Abstract
The aim of this study was to investigate the anti-tumor activity of KBH-A42, a novel synthetic histone deacetylase (HDAC) inhibitor. KBH-A42 was shown to significantly suppress the proliferation of all 14 human cancer cell lines tested. Among these cell lines, the human leukemia cell line K562 was the most sensitive, whereas the UM-UC-3 bladder cancer cells were the least sensitive. Additionally, in a human tumor xenograft model using Balb/c nude mice, KBH-A42 was shown to significantly inhibit the growth of K562 tumors, although it only slightly inhibited the growth of UM-UC-3 tumors. The results of flow cytometry analysis and caspase 3/7 activation assays showed that the growth inhibition of K562 cells by KBH-A42 was mediated, at least in part, by the induction of apoptosis, but its growth inhibitory effects on UM-UC-3 cells were not mediated by apoptotic induction. In an effort to gain insight into the mechanism by which KBH-A42 inhibits the growth of cancer cells, a microarray analysis was conducted. Four genes were selected from the genes that were down-regulated or up-regulated by KBH-A42 and confirmed via reverse transcription-polymerase chain reaction as follows: Harakiri (HRK), tumor necrosis factor receptor superfamily, member 10b (TNFRSF10B), PYD and CARD domain containing protein gene (PYCARD) and tumor necrosis factor receptor superfamily, member 8 (TNFRSF8). Collectively, the in vitro and in vivo results suggested that KBH-A42 exhibits anti-cancer activity, but various types of cells may be regulated differentially by KBH-A42.
Keywords: histone deacetylase inhibitor, cell cycle arrest, apoptosis, microarray
Introduction
The anti-cancer potential of histone deacetylase (HDAC) inhibitors has been broadly acknowledged (1,2). These compounds block HDAC activity, resulting in profound increases in the acetylation state of the chromatin, which in turn affects chromatin structure and the regulation of gene expression (3). Previous studies using cDNA arrays showed that the expression of as many as 7–10% of genes were altered in cell lines of leukemia, multiple myeloma, and carcinomas of the colon, bladder, kidney, prostate and breast, when cultured for up to 48 h with butyrate, TSA, MS-275, vorinostat or FK228 (depsipeptide), using a 2-fold change as the cut-off value (4–8). The patterns of alterations of gene expression are similar for different HDAC inhibitors, but definite differences were clearly induced by different agents in various transformed cell types (9). The development of HDAC inhibitor resistance is a major concern, as is the case with any novel anti-tumor therapy. In preclinical studies, resistance to HDAC inhibitor-induced transformed cell death was noted in human bladder carcinoma cells (T24) and prostate cancer cells (PC3) (10,11). However, the mechanisms underlying HDAC inhibitor resistance remain to be clarified.
In this study, we evaluated the functional effects of KBH-A42 on the growth of various cancer cell types (12,13). Additionally, K562 (leukemia) cell lines were the most sensitive to KBH-A42, and UM-UC-3 (bladder cancer) cells were the least sensitive. Furthermore, in a human tumor xenograft model using Balb/c nude mice, KBH-A42 was shown to significantly inhibit the growth of K562 tumors, but slightly inhibited the growth of UM-UC-3 tumors. In an effort to determine the reason for the differential response of K562 and UM-UC-3 cells to KBH-A42, cDNA microarray analyses were conducted and confirmed by reverse transcription-polymerase chain reaction (RT-PCR) on the two cell types.
Materials and methods
Chemicals, cell lines and animals
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. KBH-A42 was synthesized and supplied by Dr Gyoonhee Han at Yonsei University (Seoul, Republic of Korea). KBH-A42 was dissolved in dimethyl sulfoxide (DMSO) and freshly diluted in culture media for all in vitro experiments. Female BALB/c-nu mice were purchased from SLC (Hamahatsu, Japan) and maintained as previously described (14). All animals were permitted to acclimate to the local environment for at least 1 week prior to use. The cell lines CaSki, HeLa, Hep 3B, SNU709, A549, AsPC-1, PC-3, A375, LOX-IMVI, M14 and AZ521 were cultured in RPMI-1640 (Gibco BRL, Carlsbad, CA, USA); the UM-UC-3, K562 and KB-3-1 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL). All media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 2 mM L-glutamine, 100 U/ml of penicillin and 100 μg/ml of streptomycin. The cells were maintained at 37°C in 5% CO2 humidified air.
