Chinese Medical Journal 2006;119(9):713-718
Hypoxia-inducible factor-1α induces the epithelial-mesenchymal transition of human prostatecancer cells

LUO Yong,  HE Da-lin,  NING Liang,  SHEN Shu-lin,  LI Lei,  LI Xiang

LUO Yong (Department of Urology, First Hospital of Xi’an Jiaotong University, Xi’an 710061, China)

HE Da-lin (Department of Urology, First Hospital of Xi’an Jiaotong University, Xi’an 710061, China)

NING Liang (Department of Urology, First Hospital of Xi’an Jiaotong University, Xi’an 710061, China)

SHEN Shu-lin (Department of Urology, First Hospital of Xi’an Jiaotong University, Xi’an 710061, China)

LI Lei (Department of Urology, First Hospital of Xi’an Jiaotong University, Xi’an 710061, China)

LI Xiang (Department of Urology, First Hospital of Xi’an Jiaotong University, Xi’an 710061, China)

Correspondence to:LUO Yong,Department of Urology, First Hospital of Xi’an Jiaotong University, Xi’an 710061, China (Tel: 86-29-85323661. Fax:86-29-85251632. E-mail:luoyongxjtu@ sohu.com)
Keywords
prostate cancer; epithelial-mesenchymal transition; hypoxia-inducible factor-1α
Abstract

Background  Hypoxia-inducible factor-1α (HIF-1α) is a transcriptional factor that could improve the stimulation of angiogenesis and the metabolic adaptation of tumor cells to hypoxia. A recent study showed that HIF-1α could induce colon cancer cells epithelial-mesenchymal transition (EMT). However, no evidence indicates a similar correlation in human prostate cancer cells. This study was designed to evaluate the effect of HIF-1α over-expression on the EMT in human prostate cancer cells.
Methods  We selected the appropriate cell line for HIF-1α induction from those EMT negative prostate cell lines through vimentin gene detection by RT-PCR. As the result, LNCaP cell line is the best one for further experiment. LNCaP cells were transfected with recombinant plasmid pcDNA3.1(-)/HIF-1α and pcDNA3.1(-) control vector by Lipofectamine 2000 system. The positive cell colonies were confirmed by indirect immunofluorescence labeling. Then Transwell polycarbonate filter was used to analyze the invasive potency. The expression of EMT associated proteins, E-cadherin and vimentin, was detected by Western blotting.
Results  Among four of the EMT negative cell lines, LNCaP was the only one expressed the vimentin gene but not the associated protein. The expression level of HIF-1α in LNCaP/HIF-1α was distinctly higher than that in LNCaP/pcDNA3.1 and LNCaP. The cell numbers of LNCaP/HIF-1α that penetrated through the Transwell filter were higher than that of LNCaP/pcDNA3.1 and LNCaP. Compared with the LNCaP/pcDNA3.1 and LNCaP cells, the expression of vimentin was up-regulated in LNCaP/HIF-1α, whereas the expression of E-cadherin was down-regulated. 
Conclusions  Over-expression of HIF-1α stimulates the invasion potency of human prostate carcinoma cells through EMT pathway. The expression of E-cadherin and vimentin, playing established roles in EMT, could be regulated by HIF-1α in human prostate cancer cell line.

The presence of hypoxic regions in solid tumors is associated with a more malignant tumor phenotype and worse prognosis. Previous researches have proved that when tumor cells are exposed to hypoxic stress, hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcriptional complex composed of HIF-1α and HIF-1β subunits, is activated to promote the transcription of several genes, including vascular endothelial growth factor, glucose transporters (GLUTs) and glycolytic enzymes.1,2 HIF-1β, also known as arylhydrocarbon receptor nuclear translocator, is a common subunit of multiple transcriptional complexes. HIF-1α is an oxygen regulated subunit that determines HIF-1 activity. Under normoxic conditions, a majority of HIF-1α protein will be degraded by the protease complex. Following hypoxic stabilization, HIF-1α is translocated into nucleus, where it heterodimerizes with HIF-1β and activates the transcription of hypoxia inducible genes via hypoxia responsive element.

HIF-1α has become one really important transcriptional factor in tumor research. However, whether HIF-1α could improve adaptation tolerance of cancer cells to hypoxia and make the cancer more and more invasive through some new pathways, including epithelial-mesenchymal transition (EMT) pathway, has never been investigated in urological cancer upon molecular level.

