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Effects of hypoxia inducible factor-1[alpha] (HIF-1[alpha]) on the growth & adhesion in tongue squamous cell carcinoma cells.

Background & objectives: Hypoxia-inducible factor-1 alpha (HIF-1[alpha]) is a central transcriptional regulator of hypoxic response. Suppression of HIF-1[alpha] is important for exploring hypoxia-induced pathophysiological events. This study was carried out to analyze the hypoxia-induced changes of biological characteristics in the human tongue squamous cell carcinoma squamous cell carcinoma
n.
A carcinoma that arises from squamous epithelium and is the most common form of skin cancer. Also called cancroid, epidermoid carcinoma.
 cell line Tca8113 and evaluate the effects of HIF-1[alpha] on the phenotype of the tongue squamous cell carcinoma.

Methods: HIF-1[alpha] gene was silenced with synthesized short interfering ribonucleic acids (siRNA). HIF-11[alpha] expression was measured on mRNA level by real-time reverse transcription (RT)-PCR and protein level by Western blot and immunofluorescence. The cell cycle and apoptosis of Tca8113 ceils were analyzed by FACS FACS Fellow of the American College of Surgeons.

FACS
abbr.
Fellow of the American College of Surgeons



FACS

fluorescence-activated cell sorter.
. The proliferation and adhesion of Tca8113 cells were determined by MTT MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide
MTT Machine Tool Technology
MTT Microwave Theory and Techniques
MTT Mobile Task Team
MTT Multi-Table Tournament (poker) 
 colorimetric col·or·im·e·ter  
n.
1. Any of various instruments used to determine or specify colors, as by comparison with spectroscopic or visual standards.

2.
 assay.

Results: Tca8113 could survive and showed a more aggressive phenotype under hypoxic condition. Exposure to hypoxia induced a prolonged elevation of HIF-11[alpha] protein and transfection of siRNA targeting HIF-1[alpha] ([siRNA.sub.HIF-1[alpha]]) reduced HIF-1[alpha] synthesis as measured on mRNA and protein level. Under normoxic or hypoxic conditions, treatment of Tca8113 cells with [siRNA.sub.HIF-1[alpha]] induced cell apoptosis and inhibited the growth and adhesion.

Interpretation & conclusions: [siRNA.sub.HIF-1[alpha]] could attenuate To reduce the force or severity; to lessen a relationship or connection between two objects.

In Criminal Procedure, the relationship between an illegal search and a confession may be sufficiently attenuated as to remove the confession from the protection afforded by the
 the tolerance against hypoxia in Tca8113 cells and inhibit their aggressive potential. Interfering with HIF-1[alpha] pathways by siRNA strategy may provide a therapeutic target for human tongue squamous cell carcinomas.

Key words Hypoxia--HIF-1--phenotype--siRNA

**********

Oxygen supply to cells and tissues is pivotal in maintaining their function and integrity. Rapid growth of solid tumours often results in the development of a hypoxic microenvironment microenvironment /mi·cro·en·vi·ron·ment/ (-en-vi´ron-ment) the environment at the microscopic or cellular level.  (1). Adaptation to the hypoxic microenvironment leads to the selection of certain malignant tumour phenotypes, characterized by abnormal neovascularization, invasion, metastasis and resistance to chemo- and radiotherapies (2,3). Under these conditions, a wide variety of tumour- and host-derived factors are regulated. However, the molecular mechanism and the relative importance of specific factors and microenvironmental conditions for these processes have not been well characterized. Recent studies have shown the overexpression of hypoxia-inducible factor-1 alpha (HIF-1[alpha]) in common cancers with an advanced tumour grade, implying [HIF-1[alpha]] as a central transcriptional regulator of tumour microenvironment (4).

HIF-1[alpha] is a heterodimeric transcription factor composed of a strictly regulated [alpha] subunit and a constitutive [beta] subunit (HIF-11[beta]/ also called ARNT i.e., aryl hydrocarbon receptor nuclear translocator The aryl hydrocarbon (Ah) receptor is involved in the induction of several enzymes that participate in xenobiotic metabolism. The ligand-free, cytosolic form of the aryl hydrocarbon receptor is complexed to heat shock protein 90. ) (5). HIF-1[alpha] is constitutively expressed, but undergoes rapid ubiquitination and proteasomal degradation, the rate of which is directly proportional to cellular oxygen tension. Under hypoxic condition, this constitutive decay of HIF-1[alpha] is suppressed, allowing it to translocate into the nucleus where it dimerizes with ARNT (6). The consequent heterodimeric effector effector /ef·fec·tor/ (e-fek´ter)
1. an agent that mediates a specific effect.