Cell proliferation assay
Cells were plated at 0.8–1.5×104 cells/well in 96-well plates, incubated overnight and treated with KBH-A42 for 48 h. Cell proliferation assays were performed using a cell proliferation kit II (XTT Roche Applied Science Mannheim, Penzberg, Upper Bavaria, Germany) in accordance with the manufacturer’s instructions. The XTT labeling mixture was prepared by mixing 50 volumes of 1 mg/ml sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate with 1 volume of 0.383 mg/ml of N-methyldibenzopyrazine methyl sulfate. This XTT labeling mixture was subsequently added to the cultures and incubated for 2 h at 37°C. Absorbance was measured at 490 nm, with 650 nm as a reference wavelength.
Human tumor xenografts in nude mice
The K562 and UM-UC-3 cells were injected subcutaneously into female BALB/c-nu mice. When the tumor volume reached 100 mm3, the mice were distributed randomly and treated with vehicle, KBH-A42 (100 mg/kg body weight, i.p., QD), or doxorubicin (2 mg/kg body weight, i.p., Q2D) for 14 days. Following 14 days of treatment, the mice were sacrificed and all of the tumors were removed and weighed.
Apoptosis analysis
Apoptosis was analyzed using an annexin V-FITC apoptosis detection kit II (BD Biosciences, NJ, USA) in accordance with the manufacturer’s instructions. In brief, the cells were plated at 2-4×106 cells/dish in 100-mm dishes, incubated overnight and treated for 24 h with the indicated concentrations of KBH-A42. The cells were harvested, washed in phosphate-buffered saline (PBS) and combined with a binding buffer containing annexin V-FITC and propidium iodide (PI). Following 15 min of incubation in the dark, the cells were analyzed via flow cytometry using a FACSCalibur flow cytometer (BD Bioscience).
Caspase 3/7 assay
The activities of caspases 3 and 7 were determined using a Caspase-Glo 3/7™ assay (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. In brief, the cells were plated at 0.8–1.5×104 cells/well in 96-well plates, incubated overnight and then treated for 24 h with the indicated concentrations of KBH-A42. The culture supernatants were transferred to a turbid microtiter plate and mixed with equal volumes of Proluminescent caspase 3/7 substrate (Promega). Following 1 h of incubation at 37°C, luminescence was measured with a VICTOR™ light (PerkinElmer, NJ, USA).
RNA extraction
In brief, cells were plated at 2–4×106 cells/dish in 100-mm dishes, incubated overnight and treated for 24 h with KBH-A42 at the indicated concentrations. RNA was isolated with a Qiagen RNeasy Plus Mini kit according to the manufacturer’s instructions, and its quantity and purity were evaluated using the A260/280 ratio (SmartSpec™ 3000, Bio-Rad, CA, USA).
cDNA microarray and data analysis
Profiling of gene expression was analyzed using a Gene Agilent human 44K 4-plex chip (Digital Genomics, Korea) in accordance with the manufacturer’s instructions. In brief, approximately 2 μg of human total RNA was reverse-transcribed with Cy3- or Cy5-conjugated dUTP (Amersham Pharmacia Biotech, Uppsala, Sweden), respectively. After labeling the reaction for 1 h at 42°C, the Cy3- and Cy5-labeled cDNA probes were mixed together and hybridized to a microarray slide for 16 h at 60°C. The microarray was scanned with a GenePix 4000B scanner (Axon Instruments, CA, USA), and the scanned image was analyzed with GenePix v6.0 software to determine the gene expression ratio. Raw data were normalized via the locally weighted scatter-plot smoother (LOWESS) normalization method. Normalized spot intensities were calculated into gene expression ratios between the control and treatment groups.
