Affiliation of authors: Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD.
Correspondence to present address: Nicholas Mitsiades, M.D., Massachusetts General Hospital, Molecular Pathology Unit, Molecular Pathology Unit, 149 13th St., 7th Floor, Charlestown, MA 02129 (e-mail: mitsiade{at}helix.mgh.harvard.edu).
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ABSTRACT |
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INTRODUCTION |
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We have recently shown that a high percentage of the Ewing's sarcoma family tumors express Fas and biologically active tm-FasL and s-FasL. We have also shown that the shedding of s-FasL into the medium decreases substantially with MMPI treatment (28). Because co-expression of Fas and tm-FasL by Fas-sensitive tumor cells would lead to their death, we hypothesized that they may use the cleavage process to decrease their surface tm-FasL (29,30). If this hypothesis is correct, then reversing the shedding of s-FasL with an MMPI would result in accumulation of tm-FasL and cell death. We investigated this hypothesis by treating Fas-sensitive and Fas-resistant Ewing's sarcoma family tumor/neuroblastoma cell lines with synthetic MMPIs.
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MATERIALS AND METHODS |
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The Ewing's sarcoma family tumor cell lines SK-N-MC (American Type Culture Collection, Manassas, VA), TC-268 (31), and the two clones (CHP-100S and CHP-100L) of the CHP-100 cell line (32) that possess the characteristic for Ewing's sarcoma family tumors, i.e., a t(11;22) (q24;q12) translocation and the resulting EWS/Fli-1 fusion gene product, as well as the neuroblastoma IMR-32 cell line (American Type Culture Collection) were used in this study. All Ewing's sarcoma family tumor cell lines (28) and the IMR-32 neuroblastoma cell line (Poulaki V, Mitsiades N, Tsokos M: unpublished data) express FasL. Two of them (SK-N-MC and CHP-100L) are Fas sensitive, and three (CHP-100S, TC-268, and IMR-32) are Fas resistant [(33); Poulaki V, Mitsiades N, Tsokos M: unpublished data]. All cells were grown in Dulbecco's modified Eagle medium (DMEM) (BioWhittaker, Inc., Walkersville, MD) with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal calf serum (FCS) (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD), unless stated otherwise.
The MMPIs BB-3103 and A-151011 were gifts from British Biotech Pharmaceuticals Ltd. (Oxford, U.K.) and Abbott Laboratories (Abbott Park, IL), respectively. The anti-Fas antibodies CH11 and ZB4, which stimulate and inhibit Fas activity, respectively, were purchased from Panvera (Madison, WI). The isotype-matched antibody used in the ZB4 experiments as a negative control was purchased from Pharmingen (San Diego, CA). Normal immunoglobulin G (IgG)-free horse serum was purchased from Life Technologies, Inc.
Cell Survival Assay
Cell survival was estimated colorimetrically with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO) assay in all five cell lines. Cells were plated in 24-well plates and grown for 48 hours in DMEM with 10% FCS. After being washed in Hanks' balanced salt solution, the cells were treated for 48 hours with 10, 20, or 40 µM of either MMPI in DMEM supplemented with 0.05% calf serum. Subsequently, the cells were incubated with 1 mg/mL MTT in fresh medium for 4 hours at 37 °C and solubilized in a mixture of isopropanol and 1 N HCl (23 : 2, vol/vol). Dye absorbance (A) was measured with a microplate reader at 570 nm, with the use of a reference wavelength of 630 nm. Cell death was estimated with the following formula: % specific death = [A(untreated cells) A(treated cells)/A(untreated cells)] x 100%.
The MMPI-induced cell death over time was studied in the SK-N-MC cell line. Cells were grown in 24-well plates and treated with 10 µM BB-3103 for 1272 hours. Cell death was estimated with the MTT assay as above.
Generation and MMPI Treatment of a Fas-Resistant SK-N-MC Clone
The Fas-sensitive SK-N-MC cell line was incubated in medium containing 500 ng/mL of the Fas-activating antibody CH11 for 2 days and then in antibody-free medium for a week. This treatment was repeated in cycles for 1 year, leading to the isolation of a Fas-resistant clone. The levels of Fas and FasL in this clone were found to be comparable to those of the parental cell line (data not shown). This clone was treated with BB-3103 as described above.
