The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Letter
The anti-cancer drug gefitinib accelerates Fas-mediated apoptosis by enhancing caspase-8 activation in cancer cells
Yuto SekiguchiMayuka YamadaTakuya NoguchiChise NoomoteMei TsuchidaYuki KudohYusuke HirataAtsushi Matsuzawa
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2019 Volume 44 Issue 6 Pages 435-440

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Abstract

Fas/CD95 plays a pivotal role in T cell-mediated cytotoxicity. Accumulating evidence has suggested that resistance to Fas-mediated apoptosis contributes to the escape of cancer cells from immune destruction, and allows to undergo proliferation and outgrowth of cancer cells. In this study, we found that the anti-cancer drug gefitinib, a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), has an ability to enhance Fas-mediated cytotoxicity. In the presence of nontoxic concentrations of gefitinib, Fas-induced activation of caspase-8 and subsequent apoptosis was dramatically promoted, suggesting that gefitinib increases the sensitivity to Fas-mediated apoptosis. Interestingly, the effects of gefitinib were observed in EGFR or p53 knockout (KO) cells. These observations indicate that both EGFR and p53 are dispensable for the enhancement. On the other hand, gefitinib clearly downregulated heat shock protein 70 (HSP70) as previously reported. Considering that HSP70 contributes to protection of cells against Fas-mediated apoptosis, gefitinib may increase the sensitivity to Fas-mediated apoptosis by downregulating HSP70. Thus, our findings reveal novel properties of gefitinib, which may provide insight into the alternative therapeutic approaches of gefitinib for Fas-resistant tumors.

INTRODUCTION

Fas/CD95 is a member of the death receptor family that causes caspase-8-dependent apoptosis by forming death-inducing signaling complex (DISC) (Peter and Krammer, 2003). In particular, immune cells such as T cells express Fas ligand (FasL), and eliminate unnecessary or cancer cells by inducing Fas-mediated apoptosis (Puliaeva et al., 2008). However, recent evidence has indicated that a portion of cancer cells show resistance to Fas-mediated apoptosis, even though Fas is sufficiently expressed on the cell surface, leading to the failure of T cell-mediated immune intervention or chemotherapy (Ajayi et al., 2013; Månsson et al., 2002; Schett et al., 1999; Natoli et al., 1995). It is known that the nuclear factor-κB (NF-κB) signaling pathways and heat shock protein (HSP) family proteins negatively regulate apoptosis, through the upregulation of anti-apoptotic factors, and the direct physical interaction with key components of the apoptotic pathways, respectively (Kreuz et al., 2004; Ponton et al., 1996; Xanthoudakis and Nicholson, 2000; Takayama et al., 2003). Therefore, the overexpression of anti-apoptotic factors or HSP family proteins could be one of the mechanisms of the Fas resistance.

The tumor suppressor p53 is a master regulator of chemotherapeutic agent-induced apoptosis. It is well known that a substantial fraction of cancer cells has loss of function mutations in p53, which makes it difficult to eliminate cancer cells effectively by the chemotherapeutic agents during chemotherapy (Vogelstein and Kinzler, 1992; Wattel et al., 1994). Interestingly, it has been demonstrated that p53 promotes cell surface expression of Fas by stimulating the transport of Fas from the Golgi body, suggesting the possibility that p53 defective tumor cells acquire the Fas resistance (Bennett et al., 1998). Nevertheless, the molecular mechanism of the Fas resistance in cancer cells has not been fully elucidated.

Gefitinib (also known as ZD1839 or Iressa) is a molecular target drug that inhibits the kinase activity of epidermal growth factor receptor (EGFR), and exerts anti-tumor effect against various types of solid tumors through blocking EGFR-mediated signal transduction pathways (Araki et al., 2012). However, accumulating evidence has suggested that gefitinib affects the activities of signaling molecules other than EGFR, which may be responsible for the serious adverse reactions to gefitinib (Tigno-Aranjuez et al., 2010; Brehmer et al., 2005; Karaman et al., 2008). In this study, we investigated the cellular responses affected by gefitinib, and found that gefitinib dramatically enhances Fas-mediated apoptosis in cancer cells. Thus, our results provide the possibility that gefitinib is a novel candidate drug to overcome resistance to Fas-mediated apoptosis in cancer cells.

