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[Cancer Research 60, 3200-3206, June 15, 2000]
© 2000 American Association for Cancer Research

Experimental Therapeutics

Regrowth of 5-Fluorouracil-treated Human Colon Cancer Cells Is Prevented by the Combination of Interferon {gamma}, Indomethacin, and Phenylbutyrate1

Yicong Huang, Curt M. Horvath and Samuel Waxman2

Rochelle Belfer Chemotherapy Foundation Laboratory, Division of Medical Oncology [Y. H., S. W.] and, Immunobiology Center, Mount Sinai School of Medicine [C. M. H.], New York, New York 10029


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported that phenylbutyrate (PB), a differentiation agent, retarded the regrowth of fluoropyrimidine-treated HT29 cells to a greater extent in a well-differentiated subclone as compared with a poorly differentiated subclone (Y. Huang and S. Waxman, Clin. Cancer Res., 4: 2503–2509, 1998). To extend these results and to overcome the known heterogeneity of human colon carcinoma (HCC) cells, the effect of cytostatic agents reported to inhibit HCC growth [IFN-{alpha} and IFN-{gamma}, indomethacin, and PB alone or in combination] on clonogenicity and HCCs recovery from 5-fluorouracil (FUra) treatment was studied in eight different HCCs. IFN-{alpha} proved to be ineffective in all eight HCCs, whereas IFN-{gamma} induced marked growth inhibition in four HCCs that expressed wild-type K-ras. Despite large differences in HCC response to the other individual agents, strong growth inhibition was observed when PB was added in combination with indomethacin. The inhibition was even more pronounced when IFN-{gamma} was included in the regimen. Most importantly, after treatment with the combination of three agents, the clonogenic potential was severely inhibited (92–100%) in the IFN-{gamma}-sensitive cell lines, whereas in the IFN-{gamma}-insensitive cell lines, comparable loss of clonogenecity was obtained when the cells were pretreated with FUra. As known and described in detail, the three cytostatic agents inhibit different processes necessary for cell growth, thus requiring the cells to repair multiple pathways to restore growth. The induction of STAT1 DNA binding activity by IFN-{gamma} and p21WAF1 by PB, alone or in combination, correlated with growth inhibition and loss of clonogenicity. The finding that the readily reversible growth inhibition and decrease in clonogenicity of FUra-treated HCC are prolonged by subsequent treatment with the three cytostatic agents in all HCCs may be of clinical importance because FUra continues to be the most widely used cytotoxic agent in the treatment of colon carcinoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The treatment of advanced colorectal cancer has resisted most therapeutic efforts and continues to rely on the use of fluoropyrimidines (1, 2, 3) . Enhancing the efficacy of fluoropyrimidines with leucovorin appears to have improved response and may improve survival (4, 5, 6) . IFN-{alpha} has been used to potentiate the antiproliferative effect of fluoropyrimidines, but the clinical results are controversial (7) .

We have previously reported that PB,3 a differentiation inducer, enhanced growth inhibition by FUdR in well- and poorly differentiated subclones (U4 and U9, respectively) of the HT29 HCC cell line. Moreover, PB significantly decreased cellular recovery from FUdR treatment, as measured by loss of clonogenicity (8) . Cell growth inhibition by the combined treatment was associated with an increased and sustained expression of p21WAF1, secretion of transforming growth factor ß1, and an increased activity of ALP, a market for differentiation of colon carcinoma cells. However, growth inhibition by FUdR and PB was less marked and more reversible in the less differentiated U9 subclone.

