Skip to main content
Genomics Data logoLink to Genomics Data
. 2015 Jun 12;5:189–191. doi: 10.1016/j.gdata.2015.05.043

Epigenetic regulation of apoptosis and cell cycle regulatory genes in human colon carcinoma cells

Amy V Paschall 1, Kebin Liu 1,
PMCID: PMC4542010  NIHMSID: NIHMS700051  PMID: 26309812

Abstract

5-Fluorouracil (5-FU) is the standard chemotherapy for certain high risk stage 2 and all stage 3 and 4 human colorectal cancer patients. However, patients often develop chemoresistance to 5-FU. We have identified verticillin A from Verticillium-infected wild mushrooms as a potent anti-cancer agent that effectively suppresses 5-FU-resistant human colon cancer cells. Interestingly, a sublethal dose of verticillin A also acts as a potent sensitizer that overcomes human colon carcinoma cell resistance to FasL- and TRAIL-induced apoptosis. To identify verticillin A-regulated genes, we performed a genome-wide gene expression analysis and identified 1287 genes whose expression levels were either up- or down-regulated 1.5 fold. Forty-six of these genes have known function in regulation of apoptosis, and ninety genes have function in cell cycle regulation. Our recent study has identified verticillin A as a selective histone methyltransferase inhibitor. These identified genes are thus potential molecular targets for epigenetic-based therapy to overcome human colon cancer 5-FU resistance. The entire dataset is deposited in the NIH GEO database; accession number GSE51262.

Keywords: Cell cycle arrest, Histone methyltransferase, Colon cancer, Apoptosis, H3K9 methylation


Specifications
Organism/cell line/tissue Cell line: Human colon carcinoma cell line LS411N-5FU-R. LS411N-5FU-R cell line was generated by culturing LS411N cells in the presence of 5-FU. LS411N cell line was established from Dukes' type B colon carcinoma tissue of a 37 year old patient. LS411N-5FU-R cells grow in the presence of 5-FU as high as 2.0 mg/ml in the culture medium.
Sex Male colon cancer patient.
Sequencer or array type Affymetrix Human Gene 2.0 ST Array
Data format Raw and analyzed
Experimental factors Cells were cultured in the absence (control) or presence of 50 nM verticillin A for 3 days. The gene expression level of treated cells was compared to the untreated cells.
Experimental features
Consent Exempted human colon cancer cell lines.
Sample source location LS411N cell line was obtained from ATCC.

1. Direct link to deposited data

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51262.

2. Experimental design, materials and methods

The human colon carcinoma cell line LS411N was obtained from ATCC (Manassas, VA). Verticillin A was purified either from mushroom or fungus fermentation as previously described [1], [2]. To identify verticillin A-regulated genes, the 5-FU-resistant LS411N-5FU-R cells were cultured in the absence (control) or presence of verticillin A (50 nM) for three days. Total RNA was then isolated from cells using TRIzol according to the manufacturer's instructions (Life Technologies) as previously described [3], [4]. Biotinylated cDNAs were prepared according to the standard Ambion and Affymetrix protocol from 250 ng total RNA (The Ambion WT Expression Kit and GeneChip Terminal Labeling Kit, Affymetrix). Following labeling, cDNAs were hybridized for 16 h at 45 °C on Affymetrix Human Gene 2.0 ST Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. GeneChips were scanned using the Affymetrix GeneChip Scanner 3000. Intensities of arrays have been quantile-normalized using Partek Genomic Suite (v6.6). Differential expressions were calculated using ANOVA of Partek package.

The entire data set was analyzed for differentially expressed genes. Using a 1.5 fold change and p value less than 0.01, we have identified 1287 genes whose expression levels were either up- or down-regulated 1.5 fold. Among these genes, forty-six have known function in regulation of apoptosis (Table 1), and ninety have function in cell cycle regulation (Table 2). Consistent with the altered gene expression in cell cycle regulation, functional analysis revealed that a sublethal dose of verticillin A induces cell cycle arrest at G2 phase (Fig. 1A and B). Comparison of effects of verticillin A on cell cycle arrest between the parent LS411N and the LS411N-5FU-R cells indicate that development of resistance to 5-FU does not alter human colon carcinoma cell sensitivity to verticillin A in induction of cell cycle arrest (Fig. 1B). Induction of cell cycle arrest is a mechanism by which chemotherapeutic agents suppress tumor development. Our observation that verticillin A induces cell cycle arrest in 5-FU-resistant human colon carcinoma cells as effectively as in the parent cells (Fig. 1B) suggests that verticillin A is potentially an effective agent for 5-FU-resistant cancer suppression [5], [6]. In summary, our data indicate that verticillin A-regulated genes, including these involved in apoptosis and cell cycle regulation, are potential molecular targets for epigenetic-based therapy [7], [8] to overcome human colon cancer 5-FU resistance.

