Gemcitabine sensitizes lung cancer cells to Fas/FasL system-mediated killing

Abstract

Gemcitabine is a chemotherapy agent commonly used in the treatment of non-small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine-induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real-time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL-expressing cells was evaluated by flow cytometry, and caspase-8 and caspase-3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine-activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti-Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane-bound FasL (mFasL) and of mFasL-positive apoptotic H292 cells, as well as caspase-8 and caspase-3 cleavage. Moreover, gemcitabine increased CH11-induced caspase-8 and caspase-3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine-treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up-regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas-dependent apoptosis mediated by caspase-8 and caspase-3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing.

Introduction

Lung cancer is one of the leading causes of cancer-related death worldwide,¹ non-small cell lung cancer (NSCLC) accounting for more than 75% of all cases. The poor lung cancer prognosis argues for new effective approaches to control this disease.

Gemcitabine (2′,2′-difluorodeoxycytidine), a pyrimidine analogue of the anti-metabolite class, is one of the most commonly used chemotherapy agents in the treatment of NSCLC.^2–3 Gemcitabine exerts its anti-tumour effect mainly by incorporation of its triphosphate metabolite (dFdCTP) into DNA, leading to the arrest of DNA synthesis.⁴ However, the molecular mechanisms by which this drug causes cell death are not fully known and have not been extensively studied in lung cancer. We have previously demonstrated that gemcitabine induces apoptosis in NSCLC cells both directly and indirectly by increasing functionally active Fas (CD95, APO-1) expression.⁵

Fas receptor mediates apoptotic cell death when cross-linked by either its specific cognate ligand FasL (CD95L, CD178) or the prototypic agonistic anti-Fas monoclonal antibody (mAb) CH11, which leads to activation of the initiator caspase-8, which in turn activates the downstream effector caspases including caspase-3. Therefore, Fas and its ligand FasL are key regulators of the apoptotic extrinsic pathway as well as of one of the two main lymphocyte-mediated cytotoxicity mechanisms.^6–7

Upon anti-cancer drug treatment, an up-regulation of both Fas and FasL was observed in various tumour cell lines including Hodgkin’s lymphoma, Ewing’s sarcoma, colon cancer, small cell lung cancer, hepatoma, osteosarcoma and pancreatic cancer; triggering cell apoptosis via an autocrine/paracrine loop by Fas cross-linking.^8–12 Previous studies have also demonstrated that anti-cancer drugs increase the sensitivity of various tumour cell lines including prostate, colon, bladder, pancreas and brain cancer cells, to Fas-dependent killing by cytotoxic lymphocytes.^13–16 Although the expression and up-regulation of Fas and/or FasL by anti-cancer drugs and their role in tumour suppression are known phenomena,^17,18 the effects of gemcitabine on the Fas/FasL-system in NSCLC are largely unknown.

Therefore, on the basis of these previous findings, to elucidate the molecular mechanisms underlying the gemcitabine effect in NSCLC, the present study was aimed to evaluate for the first time in NSCLC, the involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing. For this purpose, we assessed in the NSCLC H292 (mucoepidermoid carcinoma) cell line, whether gemcitabine was able to: (i) up-regulate the expression of the FasL molecule; (ii) affect the activation of caspase-8 and caspase-3; and (iii) increase the sensitivity of tumour cells to the cytotoxic activity of lymphocytes.

Materials and methods

Cell line culture and treatment

NSCLC H292 (mucoepidermoid carcinoma) and COLO 699 (adenocarcinoma) cell lines were purchased from Interlab Cell Line Collection (Genoa, Italy). Cells were cultured in complete medium (RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin; Invitrogen, Carlsbad, CA), in the presence or absence of gemcitabine (0·05 μm for 24, 48, 72 and 96 hr; Eli Lilly, Indianapolis, IN). 16-HBE, an immortalized normal bronchial epithelial cell line, was also used in this study.²⁰ 16-HBE cells were maintained in minimal essential medium (Invitrogen), supplemented with 10% fetal bovine serum. Cell viability was assessed by trypan blue dye exclusion.

Quantitative real-time reverse transcription-PCR of FasL

Total RNA was extracted from H292, COLO 699 and 16-HBE cells with TRIzol Reagent (Invitrogen) following the manufacturer’s instructions, and was reverse-transcribed into cDNA, using Moloney-murine leukaemia virus-reverse transcriptase and oligo(dT)_12–18 primer (Invitrogen). Quantitative real-time PCR of FasL transcripts was carried out on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA) using specific FAM-labelled probe and primers (pre-validated TaqMan Gene expression assay for FasL, Hs00181225m1, Assays on Demand, Applied Biosystems). FasL gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase endogenous control gene. Relative quantification of gene expression was carried out with the comparative C_T method (2^−ΔΔCt)²¹ and was plotted as fold-change compared with untreated cells, which were chosen as the reference sample.

Western blot analysis of FasL

Total protein extracts from H292 cells were separated by SDS–PAGE on 4–12% gradient gels and then electroblotted onto nitrocellulose membranes. They were incubated with a mouse anti-human FasL primary mAb (clone G247-4; 1/250, 1 hr; BD Biosciences Pharmingen, San Diego, CA), and then with an anti-mouse horseradish peroxidase-conjugated secondary antibody (1/1000; 1 hr; Amersham Pharmacia Biotech In, Piscataway, NJ). Blot membranes were developed with an enhanced chemiluminescence system (Amersham Pharmacia Biotech) followed by autoradiography. Protein molecular masses were determined using calibrated pre-stained standards (Amersham Pharmacia Biotech), and the band optical density of autoradiographic films was analysed by densitometric scanning and the NIH Image analysis 1·61 program. Moreover, membranes were stripped and incubated with an anti-β-actin mAb (1/10 000; 1 hr; Sigma, St Louis, MO). FasL protein was normalized to β-actin housekeeping protein, and data were expressed as optical density ratio of FasL over β-actin.