RT-PCR
Single-strand cDNA was synthesized from 2 μg of total RNA, using AccuPower® RT premix (Bioneer, Korea). PCR amplification was subsequently performed in a reaction volume of 20 μl containing 2 μl of the appropriate cDNA, 1 μl of each set of primers at a concentration of 10 pM and Accupower PCR premix (Bioneer, Korea). The sequences of the primers used were: HRK, forward: 5′-TGCTCGGCAGGC GGAACTTGTAG-3′ and reverse, 5′-GCTTCCCCAGTCCCA TTCTGTGTTT-3′; TNFRSF10B, forward: 5′-CTTGATTGT GGCTGTGTTTGTT-3′ and reverse, 5′-GCCACCTTTATC TCATTGTCCA-3′; PYCARD, forward: 5′-CTCCTCAGTCGG CAGCCAAG-3′ and reverse, 5′-GGAGTGTTGCTGGGAA GGAG-3′; TNFRSF8, forward: 5′-CTGTGTCCCCTACCCA ATCT-3′ and reverse, 5′-CTTCTTTCCCTTCCTCTTCCA-3′. The RT-PCR products were electrophoresed on a 1.5% agarose gel (Mupid-2) and visualized via staining with ethidium bromide (GelDoc 1000, Bio-Rad), respectively.
Statistical analysis
The results were expressed as the means ± SD. One-way ANOVA followed by Dunnett’s t-test was employed for multiple comparisons using GraphPad Prism (GraphPad Software Inc., CA, USA). P<0.05 was considered to be statistically significant.
Results
Effect of KBH-A42 on human leukemia and bladder cancer cell lines
The effects of KBH-A42 on cell proliferation were assessed in 14 human cancer cell lines obtained from 9 different organs. Among them, the K562 human leukemia cells were the most sensitive to KBH-A42 (GI50 = 1.41 μM), whereas the UM-UC-3 bladder cancer cells were the least sensitive (GI50 >10 μM) (Table I). KBH-A42 inhibited the growth of K562 cells in a dose-dependent manner. However, the growth of UM-UC-3 cells was not affected at concentrations <5 μM of KBH-A42 and was inhibited by only 11.3% at 10 μM (Fig. 1A). Furthermore, to determine whether the in vitro effects of KBH-A42 corresponded to the anti-tumor effects in vivo, we investigated whether KBH-A42 affected tumor growth in a human tumor xenograft model. As shown in Fig. 1B and C, KBH-A42 (100 mg/kg, QD) or ADR (2 mg/kg, Q2D) induced a 37 or 40% inhibition of the growth of K562 tumors, respectively. However, in the UM-UC-3 tumors, KBH-A42 and ADR inhibited growth by 21 or 67%, respectively (Fig. 1B and C).
Table I.
Effect of KBH-A42 and SAHA on the proliferation of various cell lines.
| Cell line | Origin | GI50 (μM) | |
|---|---|---|---|
| KBH-A42 | ADR | ||
| UM-UC-3 | Bladder | >10 | 0.68 |
| K562 | Blood | 1.41 | 0.19 |
| CaSki | Cervix | 2.12 | 0.19 |
| HeLa | Cervix | 2.48 | 0.77 |
| KB-3-1 | Cervix | 3.54 | 0.51 |
| Hep 3B | Liver | 4.81 | 1.03 |
| SNU709 | Liver | 7.55 | 0.47 |
| A549 | Lung | 7.71 | 0.16 |
| AsPC-1 | Pancreas | 7.08 | 1.50 |
| PC-3 | Prostate | 9.88 | 0.76 |
| A375 | Skin | 4.44 | 0.11 |
| LOX-IMVI | Skin | 3.65 | 0.62 |
| M14 | Skin | 3.78 | 0.21 |
| AZ521 | Stomach | 7.49 | 0.21 |
Cells were plated at 9×103 cells/well in 96-well plates, incubated overnight and treated for 48 h with various concentrations of KBH-A42. Cell proliferation was evaluated with an XTT assay, as described in Materials and methods. GI50 values were determined via non-linear regression analysis using GraphPad Prism version 4.0 software.
Figure 1.
