Gefitinib induces anoikis in cervical cancer cells

Abstract

Gefitinib exerts anticancer effects on various types of cancer, such as lung, ovarian, breast, and colon cancers. However, the therapeutic effects of gefitinib on cervical cancer and the underlying mechanisms remain unclear. Thus, this study aimed to explore whether gefitinib can be used to treat cervical cancer and elucidate the underlying mechanisms. Results showed that gefitinib induced a caspase-dependent apoptosis of HeLa cells, which consequently became round and detached from the surface of the culture plate. Gefitinib induced the reorganization of actin cytoskeleton and downregulated the expression of p-FAK, integrin β1 and E-cadherin, which are important in cell-extracellular matrix adhesion and cell-cell interaction, respectively. Moreover, gefitinib hindered cell reattachment and spreading and suppressed interactions between detached cells in suspension, leading to poly (ADP-ribose) polymerase cleavage, a hallmark of apoptosis. It also induced detachment-induced apoptosis (anoikis) in C33A cells, another cervical cancer cell line. Taken together, these results suggest that gefitinib triggers anoikis in cervical cancer cells. Our findings may serve as a basis for broadening the range of anticancer drugs used to treat cervical cancer.

INTRODUCTION

Cervical cancer is the fourth most commonly diagnosed cancer in females, accounting for approximately 604,127 emerging cases in 2020. The disease also ranks as the highest cause of mortality in female cancer, with 341,831 deaths based on GLOBOCAN estimates (1). Chemotherapy is currently the standard treatment for cervical cancer. However, the continuous use of chemotherapy agents leads to drug resistance and reduced therapeutic efficacy (2). Therefore, novel drugs against cervical cancer need to be developed.

Gefitinib, a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), was approved by the US Food and Drug Administration as a first-line treatment for patients with metastatic non-small cell lung cancer in 2015 (3). Since then, this drug has been tested for other EGFR-positive cancer types, including cervical cancer (4, 5). A clinical trial showed that gefitinib administration has low toxicity and is effective in patients with cervical cancer (6). Response to gefitinib is higher when this drug is combined with chemoradiation than when used alone, and the toxicity of this treatment combination is acceptable (4, 7). However, these studies have limitations, such as a small number of patients (< 30 patients), and the mechanism through which gefitinib exerts its beneficial effects on cervical cancer remains unclear. Using cervical cancer cell lines in vitro, one study reported that gefitinib treatment decreases cell viability by affecting apoptosis-related molecules and inducing cell cycle arrest (8). However, another study showed that treatment with gefitinib alone does not induce apoptosis (9). To date, results about the effects of gefitinib against cervical cancer are insufficient.

Anoikis is a unique form of apoptosis resulting from cell detachment (10, 11). During anoikis, cells detach from the surface, become rounded, and fail to reattach to the surface (12). Also, cell-cell interaction is impaired (13). Reorganization of actin cytoskeleton and dysregulation of cell-extracellular matrix (ECM) adhesion molecules, including integrin β1 and focal adhesion kinase (FAK), are critical for cell detachment. Inhibition of integrin β1 induces cell detachment, and loss of cell-ECM interaction can indicate cell death-associated cell signaling (14, 15). FAK regulates cell adhesion via integrin activation, and its inhibition triggers cell detachment and apoptosis (16, 17). Meanwhile, loss of cell-cell interactions, which is mainly mediated by E-cadherin, triggers anoikis (18).

This study aimed to examine the effect of gefitinib on cervical cancer cells and elucidate the underlying mechanisms. We report that gefitinib exerts an antiproliferative effect on cervical cancer cells by inducing anoikis. Our results suggest that gefitinib can be used as a therapeutic drug against cervical cancer.

