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Originally published In Press as doi:10.1074/jbc.M405197200 on June 22, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36680-36688, August 27, 2004
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Oncogenic Ras Promotes Butyrate-induced Apoptosis through Inhibition of Gelsolin Expression*

Lidija Klampfer{ddagger}§, Jie Huang{ddagger}, Takehiko Sasazuki¶, Senji Shirasawa¶, and Leonard Augenlicht{ddagger}

From the {ddagger}Albert Einstein Cancer Center, Montefiore Medical Center, Department of Oncology, Bronx, New York 10467 and the Research Institute, International Medical Center of Japan, Toyama 1-21-1, Shinjuku-ku, Tokyo 162-8655, Japan

Received for publication, May 10, 2004 , and in revised form, June 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of Ras promotes oncogenesis by altering a multiple of cellular processes, such as cell cycle progression, differentiation, and apoptosis. Oncogenic Ras can either promote or inhibit apoptosis, depending on the cell type and the nature of the apoptotic stimuli. The response of normal and transformed colonic epithelial cells to the short chain fatty acid butyrate, a physiological regulator of epithelial cell maturation, is also divergent: normal epithelial cells proliferate, and transformed cells undergo apoptosis in response to butyrate. To investigate the role of k-ras mutations in butyrate-induced apoptosis, we utilized HCT116 cells, which harbor an oncogenic k-ras mutation and two isogenic clones with targeted inactivation of the mutant k-ras allele, Hkh2, and Hke-3. We demonstrated that the targeted deletion of the mutant k-ras allele is sufficient to protect epithelial cells from butyrate-induced apoptosis. Consistent with this, we showed that apigenin, a dietary flavonoid that has been shown to inhibit Ras signaling and to reverse transformation of cancer cell lines, prevented butyrate-induced apoptosis in HCT116 cells. To investigate the mechanism whereby activated k-ras sensitizes colonic cells to butyrate, we performed a genome-wide analysis of Ras target genes in the isogenic cell lines HCT116, Hkh2, and Hke-3. The gene exhibiting the greatest down-regulation by the activating k-ras mutation was gelsolin, an actin-binding protein whose expression is frequently reduced or absent in colorectal cancer cell lines and primary tumors. We demonstrated that silencing of gelsolin expression by small interfering RNA sensitized cells to butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7. These data therefore demonstrate that gelsolin protects cells from butyrate-induced apoptosis and suggest that Ras promotes apoptosis, at least in part, through its ability to down-regulate the expression of gelsolin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The short chain fatty acid butyrate is produced by bacterial fermentation of fiber and represents a primary energy source for normal colonic epithelial cells. Withdrawal of butyrate induces apoptosis, demonstrating that it acts as a survival factor for normal colonic epithelial cells (1, 2). In contrast, in colonic carcinoma cells butyrate induces G1 or G2 arrest, followed by differentiation and apoptosis (1–10), suggesting that the butyrate-induced signaling is different in normal epithelium and cancer tissues. Butyrate inhibits proliferation of transformed epithelial cells through up-regulation of p21 and down-regulation of c-Myc; however, the mechanisms whereby butyrate supports survival and promotes proliferation of normal epithelial cells are less well understood. These divergent effects of butyrate are known as the "butyrate paradox." The availability of alternative energy sources and different metabolic activities of normal and transformed cells have been proposed to underlie the divergent effects of butyrate (3), but molecular mechanisms that could account for the conflicting effects of this short chain fatty acid on normal and transformed colonic epithelial cells have not been determined. In this study we examined whether genetic and epigenetic changes associated with colon cancer regulate the responsiveness of cells to butyrate.

Inactivation of the tumor suppressor genes APC1 and p53 and activation of the oncogene k-ras underlie the development of the majority of colorectal cancers (11). The mutational activation of k-ras, or its downstream signaling effectors, is present in up to 50% of sporadic colorectal tumors (11, 12). The majority of the k-ras mutations are gain-of-function mutations at codon 12 and 13, and an activated k-ras has been shown to cooperate with both mutant APC and mutant p53 in the transformation of colonic epithelial cells (13, 14). Recent data suggest that mutations in k-ras may be sufficient to initiate colon tumor development (15). Accordingly, targeted expression of oncogenic k-ras in intestinal cells resulted in development of intestinal tumors in mice in the absence of detectable inactivating mutations of the tumor suppressor gene APC (16). Signaling transmitted through Ras results in activation of several downstream effectors, with the MAPK/ERK1/ERK2 and the phosphatidylinositol 3-kinase/AKT pathways playing a crucial role in modulation of the k-ras target genes which in turn regulate processes such as proliferation, differentiation and apoptosis (17–20). Regulation of cell survival by Ras is complex, complicated by the fact that activation of Ras, like several other oncogenes, can induce both pro-and antiapoptotic signaling. Ras protects from apoptosis via phosphatidylinositol 3-kinase/AKT/Rac and nuclear factor-{kappa}B signaling, and promotes apoptosis through its effectors RASSF1/Nore1/MstI (21). Raf/MEK/ERK signaling can be either anti- or proapoptotic, but the understanding of the downstream targets of Ras that mediate its effect on apoptosis is still far from complete.