Apoptosis Detection Assays
Two cell lines, one Fas sensitive (SK-N-MC) and one Fas resistant (TC-268), were further analyzed for apoptosis by the following methods:
(a) DNA ladder. Nuclear DNA from MMPI-treated and control cells was isolated with the apoptotic DNA ladder kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) following the instructions of the manufacturer. Five micrograms of DNA was subjected to electrophoresis in a 2% agarose gel, stained with ethidium bromide, and viewed with a UV illuminator.
(b) Cellular DNA fragmentation enzyme-linked immunosorbent assay (ELISA). This nonradioactive method used the DNA fragmentation ELISA kit (Boehringer Mannheim Biochemicals). Cells were labeled overnight with 5'-bromo-2'-deoxyuridine (BrdU) according to the manufacturer's instructions and subsequently treated as indicated. The amount of fragmented DNA was used as an estimate of the frequency of apoptosis among the target cells. DNA was quantified by measurement of the incorporation of BrdU, according to the manufacturer's instructions. The results were expressed as percentages of the value of control cells.
(c) Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate end-labeling (TUNEL) method. Air-dried cell cytospin preparations from cells treated as indicated were labeled with the use of the in situ cell death fluorescence kit (Boehringer Mannheim Biochemicals) following the instructions of the manufacturer and were viewed with a Zeiss standard fluorescence microscope equipped with an epifluorescence illuminator and fluorescein isothiocyanate narrow-band filter.
(d) Electron microscopy. Cells were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.4) for 24 hours, post-fixed in OsO4, and embedded in Maraglas 655 (Ladd Research Industries, Burlington, VT). Sections were stained with uranyl acetate and lead citrate and examined in a Philips CM10 electron microscope.
Effect of a Fas-Neutralizing Antibody on MMPI-Induced Apoptosis
To study the potential involvement of the Fas/FasL pathway in MMPI-induced apoptosis, we treated SK-N-MC cells with BB-3103 (10 and 20 µM) for 48 hours, in the presence or absence of 5 µg/mL of the Fas-neutralizing antibody ZB4 or an isotype-matched control antibody. The ZB4 or control antibodies were applied 1 hour before BB-3103. Cell death was evaluated with MTT as described above.
Detection of FasL and Fas on the Cell Surface
SK-N-MC and TC-268 cells that were selected for the apoptosis assays were also studied for levels of expression of Fas and FasL. The cells were cultured in 80-cm2 flasks for 2 days and incubated with 20 µM BB-3103 overnight. Subsequently, the cells were biotinylated by incubation in 0.5 mg/mL sulfosuccinimidyl-6-(biotinamido)hexanoate (Sulfo-NHS-LC-Biotin) (Pierce Chemical Co., Rockford, IL) in phosphate-buffered saline (PBS) for 30 minutes at room temperature as previously described (5). Sulfo-NHS-LC-Biotin does not cross the cell membrane because of its negative charge, ensuring that intracellular proteins are not biotinylated. Then the cells were washed three times in cold PBS, scraped, centrifuged briefly at 5000g for 10 minutes at 4 °C, and lysed for 30 minutes on ice in a lysis buffer (50 mM TrisHCl [pH 8], containing 120 mM NaCl and 1% Nonidet P-40), supplemented with the Complete-TM mixture of proteinase inhibitors (Boehringer Mannheim Biochemicals). The samples were cleared by microcentrifugation (14 000 rpm for 30 minutes at 4 °C) and assessed for protein concentration. Biotinylated proteins, representing the cell surface proteins, were precipitated with Streptavidin-agarose for 2 hours at 4 °C, subjected to electrophoresis in a 12% polyacrylamide gel containing sodium dodecyl sulfate, and electroblotted onto nitrocellulose membranes. After a 1-hour incubation in blocking solution (20% IgG-free normal horse serum in PBS), the membranes were exposed to the primary antibody (the monoclonal anti-FasL antibody G247-4 [Pharmingen]) or the polyclonal anti-Fas (N-18 [Santa Cruz Biotechnologies, Santa Cruz, CA]) overnight at 4 °C. After being washed in PBS, the respective secondary peroxidase-labeled antibody was applied at a 1 : 10 000 dilution for 1 hour at room temperature. The proteins were visualized with the enhanced chemiluminescence technique (Amersham Pharmacia Biotech, Piscataway, NJ).