To extend these results and address the heterogeneity of colon carcinomas, we used a panel of HCC cell lines derived from primary and metastatic cells with various degrees of differentiation containing wild-type or mutated K-ras and/or p53 genes. The intent of this study was to develop a therapeutic regimen that utilizes minimally myelosuppressive cytostatic agents to prevent the regrowth of a panel of HCCs after FUra treatment. The cytostatic agents we chose to study are IFN-{gamma}, INDO, and PB. IFN-{gamma} was selected because of its known ability to arrest growth of some HCCs probably due to its ability to activate STAT1 and p21WAF1 (9 , 10) ; INDO, a nonsteroidal anti-inflammatory drug, was chosen because it is known to inhibit the development of colon carcinomas in susceptible humans (11, 12, 13) and to inhibit the in vitro growth of some HCCs (14 , 15) ; and PB was included due to the activity cited above (8 , 16, 17, 18, 19, 20) . We found that sequential treatment with FUra, the most effective therapeutic agent against colon carcinomas, followed by a mixture of these cytostatic agents, inhibited the growth of eight different HCCs and substantially decreased their colony-forming ability on soft agar after the removal of the drugs. It is proposed that in vivo, the regrowth of colon carcinomas that occurs between cycles of FUra treatment may be diminished by the application of this regimen of cytostatic agents.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Growth Assays.
Seven HCC cell lines (COLO205, HCC2998, HCT15, HCT116, HT29, KM12, and SW620) were kindly provided by the NCI. U9, a subclone of HT29, was a generous gift from Dr. Eileen Friedman (Memorial Sloan-Kettering Cancer Center, New York, NY). Table 1Citation (data obtained from the NCI lists the characteristics of the HCCs studied with regard to their origin, cell-doubling time, and metastatic ability and whether or not they contain wild-type or mutated K-ras and/or p53 genes. Cells were seeded at 1 x 105 cells/ml 6-well plates containing 2 ml of complete medium [RPMI 1640 supplemented with 5 mM glutamine and 5% heat-inactivated fetal bovine serum (21) ] and placed in a humidified 5% CO2 air incubator at 37°C. After an overnight incubation to allow the cells to adhere, medium was replaced with fresh medium with and without IFN-{gamma} (Sigma), PB (a gift from the NCI), or INDO (Sigma) or a combination of the three. Medium was replaced with fresh medium with and without drugs every 3 days before harvesting. At termination of the experiment, adherent cells (90% viable as determined by trypan blue exclusion) were washed and harvested with 0.25% trypsin and 1 mM EDTA and counted in a Coulter counter. In studies with FUra, cells were allowed to adhere overnight and then cultured for 18 hr with FUra, washed, and placed in fresh medium with or without additional drugs. Cell culture studies were done in duplicate at least three times, and each data point represents the average.


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  		Table 1 Characteristics of HCC cell lines

 
Soft Agar Growth Assay.
As described in Ref. 8 , 1000 treated or untreated cells mixed with 2 ml of 0.4% agarose (SeaPlaque) were placed in 60-mm Petri dishes containing 3 ml of 1% agarose underlayer in complete medium and incubated at 37°C. Colony formation (>0.1 mm in size) was determined after 14 days of incubation.

Measure of Hydrogen Peroxide Production and Apoptosis.
The fluorogenic probe 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C2938; Molecular Probes) was added to the cell culture at 1 µM and incubated at 37°C for 1 h before drug treatment. Cells were harvested at the indicated time. Hydrogen peroxide production (i.e., C2938-positive cells) was measured as green fluorescence using a Zeiss fluorescence microscope at the blue excitation range with a 520 nm barrier filter. Total cell population in the field was counted in visible light. The percentage of C2938-positive cells was calculated. Cells were also stained with 20 µg/ml acridine orange (Sigma), and apoptotic cells were identified by using fluorescence microscopy as described in Ref. 22 .

ALP Activity.
ALP activity was measured as described previously (8) . Briefly, harvested cells were lysed in a buffer containing 25 mM Tris-HCl (pH 8), 50 mM NaCl, 0.5% sodium deoxycholate, 2% NP40, 50 µg/ml aprotinin, 1 mM P phenylmethylsulfonyl fluoride, and 50 µM leupeptin at 4°C for 10 min. Lysates were stored in liquid nitrogen until use. ALP activity was assayed by the ALP diagnostic kit (Sigma). The absorbance at 405 nm was measured at three time intervals. The ALP activity for each sample (100 µg of protein) was expressed as fold over untreated control.

Northern Blot Analysis.
Total RNA isolation was performed with TRIzol reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Twenty µg of RNA samples were electrophoresed in a 1.2% formaldehyde agarose gel and then transferred to a Hybond-N+ nylon membrane (Amersham). STAT1 and p21WAF1 cDNA (gifts from Drs. X. Y. Fu, Yale University, New Haven, CT, and J. Manfredi, Mt. Sinai School of Medicine, New York, NY) were used as probes. Blotting with ß-actin probe served as a loading control. Intensities of mRNA bands were determined using a LKB enhanced laser densitometer (Pharmacia LKB Biotechnology) and normalized with ß-actin.