2. an organ that produces an effect in response to nerve stimulation.
 molecule binds to hypoxia-responsive elements (HRE) located in the promoter and enhancer regions of hypoxia-regulated genes, causing their transactivation. Target genes include those fostering glucose uptake, glycolysis glycolysis (glīkŏl`ĭsĭs), term given to the metabolic pathway utilized by most microorganisms (yeast and bacteria) and by all "higher" animals (including humans) for the degradation of glucose. , erythropoeisis, and proliferation (5).

HIF-1[alpha] was shown to be expressed in oral squamous cell carcinoma (7). Clinical studies reported that high levels of HIF-1[alpha] in human squamous cell carcinomas seemed to be correlated with tumour resistance to radiation and chemotherapy (8). These findings implied that HIF-1[alpha] was important in angiogenesis and could contribute to tumour resistance to treatment. Nonetheless, its effects on the phenotype of human tongue squamous cell carcinoma cells under hypoxic condition have not yet been sufficiently studied. In the present study, we transfected short interfering RNA RNA: see nucleic acid.
RNA
 in full ribonucleic acid

One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic
 (siRNA) targeting HIF-1[alpha] ([siRNA.sub.HIF-1[alpha]]) into human tongue squamous cell carcinoma cell line Tca8113 to investigate its effect on the changes of biological behaviour of Tca8113 cells under hypoxic condition.

Material & Methods

The study was carried out at Laboratory Center of Qilu Hospital in Jinan of China.

Cell line and cell culture: Human tongue squamous cell carcinoma cell lines (Tca8113 cells) were purchased from Culture Collection of Chinese Academy of Science (Shanghai, China) and routinely cultured in RPMI RPMI Rapid Prototyping & Manufacturing Institute
RPMI Roswell Park Memorial Institute
RPMI Royal Park Memorial Institute (culture medium) 
 1640 containing 10 per cent foetal bovine serum (FBS FBS
abbr.
fasting blood sugar


FBS Fasting blood sugar. See Fasting glucose.
) (Hyclone, USA), penicillin (100 units/ml), and streptomycin (100 [micro]g/ml) at 37[degrees]C in a humidified air atmosphere containing 5 per cent carbon dioxide. For hypoxic condition, cells were cultured at 37[degrees]C in 94 per cent [N.sub.2], 5 per cent CO2 and 1 per cent O2 (HERAcel12401, Germany) when growing to 80 per cent confluence.

Gene silencing with short interfering ribonueleic acids (siRNA): Small interfering RNA Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology.  targeted to HIF-1 alpha ([siRNA.sub.HIF-1[alpha]]) was designed and validated by Ambion (Ambion, USA), and a siRNA targeted to an irrelevant mRNA ([siRNA.sub.Irr]) (Ambion) served as nonspecific control. The sequences of [siRNA.sub.HIF-1[alpha]] were:

Sense: 5'-GGGUAAAGAACAAAACACAtt-3'

Antisense: 5'-UGUGUUUUGUUCUUUACCCtt-3'

For HIF-1[alpha] siRNA transfection, Tca8113 cells were plated at a concentration of 2x[10.sup.5] cells per well in six-well plates. Growing to 40-50 per cent confluence, the cells were transfected with the [siRNA.sub.HIF-1[alpha]] (50nM) premixed with the Lipofectamine[TM] 2000 (Invitrogen, USA) in Opti-MEM (Invitrogen, USA) for 6 h and then cells were placed in flesh complete medium without penicillin and streptomycin for 24 h. As mock transfection, cells were exposed to Lipofectamine[TM] 2000 alone, [siRNA.sub.Irr] was used as a negative (nonspecific) control.

Real-time RT-PCR analysis for HIF-1[alpha]: The HIF 1-[alpha] mRNA expression was assayed quantitatively by real-time reverse transcription polymerase chain reaction “RT-PCR” redirects here. For real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction, see real-time polymerase chain reaction.  (real-time RT-PCR), as previously described (9) with the appropriate reagents (SYBR SYBR Synergy Brands, Inc. (stock symbol) [R] Green Real-time PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
 Master Mix) (TOYOBO CO., Japan) and light cyclear (Lightcyclear 2.0, Roche Diagnostic, Switzerland). Total RNA was prepared using TRIZOL reagent (Invitrogen, USA) according to the manufacturer's protocol, and cDNA was synthesized by the First Strand cDNA Synthesis kit (Fermentas, Lithuania). The relative amount of PCR product was calculated as threshold cycle (CT value) of the sample divided by that of human [beta]-actin. The thermocycler conditions used for amplification were cycles of 95[degrees]C for 5 sec, 60 [degrees]C for 20 sec, and 72 [degrees]C for 10 sec. The primers were commercially procured (TaKaRa, China) and the sequences were as follows:

