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Published as doi: 10.1096/fj.06-6347fje.
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(The FASEB Journal. 2006;20:2570-2572.)
© 2006 FASEB

Cellular caspase-8-like inhibitory protein (cFLIP) prevents inhibition of muscle cell differentiation induced by cancer cells

Zhilong Jiang* and Paula R. Clemens*,{dagger},1

* Department of Neurology, School of Medicine, University of Pittsburgh, Pennsylvania, USA; and

{dagger} Neurology Service, Department of Veterans Affairs Medical Center, Pittsburgh, Pennsylvania, USA

1Correspondence: Department of Neurology, University of Pittsburgh, Biomedical Science Tower, South Wing, Rm. 520, 203 Lothrop St., Pittsburgh, PA 15213, USA. E-mail: pclemens{at}pitt.edu

ABSTRACT

Cachexia is a frequent complication of cancer or other chronic diseases. To investigate the pathophysiology of cancer cachexia and pursue treatment options, we developed an in vitro assay of the effects of cancer cell-produced cytokines on primary muscle cells derived from murine skeletal muscle. These studies led to the novel observation that factors secreted by cell lines from prostate cancer and melanoma significantly inhibit differentiation of primary mouse muscle cells. The expression of interleukin (IL) -1ß, TNF-{alpha}, and proteolysis-inducing factor (PIF) by cancer cells used in this study suggested their role in preventing myogenic differentiation. Both NF-{kappa}B binding and transcriptional activity were enhanced in muscle cells treated with conditioned media from cancer cells or with proinflammatory cytokines. Stable expression of IKBSR, a known repressor of NF-{kappa}B activation, and cellular caspase-8-like inhibitory protein (cFLIP) inhibited activation of NF-{kappa}B in cancer cell media-treated muscle cells with an accompanying enhancement of myogenic protein expression and differentiation. In contrast, overexpression of antiapoptotic protein Bcl-xL did not protect myoblast cells exposed to the same treatment. Instead, we observed enhanced activation of NF-{kappa}B in Bcl-xL overexpressing cells. These studies show that the in vitro system recapitulates some of the molecular events causing muscle cachexia and provides the basis for new treatment approaches.—Jiang, Z., Clemens, P. R. Cellular caspase-8-like inhibitory protein (cFLIP) prevents inhibition of muscle cell differentiation induced by cancer cells.


Key Words: cachexia • NF-{kappa}B • apoptosis

CACHEXIA OR MUSCLE wasting is a common syndrome in patients with malignant cancer (1 , 2) and other chronic diseases such as rheumatoid arthritis (3) and chronic heart failure (4) . A full understanding of the underlying mechanism of cachexia is an important step in finding novel therapeutic targets for treatment of this clinical condition. Previous studies showed that higher levels of some proinflammatory cytokines contribute to the development of cachexia (5 , 6) . For example, serum TNF-{alpha} and interleukin (IL) -1{alpha} levels are markedly increased in patients with rheumatoid arthritis and cancer (3 , 7) . Chronic administration of exogenous TNF-{alpha} to an animal model generated cachexia (8) . In addition to TNF-{alpha}, other cytokines such as IL-6, IL-1ß, IL-1{alpha}, and proteolysis-inducing factor (PIF) have been reported to contribute to the development of muscle wasting (5 , 6 , 9) .

Further studies in vitro and in vivo have provided evidence that a principal mechanism by which TNF-{alpha} and IL-1{alpha} lead to progressive muscle wasting is through the activation of transcription factor NF-{kappa}B (10 11 12) . Activated NF-{kappa}B translocates to the nucleus and inhibits expression or stabilization of myogenic regulatory factors such as MyoD (10 , 13) . MyoD is a muscle-specific basic helix-loop-helix transcription factor whose expression and binding to promoter elements result in the expression of genes in the muscle differentiation program such as myogenin and cyclin-dependent kinase (Cdk) inhibitor p21 (10) . Failure of this myogenic program results in muscle wasting.

Because phosphorylation and degradation of I{kappa}B inhibitory protein (I{kappa}B{alpha}) results in activation of NF-{kappa}B, the introduction of a phosphorylation-defective mutant of I{kappa}B{alpha}, I{kappa}B super repressor (IKBSR), can prevent NF-{kappa}B activation. Studies have shown that overexpression of IKBSR prevents NF-{kappa}B activation and ameliorates the inhibitory effect of TNF-{alpha} or IL-1 on expression and stability of MyoD in muscle cells (10 , 13) .

Cellular caspase-8-like inhibitory protein (cFLIP) is an antiapoptotic protein responsible for cell survival and cell resistance to apoptosis by inhibition of procaspase-8 processing at the death-inducing signaling complex (DISC) (14 15 16 17 18) . Bcl-xL is another antiapoptotic protein and is localized to the endoplasmic reticulum so as to prevent cytochrome c release from mitochondria (19) . Recent studies showed that both cFLIP and Bcl-xL influenced NF-{kappa}B activation and cell survival in a variety of cell lines such as 293 cells, HeLa cells, and PC12 cells, a differentiated neuronal cell line (20 21 22) . However, whether cFLIP and Bcl-xL affect NF-{kappa}B activity and survival in muscle cells has not been previously studied.

