Research ArticleExperimental Studies

Effect of Thymidine Phosphorylase Gene Demethylation on Sensitivity to 5-Fluorouracil in Colorectal Cancer Cells

MUNEYUKI KOYAMA, ERIKA OSADA, NUBUTAKE AKIYAMA, KEN ETO and YOSHINOBU MANOME
Anticancer Research February 2022, 42 (2) 837-844; DOI: https://doi.org/10.21873/anticanres.15541

Abstract

Background/Aim: Chemotherapy is used for recurrent and metastatic colorectal cancer, but the response rate of 5-fluorouracil (5-FU), the standard treatment for colorectal cancer, is low. We hypothesized that thymidine phosphorylase (TYMP) expression, a rate-limiting activating enzyme of 5-FU, is regulated by methylation of the gene promoter region, and demethylation of TYMP would increase sensitivity to 5-FU. Materials and Methods: HCT116 colon cancer cells were treated with 5-aza-2’-deoxycytidine, a demethylating agent, and changes in TYMP transcription and sensitivity to 5-FU were evaluated. Results: TYMP expression increased over 54-fold in HCT116 transfected with TYMP. The cytotoxicity of 5-FU increased up to 5.5-fold. In comparison, in HCT116 treated with 5-aza-2’-deoxycytidine, TYMP expression increased 5.8-fold. However, the cytotoxicity of 5-FU remained unchanged. Conclusion: Demethylating agent alone did not promote the cytotoxicity of 5-FU against colorectal cancer. To further increase the sensitivity to 5-FU, combination with adjuvant therapy focusing on metabolic pathways other than the TYMP pathway appear necessary.

Key Words:

According to a report by the International Agency for Research on Cancer, colorectal cancer is the third most common carcinoma in the world, with 1.8 million new cases and 880,000 deaths in 2018 (1). With the remarkable progress in surgical techniques in recent years, the 5-year survival rate for colorectal cancer detected at a relatively early stage without metastasis is about 90%, and the cure rate is increasing. However, the 5-year survival rate for patients with colorectal cancer showing distant metastasis is 12.5%, representing a poor prognosis (2). Patients with distant metastasis or recurrence of colorectal cancer are treated with chemotherapy instead of surgery, but the response rate of 5-fluorouracil (5-FU) in all patients with colorectal cancer is 20%, which cannot be considered sufficient (3).

While 5-FU is the most commonly used chemotherapy for colorectal cancer, resistance to 5-FU is an obstacle to treatment. The mechanisms of resistance to 5-FU vary widely, including changes in the expression of enzymes involved in 5-FU metabolism, such as thymidine phosphorylase (TYMP), thymidylate synthase (TYMS), and thymidylate kinase (TK), decreased apoptosis, decreased autophagy, and modulation of cellular processes such as glucose metabolism and drug delivery (1, 4, 5).

The rate-limiting metabolic activating enzyme for 5-FU is TYMP. The level of TYMP is thus an independent prognostic factor in colorectal cancer (6, 7). In cell culture and mouse models, over-expression of TYMP has been reported to increase sensitivity to 5-FU (8, 9). In addition, TYMP expression is decreased by methylation of the transcription factor binding site in the promoter region (10, 11), suggesting that methylation of the promoter region may be involved in 5-FU resistance.

On the other hand, epigenetic modifications, which regulate gene expression independent of changes in the DNA sequence, have been found to be important in carcinogenesis (12). Epigenetic modifications include: DNA methylation, in which cytosine is improperly methylated to 5-methylcytosine; histone modifications, which alter the three-dimensional structure of nucleosomes; and chromatin remodeling, which changes the structure of chromatin. In the case of colorectal cancer, such epigenetic modifications are reported to be deeply involved in the development of cancer via gene silencing (12). Colorectal cancer includes a group of tumors that show accumulation of tumor-specific methylation of the promoter region of genes, and these tumors are associated with high rates of BRAF and KRAS mutations, which play roles in the selection of molecularly targeted therapies. In recent years, it has become increasingly clear that epigenetic modifications are also important in colorectal cancer (13).

DNA methylation is one of the most studied epigenetic modifications in carcinogenesis and is regulated by the DNA methyltransferase (DNMT) family (12, 14). DNMT1 is essential for the maintenance of DNA methylation at CpG sites, mainly in the promoter region of genes, and regulates proliferation and survival of human cancer cells (14). In particular, DNMT1 has been suggested to translocate into the nucleus to promote cell proliferation by methylation in human colon cancer cells (15, 16).

