- Email: [email protected]
Butyrate sensitizes human colon cancer cells to TRAIL-mediated apoptosis
Butyrate sensitizes human colon cancer cells to TRAIL-mediated apoptosis Ambrosio Hernandez, MD, Robert Thomas, MD, Farin Smith, BS, Jennie Sandberg, BS, Sunghoon Kim, MD, Dai H. Chung, MD, and B. Mark Evers, MD, Galveston, Tex
Background. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor family, induces apoptosis in TRAIL-sensitive tumors through the activation of the caspase pathway. Sodium butyrate (NaBT) induces differentiation and apoptosis in certain colorectal cancers; the molecular mechanisms for these effects have not been clearly defined. The purpose of our study was to determine whether NaBT sensitizes TRAIL-resistant human colon cancer cells to the effects of TRAIL. Methods. Human colon cancer cells (KM12C, KML4A, and KM20) that are resistant to TRAIL treatment alone were treated with TRAIL (100 ng/mL), NaBT (5 mmol/L), or a combination of these agents and harvested for total RNA and protein. Western blots were performed to assess intracellular expression of Flice-like inhibitory protein (FLIP), a caspase inhibitor. Percent-specific apoptosis, relative caspase-3 activity, and Annexin-V immunofluorescence were determined at 24 and 48 hours. Cell cycle–related gene expression was assessed by RNase protection. Results. Treatment with NaBT for 24 and 48 hours decreased FLIP protein expression in all cell lines. Furthermore, NaBT sensitized these resistant cancer cells to the effects of TRAIL with significant increases noted in cell death, caspase-3 activity, and Annexin-V staining compared with NaBT alone. Conclusions. Our findings suggest that the reduction of FLIP protein levels by NaBT renders TRAILresistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL with agents (such as NaBT, which target proteins that prevent cell death) may provide a more effective and less toxic regimen for the treatment of resistant colon cancers. (Surgery 2001;130:265-72.) From the Department of Surgery, The University of Texas Medical Branch, Galveston, Tex
COLORECTAL CANCER IS the third most common cause of cancer-related deaths in the United States in both men and women.1 It is estimated that approximately 130,000 new cases of colorectal cancer will be diagnosed this year.1 The prognosis for colon cancer is highly dependent on the stage of the disease. Colon cancer, when localized to the bowel wall, is a highly curable disease. Unfortunately, Stage IV disease (ie, distant metastases) is associated with a 5-year survival rate of less than 5%. For all but Stage IV disease, survival rates have improved in the last 40 years.2 The development of more novel therapeutic agents is required if a further improvement in the survival rate is to be realized. Supported by grants R01 AG10885, R01 DK48498, P01 DK35608, and T32 DK07639 from the National Institutes of Health. Presented at the 62nd Annual Meeting of the Society of University Surgeons, Chicago, Ill, February 8-10, 2001. Reprint requests: B. Mark Evers, MD, Department of Surgery, The University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-0536. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 11/6/115897 doi:10.1067/msy.2001.115897
Tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) is a recently characterized class II integral membrane protein that was initially identified on the basis of the homologic features of its extracellular domain to tumor necrosis factor and FasL.3 TRAIL binds its cell receptors (DR4 or DR5), which are linked to the “death domains,” to trigger apoptosis through the caspase cascade.3 TRAIL was initially reported to preferentially induce apoptosis in malignant cells, which provided the possibility of the use of TRAIL in the treatment of a number of cancers, including colorectal cancer.3-6 Recent studies, however, have demonstrated that an increasing number of cancers are resistant to TRAIL.7 One proposed mechanism for this resistance is the presence of “decoy” receptors that have been identified and shown to bind TRAIL but that are not linked to the apoptotic cascade. It is becoming increasingly apparent that other mechanisms for this resistance are more likely (such as the presence of intrinsic proteins that can act as inhibitors of apoptosis).1,8 A recently identified inhibitor of apoptosis has been described and designated Flice-like inhibitory protein (FLIP).2 FLIP structurally resembles casSURGERY 265
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Fig 1. Western blot analysis of FLIP expression after NaBT treatment. Western immunoblot analysis of protein extracts (50 µg) from KM colon cancer cells that were treated with NaBT (5 mmol/L) for 24 and 48 hours. Blots were probed for FLIP expression with a human anti-FLIP antibody. The blot was then stripped and reprobed with β-actin.
Fig 2. JAM assays. The KM colon cancer cells were plated in 96-well plates, labeled with 3H-thymidine, and treated with human recombinant TRAIL (100 ng/mL), NaBT (5 mmol/L), or the combination of TRAIL and NaBT for 24 and 48 hours. Apoptosis (percent specific apoptosis) was determined and compared with cells treated with vehicle (control) or NaBT alone. (Data are expressed as mean ± SEM; *P < .05 vs control; †P < .05 vs NaBT alone.)
pase-8 but lacks proteolytic activity, thus functioning as a dominant negative inhibitor of caspase-8, (Flice).9-11 A critical role for FLIP in the resistance of certain cancers to TRAIL-mediated cell death has been demonstrated with a reduction in cellular levels of FLIP that are associated with increased sensitivity of cancers to the effects of TRAIL.6,12 Another potentially novel agent that could serve as a useful adjunct to the treatment of metastatic colorectal cancer is the short-chain fatty acid sodium butyrate (NaBT), which is produced in the colon by the breakdown of dietary fiber.13,14 Our laboratory13,15 and others14 have shown that the NaBT treatment of certain colon cancers induces differentiation and eventual apoptosis by mechanisms that include G1 cell cycle arrest and stimulation of cell cycle–related proteins and proteins that are important for apoptosis. The purpose of our present study was to determine whether NaBT could “sensitize” TRAIL-mediated colon cancer cells to the effects of TRAIL and define the molecular mechanism responsible for this effect. MATERIAL AND METHODS Material. Tissue culture media and reagents were obtained from GIBCO-BRL (Grand Island,
NY). NaBT and the 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay were purchased from Sigma Chemical Company (St Louis, Mo). Total RNA was isolated with the use of RNAzol (Biotex, Houston, Tex) and digested with RNase free DNase I (Clontech, Palo Alto, Calif). RNase protection assay (RPA) was performed with the HybSpeed RPA Kit (Ambion, Austin, Tex) according to the manufacturer’s instructions. The human cell cycle complementary DNA template for RPA was purchased from PharMingen (San Diego, Calif). The concentrated protein assay dye was purchased from Bio-Rad laboratories (Hercules, Calif). Immobilon-P nylon membranes for Western blots were purchased from Millipore (Bedford, Mass), and x-ray film was purchased from Eastman Kodak (Rochester, NY). The enhanced chemiluminescence system for Western immunoblot analysis was obtained from Amersham (Arlington Heights, Ill). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). TRAIL apoptosis kit (17-226) was purchased from Upstate Biotechnology (Lake Placid, NY). Caspase-3 assay substrate, Ac-Val-Ala-Asp-AFC was purchased from Enzyme Systems Products (Livermore, Calif).
