HIF-1α effects on angiogenic potential in human small cell lung carcinoma
- Jun Wan†1,
- Huiping Chai†1Email author,
- Zaicheng Yu1Email author,
- Wei Ge1,
- Ningning Kang1,
- Wanli Xia1 and
- Yun Che1
© Wan et al; licensee BioMed Central Ltd. 2011
Received: 4 May 2011
Accepted: 15 August 2011
Published: 15 August 2011
Hypoxia-inducible factor-1 alpha (HIF-1α) maybe an important regulatory factor for angiogenesis of small cell lung cancer (SCLC). Our study aimed to investigate the effect of HIF-1α on angiogenic potential of SCLC including two points: One is the effect of HIF-1α on the angiogenesis of SCLC in vivo. The other is the regulation of angiogenic genes by HIF-1α in vitro and in vivo.
In vivo we used an alternative method to study the effect of HIF-1a on angiogenic potential of SCLC by buliding NCI-H446 cell transplantation tumor on the chick embryo chorioallantoic membrane (CAM) surface. In vitro we used microarray to screen out the angiogenic genes regulated by HIF-1a and tested their expression level in CAM transplantation tumor by RT-PCR and Western-blot analysis.
In vivo angiogenic response surrounding the SCLC transplantation tumors in chick embryo chorioallantoic membrane (CAM) was promoted after exogenous HIF-1α transduction (p < 0.05). In vitro the changes of angiogenic genes expression induced by HIF-1α in NCI-H446 cells were analyzed by cDNA microarray experiments. HIF-1α upregulated the expression of angiogenic genes VEGF-A, TNFAIP6, PDGFC, FN1, MMP28, MMP14 to 6.76-, 6.69-, 2.26-, 2.31-, 4.39-, 2.97- fold respectively and glycolytic genes GLUT1, GLUT2 to2.98-, 3.74- fold respectively. In addition, the expression of these angiogenic factors were also upregulated by HIF-1α in the transplantion tumors in CAM as RT-PCR and Western-blot analysis indicated.
These results indicated that HIF-1α may enhance the angiogenic potential of SCLC by regulating some angiogenic genes such as VEGF-A, MMP28 etc. Therefore, HIF-1α may be a potential target for the gene targeted therapy of SCLC.
Hypoxia inducible factor-1 alpha (HIF-1α) is a member of the HIF-1 gene family, it is highly expressed in hypoxic conditions and degraded in normoxic condition [1, 2]. HIF-1α activation is a common feature of tumors [3, 4]; it is generally more pronounced in aggressive tumors  and can be an independent predictor of poor prognosis in certain types of cancer . This is primarily due to the fact that HIF-1α plays a major role in the development of a characteristic tumor phenotype influencing growth rate, angiogenesis, invasiveness, and metastasis. Of these characteristics, angiogenesis is the most significant because it is essential for the other biological characteristics . Several investigation about the angiogenesis of some kinds of malignant tumors such as breast and prostate cancer , head and neck cancer  have demonstrated that it is an intricate multistep and temporally ordered process that involves a great number of genes, modifiers and pathways regulated by HIF-1α. Some of these genes are directly induced by HIF-1α, such as NOS(nitric oxide synthases), angiogenic and vascular growth factors(VEGF) and urokinasetype plasminogen activator receptor (uPAR). Others are indirectly regulated by HIF-1α and might be influenced by secondary mechanisms. SCLC exhibits high expression levels of HIF-1α [10, 11] and early hematogenous metastasis to other organs, such as brain, kidney, and liver, which relies on tumor angiogenesis . However, the effect of HIF-1α on the angiogenic potential and regulation of angiogenic gene expression levels that influence this biological process have not been previously reported. In our study, we will use appropriate experimental methods to investigate these points.
