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10.1245/s10434-006-9285-4
Annals of Surgical Oncology 14:1752-1762 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Effect of Combined Therapy With Low-Dose 5-Aza-2'-Deoxycytidine and Irinotecan on Colon Cancer Cell Line HCT-15

Megumi Ishiguro, MD, Satoru Iida, MD, PhD, Hiroyuki Uetake, MD, PhD, Shinji Morita, MD, Hiroshi Makino, MD, Keiji Kato, MD, Yoko Takagi, Masayuki Enomoto, MD, PhD and Kenichi Sugihara, MD, PhD

Department of Surgical Oncology, Tokyo Medical and Dental University, Graduate School, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

Correspondence: Address correspondence and reprint requests to: Megumi Ishiguro, MD; E-mail: ishiguro{at}ndmc.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Aberrant promoter hypermethylation is an epigenetic change that silences the expression of crucial genes, resulting in inactivation of the apoptotic pathway in various cancers. This hypermethylation can be restored by the demethylating agent 5-aza-2'-deoxy-cytidine (DAC). DAC might increase the tumor sensitivity to chemotherapy through deme-thylation and restoration of gene expression. We investigated the effect of combined therapy with DAC and irinotecan (CPT-11) on the human colon cancer cell line HCT-15.

Methods: Human colon cancer cell line HCT-15 was treated with DAC and/or CPT-11 both in vitro and in vivo. The changes in mRNA expression of several apoptosis-related genes were investigated by reverse transcriptase–polymerase chain reaction (PCR). Promoter methylation was detected by methylation-specific PCR and combined bisulfite restriction analysis. Suppression of tumor growth was observed during the treatment with DAC and/or CPT-11 and apoptosis in the tumors was investigated by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay.

Results: Promoter methylation of p14ARF, p16INK4a, BNIP3, and XAF1 was confirmed, and DAC restored mRNA expression of these genes. Demethylation and restoration of gene expression was observed with low-dose DAC, and demethylation status was sustained for several weeks. Combined therapy with DAC and CPT-11 produced marked suppression in tumor growth compared with DAC or CPT-11 alone, both in vitro and in vivo.

Conclusions: Pretreatment with low-dose DAC may have the potential to be used as a "biosensitizer" of DNA-damaging agents such as CPT-11 when the apoptotic pathway is inactivated as a result of aberrant promoter methylation in the cancer.

Key Words: Methylation • DAC • CPT-11 • Chemosensitivity • Apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancer (CRC) is one of the most common malignancies worldwide.1 Chemotherapy is an important treatment strategy for metastatic CRC. One of the key chemotherapy drugs for treating metastatic CRC is irinotecan (CPT-11), a semisynthetic derivative of camptothecin and a potent DNA–topoisomerase I (topo-I) inhibitor that forms stable topo-I DNA-cleavable complexes and inhibits the progression of the replication fork.2,3 CPT-11 has no cross-resistance with 5-fluorouracil (5-FU) and functions through a novel molecular mechanism.4–6 The CPT-11–5-FU–leucovorin combination is now approved as one of the standard chemotherapies for treating metastatic CRC.4,7,8

Several predictive factors of chemosensitivity have been investigated. The specific target enzyme or metabolic enzyme of the chemotherapeutic agent could be one of the important predictive factors of chemosensitivity. Regarding 5-FU–based chemotherapy, the target enzyme TS (thymidilate synthase) and the metabolic enzyme DPD (dihydropyrimidine dehydrogenase) have been reported to be predictive factors,9 whereas predictive factors of CPT-11 have not been sufficiently investigated.

In contrast, inactivation of the apoptotic pathway has been considered to be associated with chemoresistance.10 Apoptosis induced by chemotherapy may be decreased as a result of dysfunction of apoptosis-related genes. Most chemotherapeutic agents such as CPT-11 and 5-FU damage DNA and induce apoptosis. When the apoptotic pathway is inactivated in the tumor, the tumor may become resistant to chemotherapy.