To determine the role of activated k-ras in butyrate-induced apoptosis and to identify its downstream targets, we used the HCT116 colorectal carcinoma cell line and two clones derived form HCT116 cells with a targeted inactivation of the activated k-ras allele, Hkh2 and Hke-3. Hkh2 and Hke-3 cells are morphologically altered, do not form colonies in soft agar, and fail to form tumors in nude mice (22), demonstrating that tumorigenicity of HCT116 cells directly depends on the presence of an activated k-ras allele.

In this study we showed that oncogenic activation of a k-ras allele is sufficient to sensitize cells to butyrate-induced apoptosis and that this is, at least in part, mediated through transcriptional down-regulation of gelsolin expression. Gelsolin is a member of a large family of actin-severing and -capping proteins (23). In addition, human gelsolin has been shown to inhibit apoptosis through its ability to block mitochondrial membrane potential and to inhibit caspase activity (24–29, 31). The role of gelsolin in apoptosis is complicated by the fact that gelsolin is also a substrate for caspase-3, caspase-7, and caspase-9 (28, 29), which cleave gelsolin between Asp-352 and Gly-353 and generate an N-terminal cleaved product with proapoptotic activity (26). The anti-apoptotic activity of gelsolin resides in its C terminus and apparently counteracts the proapoptotic activity of the N-terminal domain (27). Overexpression of gelsolin has been shown to reverse transformation of Ras-transformed cells (32, 33), pointing to potential tumor-suppressive properties of gelsolin. Although no mutations of gelsolin have been described in human cancers, its expression is reduced or absent in ~60–90% of human colon, breast, bladder, prostate, and stomach cancers (24, 34–39).

Our data demonstrate that Ras mutations may underlie the frequent down-regulation of gelsolin in primary colon cancers (40). In addition, our finding that activating mutations of k-ras or silencing of gelsolin sensitizes cells to butyrate-induced apoptosis provides a mechanism for the divergent effects of butyrate on normal and transformed epithelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Reporter Gene Assay, and Transfections—The HCT116 human cell line was derived from colorectal carcinoma and harbors an activating mutation in codon 13 of the k-ras proto-oncogene. Two isogenic clones of the HCT116 cells with a disrupted mutant k-ras allele, Hkh2 and Hke-3, were generated by homologous recombination (22). HCT116 p53–/– cells were kindly provided by Bert Vogelstein. All cells were maintained in minimal Eagle's medium supplemented with 10% fetal calf serum and antibiotics. The viability of cells was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Roche Applied Science) according to the manufacturer's instructions.

Hke-3 cells were transiently transfected with a series of deletion constructs of the gelsolin promoter in the absence or the presence of RasV12 using a calcium phosphate method (Profection Mammalian Transfection system, Promega, Madison, WI). Transfection efficiency was normalized by cotransfection with pTK-Renilla (dual luciferase reporter assay system, Promega). Results are expressed as the -fold induction of luciferase activity, calculated from the ratio between the activity of the reporter plasmid in cells transfected with an empty vector or different deletion mutants of the gelsolin promoter.

Gelsolin expression was silenced using a pool of four siRNAs directed against the coding region of gelsolin (Dharmacon, Lafayette, CO). Cells were transiently transfected with 5–100 nM gelsolin siRNA using the calcium phosphate method as described above. Immunoblotting was performed using standard procedures (41). Antibodies specific for gelsolin were obtained from Sigma, and antibodies recognizing cleaved PARP, caspase-9, and caspase-7 were purchased from Cell Signaling Technology (Beverly, MA).

cDNA Microarrays—We performed cDNA microarray analysis using microarray slides comprising 8063 cloned sequences produced by the Microarray facility at the Albert Einstein College of Medicine. Total RNA was isolated using the RNeasy Midi kit (Qiagen) as suggested by the manufacturer. 100 µg of RNA from HCT116 cells was labeled with Cy3 (channel 1) and RNA from Hke-3 or Hkh2 cells with Cy5 (channel 2) fluorescent dye. After hybridization and washing (42), slides were scanned (532 nm/Cy3 and 633 nm/Cy5), and the two images were superimposed and quantified using Scanalyze 1.41 software. Data were imported into an Access data base for further analysis. The expression profile for HCT116 cells is presented as the intensity of channel 1 corrected for the background (ch1I/B), and expression data for Hkh2 or Hke-3 cells as the intensity of channel 2 corrected for the background (ch2I/B). Only genes with a signal/background ratio >1.25 in both Hkh2 and Hke-3 clones were analyzed further. Results are expressed as the ratio between the intensity of signals in HCT116 and Hke-3 cells, or between HCT116 and Hkh2 cells. We considered sequences as potential k-ras target genes if the ratio of the signals between HCT116 and Hkh2/Hke-3 cells was greater than 1.5 (k-ras up-regulated genes) or less than 0.6 (k-ras down-regulated genes).