RESULTS AND DISCUSSION

Gefitinib induces detachment followed by caspase-dependent apoptosis of HeLa cells

The mechanism of action of gefitinib on cervical cancer remains to be established. Therefore, we first examined the effects of gefitinib treatment on human cervical adenocarcinoma HeLa cells. Gefitinib treatment markedly decreased cell viability in a dose-dependent manner, indicating the cytotoxicity of gefitinib to HeLa cells (Fig. 1A). We then investigated whether gefitinib treatment induces apoptosis in HeLa cells. Cell cycle analysis revealed that gefitinib treatment dose-dependently increased the percentage of cells in the sub-G1 phase, a hallmark of apoptosis (Fig. 1B). Moreover, results of annexin V assay indicated that gefitinib treatment increased the number of apoptotic cells in a time-dependent manner (Fig. 1C). The treatment also increased the cleavage of poly (ADP-ribose) polymerase (PARP), another apoptotic marker, in a time-dependent manner (Fig. 1D). These data suggest that gefitinib reduces cell viability by inducing apoptosis. This finding is consistent with a previous study showing that gefitinib exerts an anticancer effect on HeLa cells by promoting apoptosis (8). It prompted us to examine whether gefitinib-induced apoptosis is caspase-dependent. We found that gefitinib treatment time-dependently promoted the cleavage of both initiator caspases (caspase-8 and -9) and executioner caspases (caspase-3 and -7) (Fig. 1E). This result indicates that gefitinib induces apoptosis via intrinsic and extrinsic pathways.

Fig. 1.

Gefitinib-induced detachment and apoptosis of HeLa cells. (A) HeLa cells were treated with an indicated dose of gefitinib for 24 h. Cell viability was subsequently analyzed. The number of viable cells in HeLa cells without gefitinib treatment was set as 100%. (B) Cells were treated the same way as that in (A) and then subjected to cell cycle analysis using flow cytometry. The percentage of cells in the sub-G1 population is displayed as a bar graph. (C-F) HeLa cells were treated with 40 μM gefitinib for the indicated durations. (C) The cells were stained with annexin V-FITC and PI, and the percentage of apoptotic cells was evaluated using flow cytometry, displayed as a bar graph. (D) PARP cleavage and (E) cleaved caspase-3, -7, -8, and -9 were analyzed using western blotting. (F) Cell images were captured using an inverted microscope (×200), and the percentage of detached cells is displayed. The number of total cells (attached cells + detached cells) was set as 100%. (G, H) HeLa cells were treated with 40 μM gefitinib in the absence or presence of 20 μM Z-VAD-fmk for 24 h. (G) The detached cells were counted, and (H) the percentage of living or dead cells in total detached cells is displayed. The number of total detached cells was set as 100%. Data are presented as the mean ± SEM of three independent experiments. P-values were determined using Student’s t-test (ns: no significance, *P < 0.05, and ***P < 0.001).

Interestingly, micrographs demonstrated that the HeLa cells gradually acquired a round shape in response to gefitinib treatment (Fig. 1F, top panel). Additionally, gefitinib treatment time-dependently caused a substantial degree of cell detachment (Fig. 1F, bottom panel). These findings call into question whether cell detachment is the result of apoptosis or a contributing factor. To determine whether caspase activation is a cause or consequence of cell detachment, we treated the cells with gefitinib in the absence or presence of the pan-caspase inhibitor Z-VAD-fmk. No significant differences in the total number of detached cells were found in both groups (Fig. 1G). However, the living-to-dead ratio of detached cells after gefitinib treatment was higher in the presence than in the absence of Z-VAD-fmk (Fig. 1H). These results revealed that treatment with Z-VAD-fmk attenuated the apoptosis of the detached cells but failed to prevent gefitinib-induced cell detachment. Thus, caspase activation, which leads to apoptosis, is not a cause but rather a consequence of cell detachment.

Anoikis is a type of apoptosis that occurs due to the loss of cell-ECM and cell-cell interactions, making the cells round (19-21). In the present study, gefitinib treatment induced cell detachment, followed by apoptosis, implying that gefitinib can cause the death of HeLa cells by inducing anoikis.