HIF-1[alpha]: forward 5'-TTTGCTGAAGACACAGAAG CAAAGA-3'

reverse 5'-TTGAGGACTTGCGCTTTCAGG-3' [beta]-actin: forward 5'-TGGCACCCAGCACAATGAA-3'

reverse 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3'

Immunofluorescence assay of HIF-1[alpha]: The Tca8113 cells (8x[10.sup.4] in 1 ml culture medium) were plated onto chamber slides and allowed to attach overnight. When growing to 40 per cent confluence, the cells were transfected with 50 nM [siRNA.sub.HIF-1[alpha]] for 24 h and incubated at 1 per cent oxygen for 24 h. Then the cells on the slides were removed and fixed with 4 per cent paraformaldehyde paraformaldehyde: see formaldehyde.  for 30 min, washed with PBS PBS
 in full Public Broadcasting Service

Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural,
, and incubated with blocking solution involving non specific antigens for 30 min. Washed with PBS for three times, the samples were incubated with mouse anti-human HIF-1[alpha] (1:1000, ABCAM, Cambridge, UK) monoclonal antibodies with 0.1 per cent Tritum-X-100 overnight at 4[degrees]C. Washed with PBS for three times, the samples were subsequently incubated with second antibodies, fluorescein isothiocyanate (FITC FITC

fluorescein isothiocyanate; used as a fluorescent label for proteins, especially antibodies.
)-conjugated goat anti-mouse immunoglobulin (1:1000, SouthernBiotech, USA), in the dark for 1 h at 37[degrees]C. Expression of HIF-1[alpha] was detected with fluorescence microscopy (OLYMPUS 1X81, Olympus, Japan).

Western blot analysis West·ern blot analysis
n.
An electrophoretic procedure for separating proteins.
: Cells plated in 60 mm cell culture dishes were cultured under hypoxic condition or transfected as described for gene silencing with siRNA. The cells were washed twice with cold PBS and scraped from cell culture dishes. Cellular protein extracts were prepared by homogenization homogenization (həmŏj'ənəzā`shən), process in which a mixture is made uniform throughout. Generally this procedure involves reducing the size of the particles of one component of the mixture and dispersing them evenly  in an ice-cold buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.5% Triton X-100; 2Mm EDTA EDTA: see chelating agents. , pH 8.0; 2 [micro]g/ml aprotinin aprotinin /apro·ti·nin/ (ap?ro-ti´nin) an inhibitor of proteolytic enzymes used to reduce perioperative blood loss in patients undergoing cardiopulmonary bypass during coronary artery bypass graft. ; 5 mM dithiothreitol and 0.2 mM phenylmethylsulphonyl fluoride) for 30 min. The lysates were centrifuged at 15000 g for 20 min at 4 h and samples of the supernatants were collected and stored at -80[degrees]C. Protein concentrations were determined by bicinchoninic acid assay The bicinchoninic acid assay (also known as the BCA assay or Smith assay) is a biochemical assay for determining the total level of protein in a solution, similar to Lowry protein assay, Bradford protein assay or biuret reagent.  methods (BCA protein assay kit, Calbiochem, USA) (9). Equivalent amount of protein (100 [micro]g) was separated on 7.5 per cent polyacrylamide-SDS gel and electroblotted onto nitrocellulose nitrocellulose, nitric acid ester of cellulose (a glucose polymer). It is usually formed by the action of a mixture of nitric and sulfuric acids on purified cotton or wood pulp.  membranes. After blocking with 5 per cent non fat dry milk, the membrane was incubated overnight at 4 h with mouse anti-human HIF-1[alpha] (1:1000, ABCAM, UK) monoclonal antibodies and signals were detected by enhanced chemiluminescence (ECL).

Cell proliferation assay: The Tca8113 cells were plated in 96-well plates (6,000/well) and transfected as mentioned above. Transfected and mock transfected cells were incubated under normoxic or hypoxic condition for 24, 48, or 72 h respectively. Then cell proliferation was analyzed by MTT colorimetric assay which detects the conversion of 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma, USA) to a purple formazan product (10). MTT is a pale yellow tetrazolium salt that produces a dark blue formazan product when incubated with living cells. MTT ring is cleaved in active mitochondria in living cells. Briefly, the cells were incubated with MTT solution (1 mg/ml in RPMI 1640) for 4 h at 37[degrees]C and carefully observed to exclude crystal formation outside of cells. The medium was decanted, and the formazan crystals formed were then dissolved by the addition of dimethyl sulphoxide (DMSO DMSO dimethyl sulfoxide.