Flow cytometry analysis of FasL

Expression of membrane-bound FasL (mFasL) in H292 cells was evaluated by indirect immunofluorescence using FACSCalibur (Becton Dickinson, Mountain View, CA). Unpermeabilized suspended cells were incubated with a mouse anti-human FasL mAb (clone NOK-1; 30 min, 4°; BD Biosciences Pharmingen) followed by a secondary phycoerythrin (PE) -conjugated polyclonal rabbit anti-mouse IgG antibody (Dako, Glostrup, Denmark) in the dark (30 min, 4°). Mouse immunoglobulins (Dako) were used as negative control. In some experiments, mFasL expression was evaluated in unpermeabilized normal bronchial epithelial cell line 16-HBE treated and untreated with gemcitabine (0·05 μm), for 72 hr, in the presence or absence of malignant pleural fluid (PF; 10%). Data were expressed as percentage of positive cells.

Soluble FasL assay

Supernatants were collected from H292 cell cultures. Soluble FasL (sFasL) concentration was measured using an ELISA kit (MBL International Corporation, Nagoya, Japan) according to the manufacturer’s instructions.

Analysis of apoptosis of FasL-expressing cells

FasL-expressing apoptotic H292 cells were identified by flow cytometry using a FACSCalibur (Becton Dickinson) by means of double staining with an anti-human FasL mAb (clone NOK-1; BD Biosciences Pharmingen) followed by secondary PE-conjugated polyclonal rabbit anti-mouse IgG antibody (Dako), and an Annexin V-FITC kit (Bender MedSystems,Vienna, Austria). FasL-positive apoptotic cells were characterized by high mean fluorescence intensity of both PE-FasL and FITC-Annexin V. In some experiments, FasL-expressing apoptotic cells were evaluated in H292 and COLO 699 (lung adenocarcinoma) cell lines treated and untreated with gemcitabine (0·05 μm), for 72 hr, in the presence or absence of malignant PF (10%). Data were expressed as percentage of FasL-bearing apoptotic cells.

Immunofluorescence microscopy

Double immunofluorescence staining was performed on cytospins from H292 cells cultured in the presence of gemcitabine for 72 hr, to evaluate the co-localization of FasL and Annexin V. Cells were fixed by paraformaldehyde 4% and permeabilized by saponin 0·05%. Immunostaining was performed using the mouse anti-human FasL mAb (clone NOK-1; BD Biosciences Pharmingen). Double labelling was performed using both a secondary PE-conjugated anti-mouse polyclonal rabbit IgG antibody (Sigma-Aldrich) and FITC-conjugated Annexin V (BD Biosciences Pharmingen) in the same cytospin. Anti-fading agent with DAPI (for nucleus staining) was added. Slides were analysed using a fluorescence microscope (Axioscop 2; Zeiss, Heidelberg, Germany).

Caspase-8 and caspase-3 cleavage and activity

Caspase-8 and caspase-3 cleavage was evaluated by Western blot as described above, using a mouse anti-human caspase-8 mAb and a rabbit anti-human caspase-3 polyclonal antibody, respectively (1/1000; 4°, overnight; Cell Signaling Technology, Beverly, MA). For loading control, stripped membranes were incubated with an anti-β-actin mAb (1/10 000; 1 hr; Sigma).

Capase-8 and caspase-3 proteolytic activity was measured using commercial kits (ApoAlert Caspase Colorimetric Assay Kits; Clontech, Mountain View, CA) following the manufacturer’s instructions as previously described.²² To determine Fas-mediated caspase activation, in some experiments, agonistic anti-Fas mAb (clone CH11; MBL International, Nagoya, Japan) was added to H292 cells, at 0·2 μg/ml concentration, 3 hr before their harvesting.

Generation of lymphokine-activated killer cells

Peripheral blood mononuclear cells were isolated by centrifugation on a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient, of heparinized venous blood from healthy donors (n = 6). To generate lymphokine-activated killer (LAK) cells, peripheral blood mononuclear cells (10 × 10⁶/ml) were incubated with recombinant human interleukin-2 (1000 U/ml; R&D Systems, Minneapolis, MN) for 5 days, at 37°.

Pleural fluid collection and lymphocyte isolation

Pleural fluids were collected by therapeutic thoracentesis from patients with lung cancer (n = 6, age range 45–78 years) and total lymphocytes were isolated from PFs by centrifugation on a Ficoll-Hypaque (Pharmacia) density gradient, as previously described.²³ All subjects gave informed written consent. The study was consistent with the Helsinki Declaration.