Apoptosis—Adherent and floating cells were collected and resuspended in hypotonic buffer (0.1% Triton X-100, 0.1% sodium citrate) and stained with 50 µg/ml propidium iodide for 4 h at 4 °C. Samples were filtered through a nylon mesh (40-µm pore size) and analyzed by flow cytometry. Cell cycle distribution and the extent of apoptosis (cells with a sub-G1 DNA content) were analyzed by Modfit software.

Immunofluorescence—Cells were grown on chamber slides and were either left untreated or were treated with sulindac sulfide for 24 h. Cells were fixed in ice-cold methanol-acetic acid solution (95:5 v/v) for 20 min at –20 °C. Incubation with antibodies recognizing an activated caspase-3 (Cell Signaling Technology) was performed overnight at 4 °C. Slides were washed with phosphate-buffered saline and incubated with a secondary anti-rabbit antibody, conjugated to fluorescein isothiocyanate, for 45 min at 37 °C. Samples were examined with a fluorescent microscope and images acquired with a SPOT CCD camera and analyzed by SPOT software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activating Mutations of k-ras Sensitize Epithelial Cells to Apoptosis—The HCT116 colorectal cancer cell line harbors an activating mutation in the k-ras proto-oncogene. Two clones, Hkh2 and Hke-3, were derived from HCT116 cells by the targeted deletion of the activated k-ras allele, and both clones displayed loss of tumorigenicity in vitro and in vivo (22). We used these isogenic cell lines to examine whether k-ras mutations modulate the response to butyrate, a dietary chemopreventive agent that exerts different biological activity in normal and transformed epithelial cells. Isogenic cell lines were treated with 3 mM NaBu for 24, 48, or 72 h, and we monitored their viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. No major differences in viability among HCT116, Hkh2, and Hke-3 cells were found in the absence of butyrate, and all three isogenic cell lines ceased to proliferate in the presence of butyrate. However, we demon-strated that the viability of HCT116 cells, but not Hkh2 and Hke-3 cells, decreased markedly by the 48-h treatment with 3 mM butyrate (not shown), suggesting that oncogenic activation of k-ras modulates apoptosis in response to butyrate. To determine the role of Ras in butyrate-induced apoptosis, we treated HCT116, Hkh2, and Hke-3 cells with 2 or 5 mM NaBu for 24 h, and the extent of apoptosis was determined by measuring the percentage of cells with a sub-G1 content of DNA, using propidium iodide staining (Fig. 1A) or by staining cells with annexin V, a marker of apoptotic cells (Fig. 1B). Both experiments revealed that HCT116 cells were more sensitive to NaBu-induced apoptosis than the two isogenic clones with a disrupted mutant k-ras allele. Consistently, treatment of cells with 1 mM butyrate was sufficient to induce cleavage of PARP in HCT116 cells, but not in Hkh2 or Hke-3 cells (Fig. 1C). Treatment with 3mM butyrate induced strong accumulation of cleaved PARP in HCT116 cells, but not in Hkh2 cell. In Hke-3 cells, which are, because of clonal variations, somewhat more sensitive to butyrate than the Hkh2 cells (see also 1, A and B), cleavage of PARP occurred upon treatment with 3 mM butyrate, but to a much lower extent than in HCT116 cells. These results demonstrated that the loss of activated Ras is sufficient to confer resistance to NaBu-induced apoptosis. We showed that HCT116 cells were also sensitized to apoptosis induced by suberoylanilide hydroxamic acid, another histone deacetylase inhibitor (Fig. 1B), and to 5-fluorouracil-induced apoptosis,2 demonstrating that the increased sensitivity of HCT116 cells to apoptosis is not restricted to butyrate.



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  		FIG. 1.
Activating k-ras mutation sensitizes colon cancer cells to butyrate-induced apoptosis. A, HCT116, Hkh2, and Hke-3 cells were left untreated (control) or were treated with NaBu for 24 h as indicated. Cells were stained with propidium iodide, and the percentage of apoptosis was determined by FACS analysis. B, the isogenic cells were treated with 3 mM NaBu or 2 mM SAHA for 24 h, and the extent of apoptosis was determined by annexin V staining. C, the cleavage of PARP was determined in cells treated with 1 or 3 mM NaBu for 48 h using an antibody that specifically recognizes the cleaved product of PARP (Cell signaling). NSP, nonspecific band.