Aberrant hypermethylation of gene promoter CpG islands of cell-cycle regulator genes, apoptosis-related genes, and mismatch repair genes has been detected in various cancers.11–13 Aberrant promoter hypermethylation in cancer cells can participate in inactivation of the apoptotic pathway at several points, either upstream (i.e., p14ARF or DAPK) or downstream (i.e., caspase) of the p53-dependent or p53-independent pathways.1 This hypermethylation is the epigenetic change that silences the gene expression without altering nucleotide sequences. Such gene expression silenced as a result of hypermethylation can be restored by the demethylating agent 5-aza-2'-deoxycytidine (DAC).14 DAC traps the DNA methyltransferase enzyme in a covalent complex with the DNA, resulting in a loss of DNA methylation with each cycle of cell division.14,15 Many studies demonstrated that treatment with DAC restored the expression and the functions of a number of crucial tumor suppressor genes such as p14ARF and suppressed the growth of cancer cells in a heritable manner.16–20

Therefore, the clinical use of DAC in the treatment of cancers resistant to common anticancer drugs due to hypermethylation of tumor suppressor genes could potentially provide a novel approach to cancer treatment by restoring gene expression and increasing the sensitivity of cancer cells to common therapeutic regimens. However, the clinical efficacy of combined therapy with DAC and other chemotherapeutic agents has not been sufficiently investigated in solid tumors.

In the present study, we demonstrated the effect of combined therapy with DAC and CPT-11 on the human colon cancer cell line HCT-15, which has aberrant hypermethylation of several gene promoters. Plated cells and xenografts were treated with DAC followed by CPT-11. Then mRNA expression and methylation status of apoptosis-related genes were studied. We discuss the usefulness of DAC as a part of a combination chemotherapeutic approach.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
CPT-11 and its active metabolite SN-38 were provided by Yakult (Tokyo, Japan). SN-38 was dissolved in dimethyl sulfoxide at a concentration of 1 µM and stored at –20°C, then further diluted in culture medium just before use. CPT-11 was dissolved in sterilized distilled water at a concentration of 5 mg/mL. After sonication at 70°C for 5 minutes, it was filter-sterilized and then stored at –20°C. DAC was obtained from Sigma (St. Louis, MO) and dissolved in phosphate-buffered saline (PBS) at a concentration of 10 µM for in vitro study and .1 mg/mL for in vivo study. It was filter-sterilized and stored at 4°C.

Cell Line
The human colon adenocarcinoma cell line HCT-15 was provided by Cell Resource Center for Biomedical Research, Tohoku University (Miyagi, Japan). The HCT-15 cell line is p53 deficient as a result of a point mutation and lacks p14ARF and p16INK4a expression because of promoter hypermethylation.21,22 Cells were maintained in RPMI 1640 medium (Sigma) containing 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL of penicillin, 100 µg/mL of streptomycin, 10 mM of HEPES (Gibco-BRL, Gaithersburg, MD), and 1.0 mM of sodium pyruvate (Gibco), and incubated at 37°C in 5% CO2.

Effect of DAC on mRNA Expression and Methylation Status of HCT-15 In Vitro
Cells (.25 x 104 per well) were plated in 24-well culture plates on day –2. On day 0, the culture medium was removed and new medium containing DAC was added. The cells were treated with 0, .1, .5, 1, or 5 µM of DAC for 72 hours, according to a previous report.23 On day 3, the cells were rinsed twice with FBS-free medium and collected with trypsin–ethylenediaminetetraacetic acid (EDTA). RNA or DNA was extracted, and then the mRNA expression and methylation status of cell cycle–related genes and apoptosis-related genes were examined by reverse transcriptase (RT)–polymerase chain reaction (PCR) analysis.

To analyze the time course of mRNA expression after DAC treatment, .5 µM of DAC was added on day 0, and the medium was removed on day 3. The medium was exchanged every 3 days. Cells were collected every 3 days, and cDNA was prepared for real-time PCR analysis.

In Vitro Growth Inhibitory Activities of DAC
Cells (.25 x 104 per well) were plated on day –2. Plated HCT-15 cells were treated with four protocols, as follows: group A, untreated control; group B, DAC alone; group C, SN-38 alone; and group D, DAC followed by SN-38. On day 0, the culture medium was removed and new medium containing .5 µM of DAC (or PBS) was added. On day 3, the cells were rinsed twice with FBS-free medium, and then new medium containing .005 µM of SN-38 (or PBS) was added. The dose of each drug was set on the bases of its pharmacological dose, results of previous reports,23–25 and our preliminary experiments (data not shown). The cells were collected with trypsin-EDTA, stained with trypan blue, and counted daily. Each experiment was performed in triplicate. After the drug treatment, RNA was extracted on day 6 for RT-PCR analysis.

