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Department of Physiology and Cellular Biophysics,
The Center for Radiation Research, and
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY;
Surgical Service, John D. Dingell VA Medical Center and Wayne State University, Detroit, MI; and the
Department of Surgical Pathology, New York Presbyterian Medical Center, New York, NY
**
*
3To whom correspondence should be addressed. E-mail: nje7{at}columbia.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • butyrate • invasive colon cancer • urokinase plasminogen activator • dietary fiber • gene-nutrient interactions
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–8
) and animal (9
–14
) and cell culture models (15
–25
). Populations consuming high levels of dietary fiber or resistant starches are associated with lower incidence rates of colon cancer (26
, 27
). These observations may be related to the production of butyrate in the colonic lumen by the bacterial fermentation of dietary fiber and resistant starches. Butyrate, a four-carbon short-chain fatty acid (SCFA),4
is the predominant energy substrate for colonocytes and has been clearly shown to modulate cell differentiation, cell invasion, proteolysis, cell adhesion, cell proliferation, cell motility and cell apoptosis (15
–20
, 28
, 29
), cell cycle regulatory genes (28
, 30
–32
) and vitamin D receptor expression (29
, 30
). Previous investigations have focused largely on the epigenetic effects of physiologic concentrations of luminal SCFA using animal models and established cell culture models before the development of malignant transformation. However, relatively little is known about the mechanisms by which SCFA may mediate primary invasive human colon cancer.
The development of invasive human colon cancer depends on the capacity of tumor cells to adhere, proliferate and transmigrate the basement membrane. We previously reported that SCFA significantly inhibit human SW1116 colon cancer cell invasion by inhibiting urokinase plasminogen activator (uPA; EC 3.4.21.73) secretion and concomitantly stimulating tissue inhibitor matrix metalloproteinase (TIMP)-1 and TIMP-2 proteolytic inhibitors, which protect the basement membrane against excessive degradation (17
). Pathophysiologic differences in c-myc protein expression occur between nonmalignant and malignant primary human colonocytes treated with SCFA, suggesting that pathophysiologic alterations in SCFA utilization may exist between nonmalignant and invasive colon cancers (16
).
In this study we investigated the ability of physiologic concentrations of acetate, propionate or butyrate to inhibit primary human colon cancer cell invasion by modulating cell adhesion, F-actin polymerization, secretion of proteolytic and proteolytic inhibitors as well as the expression of tumor suppressor and cell proliferation genes using an in vitro primary invasive human cell culture model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Disposable tissue culture plastics, 8-µm inserts and Matrigel basement membrane matrix were purchased from BD Biosciences (Bedford, MA). Low glucose Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 (F12) was purchased from Cellgro (Mediatech, Herndon, VA). Fetal bovine serum (FBS), sodium pyruvate, L-glutamine, penicillin/streptomycin, gentamycin, HEPES and trypsin-EDTA solutions were purchased from Life Technologies (Gaithersburg, MD). Type II collagenase for primary colon cancer cell dissociation was purchased from Worthington Biochemical (Lakewood, NJ). Acetate, propionate, butyrate, neutral buffer formalin solution, hematoxylin and eosin were purchased from Sigma Chemical (St. Louis, MO). ELISA kits for uPA were purchased from American Diagnostica (Greenwich, CT) and kits for TIMP-1 and TIMP-2 were obtained from Amersham Life Sciences (Arlington Heights, IL). Mutant p53, Bcl-2, Bax, p21 and proliferating cell nuclear antigen (PCNA) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse or anti-rabbit rhodamine-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Rhodamine-phalloidin for F-actin studies was purchased from Molecular Probes (Eugene, OR).
Isolation of primary invasive human colonocytes.
Permission was obtained from the institutional review boards of the Columbia University College of Physicians and Surgeons and the Yale University School of Medicine to obtain human surgical specimens from patients resected for invasive colon cancer. Human specimens were thoroughly rinsed in DMEM/F12 to remove any tissue debris before the primary invasive human colon cancer cells were isolated from the surgical specimens as previously described (16
). Tumor specimens were minced until the tissues easily passed through a 30-µm cell sieve after which the cell suspension was incubated at 37°C with type II collagenase dissolved in warm DMEM/F12. After a suspension of single cells was obtained, the primary cells were gently centrifuged then resuspended in fresh warm DMEM/F12 supplemented with 10% FBS, 1% HEPES, 1% L-glutamine, 1% penicillin/streptomycin and 0.1% gentamicin (16
, 17
). The primary cell suspension was plated in tissue culture plastic flasks for 1 h to remove any remaining contaminating cells. Purified invasive colon cancer cell populations were expanded to provide sufficient cell numbers for cell proliferation and immunofluorescence staining assays described below.