In this study, we first determined the effect of secreted factors from different cancer cell lines on myoblast cell differentiation in vitro. We then investigated whether overexpression of IKBSR, cFLIP, or Bcl-xL is able to influence muscle cell differentiation in vitro and whether activation or suppression of the NF-{kappa}B signaling pathway is involved in this process.

MATERIALS AND METHODS

Cell lines and plasmids
Human embryonic kidney 293 cells [American Type Culture Collection (ATCC), Rockville, MD, USA] and human prostate cancer cell lines PC-3 and DU-145 (ATCC,), human melanoma cells Mel (Steven Rosenberg, National Cancer Institute, NIH, Bethesda, MD, USA) and murine colon cancer cells MC-38 (Steven Rosenberg, National Cancer Institute) were maintained in RPMI1640 supplemented with 10% heat-inactivated fetal calf serum, 20 mM L-glutamine, and 1% penicillin-streptomycin. Conditioned media were collected 48 h after cells were seeded in fresh RPMI1640 medium. Cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA).

A mutant I{kappa}B{alpha} cDNA IKBSR (1.1 kb) containing an N-terminal hemagglutinin tag was subcloned into pRc/CMV2 with cytomegalovirus (CMV) promoter replaced by the chicken ß-actin promoter (Joseph DiDonato, Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA). A 1.4 kb cFLIPL cDNA (Steven Graham, Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA) was subcloned into pCR3.V64-Met-Flag-stop driven by the CMV promoter (Invitrogen). A 0.6 kb rat Bcl-xL cDNA (Jun Chen, Department of Neurology, University of Pittsburgh) was subcloned into pcDNA3.1 driven by the CMV promoter (Invitrogen). For some experiments, a GFP expression vector pN1-GFP (Clontech, Palo Alto, CA, USA) was used.

Primary myoblast cells and generation of cell lines
Primary myoblast cells were prepared from the hind limb muscles of 3-wk-old C57BL/10 (C57) mice (23) . Briefly, the muscle mass was minced into a coarse slurry using razor blades. Cells were enzymatically dissociated by adding 1% collagenase D (Boehringer Mannhein, Mannheim, Germany) for 2 h at 37°C. The single cell suspension was filtered through an 85 µm nytex filter and preplated in a collagen-coated dish in myoblast growth media. After 2 h, the supernatant was withdrawn from the dish and replated in a fresh collagen-coated dish. After 4–5 serial passages, the culture was enriched with small, round myoblast cells. Cells were assayed for desmin expression and the ability to differentiate when cultured in fusion medium (DMEM with 2% horse serum) to assure that >85% of the cells in the culture were myogenic.

Stable expression of IKBSR, cFLIP, and Bcl-xL cDNAs in primary C57 myoblasts was obtained by transfection of C57 myoblast cells with each expression plasmid or an empty control vector pcDNA3.1 by Lipofectamine 2000 (LF 2000) (Invitrogen). At 48 h after transfection, cells were passaged at 1:50 of their density in the presence 0.4 mg/ml G418 (Sigma, St. Louis, MO, USA). Antibiotic resistant clones were expanded and tested for expression of the transgene by immunochemistry and Western blot analysis.

NF-{kappa}B activity assay
Collagen-coated 24-well culture plates were seeded with C57 myoblast cells (0.5x105 cells) 1 day before transfection with the NF-{kappa}B-driven GFP reporter plasmid pNF-{kappa}B-hrGFP (200 ng/well) (Stratagene, La Jolla, CA, USA) using Lipofectamine 2000. To normalize for transfection efficiency, the ß-galactosidase plasmid pCMV-ß (100 ng/well, Clontech, Mountain View, CA, USA) was added to all transfections. At 6 h after transfection, the DNA-LP 2000 complex was removed and replaced with conditioned media (1:2 dilution) or F12 media containing cytokines TNF-{alpha}, IFN-{gamma}, IL-1ß, and IL-6 (PharMingen, Rockville, MD, USA). GFP-positive and ß-galactosidase-positive cells were counted 24 h after transfection. The ß-galactosidase-expressing cells were detected by X-gal staining reagent (5 mM FeK3(CN)6, 5 mM K4Fe(CN)6 3H2O, 1 µg/µl X-gal, and 2 mM MgCl2) at 37°C for 2 h. At least 8 fields per well were counted at 100x magnification. NF-{kappa}B activity assay was presented as the ratio of GFP-positive to ß-galactosidase-positive cells.