Table 1.

Apoptosis regulatory genes.

Gene symbol RefSeq p value Fold change
SORD ENST00000267814 0.00061649 − 1.87556
ZMAT3 ENST00000311417 0.000557593 1.57282
ING3 ENST00000315870 0.000102115 − 1.51561
CLU ENST00000316403 0.00658658 1.88366
ZADH2 ENST00000322342 0.00116724 − 1.45788
MALT1 ENST00000348428 8.70E− 05 − 1.59075
DNAJB4 ENST00000370763 0.000141036 1.57462
GADD45A ENST00000370986 0.00036571 − 1.62913
SMOX ENST00000379460 0.000453598 − 1.7213
FAS NM_000043 0.00562506 1.64701
PMP22 NM_000304 0.0117398 1.80063
TP53 NM_000546 3.20E− 06 − 1.71073
OR51B5 NM_001005567 5.42E− 05 1.47933
MYBL1 NM_001080416 0.000313286 1.60353
CRYZ NM_001130042 1.48E− 06 1.48424
JDP2 NM_001135049 0.00306543 − 1.60104
BIRC3 NM_001165 1.43E− 05 2.0818
EMP1 NM_001423 8.59E− 08 3.35724
EMP3 NM_001425 0.0010598 1.45705
CNTN1 NM_001843 0.0074873 − 1.56199
EDNRA NM_001957 0.0279268 − 1.6314
FKBP4 NM_002014 9.38E− 05 1.46074
FYN NM_002037 0.000281551 − 1.71704
LIF NM_002309 0.0155574 − 1.45691
MAGEB2 NM_002364 0.00564587 1.57381
MDM2 NM_002392 4.74E− 05 1.96004
MYBL2 NM_002466 0.000287216 1.46171
ROBO1 NM_002941 0.000219031 − 1.87573
TXN NM_003329 2.62E− 06 1.47208
CBX4 NM_003655 0.00057116 − 1.65897
BAG3 NM_004281 0.000615002 1.68888
CEBPB NM_005194 3.37E− 05 − 1.55513
HDAC5 NM_005474 0.00951344 − 1.57188
DNAJB1 NM_006145 0.000604263 1.46166
FKBP9 NM_007270 0.000399435 − 1.49977
SHC2 NM_012435 0.015437 − 1.62921
CAPN6 NM_014289 0.000292268 − 1.62879
KRCC1 NM_016618 0.000625115 − 1.51118
SHC3 NM_016848 9.40E− 05 − 1.79462
FKBP10 NM_021939 0.00031242 − 1.59534
TMEM47 NM_031442 1.92E− 06 − 2.58264
IL20RB NM_144717 3.47E− 06 − 2.65592
UNC5B NM_170744 0.00192834 − 1.52166
IFNE NM_176891 0.00165318 − 2.26639
RASSF3 NM_178169 0.000187772 − 1.47727
TYMS NM_001071 0.000178443 1.46165

Table 2.

Cell cycle regulatory genes.