DMSO
n.
Dimethyl sulfoxide; a colorless hygroscopic liquid obtained from lignin, used as a penetrant to convey medications into the tissues.


DMSO,
n.
; 150 [micro]l/well). Absorbance absorbance /ab·sor·bance/ (-sor´bans)
1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol .

2.
 was measured at 570 nm using a ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.

ELISA
n.
 Reader (RT-2100C, Rayto, USA). The number of living cells correlated with the absorbance (optical density, OD). Experiments were performed in triplicate and repeated four times.

Detection of apoptosis: To analyze the apoptosis induced by [siRNA.sub.HIF-1[alpha]], Tca8113 cells were transfected and incubated under normoxic or hypoxic condition for 24 h compared with those mock transfected cells. After harvested, the cells were washed twice with PBS and resuspended in binding buffer. After incubation for 15 min with 5 [micro]l of Annexin V-FIITC antibody and 10 [micro]l of propidium iodine (50 mg/ml) at room temperature in the dark, the cells were analysed by a flow cytometer (FACSCaliRur+Sort, Becton Dickinson, USA). The presence of apoptotic cells was scored by monitoring the loss of cell membrane phospholipid phospholipid (fŏs'fōlĭp`ĭd), lipid that in its simplest form is composed of glycerol bonded to two fatty acids and a phosphate group.  asymmetry, resulting in the externalization The ability to easily connect to and transfer information between business partners. Increasingly, information systems are designed to make their data available to outside partners and customers. This type of collaboration is expected to be a vital part of IT in the 21st century. See EDI.  of phosphatidylserine to the outer membrane without loss of membrane integrity (11). Tca8113 cells undergoing apoptosis were analyzed by FACS using annexin V and PI to detect both early and late stages of apoptosis.

Cell cycle analysis: Growing to 80 per cent confluence, Tca8113 cells (2 x [10.sup.5]) were incubated under normoxic or hypoxic condition for 24 or 48 h. For analysing the cell cycle of transfected Tca8113 cells, the transfected and mock transfected cells were incubated under hypoxic condition for 24 or 48 h. Cells were washed in ice-cold PBS and harvested by trypsinization. Centrifuged at 350 g for 5 min, the cells were washed with ice-cold PBS and fixed with 70 per cent ethanol overnight at 4[degrees]C. Propidium iodine (10 [micro]g/ml) supplemented with RNaseA (200 [micro]g/ml) was added to the cells for 30 min (at 37[degrees]C) in the dark prior to FACS analysis (Becton Dickinson, USA).

Adhesion assay: The Tca8113 cells (2 x [10.sup.5]) were plated in 6-well plates. Transfected or mock transfected cells were subsequently incubated under normoxic or hypoxic condition for 24 h. Ninety six well plates coated with Matrigel (B&D, USA) were incubated at 4[degrees]C for 30 min and 37[degrees]C for 4 h. The cells in 6-well plates were trypsinized and resuspended in serum-free RPMI 1640. Cells (2x[10.sup.4]) were plated into each well of 96 well plates, and the plates were incubated at 37[degrees]C for 30 min. The medium was removed and non adherent cells were gently removed by pipette and the layer of adhered cells was gently washed with PBS. Cell adhesion was analyzed by MTT colorimetric assay. The number of attached living cells correlated with the absorbance (optical density, OD) measured by a Microplate Reader (RT-2100C, Rayto, USA) at 570 nm. Results were showed as the mean percentage of the control (control OD at 570 nm assigned as 100%). Experiments were performed in triplicate and repeated four times.

Statistical analysis: Statistical analysis was performed using the SPSS A statistical package from SPSS, Inc., Chicago (www.spss.com) that runs on PCs, most mainframes and minis and is used extensively in marketing research. It provides over 50 statistical processes, including regression analysis, correlation and analysis of variance.  11.5 software package. Student's t-test was used for statistical analysis with a significance of P<0.05.