For the in vivo study, we used the chick embryo chorioallantoic membrane (CAM) as the experimental model. CAM is an easily accessible and highly vascularized structure lining the inner surface of the egg shell that has been used to measure the invasive and angiogenic properties of tumor cell xenografts for the loss of the mature immune system in the early phase of development [13, 14]. Several studies have investigated the formation of CAM vessels at different stages of development [15–17]. In this model, tumor cells are grafted to the CAM to reproduce the tumor characteristics in vivo including tumor mass formation, angiogenesis, and metastasis. Tumor explants and tumor cell suspensions have been shown to invade the chorionic epithelium and to form visible masses within 3 d to 5 d. After implantation and transplantation, the tumors can be macroscopically observed in the CAM . Moreover, the growth and angiogenic responses of the transplantation tumors can be examined using microscopy and quantified for analysis. Therefore, the CAM model is an ideal model for cancer research [19, 20].
With regard to the possible difference of growth and angiogenic responses after transduction by HIF-1α or siHIF-1α into SCLC cells, we think that HIF-1α may regulate the expression of some genes responsible for these biological characteristics. To identify these genes and confirm if HIF-1α influence the growth, invasiveness and angiogenesis of SCLC cells by up- or down-regulation of these genes involved in these activity, first we screened human gene chips containing 54614 unique cDNA clones using cDNA prepared from mRNA of SCLC cells in all the experimental groups. After these genes were screened out we continued to measure their expression levels in the xenografts formed by SCLC cells in the CAM by Transcriptase-polymerase chain reaction (RT-PCR) and Western-blot analysis. This study investigated the effect of HIF-1α on the angiogenic potential of the SCLC cells at histological, morphological, and molecular levels. Furthermore, this study demonstrated that HIF-1α may be used as a potential target for the treatment of SCLC in the future.
Cell culture and transduction with Ad5-HIF-1α and Ad5-siHIF-1α
In vivo CAM assay
For the in vivo study, we used the CAM as an experimental vector to evaluate different tumor parameters. Four-day-old fertilized white leghorn chicken eggs (50 g-65 g) were incubated under 60% relative air humidity at 37°C and were rotated hourly with standing. On the third day of incubation, an irregular window (2 × 1.5 cm) was made on the top of the air chamber at the large, blunt end of the egg. A 21-gauge needle was used to puncture the endoconch membrane. Sterilized saline (0.1 ml) was administrated by injection to detach the endoconch membrane from the CAM. A second air chamber, called the flase air chamber (distinguished from the autospecific air chamber), was set up between these two membranes. The transduced and non-transduced cell suspensions (5 × 104 cells/μl) were gently pipetted onto the CAM surface with a transfer pipette. The eggs were then placed in the incubator. The engraftment growth was observed, and the tumor volume was calculated from day 4 to day 17 using the following formula: tumor volume (mm3) = (tumor length × width2)/2. The following three experimental groups that contained 12 samples each were used in this study: NCI-H446 group (control group), NCI-H446/Ad group, NCI-H446/Ad-siRNA group, NCI-H446/HIF-1α group, and NCI-H446/siHIF-1α group. The results were analyzed using a t-test and one-way ANOVA. The angiogenic responses were evaluated from day 8 to day 17 using a stereomicroscope connected to an image analyzer system in NCI-H446/Ad group (control group), NCI-H446/HIF-1α group, and NCI-H446/siHIF-1α group. Several parameters of angiogenesis, such as vessel area and number of vessel branches, were quantified by MIQAS quantified system analysis. For each study group, approximately 10 to 15 domains were selected for vessel quantification, and the mean values of the vessel number and vessel density were calculated.