In Vivo Growth Inhibitory Activities of DAC and Changes of mRNA Expression
HCT-15 cells were cultured then resuspended in FBS-free medium. On day –10, approximately 107 cells in .2 mL of PBS were injected subcutaneously into the right flank of 6-week-old male BALB/c (nu/ nu) mice. After 7 days, when the mean tumor diameter was 5 mm, mice were randomized into four groups (five mice per group) as follows: group A, untreated control; group B, DAC alone; group C, CPT-11 alone; and group D, DAC followed by CPT-11. The dose of each drug was set on the basis of the results of preliminary experiments (data not shown). The most effective and nontoxic regimen of DAC was reported as being three doses of DAC at intervals of 3 hours.19

On day –3, DAC (1 mg/kg) was administered intraperitoneally (i.p.) at 18:00, 21:00, and 24:00 (total dose, 3 mg/kg per mouse). On days 0, 4, and 8, CPT-11 (40 mg/kg) was administered i.p. at 18:00. Untreated control mice or mice receiving a single agent received injections of .2 mL of PBS on the days of therapy. The mice were weighed daily, and the tumor volume was determined by the following equation: tumor volume = {alpha} x {alpha} x ß/2 mm3, where {alpha} is the shortest and ß is the largest diameter of the tumor. The relative tumor volume was determined as follows: relative tumor volume = tumor volume on day x/tumor volume on day 0.

For the analysis of mRNA expression, the mice of group B were killed on days –3, 0, 3, 6, 9, and 16. The tumors were extracted, and RNA was prepared and used for real-time PCR analysis.

The protocol for this animal experiment was approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University, and the experiment was carried out following the Guidelines for Animal Experimentation in Tokyo Medical and Dental University based on the Guide for the Care and Use of Laboratory Animals.

RT-PCR
From the treated cells and xenografts, total cellular RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA) with DNase treatment, as we reported previously.26 cDNA was synthesized with Superscript II (RNase H(–) reverse transcriptase; Invitrogen, Carlsbad, CA) with 10 ng of extracted RNA. Then PCR was performed as described previously.26 We used the following PCR conditions: the initial denaturation step was at 94°C for 2 minutes, followed by 26 to 38 cycles of denaturation for 1 minute at 94°C, annealing for 1 minute at 55 to 63°C, and extension for 1 minute at 72°C. As an internal control for RT-PCR analysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were amplified from the same cDNA samples. Primer sequences are listed in Table 1Go. After amplification, PCR products were loaded onto 2% agarose gels, stained with ethidium bromide, electrophoresed, and visualized under ultraviolet illumination.


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  		TABLE 1. Candidate genes and primer sequences
 
Real-Time PCR
We selected p14ARF mRNA expression as a marker of the demethylation status because the gene promoter of p14ARF is fully methylated in HCT-15 cells and the cells lack the gene expression. p14ARF cDNA sequences were amplified by quantitative PCR by a fluorescence-based real-time detection method (ABI Prism 7300 Real Time PCR System, Taqman; Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. Nucleotide sequences of primers and probe are listed in Table 1Go. As an internal standard, each individual sample was normalized to its ß-actin content. Relative quantification of p14ARF mRNA expression was calculated by the {Delta}{Delta}Ct method27 by SDSv1.2 with RQv1.0 software (Applied Biosystems). Each experiment was performed in duplicate.

Methylation-Specific PCR
The methylation status of gene promoters was analyzed by methylation-specific PCR.28 The DNA methylation pattern in the CpG islands of the respective promoters was determined by chemical modification of unmethylated but not methylated cytosines to uracil and subsequent PCR that used specific primers for either methylated or modified unmethylated DNA. Genomic DNA was prepared,29 and extracted DNA was subjected to bisulfite treatment as described previously30 with a DNA modification kit (Chemicon International, Temecula, CA). The modified DNA was used as a template for methylation-specific PCR. Primer sequences are listed in Table 1Go. After amplification, PCR products were electrophoresed on 2% agarose gels.

Combined Bisulfite Restriction Analysis
Combined bisulfite restriction analysis is a semi-quantitative method used to measure methylation at specific methylation-sensitive restriction sites.31 Bisulfite-modified DNA was amplified by nested PCR with specific primers for XAF1. The primer sequences and PCR conditions are listed in Table 1Go. PCR products were then digested with a specific restriction enzyme, TaqI (Toyobo, Osaka, Japan), at 65°C for 2 hours, and then electrophoresed on 2% agarose gels. In the examined region within the XAF1 promoter, TaqI cleaves only the methylated alleles.