In the nucleus, 5-aza-2’-deoxycytidine, an inhibitor of methylation, induces DNMT1 proteasome degradation (17). As a result, 5-aza-2’-deoxycytidine has been approved in the United States for the treatment of refractory anemia (RA), RA with ringed blasts (RARS), RA with excessive blasts (RAEB), and myelodysplastic syndromes including chronic myelogenous leukemia (18). The proteasomal degradation caused by 5-aza-2’-deoxycytidine is selective for DNMT-1, sparing DNMT3a and 3B (16). Taking advantage of this mechanism, the synergistic effects of combining methylation inhibitors and molecularly targeted drugs have proven effective in the treatment of hematopoietic malignancies, and therapeutic results have been achieved (19). The present study investigated whether DNMT1 regulates methylation of the promoter region of TYMP to elucidate the mechanisms underlying 5-FU resistance. Because the epigenetic modification by DNMT1 is reversible, 5-FU-resistant patients with low TYMP expression could be treated with standard therapies, including 5-FU, in accordance with the guidelines.

As 5-FU is a standard treatment in colorectal cancer chemotherapy, increasing the sensitivity to 5-FU may improve the prognosis of colorectal cancer. In this study, we hypothesized that TYMP could also be suppressed by methylation. The purpose of this study was to confirm whether 5-FU sensitivity can be increased by increasing TYMP expression through the administration of 5-aza-2’-deoxycytidine, a methylation inhibitor.

Materials and Methods

Cell culture and processing. This study included five commonly used human colon cancer cell lines: HCT116, SW480, SW620, DLD-1, and RKO-Px4 (American Type Culture Collection, Rockville, MD, USA). In addition, the MC38 cell line (American Type Culture Collection), a mouse colorectal cancer cell line, was used for comparison. HCT116, SW480, SW620, and RKO-PX4 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin, while DLD-1 and MC38 were cultured in Dulbecco’s modified Eagle’s medium under the same conditions. Cultures were maintained in an incubator at 37°C and 5% CO2.

Western blotting. Concentrations of total protein were determined using the BCA™ Protein Assay Kit (Thermo Fisher Scientific, Tokyo, Japan). For each sample, 20 μg of protein per lane were used. Protein samples were fractionated using 10% sodium dodecylsulfate-polyacrylamide gels electrophoresis and transferred to nitrocellulose membranes. The nitrocellulose membranes were then blocked with Block Ace (K.A.C. Co., Kyoto, Japan) at 4°C for 1 h. Membranes were then incubated with primary antibodies overnight at 4°C and washed three times with Tris-Buffered saline + 0.05% Tween-20 for 15 min. The primary antibodies used for immunoblotting were monoclonal rabbit anti-TYMP antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA), monoclonal mouse anti-DNMT1 antibody (1:1,000; Cell Signaling Technology), and monoclonal mouse anti-GAPDH antibody (1:1,000; Gene Tex, Irvine, CA, USA). The nitrocellulose membranes were then incubated with a secondary antibody for 1 h at room temperature. Secondary antibodies were monoclonal horseradish peroxidase goat anti-rabbit (1:1,000; GE Healthcare UK, Amersham, UK) and monoclonal horseradish peroxidase goat anti-mouse (1:1,000; GE Healthcare UK).

Pierce® ECL Western Blotting Substrate (Thermo Fisher Scientific, Tokyo, Japan) and the ChemiDoc™ Touch Imaging System (WAKENKYU, Kyoto, Japan) were then used for visualization of the protein bands.

The experimental results were analyzed using Image Lab software (BIO-RAD, Tokyo, Japan).

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted from three colorectal cancer cell lines (HCT116, SW620, and SW480) using TRIZOL@Reagent (Invitrogen, Carlsbad, CA, USA). Next, 0.4 μg of RNA was analysed using an RT-PCR RNeasy Plus Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s protocol. RT-PCR was performed using SYBR® Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan) according to the instruction manual.

Forty-five cycles of 95°C for 10 s and 68°C for 30 s were performed using the LightCycler@96 System (Roche Diagnostics, Mannheim, Germany), and the mRNA expression of each sample was examined in triplicate. GAPDH was used as an internal standard.

The following primers were used. For TYMP, Forward: 5’-caaggtcagcctggtcctcgcacctg-3’; Reverse: 5’-acgaagtttcttactga gaatggaggctgtg-3’. For GAPDH, Forward: 5’-accaccagccccagcaag agcacaagag-3’; Reverse: 5’-cccctccccttcaaggggtctacatg-3’.