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Fig 3. Caspase-3–like activity. The KM cells were treated as described in Fig 2. Caspase-3–like activity was measured with the chromogenic substrate Ac-Val-Ala-Asp-AFC and normalized to 1 µg protein. (Data are expressed as mean ± SEM; *P < .05 vs control; †P < .05 vs NaBT alone.)
Cell culture. The human colon cancer cell lines KM12C, KML4A, and KM20 were provided by Dr Isaiah J. Fidler (University of Texas MD Anderson Cancer Center, Houston, Tex). The KM12C cell line was derived from a patient with a Dukes’ B colon cancer. KML4A was derived from KM12C and has been “trained” to metastasize to the liver by multiple rounds of injection into the spleens of athymic nude mice. The KM20 cell line was derived from a patient with Dukes’ D (metastatic to the liver) colon cancer. The cells were cultured in minimal essential medium supplemented with 10% fetal calf serum, 1% sodium pyruvate, 1% nonessential amino acids, and 1% minimal essential medium vitamin mixture (complete media). Protein preparation and Western immunoblot. Cells were lysed with lysis buffer A, clarified by centrifugation (10,000g for 30 minutes at 4°C) and protein concentrations that were determined with the method of Bradford.16 Western immunoblot analysis was performed as described previously.7,13 Briefly, total protein (50 µg) was resolved on a 10% polyacrylamide gel and transferred to ImmobilonP nylon membranes. Filters were incubated overnight at 4°C in blotting solution (Tris-buffer saline solution that contained 5% nonfat dried milk and 0.1% Tween 20) and then for 3 hours with the primary antibody to human FLIP or β-actin. Filters were incubated with a horseradish peroxidase–conjugated anti-rabbit or anti-goat antibody as a secondary antibody for 1 hour. After 4 final washes, the immune complexes were visualized with the use of enhanced chemiluminescence detection. JAM assay–isotope incorporation assay with 3Hthymidine. To determine cell killing, we used a JAM assay that measures percent specific apoptosis. The assay was performed as described previously.6,17,18 Briefly, tumor cells were labeled with 1 µCi of 3Hthymidine (ICN Pharmaceuticals; Costa Mesa,
Calif) for 15 hours at a density of 0.5 × 106 cells/mL. After being labeled, cells were seeded at a density of 2 × 105 cells per well into 96-well flatbottom microtiter plates (Nunc, Wiesbaden, Germany) and incubated in 250 µL of growth medium for 24 hours at 37°C. Media were removed, and either new complete media, NaBT (5 mmol/L), TRAIL (100 ng/mL), or a combination of these agents was added in a final volume of 200 µL and allowed to grow for 24 and 48 hours at 37°C. Cells were then harvested with a cell harvester (1295-001; LKB/Wallac, Freiburg, Germany), as previously described.6,17,18 Bound radioactivity was measured by scintillation counting in a 1205-betaplate counter (LKB/Wallac), and percent apoptosis was calculated. All experiments were performed in triplicate. Caspase-3 assay. Caspase-3 assays were performed according to the manufacturer’s protocol, as described previously.6 Briefly, 10 µL of protein lysate was added to 490 µL phosphate-buffered saline solution (PBS) and incubated with 20µL of Ac-Val-Ala-Asp-AFC substrate at 30°C for 30 minutes. Fluorescence recordings were obtained with a fluorometer that was adjusted to 400 nm excitation and 505 nm emission. Fluorescence units per microgram of protein (enzyme activity) were plotted as relative caspase-3–like activity per microgram of protein. Annexin-V immunofluorescence. Slides were prepared with the use of the Santa Cruz Biotechnology ABC Staining Systems standard protocol. Briefly, cells were plated on oval glass slides for 24 hours in complete media, rinsed with PBS, and incubated with 10% normal blocking serum in PBS to suppress the nonspecific binding of IgG. Blocking serum was derived from the same species in which the secondary antibody was raised. Incubation with primary antibody, Annexin-V, for 60 minutes (1.0 µg/mL in PBS with 1.5% normal blocking serum) was per-
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Fig 4. Annexin-V Immunofluorescence. KM20 cells were treated as described in Fig 2, then incubated with primary antibody (Annexin-V; 1.0 µg/mL in PBS with 1.5% normal blocking serum) for 60 minutes, followed by a brief wash and incubation with the secondary fluorescein-conjugated antibody (diluted to 1.0 µg/mL in PBS with 1.5% normal blocking serum). Cover slips with aqueous mounting medium were placed, and each slide was examined with a fluorescence microscope with the appropriate filters.
formed, followed by 2 brief washes with PBS. Secondary fluorescein-conjugated antibody (diluted to 1.0 µg/mL in PBS with 1.5% normal blocking serum) was added, followed by 2 brief washes with PBS. Cover slips with aqueous mounting medium were placed, and each slide was examined with the use of a fluorescence microscope with appropriate filters. All experiments were performed in duplicate. RPA. To determine the effects of NaBT and TRAIL on the expression of cell cycle–related genes, a multi-probe template set (PharMingen) was used. Antisense RNA probes were prepared and labeled with sodium phosphate P 32 with an RPA kit. Total RNA (35 µg) was extracted from the KM cell lines with RNAzol as described previously7,19 and hybridized with the multiprobe for the cell cycle–related genes. After digestion of the unbound RNA and precipitation of RNA/probe complexes, samples were run on a 7.5% polyacrylamide-8 mol/L urea gel. Gels were dried on blotting paper and exposed to film. Statistical analysis. Results are expressed as the
mean ± SEM. Killing rates and relative caspase-3 activity were compared with the use of a 2-tailed Student t test and considered significant if probability values were less than .05. RESULTS Decreased FLIP levels in KM colon cancer cell lines with NaBT. We have previously shown increased expression of both FLIP messenger RNA and protein in all of the KM cell lines, which may contribute to the resistance of these cells to TRAILmediated apoptosis.6 To determine the effect of NaBT on FLIP expression, we treated the KM cell lines with NaBT (5 mmol/L) for 24 and 48 hours. Protein lysates were obtained, and FLIP protein expression was determined by Western immunoblot. All KM cell lines demonstrated a dramatic reduction of FLIP protein by 24 hours, with similar results observed at 48 hours (Fig 1). Intact protein samples were confirmed by stripping the blot and reprobing with β-actin. The inhibition of FLIP expression in all 3 human colon cancer cell lines suggests the possi-
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Fig 5. RPA. KM12C, KML4A, and KM20 cells were treated with TRAIL, NaBT, or TRAIL plus NaBT. RNA was extracted at 24 hours after treatment and assessed by RNase protection for the expression of cell cycle–related genes with the use of a multiprobe human cell cycle template set. Constitutively expressed genes, L32 and GAPDH, served as loading controls.