Cytotoxicity assay of LAK cells and PF lymphocytes

Cytotoxic activity of LAK cells and PF lymphocytes against H292 cells was analysed by a flow cytometry-based assay.²⁴ Briefly, target H292 cells were pre-labelled with the green fluorescent dye 3,3′-dioctadecyloxacarbocyanine (DiO₁₈) (Molecular Probes, Eugene, OR), and co-incubated with effector LAK cells or PF lymphocytes at an effector : target ratio of 5 : 1 for 16 hr at 37°. To determine Fas-mediated cytotoxicity, in some experiments, a neutralizing anti-Fas mAb (clone ZB4; MBL International) was added to target cells, at 0·5 μg/ml concentration, 2 hr before addition of effector cells. Cells were then harvested, counterstained with the red fluorescent dye propidium iodide (PI), and analysed using FACSCalibur (Becton Dickinson). Dead target H292 cells were identified by both green DiO₁₈ and red PI fluorescence. Data were expressed as percentage of dead target cells.

Statistical analysis

Data were expressed as means ± SD and analysed by unpaired Student’s t-test or analysis of variance followed by Bonferroni Dunn’s post-hoc test. A P value < 0·05 was considered statistically significant.

Results

Gemcitabine increases FasL expression in H292 cells

Gemcitabine increases Fas expression in H292 cells⁵ and anticancer drugs increase FasL expression in Fas-expressing tumour cells.^8–12 Therefore, we first evaluated the gemcitabine ability to up-regulate FasL expression in H292 cells both at the mRNA level and the protein level.

Gemcitabine significantly increased FasL mRNA expression in H292 cells at all time-points, reaching its maximum effect at 72 hr (P < 0·0001) (Fig. 1a). At this last time-point, gemcitabine significantly increased FasL mRNA expression in COLO 699 and 16-HBE cells also (P < 0·0001 and P = 0·006, respectively) (Fig. 1b). In parallel, by Western blot, distinct bands of expected size (mol. wt 40 000) for FasL protein were detected both in untreated and in gemcitabine-treated H292 cells, at all time-points (Fig. 1c). Densitometric analysis of the bands revealed a significant increase of the optical density ratio mean value of the FasL over β-actin, in gemcitabine-treated H292 cells compared with untreated H292 cells (baseline), after 48 and 72 hr (P = 0·001 and P = 0·007, respectively) (Fig. 1d).

Figure 1.

Gemcitabine increases Fas ligand (FasL) expression in H292 cells. H292 cells were cultured with and without gemcitabine (0·05 μm) for 24, 48, 72 and 96 hr; COLO 699 and 16-HBE cells for 72 hr. (a, b) Total RNA was extracted and used for assessing FasL mRNA expression by real-time PCR (see the Materials and methods section for details). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Relative quantification of mRNA was carried out with the comparative C_T method. Data are shown as fold increase over untreated cells (baseline), at each time-point. (c) Representative Western blot showing the expression of FasL total protein at baseline level (lanes 1) and upon gemcitabine exposure (lanes 2), at the indicated time-points. β-Actin was included for normalization. (d) Densitometric analysis results expressed as ratio of FasL over β-actin. All data are shown as means ± SD. P-values (inside the figures) represent the results of unpaired Student’s t-test (n = 3).

Gemcitabine increases the cell surface expression of FasL in H292 cells

As FasL may exist in either membrane-bound (mFasL) or soluble (sFasL) form, we evaluated mFasL expression in unpermeabilized cells and sFasL levels in culture supernatants of H292 cells, by flow cytometry and ELISA, respectively. Gemcitabine significantly increased the percentage of mFasL-bearing H292 cells after 72 hr (P < 0·0001) (Fig. 2). The sFasL was not detectable in basal conditions or in gemcitabine-treated cells at all time-points (data not shown). Moreover, to further define FasL expression in H292 cells, flow cytometry analysis was performed in permeabilized cells to determine intracellular FasL. A very low percentage of H292 cells expressed intracellular FasL both in basal condition and after gemcitabine treatment at all time-points (data not shown).

Figure 2.

Gemcitabine increases the cell surface expression of Fas ligand (FasL) in H292 cells. H292 cells were cultured with and without gemcitabine (0·05 μm) for 24, 48, 72 and 96 hr. FasL expression was assessed in unpermeabilized cells by flow cytometry using a primary anti-FasL monoclonal antibody and a secondary phycoerythrin-conjugated antibody (see the Materials and methods for details). (a) Data are shown as percentage of positive cells (mean ± SD). P-value (inside the figure) represents the result of unpaired Student’s t-test (n = 3). (b) Representative histogram (overlay of FL2 fluorescence intensity) related to cell surface expression of FasL after 72 hr of cell exposure to gemcitabine. Open curve 0, negative control; closed curve 1, baseline; open curve 2, gem 0·05 μm.

Gemcitabine induces apoptosis of mFasL-expressing H292 cells

To determine whether mFasL expression was associated with cell apoptosis, we analysed by flow cytometry the apoptosis of H292 cells expressing mFasL, in the presence and absence of gemcitabine. Gemcitabine significantly increased the percentage of mFasL-bearing apoptotic cells after 72 hr (P = 0·002) (Fig. 3a). Moreover, gemcitabine induces mFasL expression also in Annexin V-negative cells, i.e. live cells (P = 0·01, P = 0·002, P = 0·04 at 48, 72 and 96 hr, respectively). (Fig. 3b). The exposure to gemcitabine reduced the total number of cells (Table 1).

Figure 3.

