A novel role for 3, 4-dichloropropionanilide (DCPA) in the inhibition of prostate cancer cell migration, proliferation, and hypoxia-inducible factor 1alpha expression
© Jiang et al; licensee BioMed Central Ltd. 2006
Received: 16 May 2006
Accepted: 02 August 2006
Published: 02 August 2006
The amide class compound, 3, 4-dichloropropionanilide (DCPA) is known to affect multiple signaling pathways in lymphocyte and macrophage including the inhibition of NF-κB ability. However, little is known about the effect of DCPA in cancer cells. Hypoxia-inducible factor 1 (HIF-1) regulates the expression of many genes including vascular endothelial growth factor (VEGF), heme oxygenase 1, inducible nitric oxide synthase, aldolase, enolase, and lactate dehydrogenase A. HIF-1 expression is associated with tumorigenesis and angiogenesis.
We used Transwell assay to study cell migration, and used immunoblotting to study specific protein expression in the cells.
In this report, we demonstrate that DCPA inhibited the migration and proliferation of DU145 and PC-3 prostate cancer cells induced by serum, insulin, and insulin-like growth factor I (IGF-I). We found that DCPA inhibited HIF-1 expression in a subunit-specific manner in these cancer cell lines induced by serum and growth factors, and decreased HIF-1α expression by affecting its protein stability.
DCPA can inhibit prostate cancer cell migration, proliferation, and HIF-1α expression, suggesting that DCPA could be potentially used for therapeutic purpose for prostate cancer in the future.
The genetic alterations in human cancer are a major focus of cancer research over the past two decades. Many genetic alterations such as activation of oncogenes and inactivation of tumor suppression genes lead to the increased expression of Hypoxia-inducible factor 1 (HIF-1) [1–4]. HIF-1 is a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits [5, 6]. HIF-1 regulates the expression of many genes including vascular endothelial growth factor (VEGF), heme oxygenase 1, inducible nitric oxide synthase, aldolase, enolase, and lactate dehydrogenase A . HIF-1 activity correlates with tumorigenesis and angiogenesis when wild type and HIF-1-deficient cell lines levels were injected into nude mice [1, 8]. HIF-1 is overexpressed in many human cancers including prostate cancer . HIF-1 expression in prostate cancer cells is induced by growth factors, and inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors and the tumor suppressor PTEN [2, 10].
The amide class compound, DCPA is a dichlorinated ring compound of low molecular weight. Previous investigations on the effects of this compound on lymphocyte and macrophage signaling pathways, reveal that this compound reduces NF-κB DNA binding ability . This correlates with the down regulation of the production of a number of proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-6, IL-1β [11–13] as well as IL-2 [14–17] DCPA also inhibits activation of key components of the Ras pathway . The noted down regulation of the signaling pathways leads us to speculate that DCPA may also affect these pathways in cancer cells in a manner that could be exploited to provide a mode of chemotherapy against these tumors. In this study, we show that DCPA inhibited serum and growth factor-induced cell migration and proliferation, and inhibited HIF-1 expression induced by serum and growth factors in prostate cancer cells. This study indicates that DCPA or its analog may represent a new therapeutic agent for cancer treatment due to its relatively low toxicity.
The human prostate cancer cell line, DU145, was cultured in minimum essential medium (MEM) with Earle's salts and glutamine (Gibco BRL, Grant Island, NY), supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 3% chicken serum (Gibco BRL, Grant Island, NY). The human prostate cancer cell line, PC-3, was cultured in F-12K nutrient mixture (Kaighn's modification) (Gibco BRL), supplemented with 10% FBS and 1% chicken serum. The cells were incubated at 37°C in 5% CO2 incubator.
Cell migration assay
The DU145 and PC-3 cells were cultured in normal medium overnight, then switched to serum-free MEM medium in the presence or absence of DCPA for 18 h. The cells were harvested in Hanks balanced salt solution with 5 mM EDTA and 25 mM HEPES, pH 7.2, washed, and resuspended in serum-free medium (SFM). Cell migration was assayed using a polystyrene Transwell plate (Corning Costar, Cambridge, MA) with 6.5 mm diameter wells and pore sizes of 8.0 μm. The wells were coated with 10 μg/ml collagen type 1 (Upstate Biotechnology, Lake Placid, NY) in 1× PBS buffer at 4°C overnight. The excess collagen was removed from the bottom chambers after the incubation, and replaced with 250 μl serum-free medium in the absence or presence of DCPA (150 μM) or 10% FBS. The cells were counted and diluted to 400,000 cells per ml in SFM, and 250 μl of the cells were added to the top of each well. For the DCPA treatment, the cells were incubated with SFM with 150 μM DCPA 20 min prior to the addition of 100,000 cells to each well in the presence or absence of 10% FBS in the bottom chambers, followed by the addition of 10% serum, 200 nM insulin, or 2 nM insulin-like growth factor I (IGF-I) for 6 h. The wells were incubated at 37°C in 5% CO2incubator for 6 h. The wells were removed, and the cells on the top of the well were wiped out with a cotton swab, stained with 1% crystal violet in 0.1 M borate containing 2% ethanol, pH 9.0 for 20 min. The migrated DU145 and PC-3 cells were rinsed with water, dried, and counted using light microscope with 10× magnification.