bility that NaBT may sensitize these cancer cells to TRAIL treatment. Sensitization of colon cancer cells to TRAIL treatment with NaBT. With the use of JAM assays, which quantify the percent of cellular apoptosis, we next determined, with the use of NaBT, whether the decrease in FLIP levels sensitizes the KM cells to TRAIL-mediated cell death (Fig 2). After the labeling of the KM cell lines with 3H-thymidine, cells were cultured in complete media and treated with either TRAIL (100 ng/mL), NaBT (5 mmol/L), or the combination of TRAIL and NaBT for 24 and 48 hours. As we have previously shown,6 all 3 KM cell lines are resistant to TRAIL-mediated cell death. An increase in cell death was demonstrated in the presence of NaBT alone; the combination of NaBT with TRAIL significantly enhanced cell death in the KM cell lines compared with NaBT alone. These findings suggest that NaBT treatment sensitizes these colon cancer cells to the effects of TRAIL. To confirm that the enhanced cell death that we observed was mediated through the conventional
caspase pathway, we measured the relative caspase3–like activity of KM cell lysates after treatment (Fig 3). Caspase-3 activity was increased in the presence of NaBT alone, compared with untreated cells. Similar to the enhanced cell death noted by the JAM assays, caspase-3 activity increased with the combination of TRAIL and NaBT compared with NaBT alone. As a further assessment of apoptosis, KM cells were treated with NaBT, TRAIL, or a combination of NaBT and TRAIL (as previously described), and immunofluorescent studies for Annexin-V were performed. Annexin-V detects changes in the position of phosphatidylserine in the cell membrane, which redistributes to the outer layer of the membrane and becomes exposed to the extracellular environment thereby allowing early detection of apoptosis.20 Increased Annexin-V immunofluorescence was detected with NaBT alone in KM20 cells; however, the combination of NaBT and TRAIL resulted in increased fluorescence, compared with NaBT alone (Fig 4). Similar results were obtained
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in the KM12C and KML4A cell lines (data not shown). These findings provide additional evidence that NaBT can sensitize these cells to the apoptotic effects of TRAIL, thus suggesting that the combination treatment is more effective in cell killing. NaBT effects on the expression of cell cycle–related genes in the KM cell lines. To further delineate other potential proteins that may contribute to NaBT sensitization of these colon cancer cells to the effects of TRAIL, we performed an RPA using a labeled multiprobe that can simultaneously detect the expression of a number of cell cycle–related genes (Fig 5). Treatment of all 3 cell lines with either NaBT alone or NaBT plus TRAIL resulted in the induction of the cyclin-dependent kinase inhibitor p21waf1. Similar to p21waf1, 2 other cyclin-dependent kinase inhibitors, p57 and p14/15, demonstrated increased expression after NaBT alone or NaBT plus TRAIL treatment. This induction of p21waf1 is consistent with previous findings from our laboratory15 and other laboratories14,21 with the use of different colon cancer cells. Levels of p53 expression were actually decreased with NaBT alone or NaBT plus TRAIL, which suggests that the induction of p21waf1 occurs through a p53-independent pathway. Consistent with previous studies,22 we also noted a decrease in the expression of p16, another cyclin-dependent kinase inhibitor. Therefore, these findings identify other potential targets for NaBT treatment which, in addition to decreased FLIP expression, may contribute to the sensitization of these cells. DISCUSSION The mechanisms responsible for the resistance of certain cancers to the effects of chemotherapeutic agents are not completely understood. In our present study, we demonstrate sensitization of resistant human colon cancer cells to TRAIL-mediated cell death by NaBT treatment, which inhibits the expression of FLIP, an endogenous caspase pathway inhibitor. These findings suggest that the addition of the short-chain fatty acid NaBT may be useful as an adjuvant agent in the treatment of colorectal cancers that are resistant to the effects of TRAIL treatment alone. TRAIL is a unique member of the tumor necrosis factor family that, like FasL, is a type II membrane protein capable of inducing cell death in a variety of cell types.3 Initial preclinical reports suggested that normal tissues were resistant to TRAILmediated apoptosis, whereas cancer cells were preferentially killed by TRAIL.3-6 In fact, clinical trials that incorporated TRAIL treatment have been sug-
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gested. However, it is becoming increasingly apparent that a number of cancers (including breast, lung, prostate, and bladder cancer cells) are resistant to TRAIL-mediated apoptosis.7 Previously, we have shown that human colon cancer cell lines (KM12C, KML4A, and KM20) are resistant to the effects of TRAIL treatment, despite the expression of both DR4 and DR5 receptors.6 Similar to our findings, other colon cancer cell lines (eg, HT-29, SW620) have been shown to be resistant to the effects of TRAIL.4,6 A possible explanation for the resistance of cells to TRAIL treatment is the existence of decoy receptors that bind TRAIL but are not linked to the apoptotic cascade. Decoy receptors have been identified on normal cells and on some cancer cells. Our laboratory7 and other laboratoriess12,23,24 have previously identified both functional and decoy receptors in TRAIL-resistant cancer cell lines; however, the decoy hypothesis cannot entirely explain the resistance of these cells to TRAIL treatment. An alternative hypothesis involves the differential expression of intracellular inhibitors of the apoptotic process. Support of this hypothesis is provided by the finding of increased levels of FLIP, a recently identified protein homologue to caspase-8 that lacks catalytic activity, in TRAIL-resistant melanoma cell lines.12 FLIP levels decreased when cells were cultured with either actinomycin D or cycloheximide paralleled by an increase in their sensitivity to the effects of TRAIL. Similarly, we have demonstrated increased FLIP levels in the KM cell lines; FLIP levels were reduced after treatment with either actinomycin D or cycloheximide, which resulted in increased TRAIL-mediated cell death.6 Although actinomycin D and cycloheximide are capable of sensitizing various TRAIL-resistant cancer cells to TRAIL-mediated cell death, these agents are toxic to nontransformed cells, thus limiting their use as adjuvant therapy for advanced or metastatic cancers. It is for this reason that novel, effective, and relatively nontoxic adjuvant therapies are needed for the treatment of advanced colorectal cancer. NaBT is produced by bacterial fermentation of dietary fiber in the colon. Our laboratory6 and other laboratories13,15,21 have demonstrated that NaBT induces growth arrest and differentiation and, in certain cell types, triggers apoptosis. However, the molecular mechanisms that mediate these effects remain largely unknown. In our present study, we report a reduction of FLIP protein with NaBT treatment, which rendered these colon cancer cell lines sensitive to TRAILmediated cell death. Our findings further support a role for the modulation of intracellular levels of