MATERIALS AND METHODS

Cell culture and reagents

Human fibrosarcoma cell line HT1080, human lung adenocarcinoma cells line A549, and mouse embryonic fibroblasts (MEF) were grown in Dulbecco’s Modified Eagle Medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin-streptomycin solution, at 37°C under a 5% CO2 atmosphere. All reagents were obtained from commercial sources; gefitinib (Wako, Tokyo, Japan), FasL (Enzo Life Science, Farmingdale, NY, USA), CH-11 (MBL, Aichi, Japan), Z-VAD-fmk (Peptide Institute, Osaka, Japan). The antibodies used were against caspase-8 (Enzo Life Science), p53 (Cell Signaling, Danvers, MA, USA), Fas, HSP70, EGFR, inhibitor of κBα (IκBα) (Santa Cruz, Dallas, TX, USA), and β-actin (Wako).

Colorimetric cell viability assay

Cell viability assay was performed as described previously (Noguchi et al., 2018). Cells were seeded on 96-well plates. After indicated stimulation, cell viability was determined using Cell Titer 96 Cell Proliferation Assay (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The absorbance was read at 492 nm using a microplate reader. Data are normalized to control (100%) without stimulus.

Immunoblot

Cells were lysed with the 1% Triton X-100 buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton-X100, 10% Glycerol, and 1% protease inhibitor cocktails (Nacalai Tesque, Kyoto, Japan)]. After centrifugation, the cell extracts were resolved by SDS-PAGE and analyzed as described previously (Hirata et al., 2017). The blots were developed with ECL (Merck Millipore, Burlington, VT, USA).

Generation of knockout cell lines

Knockout cells were generated using Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system as described previously (Noguchi et al., 2018). Guide RNAs (gRNAs) were designed to target a region in the exon 3 of p53 gene (5’-ATCTGAGCAGCGCTCATGGTGGG-3’), and the exon 4 of EGFR gene (5’- CTTTCTCAGCAACATGTCGATGG-3’) using CRISPRdirect. gRNA-encoding oligonucleotide was cloned into lentiCRISPRv2 plasmid (addgene, Watertown, NY, USA), and knockout cells were established as previously described (Hirata et al., 2017). To determine the mutations of p53 and EGFR in cloned cells, genomic sequence around the target region was analyzed by PCR-direct sequencing using extracted DNA from each clone as a template and the following primers: 5’-AGAGACCCCAGTTGCAAACC-3’and 5’-CCCTGCCCTCAACAAGATGT-3’ for p53, and primers: 5’-GGGCTAATTGCGGGACTCTT-3’ and 5’-CCCAGTGCTGTAGAGCTGTC-3’ for EGFR.

Quantitative real-time PCR

Total RNA was extracted using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan) and reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Template cDNA was amplified by quantitative real-time PCR as described previously (Kudoh et al., 2018). Primers used for qRT-PCR; 5’-ATGAACCAGACTGCGTGCCCTG-3’ and 5’-AAGAAGAAGACAAAGCCACCCC-3’ for Fas, and 5’- AACAGCCTCAAGATCATCAGC-3’ and 5’-GGATGATGTTCTGGAGAGCC-3’ for GAPDH. Each gene expression levels were normalized to that of GAPDH.

NF-κB reporter assay

NF-κB reporter assays were performed essentially as described (Noguchi et al., 2016). Cells were transfected using Polyethylenimine “Max” (Polysciences, Warrington, PA, USA) with a plasmid mix containing a NF-κB luciferase reporter plasmid, a renilla luciferase plasmid for normalization, and an empty plasmid. After 24 hr, cells were treated with FasL for 6 hr. Firefly and renilla luciferase activities were quantified with dual luciferase reporter assay system (Promega).

