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,1
* Department of Neurology, School of Medicine, University of Pittsburgh, Pennsylvania, USA; and
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
SPECIFIC AIMS
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. 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-
B signaling pathway is involved in this process.
PRINCIPAL FINDINGS
1. 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 mouse hind limb muscle tissue. Exposure to conditioned media from human prostate cancer, PC-3, or human melanoma (Mel) cells diluted 1:1 with growth media prior to differentiation inhibited differentiation, as assessed by myotube formation and myosin expression. Exposure to conditioned media from another human prostate cancer cell line, DU-145, had less 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 only to growth media prior to differentiation. 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 formation of myotubes from myoblasts compared with conditioned media from 293 control cells. We showed that the inhibitory effect of conditioned media from PC-3 cells on muscle cell differentiation in vitro was dose dependent. We wondered whether a component of the effect on differentiation could be cytotoxicity. 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. These results demonstrated an in vitro cell culture system reflecting elements of cancer cachexia that could be exploited in order to understand the molecular mechanisms of cachexia and to develop treatment strategies.
2. Interleukin 1ßbeta; (IL-1ßbeta;), TNF-
, and proteolysis-inducing factor (PIF) are expressed by PC-3 cells
As assessed by RT-PCR of the tumor cell lines, PC-3 cells expressed IL-1ßbeta;, TNF-, and PIF transcripts, suggesting these cytokines 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-1ßbeta; or TNF-, alone or both together, decreased LDH release and enhanced expression of myosin, myoD, and myogenin from muscle cells compared with exposure to untreated conditioned media from PC-3 cells.
3. 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 IB super repressor, IKBSR, a mutated form of IB that prevents activation of NF-B and promotes muscle cell differentiation. With IKBSR as positive control, we tested whether cFLIP or Bcl-xL would promote muscle cell differentiation. Muscle cell differentiation assessed by myosin expression was partially preserved after exposure to PC-3, Mel, and DU-145 conditioned media by stable expression of cFLIP and IKBSR. 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. To assess the effect of IKBSR, cFLIP, and Bcl-xL on cytotoxicity, LDH release from stably transduced muscle cells was measured after exposure to conditioned media from tumor cell lines. Cells overexpressing either a negative control plasmid or IKBSR had increased levels of LDH release induced by exposure to conditioned media from PC-3 cells, but cells overexpressing cFLIP or Bcl-xL were protected from cytotoxicity induced by the PC-3 cell supernatant.
4. Stable expression of cFLIP, but not Bcl-xL, by muscle cells inhibits activation of NF-B
We observed NF-B activation by gel shift analysis in nuclear extracts of differentiating muscle cells when exposed to cytokine IL-1ßbeta; or conditioned media from PC-3 cells at a higher level than when exposed to conditioned media from normal control 293 cells (Fig. 1
A). Stable expression of cFLIP protected muscle cells from NF-B activation upon exposure to IL-1ßbeta; 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-B activation with exposure to conditioned media from 293 cells and an even higher level of NF-B activation with exposure to conditioned media from PC-3 cells.
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NF-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-B (Fig. 1B
). Increased NF-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-, IFN-
, IL-1ßbeta;, or IL-6). Stable overexpression of IKBSR or cFLIP prevented NF-B-induced transcriptional activation induced by tumor cell conditioned media or inflammatory cytokines. Consistent with gel shift analysis of NF-B activation, stable overexpression of Bcl-xL enhanced NF-B-induced transcriptional activation in this assay in the setting of exposure to PC-3 cell conditioned media (Fig. 1C
).
5. Stable expression of cFLIP or Bcl-xL by muscle cells inhibits 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. Stable overexpression of cFLIP or Bcl-xL inhibited the PC-3 or Mel cell conditioned media-induced increase of caspase-3 activity and caspase-3 cleavage as demonstrated by Western blot analysis. 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.
CONCLUSIONS AND SIGNIFICANCE
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 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-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- and IL-1ßbeta; transcripts. We observed a direct correlation between the inhibition of myogenic differentiation in the in vitro assay and expression of IL-1ßbeta; transcripts by specific human cancer cell lines tested. We also observed a direct correlation between NF-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-B activation within primary muscle cells. Failure of myogenic differentiation and the associated activation of NF-B were prevented by stable expression of either IKBSR or cFLIP, but not by Bcl-xL (Fig. 2
).
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Earlier studies demonstrate that activation of NF-B plays a key role in cancer-induced cachexia. Consistent with previous studies, we found that exposure to secreted factors from PC-3 and Mel cancer cell lines resulted in activation of NF-B in treated myoblast cells, as demonstrated by higher binding activity of nuclear NF-B to consensus NF-B oligonucleotides and higher levels of NF-B transcriptional activity.
cFLIP and Bcl-xL are important antiapoptotic molecules. We investigated whether cFLIP and Bcl-xL influenced NF-B activity and myogenic differentiation in primary cells derived from skeletal muscle. We noted that cFLIP overexpression inhibited IL-1ßbeta; or PC-3 or Mel media-induced NF-B activation in myoblast cells, thus promoting myogenic differentiation of treated myoblast cells as shown by enhanced myotube formation and muscle-specific protein expression. Stable expression of cFLIP yielded results similar to stable expression of IKBSR. Both IKBSR and cFLIP inhibited NF-B activation as determined by levels of nuclear NF-B binding activity and nuclear NF-B-mediated transcription. In contrast, overexpression of Bcl-xL enhanced NF-B activation in myoblast cells, with more increases upon exposure to conditioned media from PC-3 cells. Further studies are required to understand the intricacies of signaling cascades that underpin the contrasting results with cFLIP and Bcl-xL.
This study provides an in vitro assay that demonstrates secretion of cachexia-inducing factors by certain cancer cell lines that result in inhibition of myogenic differentiation by activation of NF-B. Overexpression of cFLIP in muscle cells inhibits NF-B-mediated and apoptotic pathways, preventing tumor media-induced inhibition of myogenic differentiation and cytotoxicity. These findings point to the potential to design novel molecular therapeutic treatments of cancer-induced muscle wasting.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6347fje
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