TUNEL Assay
Drug-dependent apoptosis was evaluated with ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon International) according to the manufacturer’s protocol. For in vitro study, cells (.25 x 104 per well) were seeded in Biocoat Cellware Poly-D-Lysine 2-well culture slides (Becton Dickinson, Sparks, MD) on day –2 and treated with .5 µM of DAC (from days 0 to 3), followed by .005 µM of SN-38 (from days 3 to 6). On day 6, the slides were washed with PBS and fixed for 15 minutes with 10% neutral buffered formalin, then used for TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay. For in vivo study, treated mice were killed and the tumors were extracted on day 12. The tumors were fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin, and 3-µm sections were cut. Paraffin was then removed from sections, and they were rehydrated and used for TUNEL assay. A dark brown staining indicated apoptosis. Ten representative areas without inflammation or necrosis were selected. TUNEL-positive cells from 10 fields per slide (±200 magnification) were counted under optical microscopy to calculate the mean ± SD.

Statistical Analysis
All statistical analyses were carried out with the StatView Software (version 5.0). Differences between the treatment groups were analyzed by Welch’s t-test. Differences were considered statistically significant when P < .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of DAC on mRNA Expression and Methylation Status in HCT-15
The changes in the mRNA expression of apoptosis-related genes in HCT-15 cells are shown in Fig. 1aGo. No mRNA expression of p14ARF and p16INK4a was identified in untreated HCT-15 cells. Expression of these genes was apparent with .1 µM of DAC. DAC clearly enhanced mRNA expression of p14ARF, p16INK4a, BNIP3 (bcl-2/adenovirus E1B 19-kDa interacting protein 3), and XAF1 (XIAP-associated factor 1) and decreased mRNA expression of bcl-2 in a dose-dependent manner. However, the expression of bax and bcl-XL/S was unaffected by DAC. mRNA expression of topo-I, the target enzyme of CPT-11, was also unaffected by DAC.


Figure 1
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  		FIG. 1. (a) Changes in mRNA expression of apoptosis-related genes after demethylating agent 5-aza-2'-deoxycytidine (DAC) treatment. A total of 10 ng of RNA was extracted from the cells treated with 0, .1, .5, 1, and 5 µM of DAC for 72 hours and then used for reverse transcriptase–polymerase chain reaction (RT-PCR). (b) Time course of p14ARF mRNA expression after DAC treatment. A total of .5 µM of DAC was added on day 0 and removed on day 3. RNA was extracted every 3 days. Relative quantification of p14ARF mRNA expression was investigated by real-time PCR. (c) Methylation-specific PCR analyzing the changes in the methylation status after DAC treatment. Lane M indicates existence of methylated DNA; lane U, existence of unmethylated DNA. (d) Combined bisulfite restriction analysis analyzing the changes in the methylation status of XAF1 promoter after DAC treatment. Arrows indicate bands reflecting methylation of the TaqI sites.

 
The time course of p14ARF mRNA expression after DAC treatment is shown in Fig. 1bGo. In real-time PCR analysis, no mRNA expression of p14ARF was identified in untreated HCT-15 cells. p14ARF mRNA expression was apparent on day 3 and sustained for 30 days after .5 µM of DAC was removed on day 3.

By use of methylation-specific PCR and combined bisulfite restriction analysis, aberrant promoter methylation of p14ARF, p16INK4a, BNIP3, and XAF1 was detected in untreated HCT-15 cells. After DAC treatment, the promoter demethylation of these genes was confirmed by the existence of unmethylated promoter regions (Fig. 1c,dGo).

Effect of DAC and/or SN-38 on Tumor Growth and Apoptosis In Vitro
The time course of the cell proliferation in vitro through the treatment of DAC and/or SN-38 is shown in Fig. 2aGo. On day 6, the number of cells in group D was 25% of that in group A, whereas those in groups B and C were 85% and 60%, respectively. The cell proliferation was suppressed in group D compared with groups A, B, and C. No marked suppression of the cell proliferation was observed in group B compared with group A. But there was a difference in cell proliferation between group A and C.


