Background
Hypoxia is one of the most important pathological characteristics of solid tumor which is the result of imbalance between tumor cell proliferation and blood supply [
1]. As solid tumor growing, its center becomes a hypoxic area because of lacking blood and oxygen. The hypoxic status of various solid tumor has been recognized as an important determinant for the outcome of anti-cancer therapies in a number of tumors [
2].
Hypoxia-inducible factor-1 (HIF-1) was found in the 1992 when Semenza [
3] researched the expression of erythropoietin gene induced by hypoxia. Human HIF-1 has been depurated and isolated, it is a heterodimeric transcription factor composed of oxygen-dependent HIF-1α and constitutively expressed HIF-1β subunits, HIF-1 transcriptional activity is largely determined by regulated expression of the HIF-1α subunit [
4]. HIF-1α over-expression has been detected in various tumors including breast, oropharyngeal, nasopharyngeal, prostate, brain, lung, stomach cancer and so on, and has been associated with tumor aggressiveness, vascularity, treatment failure and mortality [
5‐
7]. Interestingly, HIF-1α can also over-expressed under normoxic conditions in some human tumors [
8].
In this research, we treated a human pancreatic cancer cell line (PC-2) with cobalt chloride (CoCl2) to stimulate hypoxia in vitro. Under the hypoxic condition, we observed the proliferation of PC-2 cells by MTT assay. Meanwhile, RT-PCR and Western blot analysis were conducted to measure the expression of HIF-1α on mRNA and protein level. Furthermore, we discussed the effect of hypoxic microenvironment on apoptosis and its mechanism.
Materials and methods
Reagents
Fetal bovine serum (Gibco, USA); RPMI1640 medium (Gibco, USA); 3-(4,5) -dimethylthiahiazo(-z-y1)-3,5- diphenyte- trazoliumromide (MTT) (Gibco, USA); annexin V-FITC/PI apoptosis detection kit (Becon Dickinson Facsalibur, USA); RT-PCR kit (ampliqon, Denmark); Trizol (Invitrogen, USA); HIF-1α monoclonal antibody (Santa Cruz Biotechnology, USA); 3-(5'-hydroxymethyl-2'-furyl)-1 -benzylindazole (YC-1) (Shanghai Shenggong Biological Engineering Technology&Service, China); CoCl2 (Shanghai Shenggong Biological Engineering Technology&Service, China).
Cell line and cell culture
Human pancreatic cancer cell line, PC-2, was purchased from the medical experimental animal center of the fourth military medical university. Cells were cultured in RPMI 1640 maximal medium containing 10% inactived fetal bovine serum (56°C, 30 min), 1 × 105 U/L penicillin and 100 mg/L streptomycin in a humidified atmosphere with 5% CO2 incubator at 37°C.
MTT assay for the proliferation of PC-2 cells
The proliferation of PC-2 cells was assessed using MTT dye reduction assay (Sigma, USA), which was conducted as described previously [
9]. PC-2 cells were seeded in a 96-well plate at a density of 1 × 10
4 cells/well, cultured for 12 h under 37°C in 5% CO
2, then treated with different concentration (50, 100, 150, 200 μmol/L) CoCl
2 for 24-120 h. At the end of the treatment, MTT, 50 μg/10 μL, was added and the cells were incubated for another 4 hours. Dimethylsufloxide (DMSO; 200 μl) was added to each well after removal of the supernatant. After shaking the plate for 10 min, cell viability was assessed by measuring the absorbance at 490 nm using an Enzyme-labeling instrument (EX-800 type); all measurements were performed three times. Cell growth curve was completed using time as the abscissa and
A value (mean ± SD) as the ordinate.
Detection of morphological change by transmission electron microscope
Uranyl acetate and lead citrate staining of cells were performed to detect morphological changes. Briefly, adherent PC-2 cells were treated with 200 μmol/L CoCl
2 for 48 hours. After treatment, the treated cells were digested with pancreatin and fixed with 3% glutaraldehyde precooled in 4°C for 2 hours. To make ultra-thin sections of copper, cells were washed with phoisphate-buffered salein (PBS) once, fixed with 1% osmic acid for 1 hour, dehydrated by acetone and embedded in epoxide resin. After staining with uranyl acetate and lead citrate, the sections were examined by a Hitachi-800 transmission electron microscope [
10].
Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) assay
PC-2 cells were seeded in 6 cm culture capsules and treated with concentration gradient CoCl
2 (0, 50, 100, 150, 200 μmol/L) separately for 8 h. In the group of 200 μmol/L, we selected cells at 0 h, 4 h, 8 h and 12 h point for further experiment. And then treated with 2.0 μmol/L YC-1 (0, 50, 100, 150,) for 2 h. As previously described [
11], cells collected at specified time were used to extract total RNA using the Trizol reagent following the manufacturer's instructions. 1 μgRNA synthetized cDNA through reverse transcriptase undergo listed below condition: 70°C 5 min, 42°C extended for 60 min, 95°C enzyme inactivated for 3 min and 4°C terminated reaction. Synthetical cDNA as template to carry out polymerase chain reaction. HIF-1α primer sequence (Invitrogen CO): 5'-ACTTCTGGATGCTGGTGATT-3' (sense) and 5'-TCCTCGGCTAGTTAG GGTAC -3' (anti-sense), amplification fragment was 325 bp, renaturation temperature was 55°C (cycling 35 times). β-actin, its primer sequence was 5'-GTTGCGTTACACCCTTTCTTG-3' (sense), 5'-TGCTGTCACCTTCACCGT TC-3' (anti-sense), amplification fragment was 133 bp, and renaturation temperature was 55°C (cycling 40 times). Amplification condition was below: pre-denaturized for 3 min at 95°C, denaturized for 30s at 95°C, renaturated for 30s at 55°C and extended for 30s at 72°C. PCR product was detected on agarose gel electrophoresis and ethidium bromide imaging system was used to make density index analysis. The expression intensity of HIF-1α mRNA was denoted with the ratio of the photodensity of the RT-PCR products of HIF-1α and β-actin.
Western blot analysis
As previously described [
12], cells were washed with ice-cold PBS twice and lysed with lysis buffer containing 1% NP40, 137 mM NaCL, 20 mM Tris base(pH7.4), 1 mM DTT, 10% glycerol, 10 mg/mL Aprotinin, 2 mM sodium vanadate and 100 μM PMSF. Protein concentrations were determined using the PIERCE BCA protein assay kit. Protein was separated by 10% SDS-PAGE under denaturing conditions and transferred to nitrocellulose membranes. Membranes were incubated with an mouse HIF-1α monoclonal antibody (1:1000; Santa Cruz Biotechnology), followed by incubation in goat antimouse secondary antibody conjugated with horseradish peroxidase (1:1000; Santa Cruz Biotechnology). Immunoreactive proteins were visualized using enhanced chemiluminescence detection system (Amersham Biosciences)
Apoptosis detection by FCM
Apoptotic cells were differentiated from viable or necrotic ones by combined application of annexin V-FITC and propidium iodide (PI) (BD Biosciences Clontech, USA) [
13]. The samples were washed twice and adjusted to a concentration of 1 × 10
6 cells/mL with 4°C PBS. The Falcon tubes (12 mm × 75 mm, polystyrene round-bottom) were used in this experiment, 100 μL of suspensions was added to each labeled tube, 10 μL of annexin V-FITC and 10 μL PI(20 μg/mL) were added into the labeled tube, incubated for at least 20 min at room temperature in the dark, then 400 μL of PBS binding buffer was added to each tube without washing and analyzed using FCM analysis (BD Biosciences Clontech, USA) as soon as possible (within 30 min). This assay was done quintuplicate.
Statistical analysis
All data were expressed by mean ± S.E.M. Statistical analyses were performed using SPSS 11.0 for Windows software. ANOVA (one-way analysis of variance) and Student's t-test were used to analyze statistical differences between groups under different conditions. P-value < 0.05 was considered statistically significant.