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FLIP in the possible treatment of cancers that are resistant to standard therapies. NaBT is known to alter the expression of various cell cycle–related and apoptotic proteins in other colon cancer cell lines.14,15 To define other possible genes that may contribute to the effects of NaBT in the KM colon cancer cells, we assessed the expression of various cell cycle–related genes after NaBT treatment. Cell cycle progression is regulated by a complex interaction of cyclins (the regulatory subunits) and cyclin-dependent kinase inhibitors (the catalytic subunits). The association of the cyclins with their respective cyclin-dependent kinase inhibitors leads to activation and progression through the cell cycle. Cell cycle progression can be inhibited by a class of regulators called cyclin-dependent kinase inhibitors. Two families of cyclin-dependent kinase inhibitors have been described. One family consists of p21waf1, p27kip1, and p57kip2, which are universal cyclin-dependent kinase inhibitors; the second family consists of p14/15ink4b, p16ink4a, p18ink4c, and p19ink4d, which appear to selectively bind and inhibit cyclin-dependent kinase 4 and 6. We found that, similar to previous results in other colon cancer cells,15 NaBT resulted in a marked induction of p21waf1. Moreover, 2 other cyclindependent kinase inhibitors, p57 and p14/15, demonstrated increased expression after NaBT treatment. Similar to p21waf1, these proteins have been shown to induce cell cycle arrest in other cell lines.25 Therefore, this induction, in combination with a decrease in FLIP, may contribute to the cell death associated with NaBT treatment. In contrast to p21waf1, we noted a decrease in the expression of p16 and the tumor suppressor gene p53 with NaBT treatment, which suggested that decreased p16 and p53 expression may contribute to the apoptosis that is associated with the addition of NaBT. These results are consistent with the findings by Bernard et al,22 who demonstrated that overexpression of p16 protects human leukemic lymphoblast cells from NaBT-mediated apoptosis. Therefore, multiple cellular effects, including the inhibition of FLIP, may contribute to the sensitization of these colon cancer cells to TRAIL treatment. In conclusion, the reduction of cellular levels of FLIP by NaBT, a short-chain fatty acid naturally formed in the gastrointestinal tract, results in a “priming” effect, thus rendering the KM colon cancer cells sensitive to the effects of TRAIL. The combination of TRAIL along with agents, such as NaBT, that target proteins that prevent cell death may provide a more effective and less toxic regimen for the adjuvant treatment of resistant colon cancers.
We thank Eileen Figueroa and Karen Martin for manuscript preparation. REFERENCES 1. Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics, 2000. CA Cancer J Clin 2000;50:7-33. 2. Algeciras-Schimnich A, Griffith TS, Lynch DH, Paya CV. Cell cycle-dependent regulation of FLIP levels and susceptibility to Fas-mediated apoptosis. J Immunol 1999;162:5205-11. 3. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997;277:81821. 4. Gliniak B, Le T. Tumor necrosis factor-related apoptosisinducing ligand’s antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11. Cancer Res 1999;59:6153-8. 5. Kothny-Wilkes G, Kulms D, Poppelmann B, Luger TA, Kubin M, Schwarz T. Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosisinducing ligand. J Biol Chem 1998;273:29247-53. 6. Hernandez A, Wang Q, Schwartz SA, Evers BM. Sensitization of human colon cancer cells to TRAIL-mediated apoptosis. J Gastrointest Surg 2001;5:56-65. 7. Hernandez A, Smith F, Wang Q, Wang X, Evers BM. Assessment of differential gene expression patterns in human colon cancers. Ann Surg 2000;232:576-85. 8. Kim K, Fisher MJ, Xu SQ, el-Deiry WS. Molecular determinants of response to TRAIL in killing of normal and cancer cells. Clin Cancer Res 2000;6:335-46. 9. Hu S, Vincenz C, Buller M, Dixit VM. A novel family of viral death effector domain-containing molecules that inhibit both CD-95– and tumor necrosis factor receptor-1–induced apoptosis. J Biol Chem 1997;272:9621-4. 10. Scaffidi C, Schmitz I, Krammer PH, Peter ME. The role of c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem 1999;274:1541-8. 11. Thome M, Schneider P, Hofmann K, Fickenscher H, Meinl E, Neipel F, et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 1997;386:517-21. 12. Griffith TS, Chin WA, Jackson GC, Lynch DH, Kubin MZ. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol 1998;161:2833-40. 13. Litvak DA, Hwang KO, Evers BM, Townsend CM. Induction of apoptosis in human gastric cancer by sodium butyrate. Anticancer Res 2000;20:779-84. 14. Coradini D, Pellizzaro C, Marimpietri D, Abolafio G, Daidone MG. Sodium butyrate modulates cell cycle-related proteins in HT29 human colonic adenocarcinoma cells. Cell Prolif 2000;33:139-46. 15. Litvak DA, Evers BM, Hwang KO, Hellmich MR, Ko TC, Townsend CM Jr. Butyrate-induced differentiation of Caco2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1. Surgery 1998;124:161-9. 16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 17. Atarashi Y, Kanaya H, Whieside TL. A modified JAM assay detects apoptosis induced in activated lymphocytes by FasL+ human adherent tumor cells [letter]. J Immunol Methods 2000;233:179-83. 18. Bohm C, Hanski ML, Gratchev A, Mann B, Moyer MP, Riecken EO, et al. A modification of the JAM test is necessary for a correct determination of apoptosis induced by FasL+ adherent tumor cells. J Immunol Methods 1998;217:71-8.
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19. Wang Q, Kim S, Wang X, Evers BM. Activation of NF-κB binding in HT-29 colon cancer cells by inhibition of phosphatidylinositol 3-kinase. Biochem Biophys Res Commun 2000;273:853-8. 20. Moore A, Donahue CJ, Bauer KD, Mather JP. Simultaneous measurement of cell cycle and apoptotic cell death. Methods Cell Biol 1998;57:265-78. 21. Siavoshian S, Blottiere HM, Cherbut C, Galmiche JP. Butyrate stimulates cyclin D and p21 and inhibits cyclindependent kinase 2 expression in HT-29 colonic epithelial cells. Biochem Biophys Res Commun 1997;232:169-72. 22. Bernhard D, Ausserlechner MJ, Tonko M, Loffler M, Hartmann BL, Csordas A, et al. Apoptosis induced by the
Surgery August 2001 histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts. Faseb J 1999;13:1991-2001. 23. Lotz M, Setareh M, von Kempis J, Schwarz H. The nerve growth factor/tumor necrosis factor receptor family. J Leukoc Biol 1996;60:1-7. 24. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997;277:815-8. 25. Siavoshian S, Segain JP, Kornprobst M, Bonnet C, Cherbut C, Galmiche JP, et al. Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression. Gut 2000;46:507-14.