Western Blot Analysis.
Aliquots of 100 µg of protein from each lysate were mixed with 0.5 volume of 3x protein sample buffer [30% glycerol, 15% ß-mercaptoethanol, 9% SDS, and 25 mM TrisPO4 (pH 6.8)] and heated at 95°C for 2 min before loading onto a 10% polyacrylamide gel containing 0.1% SDS. Protein transfer was carried out by a GENIE (Idea Scientific). COX2 protein was detected using polyclonal anti-hPGHS2 (Oxford Biomedical) as a primary antibody, followed by enhanced chemiluminescence (Amersham). Ten ng of native ovine prostaglandin H synthase-2 (Oxford Biomedical) were used as standard. Protein band intensities were quantified using a LKB enhanced laser densitometer.

DNA Fragmentation.
Both floating and adherent cells were harvested and washed with calcium- and magnesium-free PBS. DNA fragmentation was performed according to the instructions provided with the TACS ethidium bromide kit (Trevigen). Ten-µg DNA samples were loaded onto a 1.5% agarose gel that was run at 100 V and then stained with 0.5 µg/ml ethidium bromide for 10–15 min. The stained gel was immersed in 5 µg/ml RNase A in H2O overnight. DNA fragmentation was visualized under UV light.

Electrophoretic Mobility Shift Assay.
Electrophoretic mobility shift assays were carried out as described previously (23) . Double-stranded oligonucleotides representing the M67 SIE GAS element (5'-CATTTCCCGTAAATCAT-3') were radiolabeled by filling in protruding ends with 32P-containing nucleotide triphosphates using the Klenow fragment of DNA polymerase. Cell extracts were mixed with 1 x 105 cpm of probe for 15 min before separation on a 5% polyacrylamide gel. Gels were dried and subjected to autoradiography.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth Inhibition of HCC by Single Agents.
The sensitivity (IC50) of each cell line to growth inhibition by FUra, IFN-{gamma}, INDO, and PB as single agents is listed in Table 2Citation . The dose-dependent effects of IFN-{gamma} and INDO are shown in Fig. 1, A and BCitation , respectively, and the time course of growth inhibition by 1 mM PB is shown in Fig. 1CCitation . In four cell lines, the IC50 of IFN-{gamma} was 15–90 units/ml, whereas the others required >100 units/ml. The HCCs with the lowest sensitivity to IFN-{gamma} were also much less sensitive to FUra (Table 2)Citation . Comparison of Tables 1Citation and 2Citation reveals a correlation between the K-ras genotype and sensitivity to FUra and IFN-{gamma}, with those HCCs bearing wild-type K-ras being more sensitive to FUra and INF-{gamma} than those with mutant K-ras. KM12, despite mutant K-ras, was resistant to IFN-{gamma} growth inhibition. As individual agents, PB and INDO were less effective antiproliferative agents than IFN-{gamma}. At 60 µM, INDO effectively inhibited (80%) only COLO205 cells, was ineffective in HT29 and HCT116 cells, and inhibited the remaining cell lines by <40% (Fig. 1BCitation ). PB, at this concentration, was the least effective of the three cytostatic agents when tested singly, with 1 mM PB causing <50% growth inhibition, and one cell line (SW620) displaying resistance to PB (Fig. 1CCitation ). In general, the growth inhibition of HCC as measured by clonogenic assay on soft agar paralleled the IC50 of individual agents as listed in Table 2Citation . The four IFN-{gamma}-sensitive cell lines showed a 91–66% decrease in clonogenicity, whereas in the IFN-{gamma} insensitive lines, there was a <15% decrease in clonogenicity. FUra, INDO, and PB were weak inhibitors of clonogenicity in all HCC cell lines tested (range, 5–20% decrease).