Northern Blotting for FasL and Fas
Total RNA was denaturated, subjected to electrophoresis in 1% agarose-formaldehyde gel, blotted by capillary transfer to a nylon membrane overnight, prehybridized for 4 hours at 42 °C in digoxigenin-Easy Hyb (Boehringer Mannheim Biochemicals), hybridized overnight with the respective probe, and visualized with the digoxigenin luminescent detection kit (Boehringer Mannheim Biochemicals) according to the manufacturer's instructions. Expression of ß-actin was used as internal control for the quantification of RNA. For FasL detection, a digoxigenin-labeled probe, corresponding to nucleotides (nt) 6681558 of the human FasL gene (GenBank D38122), was used. For Fas detection, a digoxigenin-labeled probe, corresponding to nt 2571076 of the human Fas gene (GenBank M67454), was used.
Effect of MMPIs on Fas-Induced Apoptosis
To study the potential combined effect of MMPIs and Fas activation, we treated SK-N-MC cells with 10 or 20 µM BB-3103 or A-151011 for 18 hours in the presence or absence of a subtoxic dose (25 ng/mL) of the Fas-cross-linking antibody CH11. Cell death was evaluated with MTT as described above.
Effect of MMPIs on Doxorubicin-Induced Apoptosis
To study the potential combined effect of MMPIs with chemotherapeutic drugs, we treated SK-N-MC cells with doxorubicin (50 ng/mL) for 48 hours in the presence or absence of 10 or 20 µM BB-3103 (added for the last 24 hours of the experiment). Cell death was evaluated with MTT as described above.
Effect of Recombinant Soluble FasL (rs-FasL) on MMPI-Induced Apoptosis
SK-N-MC cells were pretreated with various concentrations of rs-FasL (Oncogene Research, Cambridge, MA) for 1 hour before 10 µM BB-3103 was added. Forty-eight hours later, cell death was evaluated with MTT as described above.
Statistical Analyses
To determine whether the combined in vitro treatments with MMPIs and CH11 or doxorubicin, as described above, had greater than additive effects, we performed a two-way analysis of variance (ANOVA), as recommended by Slinker (34). All other comparisons were carried out with the one-factor ANOVA method. All values represent the mean of the measurements obtained from quadruplicate wells. All P values reported are two-sided, and statistical significance was set at .05.
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RESULTS |
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The MTT assay showed that two Fas-sensitive cell lines (SK-N-MC and
CHP-100L) underwent cell death with BB-3103 (Fig. 1,
A) or A-151011 (Fig. 1,
B) at concentrations ranging from 10
µM to 40 µM. The difference between treated
and untreated cells was statistically significant in all conditions
(P<.001). On the contrary, no cell death was detected with
this method in the Fas-resistant TC-268, CHP-100S, and IMR-32 cell
lines with either MMPI (Fig. 1,
A and B). The Fas-resistant SK-N-MC
clone that we generated from the Fas-sensitive SK-N-MC cell line was
also resistant to 10 µM (1.5% death; 95% confidence
interval [CI] = 0.3%-2.6%) and 20 µM
(2.49% death;
95% CI = -0.4% to 5.31%) BB3103, in contrast to the parental cell
line (P<.001) (Fig. 1,
C).
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To further study the involvement of the Fas/FasL pathway in the
MMPI-induced cell death, we treated the Fas-sensitive SK-N-MC cells
with BB-3103 in the presence of 5 µg/mL of the Fas-neutralizing
antibody ZB4 or an isotype-matched control antibody. ZB4 reduced the
cell death that was induced by 10 µM BB-3103 from 28%
(95% CI = 25.5%-30.6%) to 7.5% (95% CI =
7.3%7.8%)
(P<.001) and the cell death that was induced by 20
µM BB-3103 from 30.2% (95% CI =
29.3%31%) to 13.3%
(95% CI = 12.2%14.5%) (P = .0076). In
contrast, the
isotype-matched control antibody did not inhibit the BB-3103-induced
cell death (Fig. 1, D).
Apoptotic Nature of Cell Death Induced by MMPIs
(a) DNA ladder. Electrophoresis of isolated DNA from the
SK-N-MC cells treated with 20 µM or 40 µM
BB-3103 revealed a DNA ladder indicating apoptosis. In contrast, no DNA
ladder was observed in the untreated SK-N-MC cells and in the Fas-resistant
TC-268 cells after the same treatment (Fig. 2, A)
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(c) TUNEL. SK-N-MC cells treated with BB-3103 or A-151011 exhibited
many strongly positive nuclei (Fig. 2, D and E). Such nuclei were scarcely
present in untreated SK-N-MC cells (Fig. 2,
C) or in MMPI-treated
TC-268 cells (Fig. 2,
F-H).