Histological assessment of transplantation tumors in the CAM
In order to identify the pathobiological characteristics of the transplantation tumors in the CAM, hematoxylin-eosin (HE) staining was used to evaluate the structure of the tumors and peripheral tissues. Neuron-specific enolase (NSE) is a specific marker of neuroendocrine tumor cells, such as SCLC cells, and is used as an important monitoring index in clinical diagnosis and therapy. Immunohistochemical analysis was performed to measure the expression of NSE. All tumor tissue sections from the paraffin blocks were deparaffinized, and endogenous peroxidases were inhibited with 0.3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was achieved using 0.05% protease XIV at 37°C for 5 min. Sections were then incubated at room temperature for 1 h with a mouse anti-human NSE primary antibody (1:40 dilution; Wuhan Boster Biological Engineering Technology Co. Ltd.), rinsed with PBS, and incubated with a biotin-conjugated rabbit anti-mouse secondary antibody at room temperature for 45 min. The sections were subsequently incubated with a streptavidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) at room temperature for 45 min. The reaction was visualized using chromogen diaminobenzidine (DAB) for 10s. Sections were counterstained with haematoxylin, dehydrated, and permanently mounted.
RNA extraction, microarray hybridization and data analysis
For the in vitro study, cDNA microarray technology was used to evaluate the change in the gene expression profile of NCI-H446 SCLC cells after transduction with Ad5-HIF-1α or Ad5-siHIF-1α and screened out the angiogenesis-related genes with differential expression. NCI-H446 cells were transduced with Ad5-HIF-1α or Ad5-siHIF-1α for 60 h. Afterwards, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with 3 ml Trizol (Invitrogen, San Diego, CA, USA). Total RNA was extracted and purified using the RNAeasy kit according to the manufacturer's protocol (Qiagen, USA). The concentration of total RNA was measured with Biophotometer (Eppendorf, Germany) and the quality of purified RNA was confirmed by agarose gel electrophoresis. cDNA was then synthesized from each RNA sample using a SuperScript kit (Invitrogen), and the cDNA was used as a template for the preparation of biotin-labeled cDNA according to the GeneChip Labeling Kit protocol. The biotin-labeled cDNA was hybridized with a GeneChip (Human Genome U133 plus 2.0), washed, and stained with phycoerythrin-streptavidin according to the manufacturer's protocol. The microarray contained 54614 human gene probe sets, each of which consisted of 11 probe pairs corresponding to a single mRNA transcript. After saved as raw image files all the datas were converted into probe sets and analyzed by the software GCOS base on the method of normalization. Annotation by Unigene database http://www.ncbi.nlm.nih.gov/unigene, gene number, gene symbol and gene description were carried out using the database http://strubiol.icr.ac.uk/extra/mokca/ and Affymetrix databases . The expression levels of angiogenic genes were presented as the ratio of the levels in the Ad5-HIF-1α group or Ad5-siHIF-1α group to the Ad5 control group. Ratio values greater than a 2-fold increase or decrease (p < 0.05) was considered to be significant expression changes. The primary data sets are all available at the following website: http://www.ncbi.nlm.nih.gov/gene
Transcriptase-polymerase chain reaction (RT-PCR) analysis
PCR reaction conditions and primer sequences
sense 5'- TTGATGGACTCCCTAAGGC-3'
Western blot analysis
On day 17 of incubation, the transplantation tumors and peripheral tissues of the CAM were harvested and homogenized in lysis buffer (50-mmol/L Tris, pH 7.4; 100-μmol/L EDTA; 0.25-mol/L sucrose; 1% SDS; 1% NP40; 1-μg/ml leupeptin; 1-μg/ml pepstatin A; and 100-μmol/L phenylmethylsulfonylfluoride) at 4°C. The protein was electrophoresed on SDS poly-acrylamide gels and transferred to a PVDF membrane. The membranes were then blocked at room temperature for 1 h with 5% non-fat milk in Tris-buffered saline containing Tween 20 (TBST) followed by incubation with rat anti-human and rat anti-chicken primary antibodies against VEGF-A (Wuhan Boster Biological Engineering Technology Co. Ltd.) overnight at 4°C. The membranes were subsequently incubated with goat anti-rat peroxidase- conjugated secondary antibodies. Immunoreactivity was detected by an enhanced chemiluminescence kit and was captured on X-ray film.
