HCT116 cells are more sensitive to FCdR than SAHA and 5-azaC

Several small molecules inhibiting epigenetic processes have been developed with an ability to inhibit cancer cells. SAHA and 5-azaC are two such small molecule inhibitors that have been approved by FDA. We tested and compared the cyto-toxicity of FCdR with SAHA and 5-azaC on HCT116 cells, as well as one novel identified H3K9 methylation inhibitor BIX01294. We found that all the drugs tested repressed the proliferation of HCT116, however, their IC50 differed considerably (Figure ​(Figure1B).1B). IC50 of FCdR was lowest between 0.025-0.05 μM, whereas for 5-azaC, BIX01294 and SAHA, it was 5 μM, 1.5 μM and 0.25 μM respectively. These findings suggested that HCT116 is much more sensitive to FCdR compared to SAHA and 5-azaC, which may prove to be of value in a clinical study.

FCdR induces G2/M arrest in HCT116 cell

Next we sought to study the effect of FCdR on cell cycle in HCT116 cells. Since drugs targeting DNA methylation are known to induce cell cycle arrest or apoptosis, we first performed cell cycle analysis by PI (Propidium Iodide) staining and analyzed cells with flow cytometry. Cells treated with 0.05 μM FCdR for 48 h showed upto 24% of cells in G2/M phase (Figure ​(Figure2A),2A), whereas treatment with 0.5 μM FCdR increased the percentage of cells in the G2M phase to 75% (Figure ​(Figure2A).2A). These results suggest that FCdR induces G2/M arrest in HCT116. To further substantiate our conclusion, we analysed the expression of cyclins by western blot (Figure ​(Figure2B).2B). Treatment with 0.5 μM FCdR for 48 h, resulted in significant increase in the total levels of cyclin B1.

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Figure 2

FCdR arrested HCT116 cells at G2/M phase.A. HCT116 cells were treated with 0.05 μM or 0.5 μM FCdR for 48 h and cell cycle was analyzed by flow cytometry. Compared with control, percentage of cells at G2/M phase increased significantly after FCdR treatment. B. HCT116 cells were treated with indicated amount of FCdR for 48 h and the cyclins checked by western blot. CyclinB1, which accumulates at G2/M phase, significantly increased after FCdR treatment. C. HCT116 p53+/+ and p53^−/− cells were treated with 0.5 μM FCdR or 375 μM 5-Fu for 48 h and apoptosis assayed by PARP and CASP3 western blot. 5-Fu treatment caused cleavage of PARP and CASP3, but not FCdR.

Persistent cell cycle arrest leads to induction of apoptosis. However, HCT116 cells treated with FCdR at concentrations of up to 0.5 μM for 48 h, did not show any obvious apoptotic phenotype as observed by light microscopy (data not shown). Flow cytometry analysis of these cells also did not show any obvious sub-G1 peak, which is a characteristic of apoptotic cells (Figure ​(Figure2A).2A). We further examined the formation of cleaved CASP3 and cleaved PARP, which are hallmarks of apoptosis. We did not detect any cleaved CASP3 or cleaved PARP by western blot whereas 5FU treatment, which induces apoptosis in HCT116 cells, resulted in cleavage of CASP3 and PARP (Figure ​(Figure2C).2C). These observations suggested that at the given concentration FCdR solely induces G2/M arrest in HCT116 and not apoptosis.

FCdR alters gene expression pattern by elevating transcription level

DNA methylation at gene promoters represses transcriptional activation and its inhibitors up regulate expression of genes. To investigate the mechanism/s involved in FCdR-induced G2/M arrest, we performed genome wide RNA sequencing (RNA-seq) of HCT116 cells treated with or without FCdR for 24 h and analyzed the alterations in gene expression. We also performed a similar experiment with 5-Fluorouracil, a widely used chemotherapeutic drug which induces DNA damage and cell cycle arrest, and used the RNA-seq profile for comparison with FCdR dataset. To reduce background signals we only considered genes, expressions of which were changed by at least two fold. We found that FCdR treatment lead to alteration in expression of a total of 1165 genes, out of which 757 were up regulated and 408 were down regulated (Figure ​(Figure3A3A and Additional file 1: Table S1). A higher number of up-regulated genes in FCdR treated cells is expected as FCdR is known to inhibit DNA methylation. In comparison, 5-Fu treatment resulted in change in expression of 3296 genes out of which, 2/3 were down regulated (Figure ​(Figure3A3A and Additional file 2: Table S2).