Our previous research has determined the characteristic of EMT in several human prostate cancer cell lines and constructed HIF-1α expression vector, pcDNA3.1(-)/HIF-1α. In the current study, we evaluated the effect of HIF-1α over-expression on the EMT in human prostate cancer cells which have been proved as EMT negative cells.

METHODS

RT-PCR for cell lines screening
Our previous study has identified four EMT negative cell lines from several prostate cancer cell lines. They are LNCaP cell line and its sublines, C4, C4-2, C4-2B. We singled out an appropriate cell line for HIF-1α induction, according to the expression of vimentin mRNA analyzed by using RT-PCR. Total RNAs were isolated from the four different cell lines at 80%-90% confluence by using Trizol (Invitrogen Inc, USA). The RNA samples were first treated with deoxyribonuclease I (Fermentas Inc, USA) and converted into cDNA using oligo(dT)18 primers and reverse transcriptase (Fermentas Inc, USA). PCR was carried out as the following conditions: vimentin, initial denaturation at 93˚C for 3 minutes, followed by 29 cycles of denaturation at 93˚C for 45 seconds, annealing at 56.1˚C for 45 seconds, and extension at 72˚C for 45 seconds; β-actin, initial denaturation at 94˚C for 3 minutes, followed by 29 cycles of denaturation at 94˚C for 45 seconds, annealing at 58˚C for 45 seconds, and extension at 72˚C for 45 seconds. Amplified products were analyzed on 1.5% agarose gel. The following are PCR primers for vimentin (sense primer, 5’-TGGCACGTCTTGACCT TGAA-3’; antisense primer, 5’-GGTCATCGTGATG CTGAGAA-3’), and β-actin (sense primer, 5’-GTG GGGCGCCCCAGGCACCA-3’; antisense primer, 5’-CTTCCTTAATGTCACGCACGATTTC-3’). All of the primers were obtained from Sangog Ltd (Shanghai, China).

Establishment of HIF-1α over-expressed cells
The human prostate cancer LNCaP cells were cultured in RPMI1640 medium with 10% FBS and penicillin (100 U/ml)-streptomycin (100 µg/ml). Recombinant plasmid pcDNA3.1(-)/HIF-1α was constructed and transfected into LNCaP cells with Lipofectamine 2000 system (Life Technologies Inc, USA). Subsequently, cells were cultured in medium containing 600 µg/ml of G418 for 4 weeks. And the transfection of pcDNA3.1(-) control vector (provided by Leland WK Chung, Emory University) was performed as that of pcDNA3.1(-)/HIF-1α expression vector. At last, 8 HIF-1α positive cell clones and 7 pcDNA3.1(-) cell clones were selected and expanded. Five of 8 HIF-1α clones continuously expressed high level of HIF-1α protein, so all the five HIF-1α clones and one of seven pcDNA3.1(-) cell clones were applied for further molecular study. Then one of the five HIF-1α positive clones and the same pcDNA3.1(-) clone were used in the transwell trial. The LNCaP cells were successfully transfected with pcDNA3.1(-)/HIF-1α expression vector and pcDNA3.1(-) control vector were designated as LNCaP/HIF-1α and LNCaP/pcDNA3.1, respectively.

Confirmation of HIF-1α expression
After culture, prostate cancer cells were fixed in 2% paraformaldehyde for 30 minutes and blocked with goat serum for 30 minutes, respectively. Cells were then incubated at 37˚C for 1 hour with rabbit anti-human HIF-1α polyclonal antibody (Santa Cruz, USA) at a dilution of 1:100. After being washed 3 times with PBS, cells were co-incubated with a fluorescence isothiocyanate (FITC) conjugated goat anti-rabbit antibody at 37˚C for 1 hour. The fluorescence staining intensity and intracellular localization were then examined under a fluorescence microscope.