Figure 2
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  		FIG. 2. (a) In vitro growth inhibition by demethylating agent 5-aza-2'-deoxycytidine (DAC) and/or SN-38. Plated HCT-15 cells were treated with four protocols, and numbers of cells were counted daily. DAC, .5 µM; SN-38, .005 µM. Open circle, group A (untreated control); solid triangle, group B (DAC alone); open triangle, group C (SN-38 alone); solid circle, group D (DAC followed by SN-38). * P = .0009, ** P = .00216, # P = .0355 (Welch’s t-test). Error bars = SD. (b) Detection of apoptosis in vitro by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay. Data shown are mean number of TUNEL-positive cells counted in 10 fields per slide on day 6 in (a). * P = .0126 (Welch’s t-test). Error bars = SD. (c) Changes in mRNA expression of apoptosis-related genes after DAC and/or SN-38 treatment. RNA was extracted on day 6 in (a), and then mRNA expression was investigated by reverse transcriptase–polymerase chain reaction.

 
The mean number of apoptotic cells on day 6 determined by TUNEL assay is shown in Fig. 2bGo. The highest mean number of apoptotic cells was observed in group D, which was different from that in group A, whereas no difference was observed in the number of apoptotic cells between group B and group A as well as between group C and group A. Numerous mitotic cells were found in groups A, B, and C, but not in group D.

mRNA expression after the treatment with DAC and/or SN-38 on day 6 is shown in Fig. 2cGo. mRNA expression of p14ARF, p16INK4a, and XAF1 was not identified in group C. The highest mRNA expression of these genes and the lowest mRNA expression of bcl-2 were observed in group D. The expression of other apoptosis-related genes and topo-I gene was unaffected by combined therapy.

Effect of DAC and/or CPT-11 on Tumor Growth and Apoptosis In Vivo
First, by use of real-time PCR, it was confirmed that DAC (1 mg/kg x3) restored p14ARF mRNA expression in xenografts as in in vitro experiments. p14ARF mRNA expression was apparent after 3 days, maximal on day 6, and then sustained until the end of the experiment (Fig. 4aGo).


Figure 4
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  		FIG. 4. (a) Real-time polymerase chain reaction (PCR) analysis of p14ARF mRNA expression of xenografts. The mice treated with demethylating agent 5-aza-2'-deoxycytidine (DAC) alone (group B in Fig. 3Go) were killed on days –3, 0, 3, 6, 9, and 16. The tumors were extracted and cDNA synthesized. Relative quantification of p14ARF mRNA expression was investigated by real-time PCR. (b) Detection of apoptosis in xenografts by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay. On day 12 in Fig. 3Go, the tumor xenografts were extracted from the mice treated with four protocols, then used for TUNEL assay. Data shown are mean number of TUNEL-positive cells counted in 10 fields per slide. P = .00037, ** P = .00042 (Welch’s t-test). Error bars = SD.

 
The relative tumor volumes in group D were far smaller compared with those of groups A, B, and C, and the growth rate remained lower on day 16, when the experiment ended (Fig. 3Go). The inhibition rate of tumor growth was highest in group D (Table 2Go). There was no difference in tumor growth among groups A, B, and C. At the dose used in this experiment, no body weight loss (Table 2Go) or lethal toxicity was observed. In the TUNEL assay, the mean number of apoptotic cells greatly increased in group D, like in the in vitro study (Fig. 4bGo).


Figure 3
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  		FIG. 3. In vivo growth inhibition by demethylating agent 5-aza-2'-deoxycytidine (DAC) and/or CPT-11. Mice were treated with four protocols and the tumor volume was investigated daily. Data shown are mean relative tumor volume of five mice. DAC, 1 mg/kg intraperitoneally x3; CPT-11, 40 mg/kg. Open circle, group A (untreated control); solid triangle, group B (DAC alone); open triangle, group C (CPT-11 alone); solid circle, group D (DAC followed by CPT-11). * P = .0062, ** P = .0004 (Welch’s t-test). Error bars = SD.

 

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  		TABLE 2. Analysis of effect of DAC and CTP-11 on tumor xenograft
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, DAC restored and increased the expression of apoptosis-related genes, including p14ARF, BNIP3, and XAF1, in HCT-15 cells. Restoration of such genes through demethylation was observed even with low-dose DAC, and the demethylation status was sustained for several weeks after DAC was removed. Furthermore, combined treatment with DAC and CPT-11 markedly suppressed tumor growth compared with DAC or CPT-11 alone.