Gel shift assay
For gel shift analysis, a nuclear extract of each sample was prepared from 3 x 106 cells treated or untreated with indicated cytokines or tumor cell conditioned media, using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL, USA). The protein concentration of each nuclear extract was measured by the bicinchoninic acid (BCA) assay (Pierce). NF-{kappa}B oligonucleotides (2 µl, 1.75 pmol/µl, oligonucleotide sequence 5'-AGTTGA GGG GAC TTT CCC AGG C-3', 3'-TCA ACT CCC CTG AAAGGG TCC G-5') (Promega, Madison, WI, USA) were 32P-labeled with 1 µl 10 µCi [{gamma}32] ATP (Amersham Biosciences, Piscataway, NJ, USA) by using the DNA 5'-end labeling system kit (Promega) in a volume of 20 µl at 37°C for 1 h. The reaction was stopped by addition of 2 µl 0.5 mol/L EDTA and 88 µl TE buffer. The binding reaction mixture (20 µl), which contained 35 fmol 32P-labeled NF-{kappa}B oligonucleotides, 10 µg nuclear extracts, 1 µg poly (dI:dC), was incubated at room temperature for 1–2 h in 1x binding buffer (Pierce). Unlabeled NF-{kappa}B oligonucleotides (1.75 pmol) were added in the binding reaction mixture as a competitor. DNA-protein complexes were resolved by electrophoresis through 5% polyacrylamide gels in 0.5x TBE buffer at 100 V for 1 h. The gel was subsequently dried and autoradiographed with intensifying screens at –80°C. Gel supershift assays were performed by addition of 2 µl of antibodies, antip65 and antip50 (200 µg/0.1 ml) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) to the reaction mixture and incubation for 45 min at room temperature.

Immunocytochemical staining for myogenic proteins
We assessed the extent of myogenic differentiation morphologically by determining the myogenic index, which is defined as the fraction of nuclei residing in multinucleated myotube cells expressing myosin, myoD, or myogenin. Briefly, C57 myoblast cells were treated with 50% conditioned media or the indicated cytokines. After 48 h, treated C57 myoblast cells were cultured in DMEM medium supplied with 2% heat-inactivated horse serum, 20 mM L-glutamine, and 1% penicillin-streptomycin (myoblast fusion medium) for 4–5 days to promote differentiation into multinucleated myotubes. Cells were washed with PBS and fixed with 4% paraformaldehyde and 0.1% Triton X-100 for 10 min, followed by washing and incubation with PBS containing 10% goat serum for 30 min. Fixed cells were incubated with rabbit antimyosin (1:300 dilution) (M-7523, Sigma) antibody (Ab) for 2 h, followed by incubation with Cy3-conjugated anti-rabbit or Cy3-conjugated anti-mouse Ab (1:300 dilution) (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA, USA) for 1 h. Cell nuclei were stained with Hoechst for 5 min. Myosin, myoD, or myogenin-positive cells and cell nuclei were visualized using fluorescence microscopy. The differentiation of myoblast cells into myotubes was scored by the ratio of nuclei in myosin-positive cells to total nuclei in one field. At least 8 fields per sample were counted at 200x magnification.

Western blot analysis for myogenic proteins
Treated C57 myoblast cells were cultured in DMEM supplied with 2% heat-inactivated horse serum, 20 mM L-glutamine, and 1% penicillin-streptomycin antibiotics for 4 days to differentiate into myotubes. Cells were washed, harvested, and lysed with cell culture lysis reagent buffer (Promega) (40 mM Tris-HCl, [pH 7.8], 300 mM NaCl, 2% (v/v) Nonidet P-40, 1% triton X-100, 1 mM DTT, 1 mM NaVO4, and complete protease inhibitor cocktail) for 30 min on ice. Cell lysates were obtained by centrifugation (10,000 g, 20 min). Protein concentrations were determined by BCA protein assay (Pierce). Cell lysates (30 µg/lane) were separated by 7.5% sodium dodecyl sulfate-PAGE and transferred to Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham Pharmacia Biotech, UK). Membranes were incubated with TBST (12.5 mM Tris-HCl pH 7.5, 68.5 mM NaCl, 0.1% Tween 20) supplied with 5% goat serum, 5% skim milk overnight at 4°C. Then membranes were incubated with primary Ab mouse antimyosin or antimyoD (Sigma) for 1–2 h, followed by incubation with horseradish peroxidase (HRP) -conjugated anti-mouse immunoglobulin (Ig) (Amersham Biosciences). The blots were developed with ECL substrate solution (Amersham Biosciences), and exposed to Kodak X-Omat Blue film (NEN Life Sciences, Inc. Boston, MA, USA).

Lactate dehydrogenase activity assay
Lactate dehydrogenase (LDH) activity was assessed by colorimetric assay according to the manufacturer’s instructions (Promega). Briefly, 50 µl cell culture media was removed from cells after treatment and mixed with 50 µl substrate diluted in assay buffer. The mixture was incubated at room temperature in the dark for 15 min, followed by the addition of 50 µl stop solution. Absorbance was read at 492 nm. A cytotoxicity index was calculated according to the manufacturer’s instructions and expressed as a fold change from the results observed when control 293 cell conditioned media was applied to muscle cells in parallel experiments.

Caspase-3 activity and cleavage analysis
Caspase-3 activity was assessed using a colorimetric caspase-3 assay kit (Sigma). Briefly, myoblast cells were harvested by centrifugation at 1400 rpm 24 h after treatment. Supernatants were removed and discarded. Cells were lysed in cold lysis buffer (50 mM HEPES, pH 7.4, 5 mM CHAPS, 5 mM DTT) for 20 min, followed by centrifugation for 20 min at 4°C. The protein content of cell lysates was determined by BCA assay. Proteins (100–200 µg) were added to the final volume of 100 µl reaction buffer in the presence of DTT and caspase-3 specific substrate. Caspase-3 inhibitor was added as a control in each reaction to confirm the specific cleavage of caspase-3 in this assay according to the manufacturer’s guidelines. Samples were incubated at 37°C overnight and analyzed at 405 nm. Caspase-3 activity was expressed as µmol pNA/h/ml.