Gefitinib induces reorganization of actin cytoskeleton and alteration of adhesion molecules in HeLa cells

Actin cytoskeleton is reorganized when cells detach from the ECM during anoikis, and actin depolymerization can be a key player in the induction of anoikis (22, 23). Therefore, we investigated whether gefitinib affects the organization of actin cytoskeleton. As shown in Fig. 2A, a well-organized actin cytoskeleton was observed throughout the cytoplasm of the untreated control cells, whereas the gefitinib-treated cells displayed a less organized actin cytoskeleton in the cytoplasm and a more organized cortical actin cytoskeleton adjacent to the plasma membrane. We also examined whether gefitinib affects actin polymerization. Results showed that gefitinib treatment decreased actin polymerization in a dose-dependent manner (Fig. 2B). c-Abl tyrosine kinase regulates actin cytoskeleton organization and actin polymerization; therefore, we investigated the cellular levels of c-Abl and phosphorylated c-Abl (24, 25). As shown in Fig. 2C, gefitinib treatment dose- and time-dependently decreased the levels of c-Abl protein and phosphorylated c-Abl (Fig. 2C). Thus, gefitinib treatment may reduce actin polymerization in HeLa cells by downregulating c-Abl. Taken together, these results suggest that reorganization of actin cytoskeleton and depolymerization of actin enhance the roundedness and detachment of cells.

Fig. 2.

Gefitinib-induced reorganization of actin cytoskeleton and alteration of cell-ECM adhesion molecules. (A) HeLa cells were treated with or without 40 μM gefitinib for 24 h and analyzed using confocal microscopy. Actin cytoskeleton was labeled with fluorescent phalloidin (red), and nuclei were stained with DAPI (blue). Selected areas of merged images (scale bars, 40 μm) are shown at high magnification (right panels, scale bars; 10 μm). (B) Actin polymerization was assayed by measuring the fluorescence intensity of phalloidin bound to polymerized actin. The RFI is the ratio of the fluorescence intensity of the gefitinib-treated cells to that of the untreated control cells. (C) HeLa cells were treated with the indicated concentrations of gefitinib for 24 h (left) or with 40 μM gefitinib for the indicated durations (right). c-Abl and phosphorylated c-Abl (Tyr245), (D) cell-ECM adhesion molecules, and (E) E-cadherin were analyzed using western blot. P values were determined by Student’s t-test (***P < 0.001).

Considering that cell detachment can be triggered by dysregulation of cell-ECM adhesion molecules (26), we examined whether gefitinib influences cell-ECM adhesion molecules (Fig. 2D). As shown in Fig. 2D, gefitinib treatment markedly decreased the levels of integrin β1 and phosphorylated FAK in a dose- and time-dependent manner, whereas the levels of Src, paxillin, talin, and α-actinin remained constant. Integrin β1 and FAK play an important role in attachment to ECM, and downregulation of these molecules causes cell detachment from the ECM, consequently triggering apoptosis (15, 27, 28). These results are consistent with those of previous studies.

As shown in Fig. 1F, the gefitinib-treated cells dislodged as single units or small aggregates, including two or three cells. Cell-cell interactions are altered when cells become rounded. Therefore, we investigated whether gefitinib affects E-cadherin, a key molecule responsible for cell-cell interactions. As shown in Fig. 2E, gefitinib treatment dose- and time-dependently decreased E-cadherin expression.

These results suggest that gefitinib induces 1) c-Abl-mediated reorganization of actin cytoskeleton and depolymerization of actin, 2) dysregulation of integrin β1 and FAK-mediated cell adhesion to ECM, and 3) impairment of E-cadherin-mediated cell-cell interaction, leading to cell detachment as single rounded units or small aggregates.

Gefitinib suppresses reattachment and cell-cell interaction of detached cells

Two additional prerequisites for anoikis are the suppression of reattachment and spreading of detached cells and the inhibition of interactions between detached cells (20). Therefore, we examined whether gefitinib affects reattachment and cell-cell interactions in a suspension of detached cells. First, when detached HeLa cells were replated with or without gefitinib, the gefitinib-treated cells showed a substantial decrease in reattachment and spreading compared with the untreated control cells (Fig. 3A). Moreover, the cells treated with gefitinib displayed a large amount of actin cytoskeleton near the plasma membrane, whereas the untreated cells exhibited a well-organized actin cytoskeleton throughout the cytoplasm (Fig. 3B). Similar to the phenomenon observed in the attached cells treated with gefitinib (Fig. 2C, D), the level of phosphorylated c-Abl and phosphorylated FAK decreased in the gefitinib-treated cells during cell reattachment and spreading (Fig. 3C, D). However, integrin β1 levels were comparable between control and gefitinib-treated cells (Fig. 3D). The gefitinib-induced impairment of reattachment and spreading promoted PARP cleavage, implying that gefitinib-induced apoptosis can be partly triggered by the improper reattachment and spreading of cells (Fig. 3E). Taken together, these data suggest that gefitinib decreases the levels of phosphorylated c-Abl and phosphorylated FAK, resulting in dysregulation of actin reorganization and cell adhesion to ECM. This phenomenon impairs cell reattachment and spreading, eventually leading to cell death.