 

To further determine whether inactivation of the Ras signaling pathways alter the responsiveness of cells to butyrate, we tested the effect of apigenin on butyrate-induced apoptosis. Apigenin is a dietary flavonoid which has been shown to reverse transformation of cells via inhibition of the MAPK kinase activity, a downstream effector of the Ras activated signaling pathway (43–45). We demonstrated that apigenin inhibited butyrate-induced apoptosis in HCT116 cells in a dose-dependent manner (Fig. 2, A and B). The extent of apoptosis in parental HCT116 cells treated with butyrate and 20 µM apigenin was reduced to levels comparable with those of butyrate-induced apoptosis in Hkh2 and Hke-3 cells (Fig. 2, A and B). In contrast, apigenin did not have a significant effect on butyrate-induced apoptosis in Hkh2 and Hke-3 cells. The data in Figs. 1 and 2 suggest that the acquisition of a k-ras mutation confers upon colon cells the ability to respond to the physiological regulator of homeostasis, butyrate, by undergoing apoptosis. Conversely, reversal of k-ras-mediated transformation by genetic inactivation of the mutant k-ras allele or pharmacological inhibition of Ras signaling is sufficient to protect cells from butyrate-induced apoptosis.



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  		FIG. 2.
Apigenin (Apig) inhibits butyrate-induced apoptosis in cells containing an activated k-ras mutation. A, HCT116, Hkh2, and Hke-3 cells were left untreated (Ctrl) or were treated with 4 mM butyrate in the absence or the presence of 5, 10, or 20 µM apigenin (A) as indicated. The extent of apoptosis was determined 24 (open bars) or 48 h (hatched bars) after treatment. B, HCT116 and Hkh2 cells were treated with 4 mM butyrate, 10 µM apigenin, or a combination of both agents, and the photographs were taken 24 h after treatment using a Tripix advanced digital camera system.

 
These data therefore established a basis for the differential response of normal and cancer cells to butyrate.

Activating Mutation of k-ras Modulates the Expression of Genes Involved in Apoptosis, Including Gelsolin—To define the pathway whereby k-ras signaling promotes butyrate-induced apoptosis, we performed cDNA microarray analysis on the isogenic cell lines that differ by the presence of the mutant k-ras. We considered sequences as potential k-ras target genes if the ratio of the signals between HCT116 and Hkh2 or Hke-3 cells was greater than 1.5 (k-ras up-regulated genes) or less than 0.6 (k-ras down-regulated genes). We identified several sequences that are expressed differentially in HCT116 cells compared with the two clones with a disrupted mutant k-ras allele, among them several known k-ras target genes, such as epiregulin (46), glucose transporter (47), fibronectin (17), and vascular endothelial growth factor (48) (data not shown). In addition, we demonstrated that a family of interferon-responsive genes is negatively regulated by Ras signaling through Ras-mediated inhibition of STAT1/STAT2 expression (49).

Analysis of the Ras target genes revealed that several genes that modulate apoptosis were also regulated by k-ras signaling (Fig. 3). Bag-1, an antiapoptotic member of the BCL-2 family (50) and gelsolin, an actin-binding protein (23), were down-regulated by an activated k-ras. In contrast, the expression of NIP1, NIP2, and NIP3, a family of proapoptotic proteins, was up-regulated in cells containing the activating k-ras mutation. NIP proteins bear a limited homology to the BCL-2 homology domain 3 and regulate apoptosis by interacting with BCL-2 and BCL-x (51, 52). Another gene up-regulated by oncogenic k-ras was PHLDA1 (pleckstrin homology-like domain, family A), which is a human homolog of the mouse gene TDAG51, which plays a crucial role in activation-induced apoptosis of T cells (53). Activation of NIP3 expression by k-ras has been demonstrated recently in fibroblasts (17); however gelsolin, Bag-1, NIP, NIP2, and PHLDA1 represent potentially novel target genes of activated k-ras. These data demonstrated that genes with known proapoptotic functions, such as NIP1, NIP2, NIP3, and PHLDA1, are up-regulated by activated k-ras, whereas k-ras signaling leads to inhibition of genes with an antiapoptotic function, such as Bag-1 and gelsolin. Such changes may underlie the increased susceptibility of HCT116 cells to butyrate, compared with Hkh2 and Hke-3 cells.



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  		FIG. 3.
Differential expression of apoptosis-related genes in HCT116, Hkh2, and Hke-3 cells. RNA isolated from HCT116 cells was labeled with Cy3 (ch1) and RNA from Hke-3 or Hkh2 cells with Cy5 (ch2) fluorescent dye. Expression profile for HCT116 cells is presented as the intensity of channel 1 corrected for the background (ch1I/B), and expression data for Hkh2 and Hke-3 cells as the intensity of channel 2 corrected for the background (ch2I/B). Results are expressed as the ratio between intensity of signals in HCT116 and Hke-3 cells (A) or HCT116 and Hkh2 cells (B). ND (not done): NIP1 cDNA was not included on the slide we used for this particular experiment. We have repeated the experiment with Hkh2 cells and obtained similar results (not shown).