Effect of KBH-A42 on the growth of K562 and UM-UC-3 cells in vitro and in vivo. (A) Cells were treated with the indicated concentrations of KBH-A42 for 24 h. Cell proliferation was determined with a XTT assay. (B) BALB/c-nu mice were injected s.c. with K562 or UM-UC-3 cells. When the tumor volumes reached 100 mm3, the mice were treated with vehicle, KBH-A42 (100 mg/kg, QD) or ADR (2 mg/kg, Q2D) via i.p. injection for 14 days. On day 14, the mice were sacrificed and tumor weights were measured. Tumor weights were expressed as the means ± SD of each group. Significance was determined via an ANOVA test vs. the vehicle group (*P<0.05). (C) A representative tumor image.
Induction of apoptosis and activation of caspases by KBH-A42 in K562 and UM-UC-3 cells
To elucidate the mechanism underlying cell growth inhibition by KBH-A42, the apoptosis profiles were analyzed via flow cytometry. To detect apoptotic induction by KBH-A42, the cells were stained with annexin V and PI. Whereas KBH-A42 induced apoptosis by as much as 36% at 10 μM in the K562 cells, a 3.5% induction of apoptosis was noted in the UM-UC-3 cells even at 10 μM (Fig. 2A and B). These results were consistent with the results shown in Fig. 2C, whereas KBH-A42 induced the activation of caspases 3/7 in K562 cells, but not in UM-UC-3 cells.
Figure 2.
Apoptosis analysis by KBH-A42 in K562 and UM-UC-3 cells. Cells were treated for 24 h with KBH-A42 at the indicated concentrations. (A) Cells were stained with propidium iodide and a FITC-conjugated antibody directed against annexin V, and then analyzed by flow cytometry. (B) Results of flow cytometry are expressed as the means ± SD of triplicate determinations. (C) The culture supernatants were collected, and the activity of caspases 3/7 was determined. Significance was determined via an ANOVA test vs. DMSO-treated controls (*P<0.05).
Analysis of gene expression changes caused by KBH-A42 treatment
To gain insight into the mechanism by which KBH-A42 inhibits cell growth, the mRNA expression profiles of K562 and UM-UC-3 cells were analyzed in the presence and absence of KBH-A42. Total RNAs were isolated from K562 and UM-UC-3 cells that were either left untreated or treated with 5 μM KBH-A42 for 24 h, labeled and hybridized to arrays (Agilent human 44K 4-plex chip), and at least a 2-fold difference was detected between the untreated and treated groups with regard to apoptosis-related genes. Among the 139 apoptosis-related genes assessed, 14 (10.07%) and 17 (12.23%) genes were up-regulated (ratio >2.0) in the K562 and UM-UC-3 cells, and 34 (24.46%) and 42 (30.22%) genes were down-regulated (ratio <0.5) in the K562 and UM-UC-3 cells. The most prominently up-regulated genes showing at least a two-fold difference between the K562 and UM-UC-3 cells treated with KBH-A42 were: activity-regulated cytoskeleton-associated protein (ARC), baculoviral IAP repeat-containing 3 (BIRC3), CD70 molecule (CD70), CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD), harakiri (HRK), myeloid cell leukemia sequence 1 (MCL1), tumor necrosis factor receptor superfamily, member 10b (TNFRSF10B) and tumor necrosis factor receptor superfamily, member 25 (TNFRSF25) (Table II). The most profoundly down-regulated genes that also evidenced at least a 2-fold difference between K562 and UM-UC-3 cells were: B-cell CLL/lymphoma2 (BCL2), BCL2-like 13 (BCL2L13), caspase recruitment domain family, member 6 (CARD6), insulin-like growth factor 1 receptor (IGF1R), PYD and CARD domain containing protein gene (PYCARD), RPA interacting protein, transcript variant 2 (RIP), TRAF family member-associated NF-κB activator (TANK), tumor necrosis factor receptor superfamily, member 8 (TNFRSF8) and tumor protein p53 binding protein 2 (TP53BP2) (Table II).
Table II.
