Contributed equally
Colorectal cancer (CRC) is one of the most common malignant diseases and frequent cause of cancer deaths in the world. In spite of the significant advances in conventional therapeutic approaches to CRC, most patients ultimately die of their disease. There is a need to develop novel preventive approaches for this malignancy. This study was carried out to investigate the anticancer effect of MHY218, a hydroxamic acid derivative, in HCT116 human colon cancer cells. Treatment of cells with MHY218 resulted in growth inhibition and induction of apoptosis in a concentration-dependent manner. MHY218 induced G2/M phase arrest in the cell cycle progression which was observed by flow cytometry analysis, and a decrease in the protein expression of cyclin B1 and its activating partners Cdc25C and Cdc2. MHY218 also caused an increase in the expression levels of p21
Colorectal cancer (CRC) is a cancer from uncontrolled cell growth in the lining of the large intestine, such as colon and rectum. CRC is the fourth most common cancer in the world, but it is more common in developed countries. CRC is the third most common cancer and the fourth leading cause of cancer-related death in Korea and the incidence of CRC have been increased rapidly over the past few decades (
Progressive inhibition or evasion of apoptosis has been found during the transformation of colorectal epithelium to carcinoma (
Nuclear factor-κB (NF-κB), a transcription factor, controls the expression of genes involved in tumor cell growth, proliferation, angiogenesis, invasion and survival. NF-κB is composed of two subunits, p65 and p50, and is normally sequestered in the cytosol by an inhibitory protein, IκBα. Exposure of cells to a variety of extracellular stimuli leads to the rapid phosphorylation, ubiquitination, and ultimately proteolytic degradation of IκBα, resulting in the release of NF-κB from its inhibitory protein to translocate to the nucleus where it regulates transcription of various genes (
Hydroxamic acids are known as iron chelators and microbial siderophores that show diverse biological activities such as antibacterial, antifungal, antitumor and anti-inflammatory properties (
The simplified code name and structure of MHY218 [N1-hydroxy-N8-(4-phenoxyphenol)octanedianide] used in this study is shown in
HCT116 human colon cancer cells (p53 wild-type) were obtained from American Type Culture Collection (Mansssas, VA, USA) and were cultured in RPMI-1640 (Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone), 2 mM glutamine (Sigma-Aldrich), 100 U/ml penicillin (Hyclone), and 100
Cell viability was determined by MTT assay. For the MTT assay, HCT116 cells were seeded in a 24-well culture plate at a density of 4×104 cells/well, cultured for 24 h in the growth media, and then treated with or without various reagents for the indicated concentrations. The cells were incubated with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) at 37°C for 2 h. The formazan granules generated by the live cells were dissolved in DMSO, and the absorbance at 540 nm was monitored by using a multi-well reader.
Cells were washed with phosphate-buffered saline (PBS) and fixed with 3.7% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min at room temperature. Fixed cells were washed with PBS and stained with 4
Cells were lysed in a buffer, containing 5 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 0.5% Triton X-100, for 30 min on ice. Lysates were vortexed and cleared by centrifugation at 14,000 rpm for 20 min. Fragmented DNA in the supernatant was treated with RNase, followed by proteinase K digestion, phenol:chloroform:isoamyl alcohol mixture (25:24:1) extraction and isopropanol precipitation. DNA was separated through a 1.5% agarose gel, was stained with 0.1
The DNA content was measured following the staining of the cells with propidium iodide. The cells were treated under the appropriate conditions for 24 h, subsequently trypsinized, washed once in cold PBS, and then fixed in 70% ethanol at 4°C overnight. The fixed cells were pelleted and stained in cold propidium iodide (PI, Sigma-Aldrich) solution (50
The cells were harvested and washed with cold PBS. Total cells were lysed with the lysis buffer [40 mM Tris (pH 8.0), 120 mM, NaCl, 0.5% NP-40, 0.1 mM sodium orthovanadate, 2
The cells were washed with cold PBS and resuspended in Buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF and Protease inhibitor cocktail (Sigma-Aldrich)] and incubated on ice. After 15 min, 0.5% Nonidet P (NP)-40 was added to lyse the cells, which were vortexed for 10 sec. Cytosolic extracts were obtained after centrifuging at 12,000 rpm for 60 sec at 4°C. Nuclear extracts were resuspended in Buffer C [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 300 mM NaCl, 0.2 mM EDTA, 20% v/v glycerol, 0.5 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail] and incubated on ice for 20 min with gentle vortexing every 5 min. Nuclear cell extracts were recovered after centrifugation for 10 min at 12,000 rpm at 4°C. Protein concentration was determined by Bradford protein assay reagent (Bio-Rad, Hercules, CA, USA).