Cell invasion assays.
Primary invasive human colonocytes (1 x 106) were added to six-well Matrigel-coated 8-µm pore invasion chambers and treated with control medium or medium supplemented with acetate, propionate or butyrate at 0.01 mol/L and incubated at 37°C in humidified 5% CO2 (16
, 17
). After 12–18 h, the nonadherent primary cells were removed by rinsing with PBS (pH 7.4) and the chambers were fixed in formalin and stained with hematoxylin and eosin (17
). Primary cellular invasion indices were determined by counting 10 fields at 400X magnification using a Nikon Eclipse E800 light microscope (Melville, NY) (17
).
Cell adhesion assays.
Cell adhesion was quantitated as previously described (33
) with the following modifications. Adherent cells on the top and undersides of the invasion chamber were quantitated by counting 10 fields on both sides of the invasion chamber at 400X magnification using a Nikon Eclipse E800 light microscope. The total number of adherent primary invasive colon cancer cells was determined for each treatment and an adhesion percentage value was calculated relative to the total number of cells adherent to control chambers.
F-actin polymerization assays.
Expanded human invasive colon cancer cells (1 x 105) were plated onto round glass coverslips in a 24-well plate in cell culture medium for 24 h. Adherent cells were then treated with control medium with or without acetate, propionate or butyrate (0.01 mol/L) at 37°C in a humidified chamber containing 5% CO2 for 12–18 h. F-actin polymerization was quantitated using rhodamine-phalloidin (0.33 µmol/L) as described previously (34
) with the following modifications: nonadherent cells were removed by rinsing and adherent cells were fixed in 3.7% formaldehyde. Total cellular F-actin was quantitated by rhodamine fluorescence (excitation wavelength: 530 nm, emission wavelength: 590 nm) using a Cytofluor II fluorescence plate reader (Millipore, Bedford, MA). To normalize for cell number, nuclei were stained with YO-PRO at 5 µmol/L, fluorescence was determined (excitation wavelength: 485 nm, emission wavelength: 530 nm) and F-actin content per unit cell was calculated as the ratio of rhodamine to YO-PRO fluorescence (34
).
Protease and protease inhibitor assays.
Aliquots of primary invasive colon cancer cell–conditioned media were collected and stored at -80°C until batch analyzed by ELISA for uPA, TIMP-1 and TIMP-2 activities as described previously (17
). Individual concentration curves were calculated by plotting sample optical densities against the standard curve generated in parallel with the samples of each of the ELISA assays (17
). Within-assay and between-assay reproducibility and sensitivity were within the manufacturer’s acceptable limits for each of the ELISA assays tested.
Immunofluorescent confocal microscopy.
Colon cancer cells derived from expanded invasive colon cancer cell populations were cultured on Matrigel-coated glass chamber slides at a density of 1 x 104 cells/mL until 70% confluent. The cells were then treated for 24 h with either acetate-, propionate- or butyrate-containing cell culture medium and incubated at 37°C in humidified 5% CO2. After 24 h, the cells were washed twice with room temperature PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4). Cell cultures were covered with normal horse serum for 30 min at room temperature and then incubated overnight at 4°C with a 1:500 dilution of a primary anti-mouse monoclonal antibody for mutant p53, Bcl-2, Bax, p21 or PCNA. After being washed in buffer solution, the slides were incubated for 60 min at room temperature with anti-mouse or anti-rabbit rhodamine-conjugated secondary antibody at a 1:1000 dilution. After several 5-min washes with buffer solution, slides were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA). Control cultures were stained with either the primary or secondary antibodies alone to monitor the background staining. Cellular protein expression was quantified as previously described (35
) and viewed on Ziess Axiovert 100 TV microscope (Carl Zeiss, Thornwood, NY) using a 40X 11.3 NA objective lens equipped with a laser scanning confocal attachment (LSM 410 Carl Zeiss). Fluorescent images were collected in the rhodamine channel by an argon/krypton mixed gas laser. Composite images were generated using Adobe Photoshop version 5.0 and printed on a Kodak DS 8650 printer (Rochester, NY). The relative staining intensity of Bax, Bcl-2, p53, p21 and PCNA protein expression levels were quantitated from the cell area and cell staining intensities obtained from at least 10 randomly selected microscopy fields (35
).
Statistical analysis.