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Figure 3

FCdR induced alteration of global gene expression pattern. 0.5 μM FCdR or 375 μM 5-Fu were used to treat HCT116 cells for 24 h. Total RNA was used for gene expression pattern analysis by high throughput sequencing. A. The number of differentially expressed genes was calculated. FCdR caused changes in expression of 1165 genes, out of which 757 were up-regulated and 408 were down-regulated. 5-Fu caused changes in expression of 3295 genes, out of which 1103 were up-regulated, and 2193 were down-regulated. FCdR-treated cells have a much higher percentage of up-regulated genes. B. Differentially expressed genes involved in top 6 pathways (p53 signaling pathway, colorectal cancer, nucleotide excision repair, DNA replication, cell cycle, pathways in cancer) were subjected to cluster analysis. C. The change in expression of 45 genes was confirmed by real time PCR. (* p value < 0.05; ** p value < 0.01).

Next we looked at alterations of signaling pathways, and found many of them to be altered in cells treated with FCdR (Table ​(Table1).1). The pathways, which were significantly altered were also related with cancer, including p53 signaling, DNA repair, DNA replication, cell cycle (Table ​(Table1).1). We validated the altered expression of 45 genes involved in these pathways by reverse transcription followed by quantitative PCR (Figure ​(Figure3C).3C). We found that more than 90% of these genes were similarly altered as in our high throughput sequencing dataset.

We performed cluster analysis of differentially expressed genes involved in pathways, which were altered the most, including- p53 signaling pathway, colorectal cancer, nucleotide excision repair, DNA replication, cell cycle, pathways in cancer. We observed that both FCdR and 5-Fu treatment lead to similar changes in genes involved in DNA replication, DNA damage repair and p53 pathway (Figure ​(Figure3B).3B). Expression of a number of genes involved in DNA replication and repair were reduced in cells with both drugs. p53 target genes such as MDM2, CDKN1A/p21, SFN/14-3-3σ, and SERPINE1/PAI were also found to be activated in both samples, though in comparison to FCdR, 5-Fu treatment resulted in stronger up-regulation of these p53 targets (Figures ​(Figures3B3B and ​and4A).4A). Among the genes up-regulated by FCdR, we also found several well-known proto-oncogenes, such as HRAS, CMYC and ERBB2 (Figure ​(Figure3B3B and C). Increased expression of these genes might have implications in cancer treatment. Interestingly, we also observed that the receptor of TRAIL, TRAILR2, and the two decoy receptors, TRAILR3 and TRAILR4, were overexpressed (Figure ​(Figure3C).3C). TRAIL is a potential drugable protein which is known to induce apoptosis in many cancer cell lines but not in normal cells. It will be interesting to look at the effect of cancer treatment combining FCdR with TRAIL.

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Figure 4

FCdR treatment activates p53 pathway.A. Differentially expressed genes of p53 signaling pathway found in high throughput RNA sequencing were confirmed by real time PCR. B. HCT116 wild type cells were treated with indicated amounts of FCdR for 24 h and p53 was analyzed by western blot. C. HCT116 p53^+/+ and p53^−/− cells were verified by western blot. D. p53^−/− cells were treated with 0.5 μM FCdR for 24 h and expression of indicated p53 downstream genes assayed by real time PCR. These genes were not activated with p53 deficiency. E. p53^−/− cells were treated with FCdR for 48 h and cell cycle was analyzed. FCdR was still able to induce G2/M arrest without p53. (* p value < 0.05; ** p value < 0.01).

FCdR treatment activated p53 signaling pathway in HCT116

Our gene expression analysis of FCdR treated HCT116 cells suggest that FCdR activates p53 signaling pathway, which is the most important pathway inhibiting tumorigenesis. We further tested and confirmed the activation of p53 pathway by RTPCR analysis of mRNA levels of p53 target genes. We tested 11 p53 downstream genes and found that all were significantly elevated in expression (Figure ​(Figure4A).4A). As the activation of p53 involves stabilization of p53 protein, we analysed and found that the amount of p53 protein significantly increased after FCdR treatment, combined with the discovery that multiple p53 target genes increased their expression, suggesting that FCdR probably activates p53 pathway (Figure ​(Figure4B4B).

In order to investigate if p53 signaling pathway is responsible for cell cycle arrest caused by FCdR treatment, we performed FCdR treatment in a p53 kncokout HCT116 cell line. We first verified the absence of p53 protein in these cells by western blot (Figure ​(Figure4C).4C). These cells, when treated with FCdR at a concentration of 0.5 μM, did not activate p53 target genes, including GADD45A, GADD45B and 14-3-3σ (Figure ​(Figure4D).4D). To our surprise, FCdR was still able to induce G2/M arrest in these cells in the absence of p53 (Figure ​(Figure4E).4E). Compared with parental HCT116 cells (Figure ​(Figure2A),2A), these cells showed G2/M arrest and similar distribution profile of other phases of cell cycle Also, cyclin B1 accumulation was comparable to parental cells (Figure ​(Figure5A).5A). Taken together, above observations suggest that the G2M arrest observed in FCdR treated cells is not a consequence of activation of the p53 pathway.