Gemcitabine induces apoptosis of membrane-bound Fas ligand (mFasL) -expressing H292 cells. H292 cells were cultured with and without gemcitabine (0·05 μm) for 24, 48, 72, 96 and 120 hr. The mFasL-expressing apoptotic and live cells were identified by flow cytometry using a primary anti-FasL monoclonal antibody (mAb) followed by a secondary phycoerythrin (PE) -conjugated antibody, and FITC-conjugated Annexin V (see the Materials and methods for details). (a, b) Data are shown as percentage of mFasL-bearing apoptotic and live cells, respectively (mean ± SD). P-values (inside the figures) represent the results of unpaired Student’s t-test (n = 3). (c) Representative dot plots showing mFasL-expressing apoptotic cells in the upper right quadrant (PE-FasL and FITC-Annexin V positive), FasL-expressing viable cells in the upper left quadrant (PE-FasL positive and Annexin V negative), apoptotic cells in the lower right quadrant (FITC-Annexin V positive and PE-FasL negative), and viable cells in the lower left quadrant (double negative). (d) Immunofluorescence analysis in H292 cells treated with gemcitabine for 72 hr, showing the co-localization (yellow/orange overlay image) of FasL (red) and Annexin V (green). Blue nuclear staining by DAPI. Original magnification: × 400. Representative experiment.

Table 1.

Effect of gemcitabine (Gem) on the number and viability of non-small-cell lung cancer H292 cells

Expt conditions	Time of incubation	Cell number (× 106)	Cell viability (%)
Baseline	24 hr	1·23 ± 0·25	96 ± 3·20
Gem (0·05 μm)	24 hr	0·83 ± 0·42	95 ± 3·30
Baseline	48 hr	1·80 ± 0·35	95 ± 0·58
Gem (0·05 μm)	48 hr	0·63 ± 0·35	93 ± 3·20
Baseline	72 hr	2·22 ± 0·83	94 ± 2·12
Gem (0·05 μm)	72 hr	0·48 ± 0·31	91 ± 2·94
Baseline	96 hr	3·40 ± 1·00	94 ± 3·20
Gem (0·05 μm)	96 hr	0·45 ± 0·22	82 ± 9·73
Baseline	120 hr	2·25 ± 0·33	84 ± 0·92
Gem (0·05 μm)	120 hr	0·35 ± 0·28	81 ± 0·68

Data are expressed as mean ± SD of three experiments.

Figure 3(d) shows a double immunofluorescence analysis from H292 cells treated with gemcitabine for 72 hr, demonstrating the co-localization of FasL and Annexin V in the majority of cells.

Gemcitabine induces apoptosis of mFasL-expressing H292 and COLO699 cells and enhances mFasL expression in 16HBE cells in the presence of malignant PF

As malignant PFs are protective for cancer cells,^23–25 we investigated whether gemcitabine affected Fas/FasL system-mediated lung cancer cell apoptosis in the presence of malignant PF. We found that also in the presence of malignant PF, gemcitabine increased the apoptosis of both H292 and COLO699 cells bearing mFasL. As expected, COLO 699 cells showed a lower sensitivity to gemcitabine effect when compared with H292 cells, because COLO 699 cells are characterized by a high-grade of malignancy (Fig. 4a,b). Moreover, we found that gemcitabine increased FasL expression in 16-HBE cells both in the presence and the absence of malignant PF (Fig. 4c).

Figure 4.

Gemcitabine induces apoptosis of membrane-bound Fas ligand (mFasL) -expressing H292 and COLO699 cells and enhances mFasL expression in 16HBE cells in the presence of malignant pleural fluid (PF). H292, COLO699 and 16HBE cells were cultured with and without gemcitabine (0·05 μm) for 72 hr, in the presence or absence of malignant PF (10%). FasL-expressing apoptotic cells were identified by flow cytometry using a primary anti-FasL monoclonal antibody (mAb) followed by a secondary phycoerythrin-conjugated antibody, and FITC-conjugated Annexin V (see the Materials and methods section for details). Expression of mFasL was assessed in unpermeabilized 16HBE cells by flow cytometry using a primary anti-FasL mAb and a secondary PE-conjugated antibody (see the Materials and methods section for details). (a) and (b) Dot plots from representative experiments (n = 2) in H292 and COLO699, respectively, cells showing mFasL-expressing apoptotic cells in the upper right quadrant (double-positive). (c) Histograms (FL2 fluorescence intensity) related to mFasL expression in 16HBE cells, from representative experiments (n = 2).

Gemcitabine induces caspase-8 and caspase-3 cleavage in H292 cells

To further investigate the ability of gemcitabine to induce cell apoptosis by Fas/FasL pathway, we analysed its ability to activate caspase-8 and caspase-3, because these caspases are effector molecules in the Fas/FasL death signalling pathway. We assessed by Western blot the cleavage of caspase-8 and caspase-3 in H292 cells treated and untreated with gemcitabine. Caspase-8 and caspase-3 were clearly cleaved in H292 cells after 48 and 72 hr of cell exposure to gemcitabine, as shown by the caspase-8 active fragment (mol. wt 18 000) and caspase-3 active fragments (mol. wt 17 000 and 12 000), respectively (Fig. 5).

Figure 5.

Gemcitabine induces caspase-8 and caspase-3 cleavage in H292 cells. H292 cells were cultured with and without gemcitabine (0·05 μm) for 24, 48, 72 and 96 hr. Representative Western blots of three independent experiments performed for caspase-8 and caspase-3 status. With respect to caspase-8, the upper band (57 000) represents the full-length caspase-8; the two intermediate bands (43 000 and 41 000) correspond to the cleavage intermediate products of activated caspase-8; the lower band (18 000) represents the caspase-8 active fragment. With respect to caspase-3, the upper band (35 000) represents the full-length caspase-3; the lower bands (17 000 and 12 000) correspond to caspase-3 active fragments resulting from cleavage. β-Actin was included as a control for protein loading. Lanes 1, baseline; lanes 2, gemcitabine 0·05 μm.