Background. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor family, induces apoptosis in TRAIL-sensitive tumors through the activation of the caspase pathway. Sodium butyrate (NaBT) induces differentiation and apoptosis in certain colorectal cancers; the molecular mechanisms for these effects have not been clearly defined. The purpose of our study was to determine whether NaBT sensitizes TRAIL-resistant human colon cancer cells to the effects of TRAIL. Methods. Human colon cancer cells (KM12C, KML4A, and KM20) that are resistant to TRAIL treatment alone were treated with TRAIL (100 ng/mL), NaBT (5 mmol/L), or a combination of these agents and harvested for total RNA and protein. Western blots were performed to assess intracellular expression of Flice-like inhibitory protein (FLIP), a caspase inhibitor. Percent-specific apoptosis, relative caspase-3 activity, and Annexin-V immunofluorescence were determined at 24 and 48 hours. Cell cycle–related gene expression was assessed by RNase protection. Results. Treatment with NaBT for 24 and 48 hours decreased FLIP protein expression in all cell lines. Furthermore, NaBT sensitized these resistant cancer cells to the effects of TRAIL with significant increases noted in cell death, caspase-3 activity, and Annexin-V staining compared with NaBT alone. Conclusions. Our findings suggest that the reduction of FLIP protein levels by NaBT renders TRAILresistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL with agents (such as NaBT, which target proteins that prevent cell death) may provide a more effective and less toxic regimen for the treatment of resistant colon cancers. (Surgery 2001;130:265-72.) From the Department of Surgery, The University of Texas Medical Branch, Galveston, Tex
COLORECTAL CANCER IS the third most common cause of cancer-related deaths in the United States in both men and women.1 It is estimated that approximately 130,000 new cases of colorectal cancer will be diagnosed this year.1 The prognosis for colon cancer is highly dependent on the stage of the disease. Colon cancer, when localized to the bowel wall, is a highly curable disease. Unfortunately, Stage IV disease (ie, distant metastases) is associated with a 5-year survival rate of less than 5%. For all but Stage IV disease, survival rates have improved in the last 40 years.2 The development of more novel therapeutic agents is required if a further improvement in the survival rate is to be realized. Supported by grants R01 AG10885, R01 DK48498, P01 DK35608, and T32 DK07639 from the National Institutes of Health. Presented at the 62nd Annual Meeting of the Society of University Surgeons, Chicago, Ill, February 8-10, 2001. Reprint requests: B. Mark Evers, MD, Department of Surgery, The University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-0536. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 11/6/115897 doi:10.1067/msy.2001.115897
Tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) is a recently characterized class II integral membrane protein that was initially identified on the basis of the homologic features of its extracellular domain to tumor necrosis factor and FasL.3 TRAIL binds its cell receptors (DR4 or DR5), which are linked to the “death domains,” to trigger apoptosis through the caspase cascade.3 TRAIL was initially reported to preferentially induce apoptosis in malignant cells, which provided the possibility of the use of TRAIL in the treatment of a number of cancers, including colorectal cancer.3-6 Recent studies, however, have demonstrated that an increasing number of cancers are resistant to TRAIL.7 One proposed mechanism for this resistance is the presence of “decoy” receptors that have been identified and shown to bind TRAIL but that are not linked to the apoptotic cascade. It is becoming increasingly apparent that other mechanisms for this resistance are more likely (such as the presence of intrinsic proteins that can act as inhibitors of apoptosis).1,8 A recently identified inhibitor of apoptosis has been described and designated Flice-like inhibitory protein (FLIP).2 FLIP structurally resembles casSURGERY 265
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Fig 1. Western blot analysis of FLIP expression after NaBT treatment. Western immunoblot analysis of protein extracts (50 µg) from KM colon cancer cells that were treated with NaBT (5 mmol/L) for 24 and 48 hours. Blots were probed for FLIP expression with a human anti-FLIP antibody. The blot was then stripped and reprobed with β-actin.
Fig 2. JAM assays. The KM colon cancer cells were plated in 96-well plates, labeled with 3H-thymidine, and treated with human recombinant TRAIL (100 ng/mL), NaBT (5 mmol/L), or the combination of TRAIL and NaBT for 24 and 48 hours. Apoptosis (percent specific apoptosis) was determined and compared with cells treated with vehicle (control) or NaBT alone. (Data are expressed as mean ± SEM; *P < .05 vs control; †P < .05 vs NaBT alone.)
pase-8 but lacks proteolytic activity, thus functioning as a dominant negative inhibitor of caspase-8, (Flice).9-11 A critical role for FLIP in the resistance of certain cancers to TRAIL-mediated cell death has been demonstrated with a reduction in cellular levels of FLIP that are associated with increased sensitivity of cancers to the effects of TRAIL.6,12 Another potentially novel agent that could serve as a useful adjunct to the treatment of metastatic colorectal cancer is the short-chain fatty acid sodium butyrate (NaBT), which is produced in the colon by the breakdown of dietary fiber.13,14 Our laboratory13,15 and others14 have shown that the NaBT treatment of certain colon cancers induces differentiation and eventual apoptosis by mechanisms that include G1 cell cycle arrest and stimulation of cell cycle–related proteins and proteins that are important for apoptosis. The purpose of our present study was to determine whether NaBT could “sensitize” TRAIL-mediated colon cancer cells to the effects of TRAIL and define the molecular mechanism responsible for this effect. MATERIAL AND METHODS Material. Tissue culture media and reagents were obtained from GIBCO-BRL (Grand Island,
NY). NaBT and the 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay were purchased from Sigma Chemical Company (St Louis, Mo). Total RNA was isolated with the use of RNAzol (Biotex, Houston, Tex) and digested with RNase free DNase I (Clontech, Palo Alto, Calif). RNase protection assay (RPA) was performed with the HybSpeed RPA Kit (Ambion, Austin, Tex) according to the manufacturer’s instructions. The human cell cycle complementary DNA template for RPA was purchased from PharMingen (San Diego, Calif). The concentrated protein assay dye was purchased from Bio-Rad laboratories (Hercules, Calif). Immobilon-P nylon membranes for Western blots were purchased from Millipore (Bedford, Mass), and x-ray film was purchased from Eastman Kodak (Rochester, NY). The enhanced chemiluminescence system for Western immunoblot analysis was obtained from Amersham (Arlington Heights, Ill). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). TRAIL apoptosis kit (17-226) was purchased from Upstate Biotechnology (Lake Placid, NY). Caspase-3 assay substrate, Ac-Val-Ala-Asp-AFC was purchased from Enzyme Systems Products (Livermore, Calif).