Normal cells express gene products that are related to the induction of apoptosis because of physiologically present biological response modifiers (BRMs). During carcinogenesis and tumor progression, expression of such gene products can be silenced by aberrant hypermethylation of promoter CpG islands. The reexpressed gene products that occur because of the demethylation of these genes by DAC might serve as a target for BRMs (administered in pharmacological doses), leading to subsequent cell death.12 We hypothesized that apoptosis might be induced to a greater degree by the administration of anticancer drugs after demethylation of apoptosis-related genes by DAC treatment.

Paz et al.11 studied 70 widely used human cancer cell lines of 12 different tumor types, including 11 CRC cell lines for CpG island promoter hypermethylation of 15 tumor suppressor genes, global 5-methylcytosine genomic content, and chemical response to the demethylating agent DAC, and found that CpG island of 11 CRC cell lines was hypermethylated in 25% to 60%. There was a great deal of 5-methylcitosine DNA content in the human cancer cell lines assessed, and the mean 5-methylcitosine DNA content was 3.3%. The highest level was found in the CRC cell line SW48 (5.6%). They also demonstrated that the 5-methylcitosine DNA content was greatly reduced in an average of 49% of cell lines after treatment with DAC. Petak et al.32 reported that Fas promoter containing 28 CpG sites was methylated in the CRC cell lines Caco2 and RKO. And they demonstrated that DAC (1 µM, 72 hours) upregulated Fas expression and increased the percentage of apoptotic cells, furthermore sensitized RKO cells to recombinant FasL-induced apoptosis. They concluded that DNA hypermethylation may be one mechanism implying loss of sensitivity to Fas-induced apoptosis in CRC cells. These studies have demonstrated that cancer cells frequently contain promoter CpG island hypermethylation of tumor suppressor genes and DAC was expected to sensitize cancer cells to anticancer drugs.

p14ARF, which is frequently inactivated in a variety of human cancers, is known to participate in the activation of the apoptotic pathway upstream of the p53-dependent pathway. In contrast, recent reports have also implicated p14ARF in p53-independent mechanisms of apoptosis induction.16,17,33 In the present study, p14ARF restored by DAC treatment might act on the p53-independent mechanisms in the apoptotic pathway because HCT-15 cells lack normal p53 expression because of a point mutation. Over-expression of BNIP3, which is a proapoptotic gene, increased the sensitivity to apoptosis induced by to-poisomerase I and II.34 In some kinds of cancers, BNIP3 was reported to be inactivated by aberrant hypermethylation.35,36 XAF1 is a 34-kDa zinc finger protein that blocks the caspase-inhibiting and antia-poptotic abilities of X-linked inhibitor of apoptosis protein. The association between the reduction of XAF1 expression and its aberrant hypermethylation has been reported.37–39 Restoration of XAF1 expression by DAC might be one of the potential mechanisms involved in the effect of DAC on CPT-11 sensitivity. Moreover, DAC might enhance another pro-apoptotic pathway through demethylation of other genes. Indeed, we confirmed aberrant hyper-methylation of other pro-apoptotic genes (i.e., DAPK, Apaf-1 and TMS1; data not shown). Taken together, our study suggests that DAC might exert an effect on the sensitivity of cancer cells to CPT-11 by restoring the silenced gene expression because of aberrant hypermethylation participating in the inactivation of the apoptotic pathway at several points.

In this study, DAC decreased bcl-2 mRNA expression in a dose-dependent manner. Violette et al.40 reported that the balance of bax, bcl-2, and bcl-XL (bcl-2 + bcl-XL/bax) could be used as a marker to predict the pro-apoptotic ability and chemosensi-tivity, regardless of the p53 status. In this study, DAC did not influence bax expression, but decreased bcl-2 expression in HCT-15, although the mechanisms involved are unknown. Therefore, DAC might shift this balance to the pro-apoptotic side and might enhance the pro-apoptotic ability in HCT-15 cells.

Tan et al.41 and Rasheed and Rubin42 reported that the expression and activity of topo-I, the target enzyme of CPT-11, correlated to the chemosensitivity of CPT-11. In contrast, Bras-Goncalves et al.43 reported that the expression level of topo-I mRNA did not correlate with the CPT-11 response of the tumor. In this study, DAC did not influence the expression level of topo-I mRNA but increased the effect of CPT-11 on growth inhibition, suggesting that the expression level of topo-I mRNA is unlikely to be associated with HCT-15 response to CPT-11, which is consistent with the report by Bras-Goncalves et al.