Detection of EMT associated proteins
Total protein was isolated from 107 of cells, by using 200 µl of ice cold lysis buffer containing 1% NP-40, 50 mmol/ L Tris (pH 7.4), 150 mmol/ L NaCl, 0.1% SDS, 0.5% deoxycholate, 200 µg/ml PMSF and 50 µg/ml aprotinin. Insoluble materials were removed by centrifugation at 15 000 g for 15 minutes at 4˚C. The concentration of the extracted protein was measured spectrophotometrically with Coomassie G-250. Aliquots of 30 µg protein from the LNCaP/HIF-1α, LNCaP/pcDNA3.1 and LNCaP were separated by 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. The membranes were initially blocked with 5% nonfat dry milk in TBS for 2 hours and then incubated with the primary antibodies (anti-E-cadherin, anti-vimentin, and anti-β-actin, Santa Cruz, USA) all at a 1:100 dilution in 5% nonfat dry milk/TBS at 4˚C for overnight. The secondary alkaline phosphatase- conjugated goat anti-rabbit antibody for E-cadherin or goat anti-mouse antibodies for vimentin and β-actin (Boshide Inc, China) were diluted to 1:500 and 1:200 in 5% nonfat dry milk/TBS (0.1% Tween-20), respectively. After being washed three times with TBS, the membranes were incubated with the secondary antibodies for 2 hours at room temperature, then they were washed with TBS again for three times, NBT/BCIP chemiluminescence (Amresco, USA) was used to detect immunopositive protein bands.

Invasion assays
For invasion assays, Transwell polycarbonate filter (8-µm pore size, Millipore, USA) was coated with 100 µl of matrigel (Sigma, USA) at a dilution of 1:20 in serum-free medium and air-dried for 24 hours. 7×104 cells in 200 µl of complete medium were seeded into the upper chamber. Six hundred µl of medium were added to the lower chamber, and the plate was incubated at 37˚C in a 5% CO2 incubator for 24, 48 and 72 hours, respectively. Cells on the lower surface of the filter were scraped with a rubber scraper into the medium from the lower chamber, pelleted, resuspended in 50 µl of medium, and counted using a hemocytometer. The experiment was performed thrice for three different cell lines.

Statistical analysis
For each condition, the cell numbers (×104) that penetrated through the Matrigel filter were presented as mean±standard deviation (SD) and analyzed using the SPSS13.0. Analysis of variance was conducted, followed by independent-samples t test. P value less than 0.05 was considered statistically significant.

RESULTS

Vimentin mRNA expression in different EMT negative cells
RT-PCR of cytoplasmic RNA from LNCaP cells and its sublines (C4, C4-2, C4-2B) was performed. The cytoskeleton protein and β-actin did not show any appreciable difference in the mRNA levels among the four cell lines. When the β-actin mRNA was used as an internal standard, the vimentin expression was detectable only in the LNCaP cells (Fig. 1). Our previous research has determined that none of these four different EMT negative cell lines express vimentin protein. This disaccord between vimentin mRNA and protein means that maybe some underlying modulations inhibit the translation from vimentin mRNA to protein. According to the result of RT-PCR, LNCaP cells were arranged for the further research, which aimed to investigate the effect of HIF-1α transfection on the expression of mesenchymal protein, vimentin.
 

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Fig.1. mRNA expression of vimentin and β-actin in four epithelial-mesenchymal transition negative prostate cancer cell lines.

Establishment of HIF-1α over-expressed prostate cancer cells
We established HIF-1α over-expression cancer cells, LNCaP/HIF-1α, as described above. Indirect immunofluorescence labeling showed a stronger signal of HIF-1α in LNCaP/HIF-1α than that in LNCaP/pcDNA3.1 and LNCaP. Furthermore, we observed that the expression of HIF-1α was located in the cytoplasm. (Fig. 2)


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Fig.2. Hypoxia-inducible factor-1α expression analyzed by immunofluorescence staining. A: LNCaP; B: LNCaP/pcDNA3.1; C: LNCaP/HIF-1α.

Effects of HIF-1α over-expression on EMT associated proteins
EMT process involves losing of epithelial phenotype and obtaining some specific characteristics of mesenchymal cells. As observed, the expression of E-cadherin was down-regulated in the LNCaP/HIF-1α, compared with the LNCaP/ pcDNA3.1 and LNCaP cells. Moreover, the expression of vimentin was up-regulated in LNCaP/HIF-1α cells, whereas its expression was markedly absent in LNCaP/ pcDNA3.1 and LNCaP cells. (Fig. 3) All the results of Western blotting in five HIF-1α positive cell clones were the same.


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Fig.3. The expression of epithelial-mesenchymal transition associated proteins detected by Western blotting in three different cell lines. Lane 1: LNCaP; lane 2: LNCaP transfected with pcDNA3.1 control vector; lanes 3-7: LNCaP transfected with pcDNA3.1/HIF-1α. HIF-1α: hypoxia-inducible factor-1α.