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  		Table 2 Drug sensitivities of HCC cell linesa

 


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  		Fig. 1. Dose-response curves of INF-{gamma} and INDO and the effect of PB and their combination on cell growth inhibition. Cells were cultured as described in "Materials and Methods" and incubated with different concentrations of (A) INF-{gamma} and (B) INDO for 6 days before counting. C, time course of 1 mM PB on cell growth. D, cells were treated with PB (1 mM) and INDO (30 µM) for the indicated time. E, cells were treated with PB (1 mM), INDO (30 µM), and INF-{gamma} (25 units/ml) for the indicated time.

 
Effect of Cytostatic Agent Combinations.
In contrast to the poor-to-moderate growth-inhibitory effects of INDO and PB when tested singly, the combination of INDO and PB inhibited the growth of all HCC cell lines by about 80% (Fig. 1DCitation ). This enhanced effect of the two agents was also observed in cell lines in which one of the individual agents was completely ineffective. Thus, SW620 cell growth was not inhibited by PB (1 mM) and was inhibited ~25% by INDO (30 {Gamma}M), but the combined treatment caused ~80% growth inhibition.

The addition of IFN-{gamma} to INDO and PB resulted in an even greater inhibitory effect or ~90% growth inhibition of all HCC cell lines (Fig. 1ECitation ). These results demonstrate that it is possible to resolve the heterogeneity of HCCs into a strikingly uniform growth response by appropriate combinations of cytostatic agents.

Regrowth after Removal of Cytostatic Agents.
To test the potential for inhibiting regrowth, HCC cell lines were treated with a combination of IFN-{gamma}, INDO, and PB for 6 days, washed, and then plated in soft agar to determine the clonogenic potential of the treated HCCs. In the four IFN-{gamma}-sensitive cell lines, this resulted in the growth of only 0–8% colonies, relative to the number of colonies formed by untreated cells (Table 3Citation , column A). The colony-forming ability of IFN-{gamma}-insensitive lines was also reduced by pretreatment with the cytostatic agent combination, but to a lesser extent [13–38% relative to untreated cells (Table 3Citation , column B)]. If, however, the IFN-{gamma}-insensitive HCCs were sequentially treated with FUra (at IC50 for 18 h) followed by a combination of the three cytostatic agents, the number of colonies formed was reduced to 4–14% of untreated cells (Table 3Citation , column C).


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  		Table 3 Colony formation on soft agar after removal of drugs

 
Molecular Correlates of Growth Inhibition by IFN-{gamma}, INDO, and PB.
The antiproliferative effect of IFN-{gamma} in HT29, COLO205, and to a lesser extent, U9 cells, was associated with the induction of STAT1 DNA binding activity and p21WAF1 expression (Figs. 2Citation and 3Citation ). The intensity of STAT1 DNA binding as determined by electrophoretic mobility shift assay was most evident in HT29 cells, intermediate in HCT116 cells, and less evident in SW620 and KM12 cells, which correlated with IFN-{gamma}-induced growth inhibition (Fig. 2Citation ). IFN-{alpha}, even at 20-fold higher concentrations, failed to inhibit the growth of HCC cells (Table 2)Citation or to induce STAT1 DNA binding (Fig. 2Citation ). IFN-{gamma} sensitivity also correlated with the induction of p21WAF1 expression, which was much lower in the IFN-{gamma}-insensitive HCCs (KM12 and SW620). PB induced p21WAF1 expression in some IFN-{gamma}-sensitive HCCs and enhanced p21WAF1 induction by IFN-{gamma} in others (Fig. 3Citation ).



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  		Fig. 2. IFN induction of STAT1 DNA binding activity. Gel mobility shift assays were carried out as described in "Materials and Methods." Cells were untreated or treated with IFN-{alpha} (500 units/ml) or IFN-{gamma} (100 units/ml) as indicated for 30 min at 37°C.

 


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  		Fig. 3. Induction of p21WAF1 mRNA by IFN-{gamma}. Cells were treated as described in the Fig. 2Citation legend for 1 or 3 days with IFN-{gamma} alone or with IFN-{gamma} and PB. A, Northern blot of induction of p21WAF1 mRNA; B, p21WAF1 induction normalized with ß-actin and expressed as folds over control.