Gemcitabine increases CH11-mediated caspase-8 and caspase-3 activation in H292 and COLO 699 cells

Gemcitabine increases apoptosis of H292 cells following the binding of Fas receptor by CH11 mAb.⁵ To verify whether this Fas/FasL pathway triggering was associated with activation of the caspase-8/caspase-3 axis, we reproduced the effect of FasL on Fas activation using an agonist of Fas (CH11) and analysed the ability of gemcitabine to affect the caspase-8 and caspase-3 activation following Fas binding by CH11. Caspase-8 and caspase-3 were clearly cleaved in the presence of CH11 mAb, alone or combined with gemcitabine, in H292 cells (Fig. 6a,c) as shown by the caspase-8 active fragment (mol. wt 18 000) and caspase-3 active fragments (mol. wt 17 000 and 12 000), respectively. Caspase-8 and caspase-3 were clearly cleaved in COLO 699 cells after cell exposure to gemcitabine and CH11 mAb, alone or in combination, as shown by the caspase-8 active fragment (mol. wt 18 000) and caspase-3 active fragments (mol. wt 17 000 and 12 000), respectively (Fig. 7a). Gemcitabine and CH11 mAb, alone or in combination, significantly increased caspase-8 proteolytic activity (P = 0·004, P = 0·0002, P < 0·0001, respectively) in H292 cells (Fig. 6b). Gemcitabine alone or combined with CH11 mAb significantly increased caspase-3 proteolytic activity (P = 0·0008 and P < 0·0001, respectively) in H292 cells (Fig. 6d). Moreover, gemcitabine and CH11 mAb, alone or in combination, significantly increased caspase-8 proteolytic activity (P = 0·001, P = 0·001, P = 0·0005, respectively) and caspase-3 proteolytic activity (P = 0·006, P = 0·007, P = 0·007, respectively) in COLO 699 cells (Fig. 7b). Importantly, gemcitabine acted synergistically with CH11 mAb to induce caspase-8 and caspase-3 activation both in H292 cells (Fig. 6b,d) and in COLO 699 cells (Fig. 7b). Finally, gemcitabine and CH11 mAb, alone or in combination, neither cleaved caspase-8 and caspase-3 nor increased their proteolytic activity in 16-HBE cells (data not shown).

Figure 6.

Gemcitabine increases CH11-mediated caspase-8 and caspase-3 activation in H292 cells. H292 cells were cultured with and without gemcitabine (0·05 μm) for 48 hr and exposed to CH11 monoclonal anitbody (mAb; 0·2 μg/ml) 3 hr before their harvesting. (a, c) Caspase-8 and caspase-3 status was analysed by Western blot in comparison to untreated cells (baseline). In (a), the upper band (57 000) represents the full-length caspase-8; the two intermediate bands (43 000 and 41 000) correspond to the cleavage intermediate products of activated caspase-8; the lower band (18 000) represents the caspase-8 active fragment. In (c) the upper band (35 000) represents the full-length caspase-3; the lower bands (17 000 and 12 000) correspond to caspase-3 active fragments resulting from cleavage. β-Actin was included as a control for protein loading. Representative Western blots of three independent experiments. (b, d) Caspase-8 and-3 proteolytic activity was assessed using IETD-pNA and DEVD-pNA respectively, as a substrate. Colorimetric quantification of protease activity was performed using a spectrophotometer at 405 nm. The irreversible inhibitors IETD-fmk and DEVD-fmk for caspase-8 and caspase-3, respectively, were used for investigating the colorimetric assay specificity. Data are shown as protease activity versus untreated cells (baseline) (mean ± SD). Overall comparison was made by analysis of variance (P < 0·0001). P values (inside the figures) represent the results of Bonferroni Dunn’s post-hoc test (n = 5).

Figure 7.

Gemcitabine increases CH11-mediated caspase-8 and caspase-3 activation in COLO 699 cells. COLO 699 cells were cultured with and without gemcitabine (0·05 μm) for 72 hr and exposed to CH11 monoclonal antibody (mAb; 0·2 μg/ml) 3 hr before their harvesting. (a) Caspase-8 and caspase-3 status was analysed by Western blot in comparison to untreated cells (baseline). At the top, the upper band (57 000) represents the full-length caspase-8; the intermediate band (43 000) corresponds to the cleavage intermediate product of activated caspase-8; the lower band (18 000) represents the caspase-8 active fragment. In the centre, the upper band (35 000) represents the full-length caspase-3; the lower bands (17 000 and 12 000) correspond to caspase-3 active fragments resulting from cleavage. At the bottom, β-actin was included as a control for protein loading. Representative Western blots of three independent experiments. (b) Caspase-8 and caspase-3 proteolytic activity was assessed using IETD-pNA and DEVD-pNA respectively, as a substrate. Colorimetric quantification of protease activity was performed using a spectrophotometer at 405 nm. The irreversible inhibitors IETD-fmk and DEVD-fmk for caspase-8 and caspase-3 respectively, were used for investigating the colorimetric assay specificity. Data are shown as protease activity versus untreated cells (baseline) (mean ± SD). P-values (inside the figures) represent the results of unpaired Student’s t-test (n = 3).