RESULTS AND DISCUSSION

Gefitinib enhances Fas-mediated apoptosis independently of EGFR

To explore the cellular responses affected by gefitinib, we first examined gefitinib-induced cytotoxicity to determine nontoxic concentration of gefitinib. As shown in Fig. 1A, viability of both A549 cells and MEFs was not affected at concentration up to 30 μM of gefitinib. Interestingly, in the presence of nontoxic concentration of gefitinib (10 or 20 μM), we found that cell death stimulated by both soluble FasL or Fas agonistic monoclonal antibody CH-11 was drastically enhanced in A549 cells (Fig. 1B). Similar results were observed in HT1080 cells (Fig. 1C). Moreover, the enhancement of Fas-mediated cell death was completely suppressed by co-treatment with Z-VAD-fmk, a pan-caspase inhibitor, indicating that gefitinib has an ability to enhance Fas-mediated apoptosis in cancer cells (Fig. 1D). Thus, we next examined whether the inhibitory effect of gefitinib on EGFR is required for the enhancement of apoptosis. To this end, we established EGFR KO A549 cells by using CRISPR/Cas9 system, and found that gefitinib enhances Fas-mediated apoptosis in EGFR KO A549 cells to a similar extent as control cells (Fig. 1E and 1F). These observations suggest that the enhancement of Fas-mediated apoptosis triggered by gefitinib occurs independently of EGFR.

Fig. 1

Gefitinib enhances Fas-mediated apoptosis independently of EGFR. (A) A549 cells and MEFs were treated with the indicated concentrations of gefitinib for 48 hr, and then subjected to cell viability assay. Data shown are the mean ± SD. (B) A549 cells pretreated with the indicated concentrations of gefitinib for 24 hr were treated with 200 ng/mL CH-11 or 200 ng/mL FasL for 24 hr, and then subjected to cell viability assay. Data shown are the mean ± SD. Significant differences were determined by one-way ANOVA, followed by Tukey-Kramer test; ** P  <  0.01, *** P <  0.001. (C) HT1080 cells pretreated with 20 µM gefitinib for 1 hr were treated with 50 ng/mL FasL for 12 hr, and then subjected to cell viability assay. Data shown are the mean ± SD. Significant differences were determined by one-way ANOVA, followed by Tukey-Kramer test; *** P < 0.001. (D) A549 cells pretreated with the indicated concentrations of gefitinib for 48 hr were treated with 200 ng/mL FasL for 24 hr, and then subjected to cell viability assay in the presence or absence of 20 µM Z-VAD-fmk. Data shown are the mean ± SD. Significant differences were determined by one-way ANOVA, followed by Tukey-Kramer test; ** P < 0.01, *** P < 0.001. (E) A549 cells were subjected to immunoblotting with the indicated antibodies. (F) A549 cells pretreated with 20 µM gefitinib for 24 hr were treated with 200 ng/mL FasL for 24 hr, and then subjected to cell viability assay. Data shown are the mean ± SD. Significant differences were determined by one-way ANOVA, followed by Tukey-Kramer test; *** P < 0.001.

Gefitinib enhances Fas-mediated apoptosis independently of p53

A previous report has demonstrated that gefitinib induces p53-dependent upregulation of pro-apoptotic molecules such as Fas (Chang et al., 2008). We therefore speculated that gefitinib enhances Fas-mediated apoptosis through the p53-dependent upregulation of Fas. However, we could not observe the gefitinib-dependent upregulation of Fas at both mRNA and protein levels in A549 cells (Fig. 2A and 2B). To further evaluate the involvement of p53 in the enhancement of Fas-mediated apoptosis triggered by gefitinib, we established p53 KO A549 cells, and checked loss of p53 protein in the presence of cisplatin because cisplatin can stabilize p53 protein (Fig. 2C). We then found that gefitinib significantly enhances Fas-mediated apoptosis in p53 KO cells, although the enhancement in p53 KO cells (clone #2) was lower than that in clone #1 for unknown reasons (Fig. 2D). Accordingly, these observations suggest that the contribution of p53 to the enhancement of Fas-mediated apoptosis triggered by gefitinib is relatively small at least in A549 cells.

Fig. 2

Gefitinib enhances Fas-mediated apoptosis independently of p53. (A) A549 cells were treated with 20 µM gefitinib for the indicated periods. The mRNA levels were measured by RT-PCR. Data shown are the mean ± SD. (B) A549 cells were treated with 20 µM gefitinib for the indicated periods. Cell extracts were subjected to immunoblotting with the indicated antibodies. (C) A549 cells were treated with 20 µM cisplatin for 36 hr in order to stabilize p53. Cell extracts were subjected to immunoblotting with the indicated antibodies. (D) A549 cells pretreated with 20 µM gefitinib for 24 hr were treated with 200 ng/mL FasL for 24 hr, and then subjected to cell viability assay. Data shown are the mean ± SD. Significant differences were determined by one-way ANOVA, followed by Tukey-Kramer test; *** P < 0.001.