The cells were treated with the appropriate conditions, harvested, and washed with cold PBS. Total cells lysates were lysed in lysis buffer [40 mM Tris (pH 8.0), 120 mM, NaCl, 0.5% NP-40, 0.1 mM sodium orthovanadate, 2
For RT-PCR analysis, total RNA was extracted from cultured cells using a TRIzol reagent as described by the manufacturer (Invitrogen), PCR amplification cDNA was prepared using a Bioneer RT/PCR PreMix containing 1 U Taq DNA polymerase, 250
The activity of NF-κB was examined using a luciferase plasmid DNA, pTAL-NF-κB that contains a specific binding sequence for NF-κB (BD Biosciences Clontech, CA, USA). Transfection was carried out using TransIT-LT1 transfection reagent (Promega, Madison, WI, USA). Briefly, HCT116 cells were seeded in 6-well plates. When cultured cells reached ∼50% confluence, cells were treated with 2
Zymography was used to semiquantitatively determine the gelatinolytic activity of matrix metalloproteinase-9 (MMP-9) secreted into culture media. Equal amount of conditioned culture media from equal number cells were applied to SDS-PAGE containing 0.25% gelatin. The gel was incubated with the renaturing buffer (2.5% Triton X-100) with gentle agitation for 30 min at room temperature. The gel was washed once for 10 min, 2 times with distilled water and incubated overnight at 37°C with the Developing buffer [1 M Tris-HCl (pH 7.5), 1 M CaCl2, 10% NaN3, 1 M NaCl]. The gel was stained with 0.5% (w/v) Coomassie Blue R-250 for 30 min at room temperature and then destained with the destaining solution (methanol:acetic acid:distilled water = 50:10:40). Areas of protease activity appeared as clear bands against a dark blue background where the protease had digested the substrate. The product of zymography was visualized with a White light transilluminator (BioSpectrum® Imaging System, Upland, CA, USA).
Results were expressed as the mean ± SD of three separate experiments and analyzed by Student’s t-test. Means were considered significantly different at p<0.05 or p<0.01.
To investigate the effects of MHY218 on the viability of HCT116 cells, the MTT assay was performed. As shown in
To investigate whether the inhibition of HCT116 cell growth was mediated, at least in part, by regulating the cell cycle, flow cytometry analysis of PI-stained HCT116 nuclei was performed. This flow cytometry analyses data showed that MHY218 treatment induced the accumulation of cells in G2/M phase of the cell cycle, with the occurrence of sub-G1 peak, indicating DNA degradation through either necrosis or apoptosis (
To assess the effect of MHY218 on the intracellular protein expression levels of G2/M phase in the cell cycle, we performed western blot analysis. As shown in
To assess whether there are any morphological changes in MHY218-treated HCT116 cells, we examined the cells by phase-contrast light microscopy after 24 h of incubation with or without MHY218. Under the light microscope, untreated HCT116 cells spread regularly in the culture plate and grew to near confluence (
To determine whether the expression levels of apoptosis-related proteins were modulated by MHY218, western blot analysis was performed. The expression level of Bax protein was markedly upregulated, but Bcl-2 was downregulated in a concentration-dependent manner (
NF-κB regulates the expression of a wide variety of genes involved in tumor cell growth and survival. We examined whether MHY218 has the potential to inhibit NF-κB activation in MHY218-treated HCT116 cells. After 24 h exposure with the indicated concentration of MHY218, the levels of nuclear NF-κB p65 and NF-κB p50 were examined, with western blot analysis, because the nuclear translocation of the NF-κB subunits p65 and p50 is essential for NF-κB activation. As shown in
To further investigate the effect of MHY218 on expression level of the COX-2 protein, an NF-κB downstream target gene, in HCT116 cells, the RT-PCR and western blot analyses showed no change in COX-2 mRNA (
This study was conducted to investigate the effects of MHY218 on HCT116 human colon cancer cells. MHY218 induced cell cycle arrest and apoptosis in HCT116 human colon cancer cells. MHY218 also modulated the activity of NF-κB and downregulated the expression levels of COX-2, MMP-9 and 5-LOX protein in HCT116 cells.