Gemcitabine enhances the H292 cell sensitivity to Fas-mediated cytotoxic activity of LAK cells

Gemcitabine increases Fas receptor expression in H292 cells⁵ and anti-cancer drugs increase the sensitivity of tumour cells to Fas-mediated killing by cytotoxic lymphocytes.^13–16

To verify whether increased Fas expression in response to treatment with gemcitabine was associated with tumour cell killing by cytotoxic lymphocytes, we assessed the cytotoxic activity of LAK cells against H292 cells treated and untreated with gemcitabine.

LAK cells effectively killed untreated H292 cells (dead target cell mean percentage 60%). The cytotoxic effect of LAK cells was significantly increased when H292 cells were pre-treated with gemcitabine (dead target cell mean percentage 95%; P < 0·0001) (Fig. 8).

Figure 8.

Gemcitabine enhances the H292 cell sensitivity to Fas-mediated cytotoxic activity of lymphokine-activated killer (LAK) cells. H292 cells were cultured with and without gemcitabine (0·05 μm) for 48 hr and exposed to ZB4 monoclonal antibody (mAb; 0·5 μg/ml) 2 hr before addition of effector cells. LAK cells were obtained from healthy donors (n = 6) and a flow cytometry-based cytotoxic assay against H292 cells was performed (see the Materials and methods for details). (a) Data are shown as percentage of dead target cells (mean ± SD). Overall comparison was made by analysis of variance (P < 0·0001). P-values (inside the figure) represent the results of Bonferroni Dunn’s post hoc test. (b) Representative dot plots showing dead H292 cells (DiO₁₈ and PI positive) in the upper right quadrant.

Gemcitabine effectively killed H292 cells (dead target cell mean percentage 35%; P < 0·0001 gemcitabine-treated H292 cells versus untreated H292 cells). LAK cells significantly increase the percentage of dead gemcitabine-treated H292 cells (dead target cell mean percentage 95%; P < 0·0001; LAK + gemcitabine-treated H292 cells versus gemcitabine-treated H292 cells).

To assess the role of the Fas/FasL pathway in H292 cell killing by LAK cells, in some experiments gemcitabine-treated H292 cells were incubated with the anti-Fas ZB4 mAb, which inhibits the interaction between Fas present on H292 cells and FasL present on LAK cells. Fas neutralization significantly reduced the percentage of dead target cells (mean percentage 64%; P < 0·0001) (Fig. 8).

In addition, to verify whether enhanced Fas-dependent cytotoxicity of LAK cells was due to an increased expression of FasL on their surface following co-incubation with gemcitabine-treated H292 cells, we analysed FasL expression in LAK cells, by flow cytometry. Freshly isolated LAK cells expressed FasL. When co-incubated with either untreated or gemcitabine-treated H292 cells, they showed a slight increase of FasL expression both in presence and absence of gemcitabine (data not shown).

On the other hand, as the tumour cells may use FasL to kill cytotoxic lymphocytes, we assessed the cytotoxic activity of both gemcitabine-treated and untreated H292 cells against LAK cells. We found that the percentages of dead LAK cells were much lower than those of dead H292 cells (dead LAK cell mean percentage 8% in absence of gemcitabine, P < 0·0001 LAK cells versus H292 cells; dead LAK cell mean percentage 9%, in presence of gemcitabine, P < 0·0001 LAK cells versus H292 cells).

Gemcitabine enhances the H292 cell sensitivity to Fas-mediated cytotoxic activity of malignant PF lymphocytes

Finally, as lymphocytes from malignant PFs show a reduced cytotoxic activity,²³ we investigated the ability of gemcitabine to affect the H292 cell sensitivity to cytotoxic activity of these lymphocytes. As expected, malignant PF lymphocytes showed a lower cytotoxic activity against H292 cells when compared with LAK cells (dead target cell mean percentage 24%; P < 0·005 PF lymphocytes versus LAK cells) but, interestingly, also these cells showed a significantly increased cytotoxic activity against gemcitabine-treated H292 cells (dead target cell mean percentage 66%; P < 0·0001) (Fig. 9). Gemcitabine effectively killed H292 cells (dead target cell mean percentage 42%; P < 0·0004 gemcitabine-treated H292 cells versus untreated H292 cells). PF lymphocytes significantly increase the percentage of dead gemcitabine-treated H292 cells (dead target cell mean percentage 66%; P = 0·02; PF lymphocytes + gemcitabine-treated H292 cells versus gemcitabine-treated H292 cells). Moreover, anti-Fas ZB4 mAb addition significantly reduced the malignant PF-lymphocyte cytotoxic activity against gemcitabine-treated H292 cells (dead target cell mean percentage 46%; P = 0·01) (Fig. 9).

Figure 9.

Gemcitabine enhances the H292 cell sensitivity to Fas-mediated cytotoxic activity of malignant pleural fluid (PF) lymphocytes. H292 cells were cultured with and without gemcitabine (0·05 μm) for 48 hr and exposed to ZB4 monoclonal antibody (mAb) (0·5 μg/ml) 2 hr before addition of effector cells. PF lymphocytes were obtained from patients with lung cancer (n = 6) and a flow cytometry-based cytotoxic assay against H292 cells was performed (see the Materials and methods for details). (a) Data are shown as percentage of dead target cells (mean ± SD). Overall comparison was made by analysis of variance (P < 0·0001). P-values (inside the figure) represent the results of Bonferroni Dunn’s post hoc test. (b) Representative dot plots showing dead H292 cells (DiO₁₈ and PI-positive) in the upper right quadrant.