 
The fact that oncogenic activation of Ras inhibited gelsolin expression was of particular interest to us because the expression of gelsolin is known to be reduced or lost in colon cancer (40). To determine whether oncogenic Ras inhibits gelsolin expression at the transcriptional level we transfected Hke-3 cells with several deletion mutants of the gelsolin promoter fused to the luciferase reporter gene (54). Five different deletion constructs of the gelsolin promoter (Fig. 4A) were transfected into Hke-3 cells together with an empty vector or with a plasmid directing the expression of mutant Ras. The luciferase activity of the chimeric constructs was compared with the activity of the empty plasmid pGL-3 basic.



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  		FIG. 4.
Transcriptional inhibition of the gelsolin promoter by RasV12. A, Hke-3 cells were transfected with deletion mutants of the gelsolin promoter as represented schematically in the absence (CTRL) or the presence of the expression plasmid coding for RasV12 (B). The results are expressed as a relative promoter activity, calculated as a -fold induction over an empty plasmid (BASIC).

 
We showed that the activities of the full-length gelsolin promoter (–2524/LUC-1) and the –872/LUC-3 deletion construct were markedly down-regulated by mutant Ras (Fig. 4B), demonstrating that Ras signaling inhibits the expression of gelsolin at the transcriptional level. In contrast, the activities of the LUC-5 (–167) and LUC 7 (–109) were not as greatly affected by the mutant Ras, suggesting that the major element(s) responsible for the negative regulation of gelsolin promoter by RasV12 is located in the region between –167 and –872.

Our data therefore suggest that k-ras activation is sufficient for the inhibition of gelsolin expression, underlying the frequently reduced gelsolin expression in cancer cell lines and primary tumors (34–40).

We next examined whether the levels of gelsolin protein are also reduced in cells harboring an activated k-ras allele. We showed that the constitutive expression of gelsolin (0, Fig. 5A) was low in HCT116 cells, which harbor an activated k-ras allele, and that in two clones with deletion of the mutant Ras allele the basal level (0) of gelsolin was increased relative to parental HCT116 cells (11-fold and 5.5-fold, for Hkh2 and Hke-3 cells, respectively). However, we found that treatment of HCT116 cells with butyrate restored the expression of gelsolin to levels comparable with those in Hkh2 and Hke-3 cells. The induction of gelsolin expression by butyrate was evident also in Hkh2 and Hke-3 cells; however, because of elevated basal expression the induction was not as marked as in the HCT116 cells. This result demonstrated that Ras mutations do not interfere with butyrate-induced gelsolin expression. Consistently, we demonstrated that butyrate activates gelsolin promoter activity in both HCT116 and Hke-3 cells and that the –109 region of the gelsolin promoter appears to be sufficient to confer the inducibility by butyrate (Fig. 5B).



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  		FIG. 5.
Butyrate induces gelsolin expression at the transcriptional level. A, expression of gelsolin in HCT116, Hkh2, and Hke-3 cells. Cells were left untreated or were treated with 1 or 3 mM NaBu for 48 h. Expression of gelsolin was determined by immunoblotting using an anti-gelsolin monoclonal antibody (Sigma). B, deletion constructs of the gelsolin promoter as shown in Fig. 4A were transfected into HCT116 and Hke-3 cells and left untreated or were treated with 3 mM butyrate for 8 h. The results are expressed as a -fold induction over untreated cells.

 
Butyrate induces apoptosis, at least in part, through the activation of caspase-3 (55, 56), a caspase that has been shown to cleave gelsolin (26) and generates a cleaved product of gelsolin with proapoptotic activity. We did not detect significant activation of caspase-3 in HCT116, Hkh2, or Hke-3 cells upon butyrate treatment, suggesting that in these cells butyrate induces apoptosis in a caspase-3-independent manner. Like-wise, butyrate treatment did not induce significant cleavage of gelsolin (not shown).

Silencing of Gelsolin Expression Promotes Butyrate-induced Apoptosis—What is the role of gelsolin in butyrate-induced apoptosis? Overexpression of proteins often yields ambiguous results, in particular in the case of gelsolin because overexpression of full-length protein also leads to an increased amount of cleaved gelsolin product upon apoptotic insult (57). To avoid difficulties with ascribing the biological outcome to the increased levels of full-length protein or to the increased amounts of the cleaved product, we pursued the role of gelsolin in apoptosis by silencing its expression through siRNA. Hke-3 cells were transfected with a pool of four RNA duplexes directed against the coding region of gelsolin as described under "Material and Methods." Cells were transfected with 25 or 50 nM siRNA, and the expression of gelsolin protein was examined 48 h after transfection in both untreated cells and in cells treated with 3 mM butyrate. As shown in Fig. 6A, we achieved nearly complete inhibition of both basal and inducible gelsolin expression. The expression of c-Myc, its inhibition by butyrate, and the induction of p21 by butyrate were not affected by gelsolin siRNA (Fig. 6B), demonstrating the specificity of gelsolin siRNA.