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Figure 5

DNA damage response is responsible for FCdR-induced cell cycle arrest.A. Cells were treated with indicated amount of chemicals for 12 h and damaged DNA was detected by alkaline comet assay. B. Olive tail moment in the previous assay was calculated according to manufacturer’s method and the statistic results were shown. C. HCT116 p53^+/+ and p53^−/− cells were treated with FCdR. Markers for DNA damage response (pH2AX, pATM and pCHK1) and cell cycle (Cyclin B1, Cyclin D1 and Cyclin E1) were analyzed by western blot. Histone H3 and β-ACTIN were used as loading controls. D. HCT116 p53^+/+ and p53^−/− cells were treated with FCdR for 8 h and immunofluorescence staining was performed to show FCdR induced H2AX phosphorylation in both cell lines. E. Three drugs were used to treat HCT116 for 8 h and DNA damage responses were investigated by western blotting. FCdR and 5-azaC were able to induce phosphorylation of H2AX, ATM and CHK1, but not SAHA. H3 was used as control. F. The inhibitory effect of LY294002 to FCdR induced DNA damage response was assayed. G. Addition of 50 μM LY294002 restored the G2/M arrest induced by 0.1 μM FCdR. (* p value < 0.05).

MTT assay

Cells were split at 1×10³ cells per well in 96-well plate. After 24 h cells were treated with drugs and cultured for 72 h. 25 μg MTT was then added to each well and cells incubated for 4 h at 37°C. The medium with the formazan sediment was dissolved in 50% DMF and 30% SDS (pH4.7). The absorption was read at 570nM. P value was calculated by t test.

Cell cycle assay

Cells were split at 2-3×10⁵cells per well in 6-well plates. After 12-14 h cells were treated with drugs and cultured for 48 h. Cells were harvested by 0.05% Trypsin-EDTA digestion and centrifuged after PBS wash. Cells were fixed overnight with 70% ethanol. Flow cytometry analysis was performed after PI staining (50ug/mL) and RNase digestion (100ug/mL) at 37°C for 30 min.

Western blot

Approximately 2×10⁶ Cells were lyzed in 200ul 1×SDS loading buffer () and boiled at 95°C for 10 min. 5-10 μl sample was loaded to SDS PAGE gel for each lane and the separated proteins were transferred to nitrocellular membrane. The membrane was blocked in 5% milk and hybridized with indicated first antibody over night and second antibody for 1 h before developing.

Immuno-fluorescence staining

Cells were cultured on cover slips, washed twice with PBS and then fixed with chilled methanol. Cells were then washed three times with PBS and blocked in PBS with 1% BSA for 10 min. Cells were incubated with primary and secondary antibodies for one hour, respectively. Samples were mounted with prolong anti-fade kit (Invitrogen) and observed on a fluorescent microscope.

Reverse transcription and quantitative PCR

Cells were scraped and collected by centrifugation. Total RNA was extracted with RNA extraction kit (Yuanpinghao) according to manufacturer’s protocol. Approximately 1ug of total RNA was used for reverse transcription with a first strand cDNA synthesis kit (Toyobo). The amount of mRNA was assayed by quantitative PCR. β-actin was used to normalize the amount of each sample. Assays were repeated at least three times. Data shown were average values ± SD of one representative experiment. P value was calculated by t test.

Alkaline comet assay

OxiSelect Comet assay kit was purchased from Cell Biolabs and comet assay was performed according to the manufacturer’s protocol. Briefly, cells were split at 2-3×10⁵ cells per well in 6-well plate and cultured for 12 h. Drugs were added to the medium and cells were treated for indicated time. Individual cells are mixed with molten agarose and then treated with lysis buffer and alkaline solution. Following electrophoresis, the samples were dried and stained with a DNA dye, then observed with fluorescent microscope. The tail length of each cell was measured manually and the tail DNA percentage was quantified by using Quantity One software (Bio-rad). Then the Olive tail moment was calculated according to the following formula: Tail DNA% X Tail moment length, as suggested by provided manual. Data shown were average values ± SD. P value was calculated by t test.

We thank Dr. Man Mohan in Stowers Institute for Medical Research for editing the manuscript.

This work was supported by grants from the National Basic Research Program of China (973 Program, 2011CB504206, 2012CB518700), the National Natural Science Foundation of China to Min Wu (30971502 and 91019013) and Lianyun Li (30971501, 31200653 and 30921001), Program for New Century Excellent Talents in University to Min Wu (NCET-11-0410).