Effects of HIF-1α over-expression on cell growth in vitro
In order to invade surrounding tissue, the cancer cells of epithelial origin must degrade the underlying basement membrane. To assay the effects of HIF-1α over-expression on invasion, LNCaP cells were stably transfected with HIF-1α expression vector. Transfected cells were seeded onto a filter that was coated with Matrigel, an experimental basement membrane, and exposed to normoxic condition for 24 hours. The numbers of cells that digested Matrigel and migrated through the 8 µm pores in the filter were counted after 24, 48 and 72 hours. The cell numbers of LNCaP/HIF-1α that penetrated through the Transwell polycarbonate filter were higher than that of LNCaP/pcDNA3.1 and LNCaP at every time point (Table), which suggested that transfection of cells with HIF-1α expression vector resulted in a highly significant increase in invasion potency.


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Table. The cell numbers penetrated through the Matrigel filter (×104)

DISCUSSION

One aspect of tumor biology is the capacity for primary malignant epithelial cells to exit their site of origin, degrade the surrounding extracelluar matrix, migrate into blood vessel and invade secondary organs.3 In order to carry out this invasive process, tumor cells must modify their phenotype first by altering the expression of a new repertoire of cell surface receptors, growth factors, proteases and adhesion molecules that participate in the activation of particular signaling pathways needed to facilitate cell proliferation and migration.4 Tumor cells must first undergo an important process known as EMT.5,6 EMT is a normal physiologic process by which epithelial cells loose their relatively strong adhesiveness, become more motile and assume a spindle-shaped morphology resembling cells of mesenchyme origin.7 These processes normally occur during embryonic development and organogenesis, tissue growth, and wound healing and repair.8 However, EMT could reappeared and participate in tumor occurrence and development. In tumor pathophysiological process, EMT always involves altered expression of E-cadherin on the surface of epithelial cancer cells.9,10 E-cadherin, which always executes loss of cell-cell contacts and exitting original site of cancer cells, is one of the major cadherin proteins associated with tumor invasion. Cells in the center of a prostate cancer maintain an epithelial phenotype, whereas cells at the invasive front exhibit a mesenchymal phenotype characterized by increased expression of vimentin.11 Reprogramming of intermediate filament expression leading to the production of vimentin, either alone or in combination with specific keratins, promotes tumor cell invasion.12 EMT facilitates migration and invasion of epithelial tumor cells and has recently been suggested as an index of aggressiveness and increased metastasis potential in different types of malignant tumors.13,14

Recently, several further studies concluded that HIF-1α over-expression also inhibits apoptosis by down-regulating the P53-, BAX-, and caspase- dependent cell death pathways,15 increases cell proliferation via regulating cell cycle-associated proteins16 and improves high expression of several growth factors.17 All these mechanisms elucidate that tumor cells can survive in the hypoxia condition. Krishnamachary et al18 examined the effect of HIF-1α over-expression in colon cancer cells and noted that HIF-1α over-expression renders these cells resistant to hypoxia via EMT. Manotham19 also found that hypoxia plays an important role in progression of kidney fibrotic disease through inducing tubular EMT. However, up to now no data has been published about how this process operates on human prostate cancer cells.

In this study, we successfully established HIF-1α over-expressing prostate cancer cells (LNCaP/ HIF-1α), and firstly demonstrated that HIF-1α over- expression could regulate the expression of EMT associated genes, such as E-cadherin and vimentin, in human prostate cancer. As the consequence of gene alteration, loss of adhesiveness and increasing motility were observed in LNCaP cells, which were just the defining features of EMT cells.

The data presented in this article provide a molecular basis for clinical and experimental evidence associating tumor invasion with HIF-1α over- expression. Combined with its well-established roles in regulating angiogenesis and metabolic adaptation, these results add yet another dimension to the multifaceted involvement of HIF-1α in tumor progression. The coordinated activation by HIF-1 of a large battery of target genes, the protein products perform diverse but related functions contributing to tumor invasion, suggesting that HIF-1 inhibitors may have therapeutic utility as anticancer agents.20 But these results should be interpreted further, since the data presented here do not distinguish between direct and indirect regulation of the identified target genes by HIF-1. Additional studies are required to identify, within such target gene, a functional HIF-1 binding site to conclude that HIF-1 directly regulates gene expression.

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