Transfection. Human TYMP or enhanced green fluorescent protein (EGFP) amplified by PCR was cloned into EcoRI site of pIRES neo (Clonech, Gene bank accession U89673). In this vector, expression of TYMP, EGFP and the neomycin resistance gene (NeoR) was driven by the CMV immediate-early promoter. The vector was transfected into HCT116 cells using Lipofectamine® LTX Reagent (Thermo Fisher Scientific). Selection of cell clones was performed after treatment with 800 μg/ml of G418 sulfate (Geneticin; Thermo Fisher Scientific) after transfection. Three independent clones of each transfection were used for following experiments.

Cytotoxic assay. The HCT116 human colorectal cancer cell line was seeded onto 96-well plates (code: 3860-096) at a concentration of 0.5×104 cells/50 μl medium/well. Cells were incubated with 5-FU or 5-aza-2’-deoxycytidine (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at various dilutions for 72 h at 37°C and 5% CO2. After incubation, cells were fixed with 30 μl of 25% glutaraldehyde, washed three times with running tap water, and then stained with 200 μl of 0.05% methylene blue for 15 min. After staining, cells were washed three times with running tap water, 50 μl of 0.33 N hydrochloric acid was added, and absorbance was measured at a wavelength of 595 nm using a microplate reader 680XR (BIO-RAD, Tokyo, Japan) as previously described (20).

Results

Expression of DNMT-1 was examined in five human colon cancer cell lines by western blotting. DNMT-1 was expressed in all colon cancer cell lines (Figure 1). The following experiments were performed using HCT116 cells, which showed reduced expression of different tumor suppressor genes due to methylation of their promoter regions (17, 21).

Figure 1.
Figure 1.

DNMT1 expression in five colorectal cancer cell lines. GAPDH was used as an internal control. DNMT1 expression was observed in all the colorectal cancer cell lines analysed.

First, we used RT-PCR to examine TYMP transcription levels in HCT116 cells and SW620 cells as a control. TYMP levels were 520-fold higher in HCT116 cells compared to those in SW620 cells (Figure 2A).

Figure 2.
Figure 2.

Comparison of expression levels of TYMP between HCT116 and SW620 cells. (A) High transcription of TYMP in HCT116 cells. Transcription of TYMP was determined using quantitative PCR. Levels were normalized to those of GAPDH and compared between HCT116 and SW620 cells. Transcription of TYMP was higher in HCT116 than in SW620. (B) High TYMP expression in HCT116 cells. TYMP was highly expressed in HCT116 cells compared to SW620 cells. GAPDH was used as an internal control.

Since transcription of TYMP was found to be increased, we next examined TYMP protein expression in HCT116 and SW620 by western blotting. TYMP protein levels in HCT116 cells were 2,710-fold higher than those in SW620 cells (Figure 2B).

A cell line stably over-expressing TYMP was then established to investigate possible changes in the sensitivity to 5-FU.

All three clones of HCT116 cells transfected with TYMP showed 54- to 124-fold higher expression of TYMP than non-transfected wild type cells. The control EGFP-transfected HCT116 cells did not show TYMP over-expression (Figure 3).

Figure 3.
Figure 3.

TYMP expression in HCT116 transfected with TYMP or EGFP. TYMP expression was higher in HCT116 transfected with TYMP than in the wild type or HCT116 cells transfected with EGFP. GAPDH was used as an internal control.

Cytotoxic assay of 5-FU in transfected HCT116 cell lines. In the three EGFP-transfected clones of HCT116 cells, as a control, the half maximal inhibitory concentration (IC50) values of 5-FU were 15.1 μM, 14.3 μM, and 8.3 μM, respectively, compared with 4.63 μM in HCT116 non-transfected cells. No decrease in the IC50 value of 5-FU was observed (Figure 4A). However, the three clones of TYMP-over-expressing HCT116 cells showed a marked decrease in the IC50 of 5-FU at 1.6 μM, 2.0 μM, and 3.14 μM compared with the IC50 of 8.8 μM in HCT116 non-transfected cells (Figure 4B). This result suggests that 5-FU resistance is affected by the levels of TYMP expression.

Figure 4.
Figure 4.

