intensity of formazan was measured at 565 nm using a plate reader (Molecular Devices, VERSAmax). The relative percentage of cell viability was calculated by dividing the absorbance of treated cells by that of the control in each experiment.

2.5 Cell cycle analysis

The cell cycle progression after treatment with gefitinib was measured by flow cytometer. The cells were plated at a density of 1 × 10⁶ cells per 60-mm Petri dish in complete medium for overnight. Then the cells were treated with 0-60 μM gefitinib for 24 h. At the end of treatment, the cells were collected and fixed with ice-cold 70%

ethanol overnight at −20 °C. After centrifugation, the cell pellets were treated with 4 μg/ml PI solution containing 1% Triton X-100 and 100 μg/ml RNase at 37 °C for 30 min (in the dark). After re-centrifugation, the cells resuspended in 1 ml ice-cold PBS.

To avoid cell aggregation, the cell solutions were filtrated through nylon membrane (Becton-Dickinson, San Jose, CA). Subsequently, the samples were analyzed by flow cytometer. A minimum of ten thousand cells was analyzed for DNA content, and the percentage of cell cycle phases was quantified by a ModFit LT software (Ver. 2.0, Becton-Dickinson).

2.6 Annexin V and PI assay

The level of apoptosis was determined by annexin V-PI staining analysis. The annexin V-PI staining kit (BioVision, Mountain View, CA) was used to examine the cells by incubated with fluorescein isothiocyanate (FITC)-conjugated-annexin V and PI according to the manufacturer's instruction. The cells were cultured in 60-mm Petri dish at a density of 1 × 10⁶ cells for overnight. After treatment with or without gefitinib for 24 h, the cells were washed with PBS. The cells were trypsinized and collected by centrifugation at 1500 rpm for 5 min. Thereafter, the cells were incubated with 500 μL of annexin V-PI labeling solution (containing 5 μL of annexin V-FITC and 5 μL of PI) at 25 °C in the dark for 5 min. Finally, the samples were analyzed by flow cytometer using CellQuest software (FACScan, Becton-Dickinson, San Jose, CA). The cells showed annexin V(+)/PI(−) and annexin V(+)/PI(+), which indicated at early and late apoptosis, respectively.

2.7 Observation of living cell image

To examine the effect of gefitinib on cell death. After the cells were plated at a density of 2 × 10⁵ cells/p35 Petri dish for 24 h, the cells were exposed to 0 or 60 μM gefitinib. And then the cell morphology was observed under an imaging system of living cell observation in inverted microscope (OLYMPUS, IX71, Japan) for 24 h.

2.8 Mitochondrial membrane potential assay

Mitochondrial function was evaluated by the cells stained with the mitochondrial sensitive probe DiOC6. Lipophilic cation DiOC6 accumulated in the mitochondrial matrix driven by the electrochemical gradient. The cells were cultured in 60-mm Petri dish at a density of 1 × 10⁶ cells for overnight. After treatment with or without gefitinib, the cells were washed with ice-cold PBS. The cells were trypsinized and collected by centrifugation. Then cell pellets were incubated with 50 nM DiOC6 in complete medium at 37 °C for 30 min (in the dark). Finally, the cell pellets were collected by centrifugation and resuspended in 1 ml ice-cold PBS and analyzed by flow cytometer (FACScan, Becton Dickinson, San Jose, CA).

2.9 Western blot

The cells were plated at a density of 6 × 10⁶ cells per p100 Petri dish in complete medium for overnight. Then the cells were treated with 0-60 μM gefitinib for 24 h. At the end of treatment, the cells were lysed in the icecold cell extract buffer (pH 7.6) containing 0.5 mM DTT, 0.2 mM EDTA, 20 mM HEPES, 2.5 mM MgCl2, 75mM NaCl, 0.1 mM Na3VO4, 50 mM NaF, 0.1% Triton X-100. The protease inhibitors including 1 μg/ml aprotinin, 0.5 μg/ml leupeptin, and 100 μg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride were added to the cell suspension. The protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL). The total cellular protein extracts were prepared. And they were separated on 6-12% sodium dodecyl sulfate-polyacrylamide gels, and electrophoretic transfer of proteins onto polyvinylidene difluoride membranes. The membranes were sequentially hybridized with primary antibody and followed with a horseradish peroxidase-conjugated secondary antibody.

The protein bands were visualized on the X-ray film using the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). Western analyses of various phospho-EGFR, total EGFR, caspsae-3, PARP, securin, ATF3 and actin were performed using specific antibodies. To verify equal protein loading and transfer, actin was used as the protein loading control. A gel digitizing software, Un-Scan-It gel (Ver. 5.1, Silk Scientific, Inc.), was used to analyze the intensity of bands on X-ray film by semi-quantification.

2.10 Immunofluorescence staining and confocal microscopy

To view the localization and expression of proteins after gefitinib treatment, the cells were subjected to immunofluorescence staining and confocal microscopy. The cells were cultured on coverslips, which were kept in 6-well plates at a density of 2 × 10⁵ per well for overnight before treatment. After treatment with or without 40 μM gefitinib for 24 h, the cells were washed with isotonic PBS (pH 7.4). Then fixation with 4% paraformaldehyde solution for 1 h at 37 °C, the cells were washed three times with PBS. And non-specific binding sites were blocked in PBS containing 10% FBS and 0.25% Triton X-100 for 1 h at 37 °C, and washed three times with 0.25% Triton X-100 in PBS. Thereafter, the cells were incubated with mouse anti-securin (1:120) or rabbit anti-ATF3 (1:200) antibodies in PBS containing 10% FBS and 0.25% Triton X-100 overnight at 4 °C, and washed three times with 0.25% Triton X-100 in PBS. Then the cells were incubated with goat mouse FITC-labeled IgG (1:120) and goat anti-rabbit Cy3-labeled IgG (1:200) in PBS containing 10% FBS and 0.25% Triton X-100 for 2.5 h at 37 °C, and washed three times with 0.25% Triton X-100 in PBS. The β-tubulin, F-actin, and nuclei were stained with the Cy3-labeled anti-β-β-tubulin, BODIPY FL phallacidin and Hoechst 33258, respectively. Finally, the samples were stored in the dark until examined under a confocal microscope (OLYMPUS, Japan) that equipped with an UV laser (405 nm), an Ar laser (488 nm), and a HeNe laser (543 nm).