(d) Electron microscopy. Apoptotic cells with nuclear chromatin and/or
cytoplasmic condensation/fragmentation were numerous in the Fas-sensitive SK-N-MC cell line
after treatment with BB-3103 (Fig. 2, J) or A-151011 (Fig. 2,
K) but were absent in the untreated cells (Fig. 2,
I). No
apoptotic cells were observed in the Fas-resistant neuroblastoma cell line IMR-32 before or after
treatment with either MMPI. The Fas-resistant Ewing's sarcoma family tumor cell line
TC-268 exhibited only scarce apoptotic cells after treatment with either MMPI (data not shown).
Levels of tm-FasL and Fas Protein and RNA on Cell Surface After MMPI Treatment
Overnight treatment with 20 µM BB-3103 increased the
surface levels of tm-FasL in both SK-N-MC and TC-268 cells (Fig. 3, A).
This finding suggests that the MMPI-induced cell death is secondary to
accumulation of tm-FasL on the cell surface. In addition, we found that
BB-3103 treatment (20 µM for 18 hours) increased the
levels of Fas on the cell membrane of both SK-N-MC and TC-268 cells
(Fig. 3,
A). However, BB-3103 treatment had no effect
on the levels of FasL RNA or Fas RNA (Fig. 3,
B), which excluded
enhanced transcription as a possible causative mechanism.
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The induction of cell death by 10 µM BB-3103 in the
SK-N-MC cells increased gradually with time and peaked at around 60
hours after initiation of treatment (Fig. 3, C), supporting the
importance of optimal protein accumulation for MMPI-induced cell death.
Effect of MMPIs on Apoptosis Induced by the Cytotoxic Anti-Fas Antibody CH11
The finding of increased surface Fas levels in MMPI-treated cells
led us to investigate whether this increase was associated with
increased sensitivity to Fas-mediated apoptosis. We found that the
survival of SK-N-MC cells treated with a subtoxic dose of the
Fas-cross-linking antibody CH11 (that activates Fas by inducing
close proximity of multiple receptor molecules) or with 10
µM BB-3103 or A-151011 was 92.6% (95% CI =
88.2%97%), 90.07% (95% CI =
87%93.1%), and 80.39% (95% CI =
79.3%81.5%), respectively. However, when CH11 and BB-3103 or
A-151011 were administered simultaneously under the same conditions,
they resulted in a significantly lower survival, i.e., 35.98% (95% CI
= 33.9%38.1%) and 37.2% (95% CI =
34.3%40.1%), respectively
(Fig. 4, A). Similar results (not shown) were
obtained when 20 µM of each MMPI was added to the same
concentration of CH11. A two-way ANOVA showed that the MMPI treatments
potentiated the effect of CH11 in a statistically significant manner
(P = .003 and P = .0018 for BB-3103 and A-151011,
respectively).
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The survival of doxorubicin-treated cells was 68.3% (95% CI =
65.6%71%); the survival of 10 or 20 µM BB-3103-treated
cells was 93.7% (95% CI = 90.6%96.9%) and
84.6% (95% CI:
82.386.9%), respectively. The survival of doxorubicin plus
BB-3103-treated cells was 50.5% (95% CI =
50.2%50.8%) and 46.3%
(95% CI = 44.9%-47.8%) for 10 and 20 µM BB-3103,
respectively (Fig. 4, B). A two-way ANOVA showed that BB-3103
potentiated the effect of doxorubicin in a statistically significant
manner (P<.001 for both concentrations of BB-3103).
Effect of rs-FasL on MMPI-Induced Apoptosis
At high concentrations (ng/mL range), rs-FasL induced apoptosis in
the SK-N-MC cell line; in contrast, at lower (subtoxic) concentrations
(0.1-1 pg/mL), it protected SK-N-MC cells from death induced by
BB-3103 (P<.001, Fig. 4, C). This protective effect could
be due to a decrease in surface Fas through binding to rs-FasL and
internalization of the complex.