Gene symbol RefSeq p value Fold change
VRK1 ENST00000216639 0.00109304 1.48503
GINS2 ENST00000253462 0.000631431 1.55066
POLA2 ENST00000265465 0.000337543 1.50477
SKP2 ENST00000274255 0.000407268 1.4572
PLK2 ENST00000274289 0.000151913 1.72602
DUSP6 ENST00000279488 7.20E− 06 − 2.10422
ACTR1B ENST00000289228 0.00108964 − 1.51729
FGF19 ENST00000294312 5.05E− 05 − 2.13694
CDC25A ENST00000302506 0.000648382 1.56293
ESCO2 ENST00000305188 0.00119695 1.53802
PSMD2 ENST00000310118 0.000687665 1.49718
DSCC1 ENST00000313655 0.00380772 1.49838
ING3 ENST00000315870 0.000102115 − 1.51561
TUBB6 ENST00000317702 0.000718394 1.62504
NR2F1 ENST00000327111 0.000840833 − 1.73635
KLHDC1 ENST00000359332 0.00198403 − 1.85646
S100A10 ENST00000368811 0.000119039 1.45917
GADD45A ENST00000370986 0.00036571 − 1.62913
JUN ENST00000371222 4.77E− 05 − 1.92902
PLK3 ENST00000372201 0.000951583 1.49099
BICC1 ENST00000373886 0.000196311 − 1.5164
TTK ENST00000509894 0.000571488 1.49117
PMP22 NM_000304 0.0117398 1.80063
TP53 NM_000546 3.20E− 06 − 1.71073
HNRNPA1L2 NM_001011724 0.0265379 − 1.55219
VEGFA NM_001025366 7.18E− 06 − 2.08509
INCENP NM_001040694 5.34E− 06 1.70379
MYBL1 NM_001080416 0.000313286 1.60353
PABPC1L NM_001124756 0.000496661 − 1.98338
JDP2 NM_001135049 0.00306543 − 1.60104
FBXL17 NM_001163315 0.00683003 − 1.45608
ELAVL2 NM_001171197 0.0107444 − 1.83695
A1CF NM_001198819 0.00310545 − 1.53498
CDKN1A NM_001220778 0.0376178 1.47916
CCNE1 NM_001238 1.80E− 05 1.89998
DTNA NM_001390 0.000846729 − 1.60955
EMP1 NM_001423 8.59E− 08 3.35724
EMP3 NM_001425 0.0010598 1.45705
CNTN1 NM_001843 0.0074873 − 1.56199
EGR1 NM_001964 4.11E− 05 − 2.05333
FKBP4 NM_002014 9.38E− 05 1.46074
MYBL2 NM_002466 0.000287216 1.46171
PCNA NM_002592 7.01E− 06 1.80038
MAPK4 NM_002747 0.00147303 − 1.53275
ROBO1 NM_002941 0.000219031 − 1.87573
TXN NM_003329 2.62E− 06 1.47208
CDKL2 NM_003948 0.0503581 1.57628
ORC1 NM_004153 1.65E− 05 1.93504
BUB1 NM_004336 4.67E− 07 1.70252
ETV5 NM_004454 4.20E− 05 − 1.65774
RPS6KA5 NM_004755 9.56E− 06 − 2.43124
ETV1 NM_004956 2.61E− 05 − 1.74844
E2F1 NM_005225 0.000685259 1.62065
JUND NM_005354 0.000591427 − 1.61353
SKIL NM_005414 6.76E− 05 − 1.71684
HDAC5 NM_005474 0.00951344 − 1.57188
UST NM_005715 0.00693733 − 1.47903
KIF20A NM_005733 0.000183352 1.66921
RASGRP1 NM_005739 0.00120733 − 1.49275
SFN NM_006142 2.54E− 05 2.33324
POLH NM_006502 2.21E− 05 2.1575
BTG2 NM_006763 4.54E− 05 1.54606
ZFP36L2 NM_006887 0.00908993 − 1.48935
RAPGEF4 NM_007023 0.010428 − 1.48297
POLI NM_007195 0.00109012 − 1.57536
FKBP9 NM_007270 0.000399435 − 1.49977
HS2ST1 NM_012262 2.62E− 05 − 1.49078
ESPL1 NM_012291 0.00490513 1.47314
SESN1 NM_014454 0.0014303 1.5657
RPS6KA6 NM_014496 0.00411955 − 1.6027
HUNK NM_014586 0.00831223 − 1.45803
MYO1D NM_015194 0.00043732 − 1.62238
TUBE1 NM_016262 0.000107931 − 2.14386
FKBP10 NM_021939 0.00031242 − 1.59534
MOB3B NM_024761 0.0171544 − 1.48453
DUSP16 NM_030640 0.00020683 − 1.46829
SESN2 NM_031459 2.47E− 05 − 2.27913
CCNB1 NM_031966 0.000656847 1.54547
GINS4 NM_032336 0.000779725 1.67209
BRSK1 NM_032430 0.00851809 − 1.63611
CORO6 NM_032854 0.000911746 1.49868
CDKN2B NM_078487 6.95E− 05 − 2.14147
MSI2 NM_138962 0.00017894 − 1.47847
SLFN5 NM_144975 8.48E− 06 − 2.23313
DBF4B NM_145663 0.000273346 1.55948
SIK1 NM_173354 0.00486314 1.49766
PTPDC1 NM_177995 2.21E− 05 − 2.35451
WDR49 NM_178824 0.0202432 1.5734
AURKA NM_198433 0.00160995 1.62216
POLQ NM_199420 3.09E− 05 1.62956