All data were normalized to control before statistical analyses were performed. All values are presented as means ± SEM. Significance was determined by one-way ANOVA followed by Student’s t test and was set at P < 0.05 for these studies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Primary human invasive colonocytes were isolated from 21 randomly selected fresh human surgical specimens with invasive colon cancer (adenocarcinoma) nearly equally from male and female patients at the New York Presbyterian Medical Center and the Connecticut Veterans Affairs Health Care System. Sex-based differences in patient responses to SCFA were not observed (data not shown). Acetate, propionate and butyrate treatments differentially inhibited cell invasion in primary human colon cancer cells (Fig. 1
). The inhibitory effects of acetate and propionate on primary cell invasion were not significantly different. Acetate inhibited cell invasion by 11.8 ± 7.4% (P < 0.05; n = 19) whereas propionate appeared only slightly more potent at 17.6 ± 6.1% (P < 0.005; n = 20). The inhibitory effects of butyrate were significantly different from those of acetate and propionate. Of the SCFA tested, butyrate was the most effective inhibitor of primary human colon cancer cell invasion, inhibiting cell invasion by 54.7 ± 5.3% (P < 0.0001; n = 21).
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SCFA treatments did not affect primary human colon cancer cell adhesion to Matrigel-coated invasion chambers (P = 0.58, n = 8), although butyrate inhibited cell adhesion by 20.6% (data not shown). Additional morphological immunofluorescence studies also suggested that F-actin polymerization in primary human colon cancer cells was not affected by SCFA (data not shown).
Cellular proteases and protease inhibitors assays.
In general, SCFA stimulated TIMP-1 and TIMP-2 activities. Acetate and propionate stimulated TIMP-1 by 15.6 ± 3.8% (P < 0.05, n = 15) and 19.4 ± 4.3%, respectively (P < 0.05, n = 13). Butyrate stimulated TIMP-1 by 29.3 ± 3.8% (P < 0.005, n = 16) and was the most potent SCFA tested. A difference in the intensity of TIMP-1 stimulation was not observed across the SCFA (Fig. 2A
). Acetate did not significantly stimulate TIMP-2. However, propionate (P < 0.0001, n = 13) and butyrate treatments (P < 0.005, n = 15) significantly stimulated TIMP-2 activity by 22.1–23.7% (Fig. 2
B).
|
). Propionate was more potent than acetate, inhibiting uPA activity in primary human invasive colonocytes by 30.8 ± 3.1% (P < 0.0005, n = 17) and 11.2 ± 2.5% (P < 0.05, n = 16), respectively.
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SCFA significantly modulated genes regulating tumor suppression, cell apoptosis and cell proliferation (Table 1
). Mutant p53 protein expression was significantly inhibited by each of the SCFA tested. Acetate was the most potent inhibitor of mutant p53, inhibiting its expression by 34.1 ± 0.8% (P < 0.0001, n = 60). Butyrate and propionate inhibited mutant p53 protein expression by 19.8 ± 1.2% (P < 0.0001, n = 20) and 22.5 ± 0.9% (P < 0.0001, n = 59). Acetate treatment was significantly different from propionate and butyrate, whereas propionate and butyrate similarly inhibited mutant p53 expression.
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. Differential stimulatory effects were observed for each SCFA tested. Acetate stimulated Bcl-2 protein expression by 83.4 ± 3.3% (P < 0.0001, n = 20). Propionate and butyrate exerted highly stimulatory effects on Bcl-2 protein expression, stimulating Bcl-2 expression by 133.3 ± 4.0% (P < 0.0001, n = 20) and 187.2 ± 3.23% (P < 0.001, n = 20). However, SCFA exerted a significant inhibitory effect on Bax, a proapoptotic Bcl-2 family protein (Table 1)
. Acetate and propionate similarly inhibited Bax protein expression by 55.7 ± 2.2% (P < 0.0001, n = 10) and 53.0 ± 3.3% (P < 0.0001, n = 10), respectively. Butyrate inhibited proapoptotic Bax protein expression by 15.4 ± 1.7% (P < 0.0001, n = 20).
Each SCFA that we tested stimulated p21 protein expression (Table 1)
. Butyrate and propionate similarly stimulated p21 protein expression 43.0 ± 3.4% (P < 0.0001, n = 11) and 35.8 ± 3.1% (P < 0.001, n = 18), respectively. Acetate was least stimulatory, elevating p21 protein expression only 20.6 ± 2.0% (P < 0.05, n = 22). Unlike p21, PNCA protein expression was discordantly affected by SCFA (Table 1)
. Acetate and propionate stimulated PCNA protein expression by 56.7 ± 2.7% (P < 0.0001, n = 27) and 14.9 ± 1.2% (P < 0.005, n = 42), respectively, whereas butyrate actually inhibited PCNA protein expression by 21.3 ± 1.0% (P < 0.0001, n = 30).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–10
, 36
, 37
) may affect the capacities of these indigestible carbohydrates to act chemoprotectively against the development of large bowel cancers (10
).