Cytotoxicity of 5-Fluorouracil (5-FU) in different cell lines. (A) Cytotoxic effects of 5-FU in HCT116 transfected with EGFP. No difference is seen in IC50 values of 5-FU compared with the wild type. ● HCT116 non-transfected cells (wild type), ■ HCT116 transfected with EGFP (clone 1), ◆ HCT116 transfected with EGFP (clone 2), ▲ HCT116 transfected with EGFP (clone 3). (B). Cytotoxic effect of 5-FU in HCT116 transfected with TYMP. A difference is seen in the IC50 of 5-FU compared with the wild type. ● HCT116 non-transfected cells (wild type), ■ HCT116 transfected with TYMP (clone 1), ◆ HCT116 transfected with TYMP (clone 2), ▲ HCT116 transfected with TYMP (clone 3).

Next, we examined whether DNA methylation is involved in the regulation of TYMP expression. We used 5-aza-2’-deoxycytidine, a methylation inhibitor, to determine whether demethylation increases TYMP transcription.

To determine the concentration of 5-aza-2’-deoxycytidine to be used in the experiment, a cytotoxic assay was performed in the HCT116 cells and a concentration of 15 μM was selected. At this concentration, transcription of TYMP was confirmed and >80% of HCT116 cells survived for 3 days in culture.

HCT116 cells were seeded on a 10-cm dish (6×106/75 cm2 flask), incubated at 37°C in a 5% CO2 atmosphere for 24 h, and then treated with 15 μM 5-aza-2’-deoxycytidine for 1-3 days.

Protein extraction was performed on day 0 before the addition of 5-aza-2’-deoxycytidine, and on days 1, 2, and 3 after the addition of 5-aza-2’-deoxycytidine, followed by western blotting. The results are shown in Figure 5. In HCT116 cells treated with 15 μM 5-aza-2’-deoxycytidine, an increase in TYMP transcription was observed on the first day after treatment. These results suggest that 5-aza-2’-deoxycytidine treatment inhibits DNMT-1-mediated methylation and increases TYMP transcription.

Figure 5.
Figure 5.

TYMP expression in HCT116 cells on 1, 2, and 3 days after 5-aza-2’-deoxycytidine treatment (15 μM). An increase in TYMP expression was observed 1 day after 5-aza-2’-deoxycytidine treatment. GAPDH was used as an internal control.

Since TYMP mRNA was found to be up-regulated by 5-aza-2’-deoxycytidine, we investigated whether this up-regulation increased sensitivity to 5-FU.

The 5-FU cytotoxic assay was performed in the presence of 15 μM 5-aza-2’-deoxycytidine treatment (Figure 6).

Figure 6.
Figure 6.

Cytotoxic effect of 5-FU in HCT116 treated with 5-aza-2’-deoxycytidine. No difference in the IC50 of 5-FU is observed compared with the wild type. ■ HCT116 non-treated with 5-aza-2’-deoxycytidine (wild type). ● HCT116 treated with 5-aza-2’-deoxycytidine.

The IC50 of 5-FU in HCT116 cells was unchanged by treatment with 5-aza-2’-deoxycytidine. The combination of 5-FU and 15 μM 5-aza-2’-deoxycytidine thus did not enhance the cytotoxicity of 5-FU in 5-FU-resistant colon cancer cell lines.

Next, we expressed the same vector in the MC38 mouse colorectal cancer cell line, and performed the cytotoxic assay under the same conditions to compare the effects of 5-FU on these cells with those on the HCT116 human colorectal cancer cell line. The results showed that MC38 cells overexpressing TYMP were 100-fold more sensitive to 5-FU than is MC38 non-transfected cells (Figure 7). This result showed a marked increase in sensitivity compared to the HCT116 human colon cancer cell line.

Figure 7.
Figure 7.

Cytotoxic effects of 5-FU in MC38 transfected with TYMP. A clear decrease in the IC50 of 5-FU is evident in MC38 transfected with TYMP compared with the wild type. ● MC38 non-transfected cells (wild type). ■ MC38 transfected with TYMP.

Discussion

TYMP is the rate-limiting activating enzyme in 5-FU metabolism. Although TYMP is expressed in all types of carcinomas, including colorectal, gastric, breast, lung, and bladder cancers, the degree of expression varies from type to type and from patient to patient (22). As 5-FU is a standard treatment in the chemotherapy of colorectal cancer, we hypothesized that increasing the sensitivity to 5-FU would be very beneficial in the treatment of colorectal cancer. The expression of TYMP may increase sensitivity to 5-FU.