Up- and down-regulated genes by KBH-A42 treatment in K562 and UM-UC-3 cells.
| GeneBank number | Gene symbol | Fold change vs. control | |
|---|---|---|---|
| K562 | UM-UC-3 | ||
| Up-regulated genes | |||
| NM_015193 | ARC | 7.71 | 2.49 |
| NM_001165 | BIRC3 | 12.25 | 2.23 |
| NM_001252 | CD70 | 5.06 | 1.13 |
| NM_003805 | CRADD | 2.52 | 1.07 |
| NM_003806 | HRK | 5.69 | 0.40 |
| NM_021960 | MCL1 | 3.43 | 0.91 |
| NM_003842 | TNFRSF10B | 5.30 | 2.65 |
| NM_148968 | TNFRSF25 | 10.62 | 7.99 |
| Down-regulated genes | |||
| NM_000633 | BCL2 | 0.35 | 1.39 |
| NM_015367 | BCL2L13 | 0.45 | 1.27 |
| NM_032587 | CARD6 | 0.18 | 0.79 |
| BC010607 | IGF1R | 0.47 | 1.26 |
| NM_013258 | PYCARD | 0.20 | 1.42 |
| NM_032308 | RIP | 0.44 | 1.60 |
| NM_133484 | TANK | 0.41 | 1.38 |
| NM_001243 | TNFRSF8 | 0.14 | 0.76 |
| NM_005426 | TP53BP2 | 0.44 | 1.23 |
Effect of KBH-A42 on the expression of apoptosis-related genes
Since KBH-A42 induced apoptosis in K562 cells but not in UM-UC-3 cells, apoptosis-related genes that were altered by KBH-A42 were selected from the cDNA microarray results and subjected to RT-PCR for confirmation. Two genes, HRK and TNFRSF10B, were up-regulated by KBH-A42, as demonstrated by the results of the microarray analysis. The expression of HRK mRNA was reduced in the UM-UC-3 cells, but increased slightly in the K562 cells. The expression of the TNFRSF10B mRNA was greatly increased as a result of KBH-A42 treatment in the K562 cells, but was unaltered in the UM-UC-3 cells (Fig. 3). The mRNA expression of PYCARD and TNFRSF8, which was down-regulated by KBH-A42 in the cDNA microarray, was re-examined and confirmed via RT-PCR. PYCARD and TNFRSF10B mRNA showed a reduced expression as a result of KBH-A42 treatment in the K562 cells, but was not detected in the UM-UC-3 cells (Fig. 3).
Figure 3.
Confirmation of the up- and down-regulated genes in a cDNA microarray experiment by RT-PCR. Total RNA was isolated from K562 and UM-UC-3 cells either left untreated or treated with KBH-A42 (1, 2.5 and 5 μM) for 24 h. RT-PCR was conducted and a total of 5 μl of PCR products were run on 1.5% agarose gel. As an internal control, GAPDH was amplified.
Discussion
In this study, we demonstrated that a novel δ-lactam-based HDAC inhibitor, KBH-A42, inhibited the growth of 14 selected cancer cell lines. The findings described have shown that KBH-A42 significantly suppressed the growth of almost all of the tested cancer cell lines, but certain cell types were more susceptible to the compound than others. Whereas the leukemia cell lines were the most sensitive to KBH-A42, the glioma and prostate cancer cell lines were weakly sensitive, and the bladder cancer cell line was the least sensitive. Our findings demonstrate the cell type-specific growth inhibitory effect of KBH-A42. In a human tumor xenograft model using Balb/c nude mice, KBH-A42 has been shown to inhibit the growth of K562 tumors significantly, but only slightly inhibited the growth of UM-UC-3 tumors.
Since HDAC inhibitors have been reported to induce apoptosis in a variety of cancer cell lines (15–17), we evaluated the effect of KBH-A42 on the apoptosis of K562 and UM-UC-3 cells. The results of the apoptosis analysis demonstrate that the growth inhibition of K562 cells by KBH-A42 was mediated, at least in part, by the induction of apoptosis, but that of UM-UC-3 cells was not. The in vitro caspase assays, which measured the involvement of the major effectors of apoptosis, showed a significant difference between the activities of caspase 3/7.