Cleaved caspase-3 fragments were further analyzed by Western blot analysis. Cellular extracts (20 µg) were electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to Hybond-ECL nitrocellulose membrane. Membranes were incubated with primary Ab rabbit anticaspase-3 (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit secondary Ab (Jackson ImmunoResearch Lab, Inc). The blots were washed three times and incubated with ECL substrate solution (Amersham Biosciences) for 1 min according to the manufacturer’s instructions and visualized with X-ray film.

RT-polymerase chain reaction (RT-PCR) for cytokine transcripts
Total cellular RNA was extracted from cells using TRIzol reagent (Invitrogen), according to the manufacturer’s instruction. Single-stranded cDNA was generated at 42°C for 1 h in a 50 µl reverse transcriptase (RT) reaction containing 100 ng of total RNA as template, 0.5 µg oligo (dT)12–18 random primers, 1 µl 10 mM dNTP mix, 1 µl 0.1 M DTT, 1 µl 25 mM MgCl2 and 1 µl SuperScript II Rnase H– reverse transcriptase (Invitrogen). Total RNA and oligo (dT)12–18 random primers were heated at 68°C for 2 min, then incubated on ice for 5 min prior to being mixed with other reagents in the RT reaction. RT products (5 µl) were used for polymerase chain reaction (PCR) amplification in a 25 µl reaction using SuperMix (Invitrogen). Primer sequences for cytokines and ß-actin were designed on the basis of published sequences in GENBANK spanning exon-exon boundaries as listed in Table 1 . These primers are cDNA-specific and do not produce PCR products with genomic DNA as a template. Amplification was carried out for 35 cycles consisting of 94°C, 45 s; 55°C, 45 s; 72°C, 1 min 30 s. PCR products were separated on a 2% agarose gel that was stained with ethidium bromide. RNA extracted from healthy donor PBMCs stimulated with 5 µg/ml PHA for 24 h was used as a positive control of some cytokine transcripts. RT reaction without reverse transcriptase was a negative control for each sample. Simultaneously, ß-actin transcripts were detected in each sample as an internal control.


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  		Table 1. Primers used to detect cytokine transcripts by polymerase chain reactiona

Statistical analysis
All values were presented as mean ± SEM in one independent experiment. Statistical significance was determined with 2-way and unpaired Student’s t test. Differences were considered statistically significant at a value of P < 0.05.

RESULTS

Conditioned media from human prostate cancer (PC-3) and human melanoma (Mel) cell lines inhibits myogenic differentiation in vitro
Primary myoblast cultures were derived from C57 mouse hind limb muscle tissue. Exposure to conditioned media from human prostate cancer (PC-3) or human melanoma (Mel) cells inhibited differentiation as assessed by myotube formation and the quantity of myosin expression (Fig. 1 A). Exposure to conditioned media from another human prostate cancer cell line, DU-145, resulted in a lesser effect on differentiation. In contrast, a murine colon cancer cell line, MC-38, showed no inhibition of muscle cell culture differentiation. Muscle cells exposed to conditioned media from normal human 293 cells (negative control) differentiated normally with an appearance similar to muscle cells exposed to differentiation media alone. Quantitative analysis of muscle cell differentiation, assessed by the ratio of nuclei in myosin-expressing myotubes to total nuclei in randomly selected fields, showed that conditioned media from PC-3, DU-145 and Mel cells significantly inhibited the formation of myotubes from myoblasts compared with conditioned media from 293 control cells (Fig. 1B ). We further showed that the inhibitory effect of conditioned media from PC-3 cells on muscle cell differentiation in vitro was dose dependent (Fig. 1C ). Although myoblasts exposed to cancer cell conditioned media remained viable and attached to the collagen-coated dish, we wondered whether a component of the effect on differentiation could be cytotoxicity. To assess this, we measured LDH release into the culture media. Higher levels of LDH release, expressed as a fold change from LDH release due to exposure to conditioned media from 293 cells, were observed after exposure to conditioned media from PC-3 and Mel cells (Fig. 1D ). These results demonstrated an in vitro cell culture system reflecting elements of cancer cachexia that could be further exploited to understand the molecular mechanisms of cachexia and to develop treatment strategies.