 
We tested whether IFN-{gamma}-induced growth inhibition was associated with increased apoptotic rates. We found that IFN-{gamma} treatment induced apoptosis in cultures of HCC2998, HT29, and COLO205 cells as measured by DNA fragmentation (Fig. 4Citation ) and acridine orange staining (data not shown). The addition of IFN-{gamma}, PB, and INDO inhibited the growth of all HCC cell lines by approximately 90% (Fig. 1DCitation ) and induced apoptosis in HCCs in which IFN-{gamma} alone was less effective (Fig. 5Citation ). The apoptosis was associated with increased H2O2 formation. Fig. 6Citation represents an example obtained by treating HT29 cells with 25 or 100 units/ml IFN-{gamma}.



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  		Fig. 4. DNA fragmentation. COLO205 and HCC2998 cells were incubated without IFN-{gamma} (Lanes 2 and 4) or with IFN-{gamma} (25 units/ml; Lanes 3 and 5) for 3 days. IFN-{gamma} treatment of HT29 for 1, 2, and 3 days, respectively, Lanes 7, 8, and 9. Lane 6, HT29 without IFN-{gamma}; Lane 1, DNA size markers. DNA was isolated and analyzed as described in "Materials and Methods."

 


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  		Fig. 5. DNA fragmentation. Cells treated with (even-numbered lanes) or without (odd-numbered lanes) the combination of 25 units/ml IFN-{gamma}, 1 M PB, and 30 µM INDO for 3 days. DNA was isolated and analyzed as described in "Materials and Methods." 1 and 2, HCT15; Lanes 3 and 4, HCT116; Lanes 5 and 6, KM12; Lanes 7 and 8, SW620; Lanes 9 and 10, U9.

 


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  		Fig. 6. Hydrogen peroxide production and apoptosis. HT29 cells were treated with fluorogenic probe (C2938) plus/minus IFN-{gamma} (25 or 100 units/ml) and harvested at the indicated times. More than 500 cells were counted from at least three fields to assay C2938-positive cells. The remaining cells were stained with acridine orange for apoptosis analysis. Solid lines, H2O2 production; dashed lines, percentage of apoptotic cells after 1,2, and 3 days of IFN-{gamma} treatment.

 
Interestingly, ALP activity, considered a marker of colon cell differentiation (24) , was induced 25–150-fold by 2 mM sodium butyrate in IFN-{gamma}-sensitive but not IFN-{gamma}-insensitive HCC cell lines (Fig. 7Citation ). This, together with our previous demonstration of the differentiation-inducing activity of PB (8) , raises the possibility that differentiation-induced terminal cell division also contributes to the growth inhibition of IFN-{gamma}-sensitive HCC cell lines treated with the combination of three cytostatic agents.



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  		Fig. 7. Induction of ALP-specific activity by sodium butyrate. Measurement of ALP activity was performed as described in "Materials and Methods." Lysates were from cells incubated with varied concentrations of sodium butyrate for 3 days. The absorbance at 405 nm was measured at three time intervals. The ALP activity for each sample (100 µg of protein) was expressed as folds over the untreated control.

 
COX2 protein levels were decreased in six cell lines treated for 3 days with INDO (Fig. 8Citation ); however, this did not correlate with growth inhibition (Fig. 1BCitation ). The most dramatic COX2 protein decrease occurred in INDO-treated HT29 cells, which were not growth inhibited. Conversely, COLO205 cells were most growth-inhibited by INDO, despite undetectable COX2 protein in untreated cells.



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  		Fig. 8. Inhibition of COX2 protein level by INDO. Cells treated with or without 60 µM INDO for 3 days were lysed as described in "Materials and Methods." Two hundred µg of protein/sample were loaded onto a 12% of SDS-polyacrylamide gel. COX2 was detected by Western blotting. Ten ng of native ovine prostaglandin H synthase-2 served as standard.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The clinical efficacy of FUra, even when augmented by leucovorin (4, 5, 6) , is diminished by the need for rest periods between treatments, allowing the surviving cells to recover, repopulate, and maintain a large tumor burden. A rapid recovery of colon cancer cells after FUdR treatment was also observed by us (8) . The heterogeneity of colon tumors (21) and HCC cell lines (Tables 1Citation and 2)Citation further hampers the development of effective treatments. As a consequence of heterogeneity, single agents have proven incapable of substantially inhibiting the growth of most HCC cell lines. However, the protocol we describe here is capable of overcoming such heterogeneity and eliciting strikingly uniform growth-inhibitory responses across an eight-HCC cell line panel. Moreover, our protocol blocks the fast cell recovery that follows the removal of FUra.