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Fig 3. Caspase-3–like activity. The KM cells were treated as described in Fig 2. Caspase-3–like activity was measured with the chromogenic substrate Ac-Val-Ala-Asp-AFC and normalized to 1 µg protein. (Data are expressed as mean ± SEM; *P < .05 vs control; †P < .05 vs NaBT alone.)
Cell culture. The human colon cancer cell lines KM12C, KML4A, and KM20 were provided by Dr Isaiah J. Fidler (University of Texas MD Anderson Cancer Center, Houston, Tex). The KM12C cell line was derived from a patient with a Dukes’ B colon cancer. KML4A was derived from KM12C and has been “trained” to metastasize to the liver by multiple rounds of injection into the spleens of athymic nude mice. The KM20 cell line was derived from a patient with Dukes’ D (metastatic to the liver) colon cancer. The cells were cultured in minimal essential medium supplemented with 10% fetal calf serum, 1% sodium pyruvate, 1% nonessential amino acids, and 1% minimal essential medium vitamin mixture (complete media). Protein preparation and Western immunoblot. Cells were lysed with lysis buffer A, clarified by centrifugation (10,000g for 30 minutes at 4°C) and protein concentrations that were determined with the method of Bradford.16 Western immunoblot analysis was performed as described previously.7,13 Briefly, total protein (50 µg) was resolved on a 10% polyacrylamide gel and transferred to ImmobilonP nylon membranes. Filters were incubated overnight at 4°C in blotting solution (Tris-buffer saline solution that contained 5% nonfat dried milk and 0.1% Tween 20) and then for 3 hours with the primary antibody to human FLIP or β-actin. Filters were incubated with a horseradish peroxidase–conjugated anti-rabbit or anti-goat antibody as a secondary antibody for 1 hour. After 4 final washes, the immune complexes were visualized with the use of enhanced chemiluminescence detection. JAM assay–isotope incorporation assay with 3Hthymidine. To determine cell killing, we used a JAM assay that measures percent specific apoptosis. The assay was performed as described previously.6,17,18 Briefly, tumor cells were labeled with 1 µCi of 3Hthymidine (ICN Pharmaceuticals; Costa Mesa,
Calif) for 15 hours at a density of 0.5 × 106 cells/mL. After being labeled, cells were seeded at a density of 2 × 105 cells per well into 96-well flatbottom microtiter plates (Nunc, Wiesbaden, Germany) and incubated in 250 µL of growth medium for 24 hours at 37°C. Media were removed, and either new complete media, NaBT (5 mmol/L), TRAIL (100 ng/mL), or a combination of these agents was added in a final volume of 200 µL and allowed to grow for 24 and 48 hours at 37°C. Cells were then harvested with a cell harvester (1295-001; LKB/Wallac, Freiburg, Germany), as previously described.6,17,18 Bound radioactivity was measured by scintillation counting in a 1205-betaplate counter (LKB/Wallac), and percent apoptosis was calculated. All experiments were performed in triplicate. Caspase-3 assay. Caspase-3 assays were performed according to the manufacturer’s protocol, as described previously.6 Briefly, 10 µL of protein lysate was added to 490 µL phosphate-buffered saline solution (PBS) and incubated with 20µL of Ac-Val-Ala-Asp-AFC substrate at 30°C for 30 minutes. Fluorescence recordings were obtained with a fluorometer that was adjusted to 400 nm excitation and 505 nm emission. Fluorescence units per microgram of protein (enzyme activity) were plotted as relative caspase-3–like activity per microgram of protein. Annexin-V immunofluorescence. Slides were prepared with the use of the Santa Cruz Biotechnology ABC Staining Systems standard protocol. Briefly, cells were plated on oval glass slides for 24 hours in complete media, rinsed with PBS, and incubated with 10% normal blocking serum in PBS to suppress the nonspecific binding of IgG. Blocking serum was derived from the same species in which the secondary antibody was raised. Incubation with primary antibody, Annexin-V, for 60 minutes (1.0 µg/mL in PBS with 1.5% normal blocking serum) was per-
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Fig 4. Annexin-V Immunofluorescence. KM20 cells were treated as described in Fig 2, then incubated with primary antibody (Annexin-V; 1.0 µg/mL in PBS with 1.5% normal blocking serum) for 60 minutes, followed by a brief wash and incubation with the secondary fluorescein-conjugated antibody (diluted to 1.0 µg/mL in PBS with 1.5% normal blocking serum). Cover slips with aqueous mounting medium were placed, and each slide was examined with a fluorescence microscope with the appropriate filters.
formed, followed by 2 brief washes with PBS. Secondary fluorescein-conjugated antibody (diluted to 1.0 µg/mL in PBS with 1.5% normal blocking serum) was added, followed by 2 brief washes with PBS. Cover slips with aqueous mounting medium were placed, and each slide was examined with the use of a fluorescence microscope with appropriate filters. All experiments were performed in duplicate. RPA. To determine the effects of NaBT and TRAIL on the expression of cell cycle–related genes, a multi-probe template set (PharMingen) was used. Antisense RNA probes were prepared and labeled with sodium phosphate P 32 with an RPA kit. Total RNA (35 µg) was extracted from the KM cell lines with RNAzol as described previously7,19 and hybridized with the multiprobe for the cell cycle–related genes. After digestion of the unbound RNA and precipitation of RNA/probe complexes, samples were run on a 7.5% polyacrylamide-8 mol/L urea gel. Gels were dried on blotting paper and exposed to film. Statistical analysis. Results are expressed as the
mean ± SEM. Killing rates and relative caspase-3 activity were compared with the use of a 2-tailed Student t test and considered significant if probability values were less than .05. RESULTS Decreased FLIP levels in KM colon cancer cell lines with NaBT. We have previously shown increased expression of both FLIP messenger RNA and protein in all of the KM cell lines, which may contribute to the resistance of these cells to TRAILmediated apoptosis.6 To determine the effect of NaBT on FLIP expression, we treated the KM cell lines with NaBT (5 mmol/L) for 24 and 48 hours. Protein lysates were obtained, and FLIP protein expression was determined by Western immunoblot. All KM cell lines demonstrated a dramatic reduction of FLIP protein by 24 hours, with similar results observed at 48 hours (Fig 1). Intact protein samples were confirmed by stripping the blot and reprobing with β-actin. The inhibition of FLIP expression in all 3 human colon cancer cell lines suggests the possi-
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Fig 5. RPA. KM12C, KML4A, and KM20 cells were treated with TRAIL, NaBT, or TRAIL plus NaBT. RNA was extracted at 24 hours after treatment and assessed by RNase protection for the expression of cell cycle–related genes with the use of a multiprobe human cell cycle template set. Constitutively expressed genes, L32 and GAPDH, served as loading controls.