The importance of epigenetic changes in carcinomas including colorectal cancer has recently been reported (12, 23, 24). In particular, many studies have investigated DNA methylation in colorectal cancer (13, 25). About 1.5% of CpG islands in the genome of tumor tissues are reportedly over-methylated, and this methylation is considered tumor-specific (17). In colorectal cancer, the CpG island methylator phenotype (CIMP) has been shown to cause carcinogenesis through the inactivation of tumor suppressor genes such as p16 and MLH1, a mismatch repair gene. CIMP accounts for 15-30% of sporadic colorectal cancers (21). Such DNA methylation is associated with DNA methyltransferase 1 (DNMT-1), which promotes tumorigenesis in the colon by silencing tumor suppressor genes and DNA repair pathways (12).

The present study identified DNMT-1 transcription in all five colon cancer cell lines. In HCT116 cell lines, expression of DYRK2, a gene that suppresses tumor cell growth, has been shown to be regulated by DNMT-1 through methylation of its promotor region (26). In this study, we found that TYMP transcription was enhanced after treatment with 5-aza-2’-deoxycytidine, a methylation inhibitor. To investigate whether increased expression of TYMP enhances the effect of 5-FU in colorectal cancer, we transfected the TYMP gene into HCT116 cell lines. In TYMP-overexpressing HCT116 cell lines, the IC50 of 5-FU was markedly decreased and sensitivity to 5-FU was increased up to 5.5-fold compared with the wild type. Kato et al. reported an 8-fold increase in sensitivity to 5-FU in lung adenocarcinoma (27), and in the present study, sensitivity of colon cancer to 5-FU was also increased.

In this study, 5-aza-2’-deoxycytidine treatment also increased expression of TYMP in HCT116 cell lines but had no effect on the sensitivity to 5-FU.

Treatment of the HCT116 cell line with 5-aza-2’-deoxycytidine resulted in a 5.8-fold increase in TYMP expression compared with the wild type. However, TYMP expression was lower than that in TYMP-transfected HCT116 cell lines, which over-expressed TYMP by 54- to 124-fold compared with the wild type.

Regarding the efficacy of 5-aza-2’-deoxycytidine and 5-FU combination therapy in colorectal cancer, Flis et al. reported synergistic effects in human colorectal cancer cells, such as SW48 and HT-29 (28). However, no reports have described similar effects in other colon cancer cells. In addition, increased expression of TYMP has not been found to be involved in this synergistic effect. On the other hand, Ikehata et al. reported a synergistic effect of 5-aza-2’-deoxycytidine and 5-FU in the SW480 cell line, and conducted experiments using SW48, HT-29, and HCT116 cell lines to examine whether the synergistic effect could be observed in other colon cancer cells. However, in contrast to Flis et al., no synergistic effect was observed in the three colorectal cancer cell lines, including the SW48 cell line and the efficacy of this treatment thus remains unclear (29). The results of our experiments support the results of Ikehata et al.

In our experiment, the IC50 of 5-FU decreased when TYMP was over-expressed, but was unchanged when 5-aza-2’-deoxycytidine was administered, despite a 5.8-fold increase in TYMP expression. This suggests that a 5.8-fold increase in TYMP expression may not be sufficient to affect sensitivity to 5-FU. When mouse colon cancer cells were used, sensitivity to 5-FU was more than 100-fold higher than that in human colon cancer cells. This result differs from our 5.5-fold change in sensitivity for human colorectal cancer and the 8-fold change in sensitivity in human lung adenocarcinoma described by Kato et al. (27). The metabolic pathway of 5-FU and its metabolites differ between mice and humans, so the cytotoxicity of 5-FU may also differ between these two species.

Other factors involved in the sensitivity of cancer to 5-FU include the presence of cancer stem cells, which are resistant to anticancer drugs and radiation due to their high self-replication and multipotency, and the expression of the yes-associated protein (YAP), which is negatively regulated by the tumor suppressor function of the Hippo pathway, which has been linked to 5-FU resistance (30). As a pathway to further increasing sensitivity to 5-FU, future research should examine not only the effects of promoting its metabolism to the active form by metabolic enzymes including TYMP, but also the effects of combinations with other adjuvant therapies.

Acknowledgements

The Authors thank Tsuyoshi Tokita and Mayumi Nomura for the expert technical support.

Footnotes

  • Authors’ Contributions

    M.K. and Y.M. made substantial contributions to the conception and design of the study. M.K. analyzed the data, wrote the manuscript, and supervised all research. E.O., N.A., and K.E. helped design the study and provided administrative support. M.K. and E.O. collected the data. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare they have no conflicts of interest regarding this study.

  • Received November 17, 2021.
  • Revision received December 28, 2021.
  • Accepted December 31, 2021.