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DISCUSSION |
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Our novel finding of MMPI-induced apoptosis in tumor cells via the Fas/FasL pathway in vitro suggests that a similar mechanism may contribute to their anticancer activity in vivo and explains recent observations of apoptotic nuclei in MMPI-treated prostatic adenocarcinoma xenografts in nude mice (43). The fact that the apoptotic action of MMPIs is mediated by Fas is supported by our finding of inhibition of MMPI-induced tumor cell death by a Fas-neutralizing antibody. Furthermore, both MMPIs induced death only in tumor cell lines with a functional Fas pathway. Fas-resistant cell lines, including a Fas-resistant clone that we generated from the Fas-sensitive SK-N-MC parental cell line, were resistant to the cytotoxic action of the MMPIs. Because in a previous study (28) we had shown that MMPIs reduce the shedding of s-FasL in Ewing's sarcoma family tumors, we hypothesized that the MMPI-induced tumor cell death in vitro was due to the accumulation of noncleaved FasL on the cell surface, as previously reported for activated T cells and FasL transfectants (5,30). In this study, we found that the MMPIs increased the surface protein levels not only of FasL but also of Fas, without affecting the messenger RNA levels. Recent work by Tanaka et al. (30) has suggested that, in some models, s-FasL may be a negative regulator of apoptosis by forming Fass-FasL complexes that are easily internalized and degraded, thus leading to decreased surface Fas levels. According to this concept, inhibition of FasL cleavage by MMPIs would lead to surface accumulation not only of FasL but also of Fas, as was shown in our study. In support of this hypothesis, exogenously added rs-FasL inhibited MMPI-induced cell death, possibly by decreasing surface Fas through binding and internalization.
The MMPI-induced enhancement in surface levels of Fas and FasL molecules would trigger apoptosis in a fashion similar to the previously reported autocrine suicide of activated T cells (11,12,44). In activated T cells, however, the increase in FasL is due to enhanced transcription; in contrast, in MMPI-treated Ewing's sarcoma family tumor cells, it is due to inhibition of FasL cleavage. The need for accumulation of optimal Fas/FasL levels prior to induction of apoptosis is supported by our finding of increased apoptosis with time after MMPI treatment.
Although increased expression of Fas and FasL may induce apoptosis in Fas-sensitive Ewing's sarcoma family tumor cells, it may not be sufficient to overcome existing inhibitory factors in Fas-resistant cells. This theory would explain why the Fas-resistant Ewing's sarcoma family tumor cell lines did not undergo apoptosis with MMPI treatment in the present study, despite equally increased expression of Fas and FasL. In accordance with these data, previous studies have shown that overexpression of Fas renders Fas-responsive cells highly sensitive to Fas-mediated apoptosis (45), whereas it has no influence on the inherent susceptibility of tumor cells to Fas-mediated cell death (46). The above data support the notion that the MMPIs would have a more pronounced anticancer effect in those Ewing's sarcoma family tumors with a functional Fas/FasL pathway. Because primary tumors are more likely to be Fas sensitive than metastatic tumors (47), earlier rather than later administration of MMPIs may be indicated.
Our finding of a potentiating effect between MMPIs and CH11 antibody suggests that MMPIs may similarly enhance the efficacy of other anticancer agents acting via the Fas pathway. Because we (48) and other investigators (49) have shown that anticancer chemotherapeutic drugs increase FasL expression by increasing its transcription, we hypothesized that inhibition of FasL cleavage by MMPIs would potentiate the apoptosis-inducing activity of chemotherapeutic drugs. Our finding that MMPIs sensitize tumor cells to cell death induced by the anticancer drug doxorubicin supports this hypothesis and holds the promise that MMPIs may potentiate the efficacy of anticancer agents. These data also suggest that MMPIs may be of value even in FasL-negative tumors, in which FasL can be increased by chemotherapeutic drugs. It is interesting that the combination of either BB-94 or BB-2516 and cisplatin has shown a strong synergistic effect in lung and ovarian cancer mouse models (42,50), and interferon and batimastat have had a synergistic effect in an ovarian cancer model in vivo (51).
The apoptosis-inducing activity of MMPIs via the Fas/FasL pathway and their combined action with the chemotherapeutic agent doxorubicin offer new insights into their mode of action. These findings also broaden the spectrum of their applications to the treatment of Fas-sensitive, FasL-positive tumors, such as Ewing's sarcoma family tumors, possibly in combination with traditional chemotherapy. Because FasL expression is restricted to a limited number of normal tissues in the human body (52-54), it is likely that the apoptosis-inducing activity of MMPIs will be accompanied by less toxicity than standard anticancer chemotherapy, which induces apoptosis in normal and malignant cells indiscriminantly. Indeed, marimastat has been generally well tolerated in clinical studies, including one study in healthy volunteers (55,56).
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NOTES |
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Manuscript received December 29, 1998; revised July 19, 1999; accepted August 6, 1999.
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