bility that NaBT may sensitize these cancer cells to TRAIL treatment. Sensitization of colon cancer cells to TRAIL treatment with NaBT. With the use of JAM assays, which quantify the percent of cellular apoptosis, we next determined, with the use of NaBT, whether the decrease in FLIP levels sensitizes the KM cells to TRAIL-mediated cell death (Fig 2). After the labeling of the KM cell lines with 3H-thymidine, cells were cultured in complete media and treated with either TRAIL (100 ng/mL), NaBT (5 mmol/L), or the combination of TRAIL and NaBT for 24 and 48 hours. As we have previously shown,6 all 3 KM cell lines are resistant to TRAIL-mediated cell death. An increase in cell death was demonstrated in the presence of NaBT alone; the combination of NaBT with TRAIL significantly enhanced cell death in the KM cell lines compared with NaBT alone. These findings suggest that NaBT treatment sensitizes these colon cancer cells to the effects of TRAIL. To confirm that the enhanced cell death that we observed was mediated through the conventional
caspase pathway, we measured the relative caspase3–like activity of KM cell lysates after treatment (Fig 3). Caspase-3 activity was increased in the presence of NaBT alone, compared with untreated cells. Similar to the enhanced cell death noted by the JAM assays, caspase-3 activity increased with the combination of TRAIL and NaBT compared with NaBT alone. As a further assessment of apoptosis, KM cells were treated with NaBT, TRAIL, or a combination of NaBT and TRAIL (as previously described), and immunofluorescent studies for Annexin-V were performed. Annexin-V detects changes in the position of phosphatidylserine in the cell membrane, which redistributes to the outer layer of the membrane and becomes exposed to the extracellular environment thereby allowing early detection of apoptosis.20 Increased Annexin-V immunofluorescence was detected with NaBT alone in KM20 cells; however, the combination of NaBT and TRAIL resulted in increased fluorescence, compared with NaBT alone (Fig 4). Similar results were obtained
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in the KM12C and KML4A cell lines (data not shown). These findings provide additional evidence that NaBT can sensitize these cells to the apoptotic effects of TRAIL, thus suggesting that the combination treatment is more effective in cell killing. NaBT effects on the expression of cell cycle–related genes in the KM cell lines. To further delineate other potential proteins that may contribute to NaBT sensitization of these colon cancer cells to the effects of TRAIL, we performed an RPA using a labeled multiprobe that can simultaneously detect the expression of a number of cell cycle–related genes (Fig 5). Treatment of all 3 cell lines with either NaBT alone or NaBT plus TRAIL resulted in the induction of the cyclin-dependent kinase inhibitor p21waf1. Similar to p21waf1, 2 other cyclin-dependent kinase inhibitors, p57 and p14/15, demonstrated increased expression after NaBT alone or NaBT plus TRAIL treatment. This induction of p21waf1 is consistent with previous findings from our laboratory15 and other laboratories14,21 with the use of different colon cancer cells. Levels of p53 expression were actually decreased with NaBT alone or NaBT plus TRAIL, which suggests that the induction of p21waf1 occurs through a p53-independent pathway. Consistent with previous studies,22 we also noted a decrease in the expression of p16, another cyclin-dependent kinase inhibitor. Therefore, these findings identify other potential targets for NaBT treatment which, in addition to decreased FLIP expression, may contribute to the sensitization of these cells. DISCUSSION The mechanisms responsible for the resistance of certain cancers to the effects of chemotherapeutic agents are not completely understood. In our present study, we demonstrate sensitization of resistant human colon cancer cells to TRAIL-mediated cell death by NaBT treatment, which inhibits the expression of FLIP, an endogenous caspase pathway inhibitor. These findings suggest that the addition of the short-chain fatty acid NaBT may be useful as an adjuvant agent in the treatment of colorectal cancers that are resistant to the effects of TRAIL treatment alone. TRAIL is a unique member of the tumor necrosis factor family that, like FasL, is a type II membrane protein capable of inducing cell death in a variety of cell types.3 Initial preclinical reports suggested that normal tissues were resistant to TRAILmediated apoptosis, whereas cancer cells were preferentially killed by TRAIL.3-6 In fact, clinical trials that incorporated TRAIL treatment have been sug-
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gested. However, it is becoming increasingly apparent that a number of cancers (including breast, lung, prostate, and bladder cancer cells) are resistant to TRAIL-mediated apoptosis.7 Previously, we have shown that human colon cancer cell lines (KM12C, KML4A, and KM20) are resistant to the effects of TRAIL treatment, despite the expression of both DR4 and DR5 receptors.6 Similar to our findings, other colon cancer cell lines (eg, HT-29, SW620) have been shown to be resistant to the effects of TRAIL.4,6 A possible explanation for the resistance of cells to TRAIL treatment is the existence of decoy receptors that bind TRAIL but are not linked to the apoptotic cascade. Decoy receptors have been identified on normal cells and on some cancer cells. Our laboratory7 and other laboratoriess12,23,24 have previously identified both functional and decoy receptors in TRAIL-resistant cancer cell lines; however, the decoy hypothesis cannot entirely explain the resistance of these cells to TRAIL treatment. An alternative hypothesis involves the differential expression of intracellular inhibitors of the apoptotic process. Support of this hypothesis is provided by the finding of increased levels of FLIP, a recently identified protein homologue to caspase-8 that lacks catalytic activity, in TRAIL-resistant melanoma cell lines.12 FLIP levels decreased when cells were cultured with either actinomycin D or cycloheximide paralleled by an increase in their sensitivity to the effects of TRAIL. Similarly, we have demonstrated increased FLIP levels in the KM cell lines; FLIP levels were reduced after treatment with either actinomycin D or cycloheximide, which resulted in increased TRAIL-mediated cell death.6 Although actinomycin D and cycloheximide are capable of sensitizing various TRAIL-resistant cancer cells to TRAIL-mediated cell death, these agents are toxic to nontransformed cells, thus limiting their use as adjuvant therapy for advanced or metastatic cancers. It is for this reason that novel, effective, and relatively nontoxic adjuvant therapies are needed for the treatment of advanced colorectal cancer. NaBT is produced by bacterial fermentation of dietary fiber in the colon. Our laboratory6 and other laboratories13,15,21 have demonstrated that NaBT induces growth arrest and differentiation and, in certain cell types, triggers apoptosis. However, the molecular mechanisms that mediate these effects remain largely unknown. In our present study, we report a reduction of FLIP protein with NaBT treatment, which rendered these colon cancer cell lines sensitive to TRAILmediated cell death. Our findings further support a role for the modulation of intracellular levels of