Fig. 1.

Fig. 1

Verticillin A mediates cell-cycle progression in 5-FU-resistant human colon carcinoma cells. A. LS411N-5FU-R cells were cultured in the presence of Verticillin A at the indicated concentrations for 24 h. Cells were then stained with PI, and analyzed by flow cytometry. A. Cells as shown in A were quantified for percentages of cells in various phases the cell cycle (as shown). Columns, mean; bars, SD.

Acknowledgments

We thank Drs. Chang-Sheng Chang and Jeong-Hyeon Choi for their excellent technical assistance in the DNA microarray data analysis, and grant support from National Institutes of Health (CA182518 and CA185909 and VA Merit Review Award BX001962.

References

  • 1.Liu F., Liu Q., Yang D., Bollag W.B., Robertson K., Wu P. Verticillin a overcomes apoptosis resistance in human colon carcinoma through DNA methylation-dependent upregulation of BNIP3. Cancer Res. 2011;71:6807–6816. doi: 10.1158/0008-5472.CAN-11-1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Figueroa M., Graf T.N., Ayers S., Adcock A.F., Kroll D.J., Yang J. Cytotoxic epipolythiodioxopiperazine alkaloids from filamentous fungi of the Bionectriaceae. J. Antibiot. 2012;65:559–564. doi: 10.1038/ja.2012.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Paschall A.V., Zhang R., Qi C.F., Bardhan K., Peng L., Lu G. IFN regulatory factor 8 represses GM-CSF expression in T cells to affect myeloid cell lineage differentiation. J. Immunol. 2015;194:2369–2379. doi: 10.4049/jimmunol.1402412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Paschall A.V., Zimmerman M.A., Torres C.M., Yang D., Chen M.R., Li X. Ceramide targets xIAP and cIAP1 to sensitize metastatic colon and breast cancer cells to apoptosis induction to suppress tumor progression. BMC Cancer. 2014;14:24. doi: 10.1186/1471-2407-14-24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Klajic J., Busato F., Edvardsen H., Touleimat N., Fleischer T., Bukholm I. DNA methylation status of key cell-cycle regulators such as CDKNA2/p16 and CCNA1 correlates with treatment response to doxorubicin and 5-fluorouracil in locally advanced breast tumors. Clin. Cancer Res. 2014;20:6357–6366. doi: 10.1158/1078-0432.CCR-14-0297. [DOI] [PubMed] [Google Scholar]
  • 6.Montagnoli A., Moll J., Colotta F. Targeting cell division cycle 7 kinase: a new approach for cancer therapy. Clin. Cancer Res. 2010;16:4503–4508. doi: 10.1158/1078-0432.CCR-10-0185. [DOI] [PubMed] [Google Scholar]
  • 7.Greer E.L., Shi Y. Histone methylation: a dynamic mark in health, disease and inheritance. Nat. Rev. Genet. 2012;13:343–357. doi: 10.1038/nrg3173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Crea F., Nobili S., Paolicchi E., Perrone G., Napoli C., Landini I. Epigenetics and chemoresistance in colorectal cancer: an opportunity for treatment tailoring and novel therapeutic strategies. Drug Resist. Updat. 2011;14:280–296. doi: 10.1016/j.drup.2011.08.001. [DOI] [PubMed] [Google Scholar]

Articles from Genomics Data are provided here courtesy of Elsevier

RESOURCES