Gefitinib enhances Fas-mediated apoptosis by promoting caspase-8 activation

We next investigated the mechanisms by which gefitinib enhances Fas-mediated apoptosis. Immunoblot analysis revealed that pretreatment with gefitinib for 48 hr increases levels of p18 fragments of caspase-8 (active form), meaning that gefitinib potentiates Fas-induced caspase-8 activation by promoting autocatalytic cleavage of caspase-8 (Fig. 3A). On the contrary, luciferase assays revealed that gefitinib does not affect Fas-induced NF-κB activation (Fig. 3B). In steady state condition, IκBα blocks the NF-κB activation by inhibiting nuclear translocation of NF-κB, whereas the phosphorylation-dependent degradation of IκBα allows to the translocation and activation of NF-κB. As shown in Fig. 3C, the degradation of IκBα was observed at 60 min after FasL treatment, which was not affected in the presence of gefitinib. These observations suggest that gefitinib promotes Fas-induced caspase-8 activation without blocking NF-κB-associated survival signals. On the other hand, recent evidence has suggested that gefitinib downregulates the expression of HSP70, which has been reported as a negative regulator of Fas-induced apoptosis (Namba et al., 2011; Schett et al., 1999). Indeed, we clearly observed gefitinib-induced downregulation of HSP70, which raises the possibility that gefitinib increases the sensitivity of Fas-mediated apoptosis by downregulating HSP70 (Fig. 3D).

Fig. 3

Gefitinib accelerates Fas-mediated apoptosis by enhancing caspase-8 activation. (A) A549 cells pretreated with 20 µM gefitinib for 48 hr were treated with 200 ng/mL CH-11 for the indicated periods. Cell extracts were subjected to immunoblotting with the indicated antibodies. (B) HT1080 cells were transfected with NF-κB-luciferase reporter vector. After 24 hr, cells pretreated with 20 µM gefitinib for 1 hr were treated with 100 ng/mL FasL for 6 hr in the presence of 10 µM Z-VAD in order to prevent apoptosis, and then subjected to NF-κB reporter assay. Data shown are the mean ± SD. (C) HT1080 cells pretreated with 20 µM gefitinib for 1 hr were treated with 100 ng/mL FasL for the indicated periods in the presence of 10 µM Z-VAD in order to prevent apoptosis, and then cell extracts were subjected to immunoblotting with the indicated antibodies. (D)(E) A549 cells were treated with 20 µM gefitinib for the indicated periods. Cell extracts were subjected to immunoblotting with the indicated antibodies (D), and the relative expression of HSP70 was quantified using Image Lab software from Bio-Rad (E).

Although the precise mechanisms by which HSP70 inhibits Fas-mediated apoptosis remain to be clarified, gefitinib-mediated downregulation of HSP70 provides a model to explain the enhancement of Fas-mediated apoptosis by gefitinib. Moreover, how gefitinib downregulates HSP70 still remains elusive. A previous report has demonstrated that an HSP70-binding ubiquitin ligase CHIP (carboxy terminus of HSP70-binding protein) both positively and negatively regulates the HSP70 expression, and thus we speculate that gefitinib unbalances the function of CHIP in the regulation of HSP70 expression (Qian et al., 2006). Therefore, although further studies are required for understanding the details of the enhancement of Fas-mediated apoptosis by gefitinib, the present study revealed that gefitinib can increase the sensitivity to Fas-mediated apoptosis regardless of the p53 mutational status, which implies that gefitinib assists T cell-based immune intervention in p53-mutated cancer cells. Thus, our results provide insight into the strategies of Fas-based cancer therapy and immunotherapy that targets p53-mutated cancer cells.

ACKNOWLEDGMENTS

This work was supported by JSPS KAKENHI Grant Numbers JP18H02567 and JP18K06622, and by MEXT KAKENHI JP17H05518 and JP15H01168. This work was also supported by the Mitsubishi Foundation, the Shimabara Science Promotion Foundation, the Japan Foundation of Applied Enzymology, the Life Science Foundation of Japan, the Fugaku Trust for Medicinal Research, and the Takeda Science Foundation.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2019 The Japanese Society of Toxicology
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