Our current findings obtained in primary human invasive colon cancer cells are consistent with previous studies in the established SW1116 cell line (17
), Moser and HT29 human colon cancer cell models (21
). In this study we isolated primary human invasive colonocytes from fresh surgical specimens resected from patients with invasive colon cancer. A primary invasive colonocyte model may represent more accurately the diverse tumor pathophysiology found in vivo. Our findings are limited by the use of only one physiologic concentration of SCFA and because the structural dynamics of the basement membrane in vivo differ from those provided by an in vitro Matrigel-based system. Despite these caveats, this study provides evidence that SCFA inhibit primary human invasive colon cancer and that the mechanism of action may differ among SCFA.
Each SCFA significantly inhibited uPA, a serine-class protease capable of cleaving components of the basement membrane as well as activating latent forms of matrix metalloproteases that are also potent extracellular matrix degrading proteases (38
, 39
). In this study using primary invasive human colonocytes, we have confirmed our previous findings that SCFA inhibited human SW1116 colon cancer invasion in vitro by inhibiting uPA and stimulating TIMP-1 and TIMP-2 activities (17
). Consistent with a role for uPA in invasion, the expression of a1-antitrypsin Portland cDNA (coding for a potent proprotein convertase inhibitor) in HT-29 cells reduced invasiveness that paralleled a decrease in uPA, uPA receptor (uPAR) and tissue plasminogen activator (40
). Also, treatment of 95D cells, a lung carcinoma strain with high metastatic potential, with monoclonal antibodies to uPA and uPAR greatly reduced the cells invasive potential (41
). The stimulation of TIMP-1 and in some cases TIMP-2 by SCFA suggests that SCFA may protect the basement membrane from the proteolytic activities of matrix metalloproteases that may be activated during invasive colon cancer (17
).
In parallel studies, we analyzed of tumor suppressor protein mutant p53 expression and cell cycle regulatory genes (p21, Bcl-2, Bax, PCNA) that are known to participate in cell proliferation, transformation and apoptotic pathways. Our results indicated that acetate was the most potent inhibitor of mutant p53 protein expression and that propionate- and butyrate-treated cells were less potent inhibitors of mutant p53. The transformed colon cancer cells were probably previously altered in the mutant p53 expression by unknown factors; thus butyrate appeared to inhibit the accumulation of mutant p53 as indicated by the results. However, acetate and propionate did not alter such accumulations. These results suggest that butyrate probably protects against invasive colon cancer through stimulation of protective TIMP. p53 is known to regulate the expression of genes encoding products that regulate proteolytic degradation of the extracellular matrix (26
), which confirms these results.
Our findings confirm that butyrate stimulates p21 expression in vitro (24
, 30
–32
, 42
44
) in a p53-independent manner (31
, 42
, 44
). Although Bcl-2 is elevated in tumor tissue compared with rat control tissues (14
, 45
), the effects of butyrate on Bcl-2 are uncertain (14
, 46
, 47
). Our findings suggest that SCFA, in particular butyrate, stimulate Bcl-2 expression but inhibit Bax in a p53-independent manner. These results indicate that among the SCFA studied, butyrate was the most potent inhibitor of proliferation as indicated by PCNA, a clinically useful marker of cancer proliferation (21
, 22
). These results corroborated previous studies that showed that cell proliferation was inhibited by butyrate (29
, 31
, 48
, 49
) in three human colonic adenocarcinoma cell lines and that propionate exhibited a weak antiproliferative effect but acetate was ineffective (23
).
These studies indicate that butyrate and acetate stimulate p21/WAF1 protein expression, compounds that are known to induce tumor cytostasis and growth inhibition and differentiation of cancer cells (27
). Because growth arrest is observed associated with enhanced expression of p21/WAF1, it can be hypothesized that SCFA might act through a p53-independent pathway of growth control (42
). The resultant low expression of Bax observed in our studies relative to that of Bcl-2 might suggest that SCFA do not stimulate primary invasive colon cancer cell apoptosis via the Bcl-2/Bax pathway. Further investigations may provide the evidence needed to elucidate the effect of SCFA on these pathways.
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FOOTNOTES |
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2 Supported by grants awarded to N.J.E. by the Cancer Research Foundation of America (CU51341601 and CU51341602) and G.M.C. by the Avon Products Foundation Breast Cancer Center (CU51470301).
4 Abbreviations used: DMEM/F12, Dulbecco’s modified Eagle’s medium/Ham’s F12; FBS, fetal bovine serum; PCNA, proliferating cell nuclear antigen; SCFA, short-chain fatty acids; TIMP, tissue inhibitor matrix metalloproteinase; uPA, urokinase plasminogen activator; uPAR, uPA receptor.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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19. Values not sharing a letter designation are significantly different, P < 0.05.