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  		FIG. 6.
Silencing of basal and inducible expression of gelsolin. A, Hke-3 cells were transfected with 25 or 50 nM gelsolin siRNA for 24 h and were either left untreated or were treated with NaBu as indicated. The levels of gelsolin and {beta}-actin were determined by immunoblotting. B, Hke-3 transfected with gelsolin siRNA were left untreated or were treated with NaBu; the levels of gelsolin, c-Myc, and p21 were determined by immunoblotting. NSP, nonspecific band. C, Hke-3 cells were transfected with 25 or 50 nM gelsolin siRNA and were treated with 3 mM NaBu for 48 h. The percent apoptosis was determined by propidium iodide staining.

 
We next examined whether silencing of gelsolin expression in Hke-3 cells modulates butyrate-induced apoptosis. The parental Hke-3 cells and Hke-3/gelsolin siRNA cells were treated with 3 mM butyrate for 48 h, and the extent of apoptosis was determined by propidium iodide staining (Fig. 6C). As shown earlier, Hke-3 cells are, because of the deletion of the mutant Ras, relatively resistant to butyrate-induced apoptosis. Silencing of gelsolin expression strongly enhanced butyrate-induced apoptosis, demonstrating that full-length gelsolin inhibits butyrate-induced apoptosis. In addition, these data suggested that oncogenic Ras promotes butyrate-induced apoptosis at least in part through transcriptional inhibition of gelsolin expression (Figs. 3 and 4).

How does gelsolin interfere with butyrate-induced apoptosis? Gelsolin has been shown, through its ability to associate with the voltage-dependent anion channel, to regulate mitochondrial permeability for cytochrome and thereby to modulate the cascade of caspase activation (27–29). Our experimental system of silenced gelsolin expression allowed us to test the hypothesis that the activity of caspases is increased in the absence of gelsolin. Hke-3 cells were either mock transfected or transfected with 25 or 50 nM gelsolin siRNA and treated with 3 mM butyrate for 24 or 48 h as indicated in Fig. 7. The expression of gelsolin, activation of caspase-9 (assessed by the appearance of the cleaved product), activation of caspase-7 (assessed by the disappearance of full-length caspase 7), and the cleavage of PARP, a caspase substrate, were determined by immunoblotting. Both concentrations of siRNA were sufficient to silence constitutive and induced gelsolin expression (Fig. 7). We did not detect the activation of caspase 9, caspase-7 (Fig. 7), or caspase 3 (not shown) in cells treated with butyrate for 24 h. In contrast, the cleavage of PARP was apparent as early as 24 h after treatment with butyrate, and it occurred predominantly in cells with silenced gelsolin expression. 48 h after treatment of cells with butyrate, we demonstrated both activation of caspase-9 and caspase-7 almost exclusively in cells with silenced gelsolin expression. Accordingly, the levels of cleaved PARP were increased significantly in gelsolin-deficient cells (Fig. 7). These data demonstrated that silencing of gelsolin expression promotes butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7, which is in agreement with reports that full-length gelsolin inhibits the activation of caspases (28, 29).



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  		FIG. 7.
Activation of caspase-9 and caspase-7 in cells with silenced gelsolin expression. Hke-3 cells were transfected with gelsolin siRNA as indicated and were either left untreated or were treated with NaBu for 24 or 48 h. The levels of gelsolin, cleaved product of caspase-9, full-length (FL) caspase-7, and the cleaved product of PARP were determined by immunoblotting.

 
Overexpression of full-length gelsolin in HCT116 did not confer significant protection from butyrate-induced apoptosis, most likely because of the presence of the cleaved product of gelsolin in these cells, which exerts proapoptotic activity and overcomes the antiapoptotic activity of full-length gelsolin (data not shown). In contrast, overexpression of gelsolin in Caco-2 cells did not result in detectable cleavage of gelsolin upon butyrate treatment and thereby protected cells from butyrate-induced apoptosis (data not shown). The level of protection was comparable with the protection conferred by BCL-2. In accordance, expression of gelsolin in the antisense direction sensitized Caco-2 cells to both butyrate and sulindac-induced apoptosis, confirming the antiapoptotic nature of gelsolin.

Silencing of Gelsolin Promotes Sulindac-induced Apoptosis— Next we determined whether gelsolin regulates apoptosis also in response to the pharmacological chemopreventive agent sulindac. The Hke-3 cells and Hke-3 cells with silenced gelsolin expression were treated with 150 µM sulindac sulfide for 48 h, and the extent of apoptosis was determined by propidium iodide staining. As shown Fig. 8A, the extent of apoptosis induced in cells with silenced gelsolin expression was significantly higher than in mock-transfected cells, demonstrating that gelsolin can protect cells from sulindac-sulfide induced apoptosis. Unlike butyrate, sulindac did not regulate the expression of gelsolin (Fig. 8B). Consistent with an increased level of apoptosis in cells with silenced gelsolin expression, cleavage of PARP was evident primarily in cells with silenced gelsolin expression (Fig. 8B). We compared the activation of caspase-3 in mock-transfected cells and in cells with silenced gelsolin expression by immunofluorescence using an antibody that specifically recognizes activated caspase-3. As shown in Fig. 8C, treatment of cells with sulindac sulfide led to the activation of caspase-3 almost exclusively in cells with silenced gelsolin expression, demonstrating that enhanced apoptosis in cells with silenced gelsolin expression is caused by the activation of caspase cascade in the absence of gelsolin. Recently, gene expression analysis of 30 colorectal cell lines revealed that high levels of gelsolin correlate with resistance to sulindac sulfide,3 confirming that gelsolin plays a significant role in the responsiveness of cells to this chemopreventive agent.