Despite their different phenotypes, about 50% of the HCC cell lines investigated here are substantially growth inhibited and undergo apoptosis when treated with IFN-{gamma} (25–90 units/ml for 6–9 days). Van der Wilt et al. (25) and Chu et al. (26) have demonstrated that IFN-{gamma} modulates the cytotoxic effect of FUra. This may result from IFN-{gamma} prevention of thymidylate synthase up-regulation, thereby enhancing the anti-DNA effect of FUra. Alternatively, FUra with lecovorin, as a result of DNA damage, may have a FAS ligand-dependent component that is synergized by IFN-{gamma} via the induction of FAS receptor in some HCC cell lines such as HT29, but not in HCT116 (27) . The mechanism by which IFN-{gamma} alone effectively inhibited cell growth is probably related to its ability to induce STAT1, which up-regulates several genes including FAS receptor, FasL expression (28) , and/or p21WAF1 expression (9) . p21WAF1 induction by IFN-{gamma} is through STAT1 response elements in p21WAF1 promoter. The subsequent G1 cell cycle arrest may enhance the effect of PB because increased apoptosis and differentiation occur more effectively in cells arrested in G1 (9) . Moreover, in some HCC cell lines that are insensitive to IFN-{gamma}, PB induced or enhanced induction of p21WAF1. Similarly, enhanced p21WAF1 expression and differentiation induction have been observed in NB4 cells [t(15; 17) acute promyelocytic cells] treated with IFN-{gamma} in combination with all-trans-retinoic acid (29) .

The apoptosis induced in IFN-{gamma}-sensitive cell lines appears to be associated with H2O2 production (Fig. 6Citation ), perhaps by a similar mechanism whereby IFN-{gamma} is known to induce H2O2 in peripheral blood monocytes. It is known that IFN-{gamma}-induced apoptosis is inhibited by caspase-1 inhibitors (28) , and we have shown that H2O2-mediated apoptosis is also blocked by a general caspase inhibitor (30) . Wild-type K-ras function in the IFN-{gamma}-sensitive cell lines may be an important signal pathway for differentiation and apoptosis (31) . K-ras expression and function may be necessary for normal colon epithelial cell differentiation, and forced expression of K-ras in colon carcinoma cells results in expression of differentiation markers, response to transforming growth factor ß1, and terminal differentiation (32) . This is consistent with the observation that the four HCC cell lines that express wild-type K-ras can be induced to differentiate by sodium butyrate (Fig. 8Citation ), as measured by the induction of ALP activity.

The concentration of IFN-{gamma} used for these in vitro studies has been used safely in the treatment of chronic granulomatous disease (33) . Moreover, this concentration of IFN-{gamma} (0.01 mg/m2) was similar to that used in patients with melanoma, in whom it was immunomodulatory and did not produce significant systematic or hematological toxicities (34) . However, most clinical studies using IFN-{gamma} as a single-agent treatment of cancer have shown minimal benefit (35) . Although IFN-{alpha} has been reported to enhance the antiproliferative effect of FUra in vitro, but not clinically (36) , it minimally inhibited the eight HCC cell lines in our panel. IFN-{alpha} up-regulates many genes to a greater extent than IFN-{gamma} except for IRF-1 and Fas ligand (26 , 37 , 38) , which may account for the greater antiproliferative effect of IFN-{gamma} in HCCs. Moreover, IFN-{alpha}, which is ineffective in inhibit the growth of HCCs did not induce STAT1 DNA binding (Fig. 2Citation ) or ISGF3 or FAS ligand 4 suggesting a lack of expression of the IFN-{alpha} receptor in some HCC cell lines.