Fig. 3.

Gefitinib-induced decrease in cell reattachment and cell-cell interaction between detached cells. (A-E) Detached HeLa cells were replated in the absence or presence of 40 μM gefitinib for the indicated durations. (A) Cell images were captured using an inverted microscope (×200), and (B) actin cytoskeleton was labeled with fluorescent phalloidin (red) (scale bars, 12 μm). (C) Levels of phosphorylated c-Abl (Tyr245), (D) integrin β1, phosphorylated FAK (Tyr397), and (E) cleaved PARP were analyzed using western blotting. (F-H) HeLa cells were seeded on poly-HEMA-coated plates with the indicated concentrations of gefitinib for 24 h. (F) Aggregation (cell-cell interaction) of cells was observed under an inverted microscope (×200). (G) Aggregated cells were collected, and cell viability was evaluated. The number of viable cells among the untreated cells was set as 100%. (H) PARP cleavage was detected using western blotting. P values were determined by Student’s t-test (***P < 0.001).

We then examined whether gefitinib influences cell-cell interactions in suspension. As shown in Fig. 3F, the untreated suspended cells formed large aggregates in suspension, whereas the gefitinib-treated suspended cells formed smaller aggregates in a dose-dependent manner. This result suggests that gefitinib treatment suppresses the interactions between detached cells in suspension. Gefitinib treatment also decreased the viability of suspended cells and promoted the cleavage of PARP (Fig. 3G, H). This finding implies that gefitinib induces cell death partly suppressing interactions between detached cells.

Gefitinib induces anoikis in C33A human cervical adenocarcinoma cells

To investigate whether the mechanism of gefitinib-induced anoikis elucidated above is limited to HeLa cells or common to other cervical cancer cells, we examined the effect of gefitinib on human cervical adenocarcinoma C33A cells. Gefitinib treatment dose-dependently reduced cell viability and promoted PARP cleavage (Fig. 4A, B). It also dose-dependently increased the number of round cells and the detachment of cells (Fig. 4C). Furthermore, gefitinib caused actin cytoskeleton reorganization and decreased actin polymerization (Fig. 4D, E). The levels of integrin β1, phosphorylated FAK, and E-cadherin decreased in the gefitinib-treated C33A cells (Fig. 4F, G). Moreover, gefitinib suppressed the reattachment of and interactions between detached cells (Fig. 4H, I). These results suggest that gefitinib-induced anoikis is a common mechanism in other cervical cancer cells and is not limited to HeLa cells.

Fig. 4.

Gefitinib-induced anoikis in C33A cells. C33A cells were treated with the indicated concentrations of gefitinib for 24 h. (A) Cell viability was assayed, and (B) PARP cleavage was analyzed using western blotting. (C) Cell images were captured using an inverted microscope (×200). (D) C33A cells were treated with or without 40 μM gefitinib for 24 h and analyzed by confocal microscopy. Actin cytoskeleton was labeled with fluorescent phalloidin (red), and the nuclei were stained with DAPI (blue) (scale bars, 12 μm). (E) Actin polymerization was assayed by measuring the fluorescence intensity of phalloidin bound to polymerized actin. (F) C33A cells were treated with the indicated concentrations of gefitinib for 24 h. Levels of integrin β1, phosphorylated FAK, and (G) E-cadherin were analyzed using western blot. (H) Detached C33A cells were replated in the absence or presence of 40 μM gefitinib, and cell images were captured at the indicated time points using an inverted microscope (×200). (I) C33A cells were plated on poly-HEMA-coated plates with the indicated concentrations of gefitinib for 24 h, and images of suspended cell aggregates were captured using an inverted microscope (×200). P values were determined by Student’s t-test (**P < 0.01, ***P < 0.001).