The treatment of HCT116 cells with MHY218 resulted in growth inhibition concentration-dependently. Flow cytometric analysis revealed that MHY218 induced G2/M phase arrest. Different classes of cyclins and their CDK control cell cycle progression. G2/M transition provides an effective checkpoint in the cell cycle progression that is regulated by cyclin B1, Cdc2 and Cdc25C (
p21
The treatment of MHY218 also induced apoptosis as demonstrated by the formation of apoptotic bodies and DNA fragmentation. Apoptosis (programmed cell death), is an important process requited for homeostasis (
The activation of effector caspase-3, in response to MHY218 treatment also resulted in cleavage of PARP in HCT116 cells. The ratio between Bcl-2 and Bax has been suggested as a primary event in determining the susceptibility to apoptosis through maintaining the integrity of the mitochondria and inhibiting the activation of caspase cascade (
Certain chemopreventive agents for CRC, such as aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), have been shown to induce apoptosis through a suppression of NF-κB activation (
COX-2 is the inducible form of cyclooxygenase that catalyzes the rate limiting step in prostaglandin synthesis from arachidonic acid and plays an important role in cancer and tumor promotion (
In conclusion, MHY218 suppressed growth of HCT116 cells by causing G2/M cell cycle arrest and apoptosis. These results suggest that MHY218-induced cell cycle arrest and apoptosis are associated with inhibition of NF-κB pathway. Taken together, these results suggested that the novel compound MHY218 may be useful in the chemoprevention and/or treatment of colon cancer.
This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2009-0083538). We thank Aging Tissue Bank for providing research information.
Chemical structures of hydroxamic acid (A) and MHY218 (B). R, an organic residue.
Effect of MHY218 on cell viability of HCT116 cells. One day after seeding, cells were treated with increasing concentration of MHY218 synthetics for 24 h. Proliferation was measured by using the MTT assay. The results are expressed as mean ± SD (n=6) and as a percentage of vehicle-treated control. *p<0.05 and **p<0.01 compared to control and MHY218-treated cells.
Effects of MHY218 on the cell cycle and the levels of cell cycle regulator proteins in HCT116 cells. (A) Exponentially growing cells at 60–70% confluence were treated for 24 h with the indicated concentrations of MHY218. The percentages of cells in sub-G1, G0/G1, S or G2/M phases of cell cycle are indicated within each histogram. (B) HCT116 cells were treated with indicated concentrations of MHY218 for 24 h, collected, lysed and then cellular proteins were separated and immunoblotted. The membranes were probed with indicated antibodies and ECL detection. Representative results from more than three independent experiments are shown. Actin was used as a loading control.