Combined treatment with INDO and PB showed that the heterogenous response to individual agents can be overcome in some HCCs. INDO, a nonsteroid anti-inflammatory drug, has proven to be effective in many in vivo experiments: it suppresses intestinal polyposis in APC knockout mice (39) , inhibits methylnitrosourea-induced rat colon tumors (40) , and inhibits dimethylhydrazine-induced intestinal tumors (41) . Sulindac, a non-steroid anti-inflammatory drug, inhibited both COX2 protein levels and the growth of colon carcinomas (42 , 43) . In the present study, INDO decreased COX2 protein levels, but this did not correlate with cell growth inhibition (Fig. 1BCitation and Fig. 8Citation ). In vitro, INDO induces growth inhibition and apoptosis in some HCCs by a prostaglandin-independent pathway (44) and also induces apoptosis by increasing the arachidonic acid concentration, which in turn stimulates the conversion of sphingomyelin to ceramide (45) . The inhibitory effect of INDO at clinically relevant concentrations is variable in the eight HCC cell lines studied here (Fig. 1BCitation ). Similarly, PB, which is known to be a differentiation agent (8) , is a modest growth inhibitor of HCC (Fig. 1CCitation ). The combination of these two agents, however, uniformly inhibits the growth of all of the HCC cell lines studied here by about 80% (Fig. 1DCitation ), thus overcoming the heterogenous response to individually applied agents. Our data regarding the mechanism of the cytostatic effect of INDO indicate that in addition to the reported inhibition of its primary target, the expression of COX2, it has to affect other processes because it inhibits growth and enhances PB in cell lines in which we could not detect any COX2 protein (Fig. 8Citation ). Moreover, a specific COX2 inhibitor was less antiproliferative than INDO and did not enhance PB better than INDO (data not shown).

The addition of IFN-{gamma} further increases the efficacy of the two agents by inhibiting the growth of all eight cell lines by about 90% (Fig. 1ECitation ), perhaps by increasing intracellular H2O2 accumulation (Fig. 6Citation ). The effect of the combination of the three cytostatic agents manifests itself by the induction of apoptosis (Fig. 5Citation ). The IFN-{gamma}-insensitive cell lines undergo apoptosis within 3 days only when IFN-{gamma} is included in addition to PB and INDO. The reports that both INDO and butyrates increase reactive oxygen species in colon cancer cells and sensitize cells to apoptosis induced by tumor necrosis factor {alpha} or Fas ligand antibody (15 , 46) provide hypothetical explanations for the additional mechanisms of antiproliferation of HCC by IFN-{gamma}, INDO, and PB.

The combination of these three cytostatic agents induced a uniform antiproliferation response in all eight cell lines and abrogated colony formation in the four IFN-{gamma}-sensitive lines but was not as effective in reducing the colony-forming capacity of the IFN-{gamma}-insensitive cell lines (Table 3)Citation . However, pretreatment with FUra followed by treatment with the combination of the three cytostatic agents reduced the colony-forming capacity in a drug-free medium to a few percent of that observed in untreated cells. Thus, by applying this protocol in vivo, it is predicted that the regrowth of colon carcinomas that occurs between cycles of FUra treatment can be prevented or at least effectively retarded.

A Phase I clinical research study to treat refractory colorectal cancer using a 24-h FUra infusion followed by PB (410 mg/kg/day, continuous infusion for 5 days) repeated weekly for several courses has been initiated (47) . This treatment has been well tolerated, with a dose limitation of reversible neurotoxicity. Preliminary data have demonstrated stablization of disease in some patients, but no measurable tumor response. The current protocol will test the combined use of INDO and PB between cycles of FUra infusion.


   ACKNOWLEDGMENTS
 
We appreciate the continuous scientific advice of Dr. George Acs and the critical reading of the manuscript by Dr. Rafael Mira-y-Lopez.


   FOOTNOTES
 
1 Supported by NIH Grant CA 59936, the Samuel Waxman Cancer Research Foundation, and The Badgeley Residual Charitable Trust. Back

2 To whom requests for reprints should be addressed, at Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1178, New York, NY 10029. Phone: (212) 241-7995; Fax: (212) 996-5787; E-mail: Waxman{at}msvax.mssm.edu Back

3 The abbreviations used are: PB, phenylbutyrate; FUdr, fluorodeoxyuridine; ALP, alkaline phosphatase; HCC, human colon carcinoma; INDO, indomethacin; FUra, 5-fluorouracil; NCI, National Cancer Institute; COX, cyclooxygenase; STAT, signal transducers and activators of transcription. Back

Received 8/26/99. Accepted 4/11/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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