Discussion
More recently, experimental and clinical studies demonstrated that intra-tumor hypoxia might be a key factor in tumor microenvironment promoting invasive growth and metastasis [
14]. The increased malignancy of hypoxic tumors has been attributed to the ability of hypoxia to select for cells with diminished apoptotic potential and to induce their clonally expansion [
15]. Since the hypoxic phenomenon in tumors was revealed, more and more evidence indicated hypoxia existed in solid tumor generally [
16].
Pancreatic cancer is common malignant tumor of digestive system which has high malignancy, difficulty in treatment and poor prognosis. And less than 10% of pancreatic cancer is resectable when being diagnosised and 5-year overall survival rate is less than 5% [
17]. During the development of pancreatic cancer, the blood can't supply the tumor nourishment, thus the tumor are hypoxic partly, while hypoxia makes the tumor cell more malignant. In this way, the rapid growth and the hypoxia are unity of opposites in tumors [
18].
CoCl
2 is a chelator which instead of Fe
2+ in hemoglobin, and then damage cell's reception of oxygen [
19]. The mechanism of CoCl
2 simulating hypoxia is similar with hypoxic microenvironment
in vivo, because they have identical signal transduction and transcription regulation. Moreover previous research demonstrated CoCl
2 correlated with proliferation and apoptosis in human carcinoma cells [
20,
21]. In our study, we treated PC-2 cells with CoCl
2 to simulate hypoxic microenvironment, MTT assay revealed along with the increased CoCl
2 concentration, the exponential phase of PC-2 cells was earlier in advanced and persisted shorter, cells grew slower and went into platform period early(Figure
1). It is reasonable to assume that the step down in PC-2 cell proliferation correlated with the increased hypoxia, hypoxic microenvironment could slow down the speed of tumor growth.
HIF-1α, a transcription factor regulating genes' expression induced by hypoxia, is a key molecular player in the hypoxic response [
22]. HIF-1α is generally resided in mammal and human tissue in hypoxic condition, it has been found over-expressed in about 70% tumor [
5‐
7]. Experiment showed that under hypoxic the transcriptive activity of HIF-1α was increasing, which indicated that hypoxic microenvironment might increase the genetic transcriptional level of HIF-1α to regulate the expression of downstream gene [
22,
23]. However, some scholars presumed hypoxic microenvironment could enhance the stability of HIF-1α [
24]. Our present research indicated HIF-1α obviously increased at both protein level and mRNA level in PC-2 cells under hypoxic microenvironment, and it was positive correlated with the hypoxic time and the density of CoCl
2. This suggested the level of hypoxia was coinciding with the expression of HIF-1α.
Whether HIF-1α can promote tumor cell apoptosis or anti- apoptosis, the opinion didn't reach unify, different research suggest converse results. Some date indicated overexpressed HIF-1α could promote apoptosis by activating Bcl-2 and Bcl-Xl or enhancing the stability of p53 [
25]. On the other hand, experiment displayed HIF-1α could up-regulate the VEGF and GLUT1 to make tumor cell resist to apoptosis, inhibition of HIF-1α could promote apoptosis [
26]. In our research, under electron microscope, PC-2 cells in hypoxic microenvironment were found in different apoptotic stage (Figure
2A-D), most were in early stage. The FCM analysis showed that the apoptotic rate of normal control group, 100 μmol/L group, 150 μmol/L group and 200 μmol/L group, was 10.77%, 34.32%, 40.17%, 52.30%, respectively. These results were consistent with Luo's research [
27].
In conclusion, our study suggested that hypoxic microenvironment can effectively induce apoptosis and influence cell proliferation in PC-2 cells, and the mechanism may be concerned with the up-regulation of HIF-1α.
Authors' contributions
DZJ and WXJ designed the research. DZJ, GJ, MXB, YK and KHF performed the experiments throughout this research. LXX, JZZ and GHT contributed to the reagents, and participated in its design and coordination. DZJ and GJ analyzed the data; DZJ and MXB wrote the paper. Co-first authors: DZJ and GJ. All authors have read and approved the final manuscript.