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FLIP in the possible treatment of cancers that are resistant to standard therapies. NaBT is known to alter the expression of various cell cycle–related and apoptotic proteins in other colon cancer cell lines.14,15 To define other possible genes that may contribute to the effects of NaBT in the KM colon cancer cells, we assessed the expression of various cell cycle–related genes after NaBT treatment. Cell cycle progression is regulated by a complex interaction of cyclins (the regulatory subunits) and cyclin-dependent kinase inhibitors (the catalytic subunits). The association of the cyclins with their respective cyclin-dependent kinase inhibitors leads to activation and progression through the cell cycle. Cell cycle progression can be inhibited by a class of regulators called cyclin-dependent kinase inhibitors. Two families of cyclin-dependent kinase inhibitors have been described. One family consists of p21waf1, p27kip1, and p57kip2, which are universal cyclin-dependent kinase inhibitors; the second family consists of p14/15ink4b, p16ink4a, p18ink4c, and p19ink4d, which appear to selectively bind and inhibit cyclin-dependent kinase 4 and 6. We found that, similar to previous results in other colon cancer cells,15 NaBT resulted in a marked induction of p21waf1. Moreover, 2 other cyclindependent kinase inhibitors, p57 and p14/15, demonstrated increased expression after NaBT treatment. Similar to p21waf1, these proteins have been shown to induce cell cycle arrest in other cell lines.25 Therefore, this induction, in combination with a decrease in FLIP, may contribute to the cell death associated with NaBT treatment. In contrast to p21waf1, we noted a decrease in the expression of p16 and the tumor suppressor gene p53 with NaBT treatment, which suggested that decreased p16 and p53 expression may contribute to the apoptosis that is associated with the addition of NaBT. These results are consistent with the findings by Bernard et al,22 who demonstrated that overexpression of p16 protects human leukemic lymphoblast cells from NaBT-mediated apoptosis. Therefore, multiple cellular effects, including the inhibition of FLIP, may contribute to the sensitization of these colon cancer cells to TRAIL treatment. In conclusion, the reduction of cellular levels of FLIP by NaBT, a short-chain fatty acid naturally formed in the gastrointestinal tract, results in a “priming” effect, thus rendering the KM colon cancer cells sensitive to the effects of TRAIL. The combination of TRAIL along with agents, such as NaBT, that target proteins that prevent cell death may provide a more effective and less toxic regimen for the adjuvant treatment of resistant colon cancers.
We thank Eileen Figueroa and Karen Martin for manuscript preparation. REFERENCES 1. Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics, 2000. CA Cancer J Clin 2000;50:7-33. 2. Algeciras-Schimnich A, Griffith TS, Lynch DH, Paya CV. Cell cycle-dependent regulation of FLIP levels and susceptibility to Fas-mediated apoptosis. J Immunol 1999;162:5205-11. 3. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997;277:81821. 4. Gliniak B, Le T. Tumor necrosis factor-related apoptosisinducing ligand’s antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11. Cancer Res 1999;59:6153-8. 5. Kothny-Wilkes G, Kulms D, Poppelmann B, Luger TA, Kubin M, Schwarz T. Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-related apoptosisinducing ligand. J Biol Chem 1998;273:29247-53. 6. Hernandez A, Wang Q, Schwartz SA, Evers BM. Sensitization of human colon cancer cells to TRAIL-mediated apoptosis. J Gastrointest Surg 2001;5:56-65. 7. Hernandez A, Smith F, Wang Q, Wang X, Evers BM. Assessment of differential gene expression patterns in human colon cancers. Ann Surg 2000;232:576-85. 8. Kim K, Fisher MJ, Xu SQ, el-Deiry WS. Molecular determinants of response to TRAIL in killing of normal and cancer cells. Clin Cancer Res 2000;6:335-46. 9. Hu S, Vincenz C, Buller M, Dixit VM. A novel family of viral death effector domain-containing molecules that inhibit both CD-95– and tumor necrosis factor receptor-1–induced apoptosis. J Biol Chem 1997;272:9621-4. 10. Scaffidi C, Schmitz I, Krammer PH, Peter ME. The role of c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem 1999;274:1541-8. 11. Thome M, Schneider P, Hofmann K, Fickenscher H, Meinl E, Neipel F, et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 1997;386:517-21. 12. Griffith TS, Chin WA, Jackson GC, Lynch DH, Kubin MZ. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol 1998;161:2833-40. 13. Litvak DA, Hwang KO, Evers BM, Townsend CM. Induction of apoptosis in human gastric cancer by sodium butyrate. Anticancer Res 2000;20:779-84. 14. Coradini D, Pellizzaro C, Marimpietri D, Abolafio G, Daidone MG. Sodium butyrate modulates cell cycle-related proteins in HT29 human colonic adenocarcinoma cells. Cell Prolif 2000;33:139-46. 15. Litvak DA, Evers BM, Hwang KO, Hellmich MR, Ko TC, Townsend CM Jr. Butyrate-induced differentiation of Caco2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1. Surgery 1998;124:161-9. 16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 17. Atarashi Y, Kanaya H, Whieside TL. A modified JAM assay detects apoptosis induced in activated lymphocytes by FasL+ human adherent tumor cells [letter]. J Immunol Methods 2000;233:179-83. 18. Bohm C, Hanski ML, Gratchev A, Mann B, Moyer MP, Riecken EO, et al. A modification of the JAM test is necessary for a correct determination of apoptosis induced by FasL+ adherent tumor cells. J Immunol Methods 1998;217:71-8.
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19. Wang Q, Kim S, Wang X, Evers BM. Activation of NF-κB binding in HT-29 colon cancer cells by inhibition of phosphatidylinositol 3-kinase. Biochem Biophys Res Commun 2000;273:853-8. 20. Moore A, Donahue CJ, Bauer KD, Mather JP. Simultaneous measurement of cell cycle and apoptotic cell death. Methods Cell Biol 1998;57:265-78. 21. Siavoshian S, Blottiere HM, Cherbut C, Galmiche JP. Butyrate stimulates cyclin D and p21 and inhibits cyclindependent kinase 2 expression in HT-29 colonic epithelial cells. Biochem Biophys Res Commun 1997;232:169-72. 22. Bernhard D, Ausserlechner MJ, Tonko M, Loffler M, Hartmann BL, Csordas A, et al. Apoptosis induced by the
Surgery August 2001 histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts. Faseb J 1999;13:1991-2001. 23. Lotz M, Setareh M, von Kempis J, Schwarz H. The nerve growth factor/tumor necrosis factor receptor family. J Leukoc Biol 1996;60:1-7. 24. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997;277:815-8. 25. Siavoshian S, Segain JP, Kornprobst M, Bonnet C, Cherbut C, Galmiche JP, et al. Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression. Gut 2000;46:507-14.