We thought that the most suitable dose of DAC was the minimum dose in which silenced gene expression was restored and cell proliferation was not affected. According to a previous report,23 induction of gene expression by DAC was observed in .1 to 1 µM of DAC in plasma level. In our study in vitro, we confirmed that demethylation and gene reexpres-sion were also observed in .1 µM of DAC (Fig. 1Go). In our preliminary experiments of growth inhibition in vitro, cell proliferation was interfered with in 1 µM of DAC but not in .1 and .5 µM of DAC compared with untreated control (data not shown). In the combined therapy with DAC and SN-38, the growth inhibition was more apparent in .5 µM of DAC compared with .1 µM of DAC (data not shown). So we determined .5 µM to be the most suitable dose of DAC for in vitro studies.

In experiments in vivo with ovarian cancer cells xenograft, Plumb et al.19 described that the most effective and nontoxic regimen was three doses of 5 mg/kg of DAC at intervals of 3 hours (total dose, 15 mg/kg). However, our preliminary experiments showed that three doses of 5 mg/kg of DAC produced adverse effects such as weight loss and severe diarrhea when combined with CPT-11 (data not shown). We also confirmed that the tumor growth was reduced when mice were treated with DAC followed by CPT-11, but there was no difference in the tumor growth between three doses of 1 and 3 mg/kg (data not shown). From these observations, we determined three doses of 1 mg/kg of DAC at intervals of 3 hours to be the best dose of DAC for in vivo studies. This dose was the minimum that did not inhibit tumor growth, but it restored and maintained the p14ARF mRNA expression, and the combined therapy of DAC followed by CPT-11 in vivo could produce synergistic growth inhibition without causing major toxicity, as in the in vitro study.

One of the most important findings in this study was the duration of the demethylation status after DAC treatment. Through our in vitro study, expression of p14ARF mRNA in HCT-15 cells was at its maximum 9 days after DAC was added and was sustained for longer than 30 days after DAC’s removal on day 3. This result may support the idea that epigenetic change can be inherited in DNA replication, implying that the demethylation status in the tumor would continue after DAC has disappeared from the plasma in vivo. Therefore, when DAC is used as a "biosensitizer," it might as well be administered before anticancer drugs.

DAC has already been used in clinical trials on hematopoietic malignancies, and a 20% to 30% response rate has been demonstrated.13,14,44,45 However, preliminary experiences with demethylating agents in solid tumors have been disappointing because the response rates have been low.13,21,46,47 Most clinical studies of DAC in solid tumors have also failed to inhibit tumor progression, and response rates have generally failed to exceed 10%.13 However, these discouraging results were in trials that used DAC alone. Therefore, to induce apoptosis after DAC treatment, some DNA damage may be required as a trigger. We hypothesized that almost all che-motherapeutic drugs and irradiation could act as a trigger and might show some synergistic effect when combined with DAC.

In solid tumors, the clinical usefulness of combined therapy with DAC and CPT-11 has not been fully investigated. Plumb et al.19 reported that DAC treatment enhanced the sensitivity of CRC xenografts to anticancer drugs including cisplatin, epirubicin, and carboplatin. Anzai et al.48 reported on the synergistic effect of DAC and topotecan in murine CRC cells, but they did not sufficiently investigate the mechanism of synergy. Pohlmann et al.49 performed a phase 2 trial of cisplatin plus DAC in patients with advanced squamous cell carcinoma of the cervix and demonstrated that this combination therapy was moderately effective. However, the regimen used in that trial produced hematologic toxicities. Previous clinical studies have seemed to adhere to maintaining a plasma concentration of DAC, which led to severe toxicity. When we recognized reexpression gene products as a target for BRMs, DAC could be used at lower doses associated with a biological effect on the target tissues. We think that the most important finding regarding DAC treatment is to sustain the gene expression in the tumor, but not to maintain a high plasma concentration of DAC. Therefore, we propose that DAC can be effectively provided at a lower dose in combination chemotherapy for solid tumors.

In summary, we observed that combined therapy with DAC and CPT-11 resulted in marked suppression of tumor growth compared with DAC or CPT-11 alone. Furthermore, restoration of genes through demethylation was observed with low-dose DAC, and the demethylation status was sustained for several weeks. Therefore, we expect that pretreatment with low-dose DAC will be useful as a "biosensitizer" of DNA-damaging agents when the apoptotic pathway is inactivated because of aberrant hypermethy-lation. Pretreatment with low-dose DAC might be an important strategy in chemotherapy for CRC.

Received for publication August 25, 2006. Accepted for publication October 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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