2.11 Transfection

The pCT-GFP2 and pCT-GFP-sec2 were employed for transfection using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's recommendations.

The cells were plated in 96-well plates at a density of 8 × 10³ per well in complete medium for overnight, then were transfected with 50 μg/mL of control or securin-expressed vectors in 50 μL/well serum-free medium for 6 h at 37 °C in a CO2 incubator according to the manufacturer’s recommendations. Then, the equal amount medium with 20% fetal bovine serum was added without removing the transfection mixture, and incubation proceeded for an additional 24 h. After transfection, the cells were subjected to cytotoxicity analysis as described above.

2.12 Statistical analysis

Each experimentwas repeated at least three times. Data from the population of cells treated with different conditions were analyzed using paired Student’s t-test. In a comparison of multiple groups, data were analyzed by one-way or two-way analysis of variance (ANOVA), and further post Tukey’s tests using the statistic software of GraphPad Prism 5 (GraphPad software, Inc. San Diego, CA). A p value of < 0.05 was considered as statistically significant in each experiment.

3. Results

3.1 Gefitinib elicits the cytotoxicity in both low level EGFR and high

We examined the cytotoxicity following treatment with gefitinib in a variety of human cancer cell lines including RKO (colon cancer), A549 (lung cancer), BFTC905 (bladder cancer), MCF7 (breast cancer) and A375 (skin cancer) cells. Gefitinib significantly induced cancer cell death via a concentration-dependent manner in these cancer cell lines (Fig. 1). The order of cytotoxic sensitivity was A375 > MCF7 >

BFTC905 > A549 > RKO cells by exposure to 10-60 μM gefitinib for 24 h. The IC50

value of gefitinib toward cultured human cancer cells lines was 31.45 μM in A375 cells, 40.82 μM in MCF7 cells, 43.49 μM in BFTC905 cells, 83.16 μM in A549 cells, and 91.37 μM in RKO cells.

We have further determined the protein expression of phospho-EGFR and total EGFR in these cencer cell lines. The total protein extracts from various cancer cell lines including RKO, A549, BFTC905, MCF7 and A375 were subjected to western blot analysis. The immunoblot analysis indicated that various human cancer cell lines contained different the protein level of phospho-EGFR and total EGFR (Fig. 2). A375, MCF7 and RKO cells did not significantly express the proteins of the phosphorylated-EGFR and total phosphorylated-EGFR; in contrast, BFTC905 and A549 cells expressed high level of EGFR. Actin was used as a loading control protein. Positive control of phospo-EGFR and EGFR were purified from proteins of A431 cell extracts.

3.2 Gefitinib induces apoptosis in both A375 and BFTC905 cancer cells

Subsequently, we determined the possible involvement of gefitinib in the regulation of cell cycle progression, the effect of gefitinib on A375 and BFTC905 cells was analyzed by flow cytometry (Fig. 3A). Gefitinib did not significantly alter the fractions of G0/G1 and G2/M phases; however, it markedly decreased the fractions of S

phase in both A375 and BFTC905 cells (Fig. 3B). Meanwhile, gefitinib increased the fraction of sub-G1 phase. Treatment with 60 μM gefitinib for 24 h increased apoptosis by 26.6% and 12.3% in A375 and BFTC905 cells, respectively (Fig. 3C).

We further assessed apoptosis from the cells that had been exposed to gefitinib by annexin V and PI staining analysis. The control cells were not significantly stained with fluorochromes; however, the annexin V (+)/PI (−) cells (early apoptosis) and annexin V (+)/PI (+) cells (late apoptosis) were increased by treatment with gefitinib 60 μM for 24 h in both A375 and BFTC905 cells (Fig. 4A). The percentages of apoptosis populations (early and late stages) were quantified. Gefitinib at 60 μM induced about 61.6% of apoptosis in A375 cells and 28.9% of apoptosis in BFTC905 cells (Fig. 4B).

3.3 Gefitinib promotes the hyperpolarization of mitochondria and

The time-lapse observation of cell death was found by gefitinib treatment.

Treatment with gefitinib significantly induced the cell disruption in A375 cells; in contrast, the control group did not induce the cell death (Fig. 5) .

To examine the induction of apoptosis pathway following gefitinib treatment, the cells were subjected to mitochondrial functional assay. Gefitinib induced the hyperpolarization of mitochondrial membrane potential (Fig. 6A). The quantified data showed that treatment with gefitinib for 24 h significantly increased the levels of DiOC6 intensities in a concentration-dependent manner (Fig. 6B). In A375 cells, gefitinib increased the fluorescence intensity of DiOC6 by about 19.1% and 51.0% at 20 μM and 40 μM, respectively.

The apoptosis-regulated proteins were also analyzed by gefitinib. The active form

The apoptosis-regulated proteins were also analyzed by gefitinib. The active form