A cDNA microarray was conducted to screen for any changes in the expression of apoptosis-associated genes. The DNA microarray technique is a powerful method, allowing for the simultaneous analysis of the expression levels of multiple genes. In this study, gene expression was profiled using cDNA microarrays on KBH-A42-treated cells. A number of apoptosis-related genes were identified from the list of the genes modulated by KBH-A42 on the cDNA microarray assays. Among them, four genes that were up- or down-regulated by KBH-A42 were selected and their expression patterns were confirmed via RT-PCR.
In the present study, 139 apoptosis-related genes whose expression was altered by KBH-A42 treatment in K562 and UM-UC-3 cells were screened out. Eight genes that were up-regulated (ratio >2.0) and showed at least a 2-fold difference between the K562 and UM-UC-3 cells were apoptosis-associated genes. In mammalian cells, the pro-survival members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1) oppose the pro-apoptotic members, which consist of the Bax group (Bax, Bak and Bok) and the BH3-only proteins (Bim, Bad, Bid, Bik, Bmf, Puma, Noxa and HRK). The BH3-only proteins monitor cellular well-being and, when activated by cytotoxic signals, engage pro-survival relatives by inserting the BH3 domain into a hydrophobic groove on their surfaces (18). The expression of HRK induced rapid cell death, which was repressed by Bcl-2 and Bcl-xL. It was determined that the deletion of the BH3 region eliminated the ability of HRK to interact with Bcl-2 or Bcl-xL and eliminated or profoundly attenuated HRK killing activity (19). In this study, HRK was slightly up-regulated in K562 cells but significantly down-regulated in UM-UC-3 cells as a result of KBH-A42 treatment. In addition, Takimoto et al previously asserted that TNFRSF10B was a viable tumor suppressor candidate, and the overexpression of TNFRSF10B induced apoptosis in tumor cells in a p53-dependent manner (20). The results of this study have shown that KBH-A42 up-regulated the expression of TNFRSF10B in K562 cells in a concentration-dependent manner, but caused no such changes in UM-UC-3 cells.
In addition, nine genes were found to be down-regulated (ratio <0.5) and exhibited more than a 2-fold difference between the K562 and UM-UC-3 cells; these cells were also associated with apoptosis. Apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain (CARD) is a protein encoded by the PYCARD gene in humans. This gene encodes for an adaptor protein composed of a PYD and a CARD domain (21). PYCARD mediates the assembly of large signaling complexes in the inflammatory and apoptotic signaling pathways, via caspase activation. In K562 cells, PYCARD was reduced in a dose-dependent manner and was not detected in UM-UC-3 cells. TNFRSF8, which is involved in apoptosis, is a member of the TNF receptor superfamily, which is capable of inducing apoptosis by activating the caspase pathway (22). In K562 cells, TNFRSF8 was reduced in a dose-dependent manner and was not detected in UM-UC-3 cells. Thus, KBH-A42 may inhibit cell growth by regulating the expression of apoptosis-related genes, and may have better efficacy in leukemia.
In conclusion, the results of this study have demonstrated that KBH-A42 inhibited the growth of cancer cells such as K562 cells under in vitro and in vivo conditions, and also that the growth inhibitory effects of KBH-A42 appear to be mediated by apoptosis. In the microarray assays, apoptosis-related genes containing ARC, BIRC3, CD70, CRADD, HRK, MCL1, TNFRSF10B and TNFRSF25 were up-regulated and BCL2, BCL2L13, CARD6, IGF1R, PYCARD, RIP, TANK, TNFRSF8 and TP53BP2 were down-regulated. Among these genes, changes in the expression of HRK, TNFRSF10B, PYCARD and TNFRSF8 mRNAs was confirmed via RT-PCR. Collectively, our results have shown that KBHA42 possesses anti-cancer properties and has a strong potential as a therapeutic candidate with improved efficacy on leukemia under in vitro and in vivo conditions. However, different cells may be regulated differentially by KBH-A42.
Acknowledgements
This study was supported by the Bio R&D program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (2008-0029594) and the KRIBB Research Initiative program.
References
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