All values were presented as means ± standard deviation (SD). The Student's t-test or one-way ANOVA was used to compare the parameters between the different study groups. P-values less than 0.05 were considered statistically significant. The statistical analyses were performed by the Windows SPSS 13.0 software.
Implantation of cells on CAM in vivo
Quantification of vessel area and the number of vessel branches around the transplantation tumor
Vessel length (pixels)
Control (n = 10 × 4)
2106 ± 143
1967 ± 113
1457 ± 135
2183 ± 156
NCI/H446(n = 10 × 4)
2452 ± 117
2564 ± 96*
2687 ± 103*
2798 ± 135*
NCI/H446/HIF-1α(n = 15 × 4)
2742 ± 83
2814 ± 154
2910 ± 137§
2994 ± 124§
NCI/H446/siHIF-1α(n = 12 × 4)
2331 ± 53#
2268 ± 106#
2236 ± 162#
2203 ± 116#
Vessel Branch points
Control (n = 10 × 4)
76 ± 5
82 ± 9
73 ± 8
89 ± 5
NCI/H446(n = 10 × 4)
92 ± 7
101 ± 11
105 ± 6*
117 ± 7*
NCI/H446/HIF-1α(n = 15 × 4)
116 ± 16
123 ± 11§
128 ± 9§
134 ± 21§
NCI/H446/siHIF-1α(n = 12 × 4)
82 ± 5#
87 ± 6#
92 ± 11#
102 ± 13#
Regulation of angiogenic gene expression by HIF-1α
The effect of HIF-1α on angiogenic gene expression
(ratio ≥ 2, p < 0.05)
Tenascin C (hexabrachion)
Interleukin 6 (interferon, beta 2)
Vascular endothelial growth factorA
Tumor necrosis factor, alpha-induced protein 6
Platelet derived growth factor C
Interleukin 1 receptor, type I
Heme oxygenase (decycling) 1
Matrix metallopeptidase 28
Matrix metallopeptidase 14
Glucose transporter 1
Glucose transporter 2
Suppressor of cytokine signaling 2
Insulin-like growth factor binding protein 3
Insulin-like growth factor 1 receptor
Cysteine-rich, angiogenic inducer, 61
RT-PCR analysis for angiogenic factors in CAM
Western blot analysis for VEGF-A expression
Gene transduction of SCLC cells by HIF-1α
With regard to SCLC, a common pulmonary solid tumor, angiogenesis regulated by HIF-1α may have an important role in determining tumor phenotypes. In order to recapitulate the effect of HIF-1α in a hypoxic environment, we overexpressed human HIF-1α in SCLC NCI-H446 cells with the gene vector Ad5-based transduction system. The type 5 adenovirus-based transduction system is a transient expression system that allows protein expression in transduced cells to reach a higher level than the level found in non-transduced cells in a short period of time, which can reduce the possibility of experimental error to some extent . According to our previous study, we used the appropriate plaque-forming unit (pfu) (MOI = 50) for a high expression level of HIF-1α  in this study. A gene-specific siRNA, which exhibited stronger suppressive effects than antisense oligonucleotides , was used to silence the expression of HIF-1α and to further confirm the effects of HIF-1α on NCI-H446 cells and transplantation tumors. The in vitro study demonstrated that cells transduced with HIF-1α grew more rapidly than control cells, and cells transduced with siHIF-1α grew more slowly than control cells. The in vivo study indicated that the tumor formation rate of the HIF-1α transduction group was significantly higher than the rate of the non-transduction and siHIF-1α transduction groups. Moreover, the average tumor growth rate in the HIF-1α gene transduction group was higher than the tumor growth rates in the non-transduction and siHIF-1α groups. Thus, these results suggest that HIF-1α may be involved in promoting the progression of SCLC. Our study further supports the previous opinion that HIF-1α is correlated with the development of an aggressive phenotype in some tumor models , and that HIF-1α has been identified as a positive factor for tumor growth .