Figure 1
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  		Figure 1. Conditioned tumor media from prostate cancer cell lines (PC-3 and DU-145) and a melanoma cell line (Mel) inhibit myogenic differentiation and increase LDH release. A) Primary myoblast cells were cultured in growth media in the presence of conditioned media (293 (noncancer cell control), PC-3, DU145, Mel, MC-38) (1:2 dilution) for 48 h and induced to differentiate in differentiation media for 4 days. Myosin protein expression was visualized by immunohistochemistry (red) and cell nuclei were stained using Hoechst (blue). B) Quantitative analysis of myoblast cell differentiation. The total number of nuclei and those in myosin-positive myotubes were scored. At least 8 fields at 200x magnification were counted for each sample. Myogenic differentiation was assessed by the ratio of nuclei in myosin-positive myotubes to total nuclei and expressed as the mean differentiation index ± SEM (n=8, *P<0.05) vs. 293 conditioned media-treated cells. C) PC-3 media inhibits myogenic differentiation in a dose-dependent manner. Cells were cultured in growth media in the presence of PC-3 media for 48 h, then induced to differentiate by culturing in differentiation media for 4 days. Myogenic differentiation index (expressed as mean± SEM, n=4) was quantitatively analyzed after detection of myosin by immunohistochemistry and nuclei by Hoechst staining. D) Analysis of lactate dehydrogenase (LDH) activity. LDH release was analyzed 48 h after myoblast cells were maintained in tumor cell conditioned media (1:2 dilution). Results are expressed as mean fold increase ± SEM (n=4, *P<0.05) vs. 293 conditioned media treated cells. Results shown are representative of at least 4 independent experiments.

IL-1ß, TNF-{alpha}, and PIF are expressed by PC-3 cells
To explore the mechanism of the inhibitory effect on muscle cell differentiation, cytokine transcripts were amplified by RT-PCR of total RNA from each tumor cell line. Primer sequences for cytokines (TNF-{alpha}, IFN-{gamma}, IL-1ß, IL-6, PIF) and internal control gene ß-actin were designed to exon sequences that span introns to avoid genomic DNA amplification. We detected production of IL-1ß transcripts from both PC-3 and Mel cells, but not from other cells used in this study (Fig. 2 A). Detectable levels of TNF-{alpha} and PIF transcripts were detected from PC-3 and Mel cells but not from other cells. IFN-{gamma} and IL-6 transcripts were not detected from any of the cell lines. These data suggested that PC-3 and Mel cells produced cytokines (interleukin-1ß, TNF-{alpha}, and PIF) that may play critical roles in the inhibitory effect on muscle cell differentiation and viability. Supporting this hypothesis, preincubating PC-3 media with different concentrations of neutralizing antibodies against human IL-1ß or TNF-{alpha} alone, or both together, effectively decreased LDH release (Fig. 2B ) and enhanced expression of myoD, myosin, and myogenin (data not shown) from muscle cells compared with exposure to untreated conditioned media from PC-3 cells.


Figure 2
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  		Figure 2. TNF-{alpha}, IL-1ß, and PIF are mediators in failure of myogenic differentiation and LDH release due to PC-3 media exposure. A) RT-PCR analysis of TNF-{alpha}, IFN-{gamma}, IL-1ß, IL-6, and PIF transcripts expressed by tumor cells. Transcripts of ß-actin were analyzed as a loading control. B) Addition of anti-TNF-{alpha} or anti-interleukin-1ß neutralizing Ab (NAB) to PC-3 cell conditioned media decreased myoblast LDH release after exposure to PC-3 cell media. LDH release from myoblast cells was measured 48 h after cells were cultured with PC-3 conditioned media (1:2 dilution) preincubated with increasing concentrations of neutralizing antibodies for 1 h at 37°C. LDH release from negative control myoblasts was measured 48 h after the cells were cultured with 293 conditioned media (1:2 dilution) without preincubation with NAB. Results are expressed as mean fold increase ± SEM LDH release vs. 293 conditioned media-treated cells that have not been treated with NAB (n=3).

To provide further evidence that cytokines were mediators of the effects observed with exposure to cancer cell conditioned media, muscle cells were treated with cytokines TNF-{alpha}, IFN-{gamma}, IL-1ß, or IL-6 at a dose of 20 ng/ml to directly determine whether these cytokines could inhibit muscle cell differentiation. Exposure to IFN-{gamma}, IL-1ß, and IL-6, either alone or in combination, impaired muscle cell differentiation (Fig. 3 A) but had only a small effect on LDH release (Fig. 3B ). Exposure to TNF-{alpha} alone or combined with the other cytokines for 48 h in vitro resulted in a cytotoxic effect demonstrated by increased LDH release (Fig. 3B ).


Figure 3
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  		Figure 3. TNF-{alpha}, IFN-{gamma}, IL-1ß, and IL-6 inhibit myogenic differentiation and induce LDH release by myoblast cells. A) Myogenic differentiation was inhibited by TNF-{alpha}, IFN-{gamma}, IL-1ß, and IL-6. Myoblast cells were treated with TNF-{alpha}, IFN-{gamma}, IL-1ß, and IL-6 alone, each at a concentration of 20 ng/ml, and maintained in differentiation media for 4 days. Control cells were not exposed to a cytokine. Myogenic differentiation was quantitatively analyzed after detection of myosin by immunohistochemistry and nuclei by Hoechst staining and expressed as the mean differentiation index ± SEM (n=4, *P<0.05). B) LDH release by myoblast cells treated with TNF{alpha}, IFN-{gamma}, IL-1ß, and IL-6 alone or in combination with the concentration of each cytokine at 20 ng/ml for 2 days. LDH release is expressed as mean fold increase ± SEM vs. untreated media cells (n=3, *P<0.05).