Results

Expression of HIF-1[alpha] in Tca8113 cells and regulation by hypoxia: HIF-1[alpha] mRNA was expressed in Tca8113 cells under normoxic condition as analysed by real-time RT-PCR. Exposed to the hypoxic environment, no significant increase in HIF-1[alpha] mRNA transcript was observed (Fig. 1A). Western blot with monoclonal anti-HIF-1[alpha] demonstrated that HIF-1[alpha] protein showed basal level expression in Tca8113 cell lines under normoxic conditions. A band of HIF-1[alpha] protein was detected at 120 kDa (Fig. 1B). Hypoxia induced a rapid and sustained accumulation of HIF-1[alpha] protein in Tca8113 cells up to 24 h (Fig. 1B)

HIF-1[alpha] expression was suppressed by [siRNA.sub.HIF-1[alpha]]: RNA interference targeting HIF-1[alpha] was performed by transfecting 50nM [siRNA.sub.HIF-1[alpha]] to Tca8113 cells for 24 h. [siRNA.sub.Irr] was used as a nonspecific control. To assess the potency of the [siRNA.sub.HIF-1[alpha]] to block HIF-1[alpha] expression, HIF-1[alpha] expression was measured on mRNA level by real-time RT-PCR and protein level by Western blot and immunofluorescence. Reduction of HIF-1[alpha] mRNA expression by approximately 90 per cent was found in [siRNA.sub.HIF-1[alpha]] treated cells, compared to [siRNA.sub.Irr] treated cells or mock transfection control in both normoxia and hypoxia (Fig. 2A). Likewise, the results of Western blot and immunofluorescence showed that hypoxia-induced HIF-1[alpha] protein was significantly (P<0.01) suppressed in Tca8113 cells treated with [siRNA.sub.HIF-1[alpha]] in comparison to mock transfection controls (Fig. 2B and 2C). These results indicated that [siRNA.sub.HIF-1[alpha]] had a gene-silencing effect targeting HIF-1[alpha] at mRNA and protein level (Table).

[FIGURE 1 OMITTED]

[siRNA.sub.HIF-1[alpha]]-induced changes of biological characteristics in Tca8113 cells:

(i) Knockdown of HIF-1[alpha] inhibits proliferation of Tca8113 cells: Transfected and mock transfected Tca8113 cells were cultured under normoxic or hypoxic condition for 24, 48, or 72 h. When the mock transfected cells were cultured under hypoxic condition for 48 or 72 h, the proliferation of Tca8113 cells was inhibited significantly compared with the normoxic control (0.593 [+ or -] 0.041 vs 0.405 [+ or -] 0.032, 0.874 [+ or -] 0.064 vs 0.255 [+ or -] 0.019, P<0.01). In the first 24 h, the proliferation of Tca8113 cells treated with [siRNA.sub.HIF-1[alpha]] was not inhibited significantly compared with the mock transfected control under normoxic condition (0.354 [+ or -] 0.039 vs 0.347 [+ or -] 0.045). However, the proliferation was inhibited significantly under hypoxic condition (0.252 [+ or -] 0.029 vs 0.322 [+ or -] 0.031, P<0.05). At 48 and 72 h, [siRNA.sub.HIF-1[alpha]] significantly inhibited the proliferation of Tca8113 cells under hypoxic and normoxic condition compare, the mock transfected control (P<0.01) (Fig. 3A).

(ii) Cell cycle analysis of [siRNA.sub.HIF-1[alpha]] treated Tca8113 cells: DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 staining by propidium iodide was done for discrimination of cells in [G.sub.0]/[G.sub.1] phase or S/[G.sub.2] phase by FACS. Hypoxia induced a significant increase of cells in [G.sub.0]/[G.sub.1] phase and a decrease of cells in the [G.sub.2]/M+S phase compared to normoxia. The cells in [G.sub.0]/[G.sub.1] phase were increased significantly under hypoxic condition for 24 or 48 h [(73.37 [+ or -] 4.01)% vs (66.59 [+ or -] 5.25)%, P<0.05; (87.62 [+ or -] 6.54)% vs (61.88 [+ or -] 4.38)%, P<0.01]. Consequently, the proliferation indices were decreased significantly (0.26 [+ or -] 0.07vs 0.33 [+ or -] 0.06, P<0.05; 0.12 [+ or -] 0.01 vs 0.38 [+ or -] 0.02, P<0.01). Cell cycle in [siRNA.sub.HIF-1[alpha]] treated Tca8113 cells and mock transfected controls were analysed. Under hypoxia for 24 or 48 h, a significant (P<0.01) decrease of cells in [G.sub.0]/[G.sub.1] phase and an increase of cells in the [G.sub.2]/M+S phase were found in [siRNA.sub.HIF-1[alpha]] treated Tca8113 cells compared to mock transfected Tca8113 cells. The proliferation index of [siRNA.sub.HIF-1[alpha]] treated Tca8113 cells was increased on an average 66.59 and 150 per cent respectively (Fig. 3B).