Induction of apoptosis in HCT116 cells by MHY218. (A) HCT116 cells were treated for 24 h with the indicated concentrations of MHY218 and morphological changes were observed by phase contrast microscopy at ×400 magnification. (B) Stained nuclei with fluorescent microscopic dye, Hoechst 33342, were then photographed with a fluorescent microscope using a blue filter at ×400 magnification. Arrows, apoptotic cells. (C) HCT116 cells were treated for 24 h with the indicated concentrations of MHY218. DNA was extracted and analyzed by 1.5% agarose gel electrophoresis in the presence of EtBr. A representative blot is shown from three independent experiments. M, marker. (D) To detect degradation of PARP, HCT116 cells were treated with indicated concentrations of MHY218 for 24 h, collected, lysed and then cellular proteins were separated and immunoblotted. The membranes were probed with PARP (116 kDa). Proteins were visualized using the ECL detection system. Representative results from three independent experiments are shown. Actin was used as a loading control.
Effects of MHY218 on Bax/Bcl-2 ratio and caspase activation in HCT116 cells. (A) HCT116 cells were treated with variable concentrations of MHY218 for 24 h and total cell lysates were prepared and immunoblotted. The membranes were detected with antibodies against Bax and Bcl-2 and ECL detection. Representative results from three independent experiments are shown. Actin was used as a loading control. (B) Bax/Bcl-2 ratio is presented in the bar graphs. (C) HCT116 cells were treated with indicated concentrations of MHY218 for 24 h, collected, lysed and then cellular proteins were separated and immunoblotted. The membranes were probed with pro-caspase-3, -8 and -9. Proteins were visualized using the ECL detection system. Representative results from three independent experiments are shown. Actin was used as a loading control. (D) Cell lysates from HCT116 cells treated with MHY218 for 24 h were assayed for
Effect of MHY218 on NF-κB translocation and TNF-α induced NF-κB activity in HCT116 cells. (A) HCT116 cells were treated with variable concentrations of MHY218 for 24 h and total cell lysates were prepared and immunoblotted. The membranes were detected with antibodies against p50 and p65 in the nuclear extract and ECL detection. Representative results from three independent experiments are shown. Histone H1 was used as a loading control. NE, nucleus extract. (B) At 24 h post-transfection with NF-κB luciferase plasmid, HCT116 cells were treated with variable concentrations of MHY218 and incubated for 1 h and then TNF-α (10 ng/ml) was treated and incubated for 6 h. Luciferase activity was measured by a luminometer. The obtained raw luciferase activity was normalized by protein concentration in each well. Results are expressed as percentages of the vehicle-treated control ± SD of triplicate experiments. The significance was determined by Student’s t-test (*p<0.05 and **p<0.01 compared with TNF-α treated cells).
Effect of MHY218 on the levels of COX-2, MMP-9 and 5-LOX in HCT116 cells. (A) After 24 h incubation with MHY218, total RNAs were isolated and reverse-transcribed. The resulting cDNAs were subjected to PCR with COX-2 primers and the reaction products were subjected to electrophoresis in a 1% agarose gel and visualized by EtBr staining. GAPDH was used as a loading control. (B) The cells were treated with variable concentrations of MHY218 for 24 h. Total cell lysates were prepared and subjected to 10% SDS-PAGE and electrophoretically transferred to PVDF membranes. Western blot analysis was conducted with COX-2 antibody and ECL kits. (C) Conditioned medium was collected from cultures following 24 h and analyzed by gelatin zymography (zymo). Western blot analysis was conducted with MMP-9 antibody and ECL kits. (D) In a parallel experiment, western blot analysis was conducted with 5-LOX antibody and ECL kits. Representative results from three independent experiments are shown. Actin was used as a loading control.
Primer sequences for RT-PCR.
Gene | Sequence of primers (5′→3′) | |
---|---|---|
Cox-2 | Sense | AGA TCA TCT CTG CCT GAG TAT CTT |
Antisense | TTC AAA TGA GAT TGT GGG AAA ATT GCT | |
GAPDH | Sense | CGG AGT CAA CGG ATT TGG TCG TAT |
Antisense | AGC CTT CTC CAT GGT GGT GAA GAC |