In the present study, we investigated the mechanisms underlying the antiproliferative effects of gefitinib on cervical cancer cells. Results showed that gefitinib treatment 1) induced cell detachment, 2) suppressed cell reattachment and spreading, and 3) inhibited cell-cell interactions, leading to caspase-dependent apoptosis. This study shows that gefitinib triggers anoikis, apoptosis upon detachment from the ECM, in cervical cancer cell lines. Our findings may serve as a reference for using gefitinib as an anticancer drug against cervical cancer.

MATERIALS AND METHODS

Materials

Gefitinib (MW 446.90), poly 2-hydroxyethyl methacrylate (poly-HEMA), and phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PARP, caspases, cell-ECM adhesion molecules, c-Abl, phosphorylated c-Abl, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against E-cadherin was purchased from BD Biosciences (San Jose, CA, USA). Antibodies against α-tubulin and Z-VAD-fmk (pan-caspase inhibitor) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell culture and gefitinib treatment

Human cervical adenocarcinoma HeLa cells (ATCC, Manassas, VA, USA) and human cervical adenocarcinoma C33A cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and streptomycin-penicillin (100 μg/ml and 100 IU/ml). The cells were incubated at 37°C in a humidified atmosphere with 5% CO₂ and then seeded into a six-well plate at a density of 2 × 10⁵/well. The cells were treated with the indicated concentrations of gefitinib for the indicated times.

Actin cytoskeleton staining

To visualize actin cytoskeleton, the cultured cells were fixed with 4% paraformaldehyde at 37°C for 30 min and then permeabilized with 0.2% Triton X-100 at room temperature (RT) for 10 min. The cells were washed twice with PBS and then stained with 5 μg/ml phalloidin-TRITC, which can bind polymerized actin or actin cytoskeleton, in PBS at RT for 30 min. Subsequently, the cells were added with mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) and then imaged under a Zeiss LSM 710 confocal laser scanning microscope (Zeiss, Heidenheim, Germany).

Measurement of actin polymerization

Polymerized actin was assessed using flow cytometry with phalloidin-TRITC staining. Cells with or without gefitinib treatment were trypsinized to prepare cell suspensions and then stained with phalloidin-TRITC. Subsequently, the fluorescence intensity of phalloidin-TRITC bound to polymerized actin was measured using flow cytometry. The relative fluorescence index (RFI) is the ratio of the fluorescence intensity of the gefitinib-treated cells to that of the untreated control cells.

Evaluation of cell adhesion and spreading

For adhesion and spreading monitoring, cells cultured in a 100 mm dish were detached via trypsinization. A suspension of 1 × 10⁶ cells was replated in six-well plates with or without 40 μM gefitinib for the indicated durations (4 and 8 h). Unbound cells were removed by aspiration, and the wells were gently rinsed once with PBS. Subsequently, the bound cells were photographed under an inverted microscope (×200).

Evaluation of cell-cell interaction in suspension culture

Assays were conducted as described previously (29). For suspension culture, a six-well plate was coated with poly-HEMA, a non-cell adhesive polymer. Specifically, 2 ml of poly-HEMA solution (20 mg/ml in absolute ethanol) was added to each well and then dried overnight at 37°C. The wells were washed twice with sterile PBS before use. The cells were seeded in poly-HEMA pre-coated 6-well plates at a density of 4 × 10⁵/well and then treated with the indicated gefitinib concentrations for 24 h. Subsequently, the aggregated cells were photographed using an inverted microscope or harvested for western blot. Furthermore, the aggregated cells underwent disaggregation through trypsinization, and the number of viable cells was quantified by trypan blue exclusion assay.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA, USA). P-values were calculated using Student’s t-tests, and statistical significance was defined as *P < 0.05, **P < 0.01, or ***P < 0.001. Data are presented as the mean ± standard error of the mean (SEM). Each experiment was conducted at least three times.

SUPPLEMENTARY METHODS

Further detailed description of the methods is provided in Supplementary Information.