[FIGURE 2 OMITTED]

(iii) Apoptosis induced by [siRNA.sub.HIF-1[alpha]] in Tca8113 cells: No significant increase in the percentage of apoptotic cells was found after the mock transfected cells were cultured under hypoxic condition for 24 h when compared with normoxic incubated cells. It was revealed that transfection with [siRNA.sub.HIF-1[alpha]] induced approximately 12.15 and 17.77 per cent of the Tca8113 cells apoptosis when incubated under normoxic or hypoxic condition for 24 h. Significant increase was found as compared to mock transfected control [(12.15 [+ or -] 1.02) per cent vs (4.68 [+ or -] 0.56) per cent; (17.77 [+ or -] 1.22) per cent vs (6.43 [+ or -] 0.55) per cent, P<0.01] (Fig. 3C).

[FIGURE 3 OMITTED]

(iv) Treatment with [siRNA.sub.HIF-1[alpha]] inhibited adhesion of Tca8113 cells: The adhesion of transfected and mock transfected Tca8113 cells under normoxic or hypoxic condition was determined by MTT colorimetric assay. Adhesion of Tca8113 cells to Matrigel-coated cell culture plates in vitro was increased significantly under hypoxic condition compared to that under normoxic condition [(100 [+ or -] 9.8)% vs (140 [+ or -] 12.2) per cent, P<0.05]. When the Tca8113 cells were treated with [siRNA.sub.HIF-1[alpha]], the adhesion of Tca8113 cells was reduced to 74 and 63.6 per cent of that of the mock transfected control under normoxic or hypoxic condition respectively (P<0.01) (Fig. 3D).

Discussion

Hypoxia exerts a biologically inducible stress and a strong selection pressure in a tumour (12). In the present study, we analyzed the proliferation, cell cycle, apoptosis and adhesion of Tca8113 cells which were correlated to the growth, survival and metastasis of tumours. When the Tca8113 cells were cultured under hypoxic condition the proliferation was inhibited. There was no significant increase in the per cent of apoprotic cells, but we observed a significant increase of cells in [G.sub.0]/[G.sub.1] phase and a decrease of cells in the S/[G.sub.2] phase which indicated the [G.sub.1]/S arrest. These findings reflected a hypoxia-induced growth arrest in Tca8113 cells, which was similar to observation in other cell types (13). The ability to adhere to Matrigel was significantly increased under hypoxic condition. Therefore, the different biological characteristics of Tca8113 cells could account for the tumour cells survival and aggressive phenotype under hypoxic condition.

It has been reported that HIF-1[alpha] is regulated by hypoxia (14). It transactives hundreds of target genes encoding proteins that are required for angiogenesis, regulation of blood vessel tone, and vascular remodelling; cell proliferation and viability; erythropoiesis erythropoiesis /eryth·ro·poi·e·sis/ (-poi-e´sis) the formation of erythrocytes.erythropoiet´ic

e·ryth·ro·poi·e·sis
n.
The formation or production of red blood cells.
 and iron metabolism; glucose transport and glycolysis (5). Therefore, HIF-1[alpha] plays a critical role as a key transcriptional factor in the survival of cells. The vast majority of human tumour cells has shown overexpression of HIF-1[alpha] (15,16). In head and neck carcinomas, it has been reported that squamous cell carcinoma cells in tumour tissues overexpress HIF-1[alpha] protein and it is associated with the microvessel vascular density and vascular endothelial growth factor Vascular endothelial growth factor (VEGF) is an important signaling protein involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature).  (VEGF VEGF vascular endothelial growth factor. ) expression (16). In the present study, we demonstrated that HIF-1[alpha] enhanced malignant phenotype of cultured human tongue squamous cell carcinoma cells line Tca8113 under hypoxic condition. Using siRNA technique, dysfunction of HIF-1[alpha] abolished the increase of aggressive potential induced by hypoxia in cultured Tca8113 cells.

The hypoxia-inducible transcription factor subtype HIF-1[alpha] was found to be upregulated in hypoxic Tca8113 cells and exhibited nuclear translocation. We demonstrated that a low level of HIF-1[alpha] mRNA and protein were constitutively expressed in Tca8113 cells under normoxic condition. These findings support the contention that maintaining a constitutive level of HIF-1[alpha] is essential to adaptive cellular functions, such as cell proliferation and survival in the hostile microenvironment of tumour (17). Exposure of Tca8113 cells to hypoxia induced a mild non significant increase in the expression of HIF-1[alpha] mRNA. HIF-1[alpha] protein increased steadily up to 24 h and exhibited nuclear translocation after exposure to hypoxia. As a key transcriptional factor of hypoxic regulation, HIF-1[alpha] was upregulated mainly on protein level in Tca8113 cells under hypoxic condition. It could be presumed that overexpression of HIF-1[alpha] allowed Tca8113 cells to resist stresses and show aggressive phenotype