References

  1. ↵
    1. Bray F,
    2. Ferlay J,
    3. Soerjomataram I,
    4. Siegel RL,
    5. Torre LA and
    6. Jemal A
    : Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68(6): 394-424, 2018. PMID: 30207593. DOI: 10.3322/caac.21492
    OpenUrlCrossRefPubMed
  2. ↵
    1. Goldstein DA,
    2. Zeichner SB,
    3. Bartnik CM,
    4. Neustadter E and
    5. Flowers CR
    : Metastatic colorectal cancer: a systematic review of the value of current therapies. Clin Colorectal Cancer 15(1): 1-6, 2016. PMID: 26541320. DOI: 10.1016/j.clcc.2015.10.002
    OpenUrlCrossRefPubMed
  3. ↵
    1. Devita VT,
    2. Hellman S,
    3. Rosenberg SA
    1. Cohen AM,
    2. Minsky BD and
    3. Schilsky RL
    : Colon cancer. In: Devita VT, Hellman S, Rosenberg SA (eds.). Cancer: Principles and Practice of Oncology (4th edition). Philadelphia, J. B. Lippincott Co, pp. 929-977, 1993.
  4. ↵
    1. Jakob C,
    2. Aust DE,
    3. Meyer W,
    4. Baretton GB,
    5. Schwabe W,
    6. Häusler P,
    7. Becker H and
    8. Liersch T
    : Thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase expression, and histological tumour regression after 5-FU-based neo-adjuvant chemoradiotherapy in rectal cancer. J Pathol 204(5): 562-568, 2004. PMID: 15538739. DOI: 10.1002/path.1663
    OpenUrlCrossRefPubMed
  5. ↵
    1. Marsh S
    : Thymidylate synthase pharmacogenetics. Invest New Drugs 23(6): 533-537, 2005. PMID: 16267625. DOI: 10.1007/s10637-005-4021-7
    OpenUrlCrossRefPubMed
  6. ↵
    1. Takebayashi Y,
    2. Akiyama S,
    3. Akiba S,
    4. Yamada K,
    5. Miyadera K,
    6. Sumizawa T,
    7. Yamada Y,
    8. Murata F and
    9. Aikou T
    : Clinicopathologic and prognostic significance of an angiogenic factor, thymidine phosphorylase, in human colorectal carcinoma. J Natl Cancer Inst 88(16): 1110-1117, 1996. PMID: 8757190. DOI: 10.1093/jnci/88.16.1110
    OpenUrlCrossRefPubMed
  7. ↵
    1. Mader RM,
    2. Sieder AE,
    3. Braun J,
    4. Rizovski B,
    5. Kalipciyan M,
    6. Mueller MW,
    7. Jakesz R,
    8. Rainer H and
    9. Steger GG
    : Transcription and activity of 5-fluorouracil converting enzymes in fluoropyrimidine resistance in colon cancer in vitro. Biochem Pharmacol 54(11): 1233-1242, 1997. PMID: 9416974. DOI: 10.1016/s0006-2952(97)00330-4
    OpenUrlCrossRefPubMed
  8. ↵
    1. Evrard A,
    2. Cuq P,
    3. Ciccolini J,
    4. Vian L and
    5. Cano JP
    : Increased cytotoxicity and bystander effect of 5-fluorouracil and 5-deoxy-5-fluorouridine in human colorectal cancer cells transfected with thymidine phosphorylase. Br J Cancer 80(11): 1726-1733, 1999. PMID: 10468288. DOI: 10.1038/sj.bjc.6690589
    OpenUrlCrossRefPubMed
  9. ↵
    1. Evrard A,
    2. Cuq P,
    3. Robert B,
    4. Vian L,
    5. Pèlegrin A and
    6. Cano JP
    : Enhancement of 5-fluorouracil cytotoxicity by human thymidine-phosphorylase expression in cancer cells: in vitro and in vivo study. Int J Cancer 80(3): 465-470, 1999. PMID: 9935191. DOI: 10.1002/(sici)1097-0215(19990129)80:3<465::aid-ijc21>3.0.co;2-6
    OpenUrlCrossRefPubMed
  10. ↵
    1. Zhu GH,
    2. Lenzi M and
    3. Schwartz EL
    : The Sp1 transcription factor contributes to the tumor necrosis factor-induced expression of the angiogenic factor thymidine phosphorylase in human colon carcinoma cells. Oncogene 21(55): 8477-8485, 2002. PMID: 12466967. DOI: 10.1038/sj.onc.1206030
    OpenUrlCrossRefPubMed
  11. ↵
    1. Guarcello V,
    2. Blanquicett C,
    3. Naguib FN and