Induction angiogenesis of SCLC cells on CAM by HIF-1α
Chicken embryos are immunodeficient during embryonic development until day 19 of incubation . Thus, CAM was first adapted by many investigators as a convenient model to evaluate many different parameters of tumor growth  and to screen antineoplastic drugs [29, 30]. Furthermore, the CAM model is an ideal alternative to the nude mouse model system for cancer research because it can conveniently and inexpensively reproduce many tumor characteristics in vivo, such as tumor mass formation, tumor-induced angiogenesis, infiltrative growth, and metastasis . This model is especially ideal to study tumor-induced angiogenesis because of its dense vascular net and rapid vascular reactivity . In this study, we have successfully established the transplantation tumor model and have clearly shown that the avian microenvironment provided the appropriate conditions for the growth of human SCLC cells, as in the case when they are transplanted into immunodeficient mice . Moreover, the stroma of the CAM may represent a supportive environment for SCLC expansion because morphologically we could see that the SCLC cells were implanted on the side facing the window, invaded across the capillary plexus and formed a visible mass on the side of the chicken embryo.
With regard to targeted therapy of solid tumors, it is important to find a therapeutic target that is widely involved in many biological processes. HIF-1α is overexpressed in many human cancers. Significant associations between HIF-1α overexpression and patient mortality have been shown in cancers of the brain, breast, cervix, oropharynx, ovary, and uterus [2, 4]. However, some scholars have suggested that the effect of HIF-1α overexpression depends on the cancer type. For example, associations between HIF-1α overexpression and decreased mortality have been reported for patients with head and neck cancer  and non-small cell lung cancer . In our study, however, HIF-1α overexpression by Ad-HIF-1α significantly enhanced the angiogenic and invasive potential of SCLC, but transduction with Ad-siHIF-1α inhibited these potentials. Angiogenesis in SCLC is a key biological characteristic and an important mediator of tumor growth rate, invasiveness, and metastasis. Thus, the inhibition of angiogenesis is an effective method for the treatment of SCLC, and many targeted therapy drugs against angiogenesis, such as bevacizumab , cedirnnib , and sorafenib , have widely been used in clinical practice. However, the therapeutic targets of these drugs are confined to VEGF-A and its receptor or signaling pathway. VEGF-A is a downstream target of HIF-1α, and it contains HREs with an HIF-1α binding site . In our study, the expression of VEGF-A and the vascular reaction in the transplantation tumor was significantly inhibited after the expression of HIF-1α was downregulated by siHIF-1α. In addition to VEGF-A, there are many angiogenic factors that are directly or indirectly regulated by HIF-1α. Therefore, we propose that targeting HIF-1α may provide a broader inhibition of tumor angiogenesis than targeting downstream angiogenesis factors of HIF-1α. In the future, we will conduct correlated research to confirm this proposal.
Angiogenic factors regulated by HIF-1α in SCLC cells transplantation tumor
In pervious study although the multitude of insights were put into individual molecular effect on angiogenesis, such as increased migration and tube formation, which may be predicted to induce angiogenesis in vitro, these analyses in isolated systems clearly have their limitations, especially when a large scale of interconnections and complexity involved in the process of angiogenesis in vivo are considered. Allowing for this the in vivo expression of angiogenesis genes selected from the in vitro microarray analysis must be confirmed. Thus, it is important to successfully establish a simple and comprehensive model to test how HIF-1α regulates angiogenesis genes. Some scholars have suggested that xenograft models of tumor cells rely more on angiogenesis than naturally occurring tumors and that the extent of angiogenesis is dependent on the site of implantation of the xenografts . CAM is essentially a respiratory membrane with a dense vascular net that maintains the blood-gas exchange. For abundant blood supply and a special anatomical position in the chick embryo, the CAM may provide more precise and convincing data for angiogenic factors than other in vivo experimental models .