Stable expression of cFLIP, but not Bcl-xL, by muscle cells partially reverses the inhibition of muscle cell differentiation induced by exposure to conditioned media from tumor cells
Muscle cells were stably transduced with the I{kappa}B{alpha} super repressor, IKBSR, a mutated form of I{kappa}B{alpha} that prevents the activation of NF-{kappa}B. Primary muscle cells were also stably transduced with cFLIP, Bcl-xL, an empty control vector pcDNA3.1, or a GFP expression vector pN1-GFP, as confirmed by immunohistochemistry and Western blot after 2 wk of antibiotic selection in culture (data not shown). Muscle cell differentiation, as assessed by the expression of myosin, was partially preserved after exposure to PC-3, Mel, and DU-145 conditioned media by stable expression of cFLIP and IKBSR (Fig. 4 A). Quantitation of muscle cell differentiation (Fig. 4B ) and Western blot analysis for myosin and MyoD protein expression (Fig. 4C ) confirmed these results. In contrast, Bcl-xL overexpression did not protect against inhibition of muscle cell differentiation induced by exposure to PC-3, Mel, or DU-145 cell conditioned media (Fig. 4A ). LDH release from stably transduced muscle cells was measured after exposure to conditioned media from tumor cell lines for 48 h prior to differentiation media for 4 days (Fig. 4D ). Cells overexpressing either a negative control plasmid or IKBSR had increased levels of LDH release induced by exposure to conditioned media from PC-3 or DU-145 cells. However, cells overexpressing cFLIP or Bcl-xL were protected from the cytotoxicity induced by these cell supernatants. cFLIP protected against cytotoxicity induced by conditioned media from Mel cells, but Bcl-xL or IKBSR did not.


Figure 4
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  		Figure 4. Overexpression of cFLIP decreases inhibition of myogenic differentiation and LDH release caused by tumor conditioned media. Myoblast cell lines with stable expression of IKBSR, cFLIP, or Bcl-xL were established by stable transfection of primary myoblast cells with a plasmid carrying the indicated transgene or with an empty plasmid pcDNA3.1 as a negative control. A) Cells were cultured in growth medium in the presence of conditioned media (1:2 dilution) (293, PC-3, DU145, Mel, MC-38) for 2 days, then induced by exposure to differentiation media for 4 days to fuse into myotubes. Myosin expression was analyzed by immunohistochemistry. B) The myogenic differentiation index was analyzed by scoring the ratio of nuclei in myosin-positive myotubes to total nuclei in randomly selected fields (200x). Results are expressed as mean ± SEM (n=8, *P<0.05) vs. myotubes transfected with empty expression vector (control). C) Western blot analysis of myosin and myoD expression in myotubes treated with 293 or PC-3 media (1:2 dilution) and cultured in differentiation media for 4 days. Whole cell extracts were prepared and 30 µg total protein was used in Western blot analysis to detect myosin and myoD protein expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a loading control. D) LDH release was assayed from cells cultured in conditioned media (1:2 dilution) for 2 days. LDH release is expressed as mean fold increase ± SEM vs. control cells (n=3, *P<0.05).

Stable expression of cFLIP, but not Bcl-xL, by muscle cells inhibits the activation of NF-{kappa}B
We observed NF-{kappa}B activation by gel shift analysis in nuclear extracts of differentiating muscle cells when exposed to cytokine IL-1ß or conditioned media from PC-3 cells at a higher level than when exposed to conditioned media from normal control 293 cells (Fig. 5 A). Stable expression of cFLIP protected muscle cells from NF-{kappa}B activation upon exposure to IL-1ß or conditioned media from PC-3 cells, similar to the protection seen with stable expression of IKBSR. However, stable expression of Bcl-xL in muscle cells resulted in higher levels of NF-{kappa}B activation with exposure to conditioned media from 293 cells, and an even higher level of NF-{kappa}B activation with exposure to conditioned media from PC-3 cells.


Figure 5
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  		Figure 5. Overexpression of cFLIP and Bcl-xL suppressed NF-{kappa}B activity in myoblasts. A) Analysis of NF-{kappa}B binding activity to a consensus NF-{kappa}B oligonucleotide. Myoblast cells were cultured for 60 min in the presence of 20 ng/ml IL-1ß or 24 h in the presence of 293 or PC-3 media (1:2 dilution). Nuclear extracts were prepared and assessed for DNA binding activity to a consensus NF-{kappa}B oligonucleotide by electromobility shift assay (EMSA). Lane markers indicate cells with stable expression of IKBSR, cFLIP, or Bcl-xL, GFP from a GFP expression plasmid or untransduced (Ctl). B) Analysis of NF-{kappa}B transcriptional activity. Myoblast cells with stable expression of IKBSR or cFLIP (Control cells are untransduced) were cotransfected with pNF-{kappa}B-hrGFP (200 ng/well) and pCMV-ß (100 ng/well). 6 h after transfection, cells were cultured in the presence or absence of conditioned media (1:2 dilution) (left panel) or cytokines (no cytokine (no cyt) or 20 ng/ml TNF-{alpha}, IFN-{gamma}, IL-1ß, or IL-6) alone (right panel). GFP-positive and ß-galactosidase-positive cells were counted 24 h after treatment. At least 8 randomly selected fields per well were counted (magnification 100x). NF-{kappa}B activity is presented as a ratio of GFP-positive to ß-galactosidase-positive cells, expressed as mean ± SEM vs. control myoblast cells. One of at least four independent experiments is shown (n=8, P<0.05). C) Analysis of NF-{kappa}B transcriptional activity in myoblast cells with stable expression of Bcl-xL cotransfected with pNF-{kappa}B-hrGFP (200 ng/well) and pCMV-ß (100 ng/well). 6 h after transfection, cells were cultured in the presence of 293 or PC-3 cell conditioned media (1:2 dilution). The GFP-positive and ß-galactosidase-positive cells were counted 24 h after treatment. At least 8 randomly selected fields per well were counted (magnification 100x). NF-{kappa}B activity is presented as a ratio of GFP-positive to ß-galactosidase-positive cells, expressed as mean ± SEM vs. control myoblast cells. One of at least 4 independent experiments is shown (n=8, P<0.05).