    4. El Kouni MH
    : Suppression of thymidine phosphorylase expression by promoter methylation in human cancer cells lacking enzyme activity. Cancer Chemother Pharmacol 62(1): 85-96, 2008. PMID: 17805539. DOI: 10.1007/s00280-007-0578-5
    OpenUrlCrossRefPubMed
  12. ↵
    1. Jin B and
    2. Robertson KD
    : DNA methyltransferases, DNA damage repair, and cancer. Adv Exp Med Biol 754: 3-29, 2013. PMID: 22956494. DOI: 10.1007/978-1-4419-9967-2_1
    OpenUrlCrossRefPubMed
  13. ↵
    1. Foran E,
    2. Garrity-Park MM,
    3. Mureau C,
    4. Newell J,
    5. Smyrk TC,
    6. Limburg PJ and
    7. Egan LJ
    : Upregulation of DNA methyltransferase-mediated gene silencing, anchorage-independent growth, and migration of colon cancer cells by interleukin-6. Mol Cancer Res 8(4): 471-481, 2010. PMID: 20354000. DOI: 10.1158/1541-7786.MCR-09-0496
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Miremadi A,
    2. Oestergaard MZ,
    3. Pharoah PD and
    4. Caldas C
    : Cancer genetics of epigenetic genes. Hum Mol Genet 16 Spec No 1: R28-R49, 2007. PMID: 17613546. DOI: 10.1093/hmg/ddm021
    OpenUrlCrossRefPubMed
  15. ↵
    1. Robert MF,
    2. Morin S,
    3. Beaulieu N,
    4. Gauthier F,
    5. Chute IC,
    6. Barsalou A and
    7. MacLeod AR
    : DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet 33(1): 61-65, 2003. PMID: 12496760. DOI: 10.1038/ng1068
    OpenUrlCrossRefPubMed
  16. ↵
    1. Chen T,
    2. Hevi S,
    3. Gay F,
    4. Tsujimoto N,
    5. He T,
    6. Zhang B,
    7. Ueda Y and
    8. Li E
    : Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells. Nat Genet 39(3): 391-396, 2007. PMID: 17322882. DOI: 10.1038/ng1982
    OpenUrlCrossRefPubMed
  17. ↵
    1. Ghoshal K,
    2. Datta J,
    3. Majumder S,
    4. Bai S,
    5. Kutay H,
    6. Motiwala T and
    7. Jacob ST
    : 5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. Mol Cell Biol 25(11): 4727-4741, 2005. PMID: 15899874. DOI: 10.1128/MCB.25.11.4727-4741.2005
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Silverman LR,
    2. Demakos EP,
    3. Peterson BL,
    4. Kornblith AB,
    5. Holland JC,
    6. Odchimar-Reissig R,
    7. Stone RM,
    8. Nelson D,
    9. Powell BL,
    10. DeCastro CM,
    11. Ellerton J,
    12. Larson RA,
    13. Schiffer CA and
    14. Holland JF
    : Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol 20(10): 2429-2440, 2002. PMID: 12011120. DOI: 10.1200/JCO.2002.04.117
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Mei M,
    2. Aldoss I,
    3. Marcucci G and
    4. Pullarkat V
    : Hypomethylating agents in combination with venetoclax for acute myeloid leukemia: Update on clinical trial data and practical considerations for use. Am J Hematol 94(3): 358-362, 2019. PMID: 30499168. DOI: 10.1002/ajh.25369
    OpenUrlCrossRefPubMed
  20. ↵
    1. Manome Y,
    2. Abe M,
    3. Hagen MF,
    4. Fine HA and
    5. Kufe DW
    : Enhancer sequences of the DF3 gene regulate expression of the herpes simplex virus thymidine kinase gene and confer sensitivity of human breast cancer cells to ganciclovir. Cancer Res 54(20): 5408-5413, 1994. PMID: 7923173.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Gao D,
    2. Herman JG and
    3. Guo M
    : The clinical value of aberrant epigenetic changes of DNA damage repair genes in human cancer. Oncotarget 7(24): 37331-37346, 2016. PMID: 26967246. DOI: 10.18632/oncotarget.7949
    OpenUrlCrossRefPubMed
  22. ↵
    1. Braybrooke JP,