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  		FIG. 8.
Silencing of gelsolin promotes sulindac-induced apoptosis. A, mock-transfected cells and cells with silenced gelsolin expression were left untreated or were treated with 150 µM sulindac sulfide for 48 h. The data represent a mean of three different experiments. B, the levels of gelsolin and cleaved PARP were determined 48 and 72 h after treatment with 150 µM sulindac sulfide. C, Hke-3 and Hke-3/gelsolin siRNA cells were left untreated or were treated with sulindac sulfide for 48 h, and cells were stained with antibody that recognizes activated caspase-3 and with DAPI to delineate nuclear morphology.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The response of normal and transformed epithelial cells to the short chain fatty acid, butyrate, is divergent: butyrate serves as an energy source for normal epithelial cells and supports their survival and proliferation, but it induces a rapid growth arrest, coupled to differentiation and apoptosis in transformed cells. This observation is known as the butyrate paradox, but the mechanisms underlying the divergent action of butyrate are not understood.

To determine whether genetic alterations associated with colon cancer modulate the responsiveness to butyrate, we used isogenic cell lines derived from the HCT116 cell line which differ only by the presence of oncogenic k-ras or p53. Targeted deletion of p53 in HCT116 did not alter the response of cells to butyrate (data not shown), which is in accord with published data demonstrating that butyrate induces growth arrest and apoptosis in a p53-independent manner (6). In contrast, this study demonstrates that targeted deletion of an oncogenic k-ras is sufficient to protect cells from butyrate-induced apoptosis. In agreement, we showed that apigenin, an inhibitor of Ras signaling and Ras-dependent transformation, inhibits the ability of butyrate to elicit the apoptotic response in HCT116 cells. These results demonstrate that during the process of cellular transformation cells acquire the ability to respond to butyrate by undergoing apoptosis. Apigenin inhibits MAPK activation, raising the possibility that activation of the MAPK pathway may be required for the proapoptotic activity of butyrate. Consistent with this hypothesis is the finding that during spontaneous differentiation of Caco-2 cells, which is accompanied by down-regulation of the MAPK activity (58), the cells become resistant to butyrate-induced apoptosis (data not shown and Ref. 59).

The expression of the oncogenic Ras induces both pro- and antiapoptotic signaling. When we compared the sensitivity of the isogenic cell lines to a variety of apoptosis-inducing agents, we found that HCT116 cells, harboring mutant Ras, are also more sensitive to apoptosis induced by chemotherapeutic agents, such as 5-fluorouracil and to growth factor-withdrawal induced apoptosis, than the two clones with the targeted deletion of the mutant Ras allele (not shown). In contrast, we showed that HCT116 cells are less sensitive to apoptosis induced by transforming growth factor-{beta} (data not shown), most likely because of a negative regulation of Smad signaling by oncogenic Ras (60). Expression of oncogenic k-ras has recently been shown to enhance apoptosis of embryonic stem cells in response to chemotherapeutic agents (61), and the expression of an activated k-ras in Caco-2 cells resulted in accelerated apoptosis in response to sulindac (62). Thus, k-ras is a member of the group of proto-oncogenes whose activation can induce not only proliferation, but also apoptosis. Several other oncogenes, such as c-myc, k-ras, E1A, E2F-1, and CDC25A have also been shown to be dual function proteins; they induce both cell proliferation and apoptosis (63–68). Proapoptotic activity of activated oncogenes may in fact represent the primary cause of elimination of mutant cells and may explain the fortunate fact that cancer is a rare disease. In addition, the proapoptotic nature of certain oncogenes underscores the need of tumor cells to acquire additional changes that counteract the proapoptotic activity of activated oncogenes, such as activation of BCL-2 in the case of c-myc-induced apoptosis (69). Because the expression of apoptotic modulators correlates with the sensitivity of tumors to cancer therapy, it is important to understand the pathways whereby oncogenes activate apoptosis and to identify the lesions that blunt their proapoptotic activity. An example of such functional collaboration has been demonstrated between c-Myc and BCL-x in the development of pancreatic cancer, where BCL-x efficiently suppresses apoptosis, induced by overexpression of c-Myc (70).