In the present study, we silenced the function of HIF-1[alpha] with the technique of siRNA to evaluate the role of HIF-1[alpha] in progressior, of cultured Tca8113 cells induced by hypoxia. Tca8113 cells were transfected with siRNA duplexes targeting HIF-1[alpha] mRNA ([siRNA.sub.HIF-1[alpha]]) followed by cultured under hypoxic condition. The efficacy of interference was assessed by the ability of the duplexes to knock down HIF-1[alpha] mRNA by real-time RT-PCR. The specificity of the siRNA duplexes was demonstrated by the transfection of [siRNA.sub.Irr]. When the Tca8113 cells were transfected with 50 nM [siRNA.sub.HIF-1[alpha]] mRNA and protein expression were significantly suppressed, when compared to [siRNA.sub.Irr] treated cells (nonspecific control) or mock transfection control in both normoxia and hypoxia.

The association of HIF-1[alpha] with proliferation has been noted before, but it is still not fully understood (18). Hypoxia is believed to arrest tumour cell proliferation (19), however, it was proved that growth of HIF-1[alpha] tumours was not retarded but was accelerated, owing to increased hypoxic stress-induced proliferation (20). In our present study, significant increase of the [G.sub.0/1] phase cells was found under hypoxia after 24 or 48 h exposure to 1 per cent oxygen. It is suggested that Tca8113 cells subjected to hypoxia arrested in [G.sub.1]. The proliferation index of [siRNA.sub.HIF-1][alpha] treated Tca8113 cells was on average 66.59 or 150 per cent increase than that in the control population. HIF-1[alpha] was the master regulator to cellular adaptation in Tca8113 cells during hypoxia, meanwhile, endoplasmic reticulum reticulum /re·tic·u·lum/ (re-tik´u-lum) pl. retic´ula   [L.]
1. a small network, especially a protoplasmic network in cells.

2. reticular tissue.
 (ER) stress could be induced and resulted in inhibition of protein translation. Inhibition of protein synthesis could induce the cells to enter the cell cycle, thus promoting mitogenic responses. Therefore, the proliferation of Tca8113 may be an integrated stress response involving an ER-generated signal and the cellular adaptation to hypoxia.

Besides the role in angiogenesis (21), HIF-1[alpha] has been reported to be related to apoptosis (22). It has been reported that under severe or prolonged hypoxic condition, the stabilization of p53 protein is HIF-1[alpha] dependent, which in turn leads to the induction of apoptosis (23). Another study showed that HIF-1[alpha] might have a protective role in limiting hypoxia-induced apoptosis (17). Pancreatic cancer cell lines that constitutively expressed HIF-1[alpha] were more resistant to apoptosis induced by hypoxia than similar cell lines that lacked constitutive expression of HIF-1[alpha] (24). In addition, it has been demonstrated that the inhibition of apoptosis promotes the mitotic mitotic

pertaining to mitosis.


mitotic activity
degree to which a cell population is proliferating; used as an index of tumor aggression.
 progression in cancer cells (25). It suggests that HIF-1[alpha] possesses dual functions in mediating apoptosis of tumour cells, antiapoptotic and pro-apoptotic function. Using siRNA to evaluate the role of HIF-1[alpha] on apoptosis of cultured human Tca8113 cell line under hypoxia, the amount of apoptotic cells were significantly increased in [siRNA.sub.HIF-1[alpha]] transfected Tca8113 cells compared to that of mock transfection or [siRNA.sub.Irr] transfection under normoxic or hypoxic condition. These results suggested that the apoptotic induction of Tca8113 cells was a specific outcome of HIF-1[alpha] loss-of function.

Adhesion of cancer cells to extracellular matrices (ECM (1) (Enterprise Change Management) See version control and configuration management.

(2) (Error Correcting Mode) A Group 3 fax capability that can test for errors within a row of pixels and request retransmission.
) is known to be involved in the haematogenous metastasis of cancer (26) and tumour progression (27). Hypoxia is known to enhance the metastatic potential and invasiveness of tumour cells. We determined the adhesion of hypoxia-exposed cultured Tca8113 cells and analysed the role of HIF-1[alpha] on this response. When Tca8113 cells were exposed to low-oxygen environments, their ability to adhere to the reconstituted basement membrane (Matrigel) was significantly increased with increased expression of HIF-1[alpha]. Treatment with [siRNA.sub.HIF-1[alpha]] significantly reduced adhesion of Tca8113 cell to Matrigel. It suggested that HIF-1[alpha] may play a major role in regulating the hypoxia-induced adhesion of Tca8113 cells to Matrigel in vitro. The potential mechanism might be transcription of downstream genes to induce a set of adhesive molecule expression, which mediated adhesion to ECM to facilitate metastasis and progression of tumour (28).