Cell viability assay
The DU145 and PC-3 cells were seeded at 5 × 105 cells per well in a 24-well plate, and incubated at 37°C in 5% CO2 incubator in the complete medium for 24 h. Then, the culture medium was removed and replaced with serum-free medium containing DCPA (25, 75 or 150 μM) or solvent alone (ETOH). A medium-only control was also included. After the culture in serum-free medium for 24 h, the cells were switched to the medium with 10% serum for 6 h. The cells were then stained with trypan blue, and counted using standard hematocytometer methods.
Cell proliferation assay
One day before the assay, DU145 and PC-3 cells were seeded at 100,000 cells per well in a six-well plate and incubated at 37°C in 5% CO2 incubator in the medium as described above. Then the cells were switched to fresh medium containing DCPA (150 μM) or solvent alone (0 μM DCPA), and the cell number was counted 24, 48, and 72 h after the treatment using standard hematocytometer methods.
Protein extraction and immunoblotting
DU145 and PC-3 cells were harvested in cold 1× PBS and lysed on ice for 30 min in RIPA buffer (150 mM NaCl, 100 mM Tris, pH 8, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA and 10 mM NaF) supplemented with 1 mM sodium vanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, and 2 mM pepstatin A. The lysates were cleared by centrifugation at 14,000 rpm for 15 min, and the supernatants were collected as total cellular protein extracts. The protein concentration in the extracts was determined using Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA). The protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes in 20 mM Tris-HCl (pH 8.0) containing 150 mM glycine and 20% (v/v) methanol. Membranes were blocked with 5% nonfat dry milk in 1× TBS containing 0.05% Tween 20 and incubated with antibodies against HIF-1α and HIF-1β . The protein bands were detected by the incubation with horseradish peroxidase-conjugated antibodies (NEN, Boston, MA), and visualized using the enhanced chemiluminescence reagent (NEN).
HIF-1α protein stability analysis
PC-3 cells were cultured in serum-free medium for 24 h, followed by the incubation with medium containing 10% fetal bovine serum for 5 h. Then the cells were treated with solvent alone or 150 μM DCPA for 1 h, followed by the incubation with 100 μM cycloheximide for 0 to 16 min. Cellular lysates were subjected to immunoblotting using antibodies against HIF-1α and HIF-1β. The intensity of HIF-1α/HIF-1β protein signals was quantified using EagleSight densitometry software (Version 3.21; Stratagene).
The results on DU145 and PC-3 cell migration and proliferation were analyzed statistically using the Student-Newman-Keuls multicomparisons test using SigmaStat Software (SPSS Inc., Chicago, IL).
Results and discussion
DCPA inhibits prostate cancer cell migration
To determine if the decreased cell migration was due to decreased cell viability, cell viabilities were determine on separate cultures treated as described above. As shown in Fig. 1C, neither cell line showed any increase in cell death over either the solvent (ETOH) or medium (nil) controls. Thus, the inhibition of cell migration by DCPA is due to the effect of DCPA on the signaling pathways associated with cell motility and not simply due to increased cell death.
DCPA inhibits cell proliferation
DCPA treatment diminishes HIF-1α expression induced by serum
In conclusion, these results demonstrated that DCPA inhibits cell migration, proliferation, and HIF-1α expression in prostate cancer cells. To further understand the molecular mechanism of DCPA in decreasing HIF-1α expression in these cells, we found that 1) DCPA specifically inhibits the induction of HIF-1α expression by serum, 2) expression of HIF-1β is not altered by the addition of serum or DCPA, 3) the effect of DCPA on HIF-1α is not due to the non-specific effect in the cells, 4) DCPA treatment decreased HIF-1α expression by affecting its stability through the proteasomal degradation pathway, and 5) DCPA also inhibited hypoxia-induced HIF-1α expression in human prostate cancer cells.
DCPA, n-3, 4-dichlorophenyl propanamide
fetal bovine serum
Hypoxia-inducible factor 1
insulin-like growth factor I
Minimum Essential Medium
vascular endothelial growth factor.
This study was supported by NIH Grants ES07512, ES011311, CA109460; and American Cancer Society Research Scholar Grant 04-076-01-TBE. We are grateful to Zongxian Cao, Jenny Z. Zheng, Linda Corum, and Cheryl Walton for their technical assistance.
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