Recent research and development for a targeted drug for SCLC has focused on inhibiting the expression of angiogenic factors, such as VEGF-A. However, the microenvironment of SCLC cell growth is largely hypoxic, and HIF-1α is the primary regulatory factor for angiogenesis. The factors that are mediated by HIF-1α and involved in angiogenesis of SCLC have not been previously reported. Therefore, in our study, we initially evaluated the effects of HIF-1α on the invasiveness of SCLC, which precedes angiogenesis. Matrix metalloproteinases (MMPs) are a family of enzymes responsible for remodeling the extracellular matrix during growth and morphogenetic processes, which are important for tumor invasiveness. In our study, two members of the MMP family, MMP-14 and MMP-28, had increased expression resulting from HIF-1α overexpression in the in vitro microarray experiment and in the CAM experiments. The increased expression of MMP-14 has been identified as a negative predictor of survival in SCLC , and the targeted drug inhibiting MMP-14 expression, marimastat , has been used in clinical studies. MMP-28 is expressed at low levels in normal lung tissue, but the expression of MMP-28 is highly increased after cancer formation . MMP-28 induces epithelial-mesenchymal transitions (EMT), which yield tumor cells with collagen-invasive properties allowing the invasion of collagen matrices . The upregulation of MMP-28 by HIF-1α enhances this ability.
The expression level of angiogenic factors is the gold standard to measure the angiogenic potential of tumors, and the inhibition of the expression of angiogenic factors is the primary treatment for SCLC. Angiogenic factors that are significantly regulated by HIF-1α in a hypoxic microenvironment are also therapeutic target points . In addition to VEGF, FGF-2 , ANG-2 , HIF-2α , and PDGFC are also involved in tumor angiogenesis. In this study, three inflammatory factors, IL-6, TNFAIP6, and IL1R1, were upregulated by HIF-1α. These inflammatory factors actively responded during the process of inflammatory angiogenesis. TNFAIP6 is the stimulating factor for TNF-α , and IL-1R1 is the receptor for IL-1 . IL-6 and VEGF-A have synergistic effects in stimulating the proliferation and invasiveness of tumors by promoting angiogenesis . Our results indicate that HIF-1α may enhance the inflammatory reaction or stimulate the secretion of coherent inflammatory factors to promote the angiogenesis of SCLC, which highlights the importance of anti-inflammation for the treatment of SCLC as some scholars have suggested . In addition, the TNC, FN1, and HMOX1 cytokines were screen out by microarray analysis. TNC is an extracellular matrix protein with angiogenesis-promoting activities, and it has specific functions in vessel formation . FN1 has been shown to be an angiogenic cytokine involved in angiogenesis during several pathological processes, such as psoriasis, diabetic retinopathy, and cancer . The overexpression of HMOX1 has been observed in liver cancer , pancreatic cancer , and melanomas . Targeting these cytokines for gene therapy of SCLC in the future requires their verification in clinical trials.
Overall, our results suggest that HIF-1α significantly promotes the growth and angiogenesis of NCI-H446 cells by upregulating the expression of angiogenic genes. Moreover, our use of the chick CAM as an in vivo experimental model further confirms the expression of these genes induced by HIF-1α. Tumor growth on the chick CAM after they were grafted with human SCLC NCI-H446 cells represents an excellent model to study human SCLC angiogenesis. This study suggests that HIF-1α may be a potential target in the treatment of SCLC. In the future, we will further investigate human SCLC progression and invasiveness, and we will screen anti-angiogenic molecules in the CAM model to further enhance the number of possible genes for SCLC targeted therapies.
We would like to thank the Research Center of the Xinhua Hospital in Shanghai for providing technical assistance and professor GenFa-Shan for the critical reading of the manuscript.
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