Our results indicated that hypoxic Tca8113 cells were more aggressive and HIF-1[alpha] overexpression regulated crucial biological response to hypoxia including proliferation, cell cycle, adhesion, apoptosis, whereas application of chemically synthesized siRNAs markedly attenuated these features under hypoxic condition. Our data suggest that [siRNA.sub.HIF-1[alpha]] could specifically block HIF-1[alpha] gene expression, induce cell apoptosis, inhibit the growth and adhesion of Tca8113 cells, consequently abolished the malignant potential of carcinoma cells. We observed the effects of HIF-1[alpha] on the adaptation of Tca8113 cells to hypoxia in vitro, however, the specific signaling pathways by which HIF-1[alpha] regulated the crucial biological response to hypoxia and the effects of HIF-1[alpha] in tongue squamous cell carcinoma in vivo need to be discovered. In conclusion, HIF-1[alpha] gene can be regarded as a good target gene in genetic therapy for human tongue squamous cell carcinoma and the use of [siRNA.sub.HIF-1[alpha]] deserving further investigations as a new therapeutic strategy for hypoxia related diseases and cancer.

Acknowledgment

This work was supported by The National Natural Science Foundation of China (Grant No.30572056). Authors thank Meixiang Yang, Shi Yan, Jintang Sun, Qianqian Shoo, Bingfeng Song and Haibao Song for their excellent technical assistance.

Received December 24. 2007

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  • James C. Dozier, Medal of Honor Recipient
  • Kimberly Dozier, CBS News correspondent
  • Lamont Dozier, musician
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Reprint requests: Dr Shanzhen Sun, Department of Pathology, Stomatology stomatology /sto·ma·tol·o·gy/ (sto?mah-tol´ah-je) the branch of medicine that deals with the mouth and its diseases.stomatolog´ic
stomatology
 College of Shandong University 44#, Wenhua Xi Road Jinan 250 012, China e-mail: songying0829@sina.com

Ying Song *(+), Wenxia Wang **, Xun Qu (#) & Shanzhen Sun *

* Department of Pathology, Stomatology College of Shandong University, Jinan; (+) Department of Pathology, Medical College of Qingdao University, Qingdao; ** Department of Periodontology periodontology,
n See periodontics.
, Stomatology College of Shandong University, Jinan & (#) Institute of Basic Medical Sciences, Qilu Hospital, Jinan, China
Table. The effect of si[RNA.sub.HIF-1[alpha]] on cell cycle of
Tca8113 cells

              Cell cycle distribution (means [+ or -] SD %)

Groups             [G.sub.0/1]                   S

Nox24         66.59 [+ or -] 5.25        32.05 [+ or -] 3.27
Nox24         73.37 [+ or -] 4.01 (#)    20.83 [+ or -] 3.22
Nox48         61.88 [+ or -]4.38         30.9 [+ or -] 2.19
Nox48         87.62 [+ or -] 6.54 (##)   10.74 [+ or -] 1.89
Mock-Nox24    76.34 [+ or -] 6.96        19.17 [+ or -] 3.11
RNAi-Nox24    58.92 [+ or -] 5.01 *      26.78 [+ or -] 1.73
Mock-Nox48    87.26 [+ or -] 9.07        10.23 [+ or -] 0.98
RNAi-Hox4R    68.2 [+ or -] 5.36 *       28.55 [+ or -] 2.23

                  Cell cycle
              distribution (means
               [+ or -] SD %)

Groups           [G.sub.2]/M              Proliferation index

Nox24         1.36 [+ or -] 0.19         0.33 [+ or -] 0.06
Nox24         5.80 [+ or -] 0.68         0.26 [+ or -] 0.07 (#)
Nox48         7.22 [+ or -] 0.56         0.38 [+ or -] 0.02
Nox48         1.64 [+ or -] 0.27         0.12 [+ or -] 0.01 (##)
Mock-Nox24    4.49 [+ or -] 0.52         0.24 [+ or -] 0.03
RNAi-Nox24    14.3 [+ or -] 1.05         0.41 [+ or -] 0.05 *
Mock-Nox48    2.51 [+ or -] 0.54         0.12 [+ or -] 0.04
RNAi-Hox4R    3.25 [+ or -] 0.20         0.31 [+ or -] 0.02 *

(#) P<0.05; (##) P<0.01; compared with the Nox group

* P<0.01 compared with the Mock group
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Author:Song, Ying; Wang, Wenxia; Qu, Xun; Sun, Shanzhen
Publication:Indian Journal of Medical Research
Article Type:Clinical report
Geographic Code:9INDI
Date:Feb 1, 2009
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