NF-{kappa}B activation was also analyzed in a transcriptional assay using a transiently transfected reporter plasmid that expresses enhanced GFP (EGFP) from a promoter element that must be activated by binding of nuclear NF-{kappa}B (Fig. 5B ). Increased NF-{kappa}B-induced transcriptional activation was observed in muscle cells when exposed to conditioned media from PC-3 or Mel cells or to inflammatory cytokines (TNF-{alpha}, IFN-{gamma}, IL-1ß, or IL-6). Stable overexpression of IKBSR or cFLIP prevented NF-{kappa}B-induced transcriptional activation induced by tumor cell conditioned media or inflammatory cytokines. Further confirmation that cFLIP prevented NF-{kappa}B activation was obtained by showing that inhibition of NF-{kappa}B activation was reversed by treatment of the cells stably overexpressing cFLIP with a small interference RNA (siRNA) to cFLIP (data not shown). Consistent with gel shift analysis of NF-{kappa}B activation, stable overexpression of Bcl-xL enhanced NF-{kappa}B-induced transcriptional activation in this assay in the setting of exposure to PC-3 cell conditioned media (Fig. 5C ).

Stable expression of cFLIP or Bcl-xL by muscle cells inhibits the activation of caspase-3
Caspase-3 activity was enhanced in muscle cells exposed to conditioned media from PC-3 or Mel cells compared with muscle cells exposed to conditioned media from normal control 293 cells (Fig. 6 ). Stable overexpression of cFLIP or Bcl-xL inhibited the PC-3 or Mel cell conditioned media-induced increase of caspase-3 activity (Fig. 6A ) and caspase-3 cleavage as demonstrated by Western blot analysis (Fig. 6B ). Stable expression of IKBSR in muscle cells did not appear to inhibit the increase in caspase-3 activity induced in muscle cells by exposure to conditioned media from PC-3 or Mel cells.


Figure 6
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  		Figure 6. Overexpression of cFLIP and Bcl-xL inhibit caspase-3 activation. Myoblast cells with stable expression of IKBSR, cFLIP, Bcl-xL, or empty vector (control) plasmid were cultured with 1:2 dilution of conditioned media from the indicated cell line (293, PC-3, or Mel) for 24 h. A) Cell extracts (100–200 µg/well) were assayed for caspase-3 specific activity, expressed as µmol pNA/h/ml. Data are presented as mean ± SEM (n=3, P<0.05) vs. control myoblast cells and represent one of three independent experiments. B) Cell extracts were assayed by Western blot for caspase-3 cleavage. After cells were maintained in conditioned media (1:2 dilution) for 24 h, total cell extracts were prepared and 20 µg of each cell extract was used for detection of cleaved caspase-3 fragments.

DISCUSSION

We report here a new in vitro cell culture assay to study the effects of cancer cell cytokines on muscle cell differentiation and use this assay to test novel gene transfer approaches for the treatment of cancer cachexia. Exposure to conditioned media from selected human cancer cell lines resulted in failure of muscle cell differentiation. A known intracellular mechanism of NF-{kappa}B activation as a cause of cancer cachexia was recapitulated in this in vitro system. Consistent with previous reports in vivo, we demonstrated that human prostate cancer PC-3 cells express PIF as well as TNF-{alpha} and IL-1ß transcripts (2) . We observed a direct correlation between inhibition of myogenic differentiation in the in vitro assay and the expression of IL-1ß transcripts by specific human cancer cell lines tested. Furthermore, we observed a direct correlation between NF-{kappa}B activation and inhibition of myogenic differentiation in the in vitro assay. Exposure to inflammatory cytokines and to conditioned media from human cancer cells each resulted in NF-{kappa}B activation within primary muscle cells. Failure of myogenic differentiation and the associated activation of NF-{kappa}B were prevented by stable expression of either IKBSR or cFLIP, but not by Bcl-xL.