In addition, freshly isolated PF lymphocytes expressed FasL. When co-incubated with either untreated or gemcitabine-treated H292 cells, they showed a slight increase of FasL expression both in the presence and absence of gemcitabine (data not shown).

Moreover, the percentages of dead PF lymphocytes were much lower than those of dead H292 cells, both treated and untreated with gemcitabine (dead PF lymphocyte mean percentage 6%, in absence of gemcitabine, P < 0·001 PF lymphocytes versus H292 cells; dead PF lymphocyte mean percentage 8·5%, in presence of gemcitabine, P < 0·0001 PF lymphocytes versus H292 cells).

Discussion

Fas and FasL expression as well as their role in anti-cancer drug-induced apoptosis has been demonstrated in a number of cell lines from haematological malignancies and solid tumours.^8–12

In a previous study, we found that gemcitabine increased the expression of functionally active Fas in three different NSCLC cell lines (H292 mucoepidermoid carcinoma, Colo699 adenocarcinoma and CorL23 large-cell carcinoma).⁵ In the present study, we demonstrate that gemcitabine increases the expression of FasL in H292 cells, leading to an autocrine and/or paracrine death mechanism involving the Fas/FasL system-mediated caspase-8 and caspase-3 activation.

We previously found that H292 cells are more sensitive to gemcitabine effects than either Colo699 or CorL23,⁵ for this reason we selected these cells as a model in the present study.

In addition, we have previously demonstrated that 0·05 μm gemcitabine concentration was the most effective concentration in up-regulating Fas and in inducing cell apoptosis among the tested concentrations (ranging from 0·5 to 0·005 μm),⁵ therefore the present experiments were performed using this drug concentration.

In this study we show that gemcitabine increases total FasL expression in NSCLC H292 cells at protein level and also at mRNA level. This indicates that gemcitabine up-regulates FasL by enhancing its transcription, i.e. by de novo gene expression. On the other hand, FasL protein may exist as either membrane-bound or soluble form. The latter is released from the cell surface following cleavage by matrix metalloproteinases (MMPs) including MMP-7 and a disintegrin and metalloproteinase protein 10 (ADAM-10). Previous studies demonstrated that synthetic MMP inhibitors may directly induce cancer cell apoptosis by inhibiting FasL shedding,²⁶ and that in primary human T cells, ADAM-10 inhibition increases T-cell cytotoxic activity and lymphocyte activation-induced cell death (AICD) by reducing FasL shedding, consequently increasing its presence on the cell surface.²⁷ In the present study we found that gemcitabine-induced FasL is almost completely in the membrane-bound form, and the majority of mFasL-bearing H292 cells undergo apoptosis. These findings suggest that the gemcitabine-induced suicide and/or fratricide cell death could be secondary to the accumulation of FasL on the tumour cell surface. These concepts are supported by immunofluorescence analyses demonstrating the co-localization of FasL and Annexin V in the majority of gemcitabine treated H292 cells.

This death scenario reflects the AICD that occurs in activated T cells following T-cell receptor stimulation.²⁸ Membrane-bound FasL, but not sFasL, is essential for AICD as well as for T-cell-mediated cytotoxicity.²⁹ The mFasL is much more potent than sFasL in promoting cell apoptosis and is the most effective activator of Fas in vivo.^26,27 It is therefore possible that gemcitabine, promoting MMP inactivation in some way, induces FasL accumulation on the surface of H292 cells, sensitizing them to apoptosis.

Moreover, gemcitabine induces mFasL expression in both Annexin V-positive and Annexin V negative cells, i.e. dead and live cells. This further supports the ability of gemcitabine to up-regulate mFasL in lung cancer cells. The exposure to gemcitabine reduced the total number of cells. It is conceivable that this effect might be related to both cell proliferation arrest and death.

In agreement with our previous findings,⁵ Gordon and Kleinerman¹⁹ demonstrated in the mice that gemcitabine delivered by aerosol up-regulates Fas expression on the cell surface of osteosarcoma cells and induces the regression of lung metastases by the FasL constitutively expressed on the lung epithelium, indicating that the lung microenvironment is an important contributor to the metastatic potential of osteosarcoma cells. In the present study, we demonstrated that gemcitabine increases the apoptosis of mFasL-bearing lung cancer cells also in the presence of malignant PF. This finding suggests that the anti-cancer activity of gemcitabine persists within a microenvironment promoting protection and growth of cancer cells, like malignant PF,^23–25 and further supports its therapeutic efficacy. In addition, we demonstrated that gemcitabine increases FasL mRNA, and mFasL expression in 16-HBE normal bronchial epithelial cells in both the presence and absence of malignant PF. This finding suggests that an additional mechanism by which gemcitabine induces lung cancer cell killing may be mediated by the enhanced ability of the resident lung cells to eliminate cancer cells by the Fas/FasL pathway, even in the presence of a cancer-protective milieu, like malignant PF.