    2. Propper DJ,
    3. O’Byrne KJ,
    4. Koukourakis MI,
    5. Patterson AV,
    6. Houlbrook S,
    7. Love SD,
    8. Varcoe S,
    9. Taylor M,
    10. Ganesan TS,
    11. Talbot DC and
    12. Harris AL
    : Induction of thymidine phosphorylase as a pharmacodynamic end-point in patients with advanced carcinoma treated with 5-fluorouracil, folinic acid and interferon alpha. Br J Cancer 83(2): 219-224, 2000. PMID: 10901374. DOI: 10.1054/bjoc.2000.1230
    OpenUrlCrossRefPubMed
  23. ↵
    1. Yang H,
    2. Cui W and
    3. Wang L
    : Epigenetic synthetic lethality approaches in cancer therapy. Clin Epigenetics 11(1): 136, 2019. PMID: 31590683. DOI: 10.1186/s13148-019-0734-x
    OpenUrlCrossRefPubMed
  24. ↵
    1. Baylin SB and
    2. Jones PA
    : Epigenetic determinants of cancer. Cold Spring Harb Perspect Biol 8(9): a019505, 2016. PMID: 27194046. DOI: 10.1101/cshperspect.a019505
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Zhu GH,
    2. Lenzi M and
    3. Schwartz EL
    : The Sp1 transcription factor contributes to the tumor necrosis factor-induced expression of the angiogenic factor thymidine phosphorylase in human colon carcinoma cells. Oncogene 21(55): 8477-8485, 2002. PMID: 12466967. DOI: 10.1038/sj.onc.1206030
    OpenUrlCrossRefPubMed
  26. ↵
    1. Kumamoto T,
    2. Yamada K,
    3. Yoshida S,
    4. Aoki K,
    5. Hirooka S,
    6. Eto K,
    7. Yanaga K and
    8. Yoshida K
    : Impairment of DYRK2 by DNMT1 mediated transcription augments carcinogenesis in human colorectal cancer. Int J Oncol 56(6): 1529-1539, 2020. PMID: 32236621. DOI: 10.3892/ijo.2020.5020
    OpenUrlCrossRefPubMed
  27. ↵
    1. Kato Y,
    2. Matsukawa S,
    3. Muraoka R and
    4. Tanigawa N
    : Enhancement of drug sensitivity and a bystander effect in PC-9 cells transfected with a platelet-derived endothelial cell growth factor thymidine phosphorylase cDNA. Br J Cancer 75(4): 506-511, 1997. PMID: 9052401. DOI: 10.1038/bjc.1997.88
    OpenUrlCrossRefPubMed
  28. ↵
    1. Flis S,
    2. Gnyszka A and
    3. Flis K
    : DNA methyltransferase inhibitors improve the effect of chemotherapeutic agents in SW48 and HT-29 colorectal cancer cells. PLoS One 9(3): e92305, 2014. PMID: 24676085. DOI: 10.1371/journal.pone.0092305
    OpenUrlCrossRefPubMed
  29. ↵
    1. Ikehata M,
    2. Ogawa M,
    3. Yamada Y,
    4. Tanaka S,
    5. Ueda K and
    6. Iwakawa S
    : Different effects of epigenetic modifiers on the cytotoxicity induced by 5-fluorouracil, irinotecan or oxaliplatin in colon cancer cells. Biol Pharm Bull 37(1): 67-73, 2014. PMID: 24172061. DOI: 10.1248/bpb.b13-00574
    OpenUrlCrossRefPubMed
  30. ↵
    1. Bauzone M,
    2. Souidi M,
    3. Dessein AF,
    4. Wisztorski M,
    5. Vincent A,
    6. Gimeno JP,
    7. Monté D,
    8. Van Seuningen I,
    9. Gespach C and
    10. Huet G
    : Cross-talk between YAP and RAR-RXR drives expression of stemness genes to promote 5-FU resistance and self-renewal in colorectal cancer cells. Mol Cancer Res 19(4): 612-622, 2021. PMID: 33472949. DOI: 10.1158/1541-7786.MCR-20-0462
    OpenUrlAbstract/FREE Full Text
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Effect of Thymidine Phosphorylase Gene Demethylation on Sensitivity to 5-Fluorouracil in Colorectal Cancer Cells
MUNEYUKI KOYAMA, ERIKA OSADA, NUBUTAKE AKIYAMA, KEN ETO, YOSHINOBU MANOME
Anticancer Research Feb 2022, 42 (2) 837-844; DOI: 10.21873/anticanres.15541
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