The underlying mechanism of the cachexia-inducing effects of proinflammatory cytokines (TNF-{alpha}, IL-1ß, or PIF) has been extensively studied recently. Activation of NF-{kappa}B participates in a key signaling pathway resulting in the inhibition of myogenic differentiation (10 , 11) . Consistent with previous studies, in this study we found that exposure to secreted factors from PC-3 and Mel cancer cell lines resulted in activation of NF-{kappa}B in treated myoblast cells, as demonstrated by higher binding activity of nuclear NF-{kappa}B to consensus NF-{kappa}B oligonucleotides and higher levels of NF-{kappa}B transcriptional activity. Results demonstrating NF-{kappa}B activation in cancer cytokine-treated muscle cells suggest that IL-1ß, TNF-{alpha}, or PIF produced from PC-3 or Mel cells activate NF-{kappa}B by phosphorylation and degradation of I{kappa}B{alpha}, thus facilitating the translocation of NF-{kappa}B to the nucleus. Shown in previous studies and confirmed in our study, expression of myoD was decreased in cancer cytokine-treated muscle cells. Other studies demonstrate that TNF-{alpha} exposure resulted in decreased myoD expression that is due to stimulatory effects on myoD protein degradation and inhibitory effects on its transcription by activated NF-{kappa}B, ultimately leading to a failure of initiation of the muscle differentiation process (13) .

Recent reports suggest that certain molecules involved in signaling pathways that result in apoptosis can promote or inhibit NF-{kappa}B activation in particular cell lines. Caspase-8, Fas-associated death domain (FADD), FLIP, TRAIL-R, TNF-R1, and procaspase-8 activate NF-{kappa}B in a variety of cells, using a TRAF2/NIK/IKKs-dependent pathway (20 , 26 27 28 29 30) that can be blocked by their inhibitors or dominant-negative mutants (22 , 28 , 31) . cFLIP and Bcl-xL are both important antiapoptotic molecules. cFLIP is a caspase-8 homologue devoid of protease activity and contains two death effector domains and a catalytically inactive caspase-like domain. cFLIP inhibits procaspase-8 processing at the DISC in most cells, thus inhibiting the extrinsic apoptotic signal pathway (14 15 16 , 28) . Bcl-xL is localized to the endoplasmic reticulum to prevent cytochrome c release from mitochondria, and thus inhibits the intrinsic apoptotic signal pathway (19) . Previous studies demonstrated the cell-specific effects of cFLIP and Bcl-xL on NF-{kappa}B activity in other cell lines (20 , 22 , 26 , 27 , 32 33 34) . We speculated that cFLIP and Bcl-xL could promote muscle cell differentiation by effects on NF-{kappa}B activation and apoptosis. Therefore, in this study we investigated whether cFLIP and Bcl-xL influenced NF-{kappa}B activity, apoptosis, and myogenic differentiation in primary cells derived from skeletal muscle.

We observed that cFLIP overexpression inhibited IL-1ß or PC-3 or Mel media-induced NF-{kappa}B activation in myoblast cells, thus promoting myogenic differentiation of treated myoblast cells, as demonstrated by enhanced myotube formation and muscle-specific protein expression. Stable expression of cFLIP yielded similar results to stable expression of IKBSR. Both IKBSR and cFLIP inhibited NF-{kappa}B activation, as determined by levels of nuclear NF-{kappa}B binding activity and nuclear NF-{kappa}B-mediated transcription.

In contrast, overexpression of Bcl-xL enhanced NF-{kappa}B activation in myoblast cells, with further increases upon exposure to conditioned media from PC-3 cells. The contrasting results with cFLIP and Bcl-xL suggest differential effects on NF-{kappa}B activation by these molecules in muscle cells. Further studies are required to understand the intricacies of signaling cascades that underpin this result. However, it is interesting to speculate that although the antiapoptotic properties of Bcl-xL may have promoted muscle cell differentiation, its ability to increase activation of NF-{kappa}B may have counteracted this beneficial effect, resulting in a net failure of protection from the detrimental effects of cancer cell cytokines on muscle cell differentiation.

We considered whether the inhibition of muscle cell differentiation induced by exposure to certain cancer cell cytokines might be in part a cytotoxic effect. Although high doses of inflammatory cytokines, such as TNF-{alpha}, result in severe cytotoxicity with loss of cell attachment (data not shown), such effects were not observed under conditions used for the studies reported here. By measuring LDH release, however, we demonstrated more subtle cytotoxicity in inflammatory cytokine or cancer cell-derived cytokine-treated muscle cells, as expressed as a fold change in LDH release compared with untreated or 293 cell conditioned media-treated cells. Furthermore, PC-3 cell media significantly increased caspase-3 activity and cleavage in muscle cells. However, overexpression of cFLIP and Bcl-xL, important antiapoptotic proteins, but not IKBSR, provided protection from cytotoxicity and this protection was accompanied by a reduction in caspase-3 activation, suggesting that activation of the apoptosis pathway was involved in the cytotoxic effect of tumor media.

Taken together, this study provides an in vitro assay that demonstrates secretion of cachexia-inducing factors by certain cancer cell lines causing inhibition of myogenic differentiation by activation of NF-{kappa}B and apoptosis. Overexpression of cFLIP in muscle cells inhibits both NF-{kappa}B-mediated and apoptotic pathways, thereby preventing tumor media-induced inhibition of myogenic differentiation and cytotoxicity. These findings point to the potential to design novel molecular therapeutics for the treatment of cancer-induced muscle wasting.

ACKNOWLEDGMENTS

This work was supported by grants from the Department of Veterans Affairs and the U.S. Army Medical Research and Materiel Command (USAMRMC) to P.R.C.

Received for publication May 25, 2006. Accepted for publication July 17, 2006.

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