Apoptosis depends on activation of caspases. The initiator caspase-8 and the effector caspase-3 are cysteine proteases involved in Fas-mediated apoptosis. In this study, we show that gemcitabine induces Fas-mediated H292 and COLO 699 cell apoptosis via caspase-8 and caspase-3 activation, acting synergistically with the CH-11 agonistic anti-Fas mAb. CH-11 induces apoptosis in cell lines with a functionally active Fas.⁵ Previously, other authors observed caspase-8 activation irrespective of whether or not chemotherapy-induced apoptosis was dependent on the Fas activation.^10–32 These different results are due to the fact that different mechanisms beyond Fas-activation, can contribute to caspase-8/caspase-3 axis activation, probably in stimulus and/or cell type-dependent manner. For instance, caspase-8, and in turn caspase-3, can be activated by other death receptors, including DR5,³⁰ or by mitochondrial amplification of the apoptotic signal,³¹ as well as through autophagy.³² Conversely, Fas can trigger cell death in the absence of active caspases.³³

Our results indicate that gemcitabine induces in NSCLC H292 and COLO 699 cell lines, a full activation of an intact Fas death signalling pathway, wherein caspase-8 and caspase-3 form an integral part by functioning as death effector molecules. By contrast, no caspase-8 and caspase-3 activation by gemcitabine was found in 16-HBE cells. In line with the findings of Kleinerman and colleagues,^19–34 these results suggest that gemcitabine has no toxic effect on lung-resident cells.

A host immune system-mediated anti-tumour activity has been shown in almost all types of cancer, as well as the immune potential of some chemotherapy agents. As the immune cells including cytotoxic T lymphocytes (CTL), natural killer (NK) and LAK cells, are capable of inducing the tumour cell death via the interaction of FasL on their surface with Fas receptor on the surface of tumour target cells, the relationship between the gemcitabine-induced Fas expression⁵ and the immune clearance of lung cancer cells was explored.

Most solid tumours are characterized by infiltration of lymphocytes, the so-called tumour-infiltrating lymphocytes (TILs), that are composed of CTLs, NK and LAK cells, and sometimes, also of regulatory T cells. TILs are frequently not able to kill autologous tumour cells. Accordingly, NK cells infiltrating human NSCLC exhibit a reduced ability to kill tumour cells.³⁵

Specific cancers including lung or breast cancer, cancer of the ovary and stomach metastasize the pleural compartment where specific subsets of tumour-associated lymphocytes, regarded like TILs, accumulate. CD8⁺ CTLs and NK cells from malignant PFs show high intracellular expression of CD94/NKG2A, undetectable perforin expression, and exhibit a reduced ability to kill tumour cells.²³

In this study, we show that gemcitabine enhances the sensitivity of NSCLC H292 cells to cytotoxic activity of LAK cells from healthy donors as well as of PF lymphocytes from patients with lung cancer, in spite of malignant PF lymphocytes showing a reduced cytotoxic activity against tumour cells. This effect is, at least in part, mediated by the Fas/FasL pathway, since the Fas neutralization partially reverses it.

In addition, as enhanced Fas-dependent cytotoxicity of immune cells could be the result of an increased expression of FasL on immune cells co-incubated with anti-cancer drug-treated tumour cells, we analysed the FasL expression in LAK cells and in PF lymphocytes. Freshly isolated LAK cells and PF lymphocytes expressed FasL. When co-incubated with either untreated or gemcitabine-treated H292 cells, they showed a slight increase in FasL expression, independently of the H292 cell treatment (data not shown). These results indicate that enhanced Fas-dependent cytotoxicity of LAK cells and PF lymphocytes against gemcitabine-treated NSCLC H292 cells was not due to an increased FasL expression on their surface, so ruling out any direct effects of gemcitabine on immune effectors.

On the other hand, FasL expression in tumour cells may provide an escape mechanism from immune surveillance, by triggering the death of Fas-expressing immune cells. Fas/FasL pathway may either contribute to immune cell-mediated and anti-cancer drug-induced tumour cell killing or protect the tumour cells from immune clearance. We found here low percentages of dead LAK cells and PF lymphocytes and much higher percentages of dead tumour cells. This suggests that gemcitabine is able to strengthen the cytotoxicity due to the activation of Fas/FasL pathway much more in tumour cells than in immune effector cells.

Our results are in line with previous results on the ability of some chemotherapy agents to sensitize various tumour cell lines to Fas-mediated cytotoxicity by LAK cells, NKT cells, CTLs^13–16 and by TILs from renal and prostate cancers.³⁶ Moreover, these results support the immune potential of gemcitabine that could be exploited with immunotherapy to enhance immune clearance of lung cancer cells.

Taken together, the results of this study demonstrate, for the first time in NSCLC, that gemcitabine aside from its direct cytotoxic effect as a nucleoside analogue, also exhibits an indirect cytotoxic effect by preparing lung cancer cells for a suicide/fratricide death as well as to be eliminated by the host immune cells via a Fas/FasL-dependent pathway.

In conclusion, the Fas/FasL system may mediate gemcitabine-induced lung cancer cell killing, indicating an active role of Fas and FasL molecules, key regulators of apoptosis and the immune system, during gemcitabine treatment of lung cancer. New strategies might be developed to improve the efficacy of gemcitabine.

Glossary

AICD

activation-induced cell death

CTLs

cytotoxic T lymphocytes

FasL

Fas ligand

LAK

lymphokine-activated killer

mAb

monoclonal antibody

mFasL

membrane-bound Fas L

MMPs

matrix metalloproteinases

NK

natural killer

NSCLC

non-small cell lung cancer

PE

phycoerythrin

PF

pleural fluid

sFasL

soluble FasL

TILs

tumour-infiltrating lymphocytes

Disclosures

The authors have no financial disclosures or competing interests.