We demonstrated that activation of K-Ras modulates the expression of several genes involved in apoptosis. One of the genes that was strongly down-regulated by oncogenic k-ras was gelsolin, an actin-binding protein with an established role in apoptosis (26, 30, 57, 71). However, several colon cancer cell lines, such as Caco-2 and HT29, which have not been reported to carry an activating mutation of k-ras, also display low levels of gelsolin, demonstrating that there are pathways other than k-ras mutations leading to the inhibition of gelsolin expression. Activation of several other oncogenes results in down-regulation of gelsolin expression, such as H-ras, SV40 (72) or Bcr/Abl,4 which may underlie the frequent down-regulation of gelsolin in tumors. We demonstrated that butyrate, a dietary chemopreventive agent, restores gelsolin expression in cells with the mutant Ras. Our results therefore established a point of convergence between the genetic changes underlying colon cancer progression, and a physiological regulator of homeostasis in epithelial cells, butyrate.

We showed that silencing of gelsolin expression through RNA interference sensitizes cells to butyrate-induced apoptosis, demonstrating that gelsolin protects cells from butyrate-induced apoptosis. Similarly, induction of p21 by butyrate protects cells from butyrate-induced apoptosis (73). Silencing of gelsolin expression resulted in enhanced activation of caspase-9 and caspase-7 and the subsequent cleavage of their downstream substrate, PARP, upon butyrate treatment. This is consistent with a report that full-length gelsolin inhibits activation of caspase-3, caspase-8, and caspase-9 because of the lack of release of cytochrome c in cells overexpressing gelsolin (28, 29). Our data, however, revealed that full-length gelsolin had no major affect on apoptosis in HCT116 cells. The lack of the protective effect of full-length gelsolin is likely the result of its cleavage in cells that overexpress gelsolin. Consistent with this, we showed that the expression of the N-terminal cleavage product exerts strong proapoptotic activity in HCT116 cells (not shown). It is therefore likely that the regulation of apoptosis by gelsolin depends on its caspase-mediated processing and that the ratio between the full-length and the cleaved product of gelsolin determines whether the cell will undergo apoptosis or survive. The expression of gelsolin is activated during differentiation of Caco-2 cells, a process accompanied by acquired resistance to butyrate (data not shown and Ref. 59). Likewise, overexpression of gelsolin in Caco-2 cells conferred significant protection from butyrate-induced apoptosis (data not shown). It is possible that accumulation of gelsolin is, at least in part, responsible for the resistance of differentiated cells to butyrate.

The significance of gelsolin down-regulation for the progression of colon cancer remains to be determined. Because oncogenic k-ras has been shown to synergize with an APC mutation in the progression of colon cancer (13), we have generated a compound mouse model, containing both an APC 1638 mutation and a gelsolin null mutation, to test the hypothesis that gelsolin is a downstream effector of k-ras signaling in the progression of colon cancer. Our preliminary data suggest that targeted deletion of gelsolin in vivo, like oncogenic k-ras, enhances APC-initiated development of intestinal tumors.5 Our data also suggest that gelsolin might have prognostic value in colon cancer patients. We demonstrated that silencing of gelsolin-sensitized cells to apoptosis was induced by a pharmacological chemopreventive agent, sulindac, suggesting that colon cancer patients with low gelsolin level may have improved response to sulindac treatment. A high level of gelsolin expression confers a significantly worse prognosis and predicts a high risk of cancer relapse in non-small cell lung cancers (30), possibly because of enhanced motility of the gelsolin-expressing lung carcinoma cells. Our data suggest that the increased susceptibility of cells with reduced gelsolin expression to undergo apoptosis may also lead to a better prognosis in colon cancer patients with low or absent gelsolin expression.


   FOOTNOTES
 
* This work was supported by a Montefiore Medical Center new research initiative award (to L. K.), American Cancer Society Institutional Research Grant ACS IRG 98-274-01 (to L. K.), NCI, National Institutes of Health Grant UO1 CA88104 (to L. A.), and Cancer Center Grant PO13330. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Albert Einstein Cancer Center, Montefiore Medical Center, 111 E. 210th St., Bronx, NY 10467. Tel.: 718-920-6579; Fax: 718-882-4464; E-mail: lklampf{at}aecom.yu.edu.

1 The abbreviations used are: APC, adenomatous polyposis coli; ERK, extracellular signal-regulated kinase; LUC, luciferase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; NaBu, sodium butyrate; siRNA, small interfering RNA; STAT, signal transducers and activators of transcription; NIP, 19-kDa interacting protein. Back

2 L. Klampfer, L.-A. Swaby, J. Huang, T. Sasazuki, S. Shirasawa, and L. Augenlicht, manuscript in preparation. Back

3 A. J. Wilson, personal communication. Back

4 L. Klampfer, unpublished data. Back

5 L. Klampfer, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Bonnie Asch for providing the gelsolin promoter constructs, Bert Vogelstein for the gift of HCT116 cells with the targeted deletion of p53 and p21, and Georg Wisniewski and Laurie-Anne Swaby for reading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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