HIF-1α effects on angiogenic potential in human small cell lung carcinoma

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Transduction of NCI-H446 cells with Ad5. Chosing transduction condition and the effect on NCI-H446 cells growth by HIF-1α. (A)Five different multiplicities of infection (MOI: 20, 30, 40, 50, and 70) were tested in the transduction experiment (60 h). The transduction efficiency was the highest when the MOI was 50 (*p < 0.05 represents MOI50 vs. MOI40; **p < 0.05 represents MOI50 vs. MOI70). (B) Transduction efficiency of NCI-H446 cells with Ad5-EGFP after 60 h (MOI = 50; 200 ×). (C) After the cells were transduced with Ad5 and Ad5-siRNA(MOI = 50), the mRNA expression level of HIF-1α was measured in the indicated time period by real-time PCR (*p > 0.05 represents NCI-H446/Ad5 group vs control group; ^▲p > 0.05 represents NCI-H446/Ad5- siRNA group vs control group;) (D)After the cells were transduced with Ad5-HIF-1α and Ad5-siHIF-1α (MOI = 50), the mRNA expression level of HIF-1α was measured in the indicated time period by real-time PCR (*p < 0.05 represents NCI-H446/HIF-1α group and NCI-H446/siHIF-1α group, 60 h vs. 48 h; ** p < 0.05 represents NCI-H446/HIF-1α group and NCI-H446/siHIF-1α group, 60 h vs. 72 h). (E) Growth curve of the cells in five groups. After transduction with Ad5 and Ad5-siRNA, the trendency of growth curve had no significant change. After transduction with HIF-1α, the growth curve of NCI-H446 cells shifted to the left with the growth of cells entering the period of logarithmic growth. After transduction with Ad5-siHIF-1α, however, the growth curve shifted to the right (*p > 0.05 represents NCI-H446/Ad5 or NCI-H446/Ad5-siRNA group vs. NCI-H446 group; **p < 0.01 represents NCI-H446/HIF-1α group vs. NCI-H446 group; ***p < 0.01 represents NCI-H446/siHIF-1α group vs. NCI-H446 group).

Figure 2

Macroscopic examination of the CAM and implanted human NCI-H446 cells. The entire experimental process from the implantation of NCI-H446 cells on the CAM and the formation of the transplantation tumor is shown. (A) Irregular window made in the egg shell of a 7-day-old chick embryo. (B) Elimination of the chick embryo in the CAM was observed. (C) The CAM was peeled for the assay. (D) Diagram of the technique for the implantation of NCI-H446 cells onto the CAM. (E) Diagram of the technique for the formation of the transplantation tumor. (F) The transplantation tumor (white mass was pointed by the tip) was formed on the side facing the chick embryo. (G-H) Histological evaluation of the transplanted tumor on the CAM by hematoxylin-eosin staining is shown:(G) The structure of the transplantation tumor and peripheral vessels (50 ×). (H) Pathological appearance of the transplantation tumor (200 ×). (I) Specific analysis was carried out by immunohistochemistry for the expression of NSE. The cellular nucleus was irregular, and positive expression for NSE was found in the intercellular substance or endochylema (400 ×).

Figure 3

Growth of the transplantation tumor. The growth curves of the transplantation tumors in the three groups are shown. Data are presented as means ± SD. (A) The growth curves of transplantation tumors in the NCI-H446/HIF-1α group shifted left, and the growth curves shifted right in the Ad5-siHIF-1α group (*p < 0.01 represents NCI-H446/HIF-1α group vs. NCI-H446 group; **p < 0.01 represents NCI-H446/siHIF-1α group vs. NCI-H446 group). (B) A transplantation tumor from the NCI-H446 group (10 d after implantation). (C) A transplantation tumor from the NCI-H446/HIF-1α group (10 d after implantation). (D) A transplantation tumor from the NCI-H446/siHIF-1α group (10 d after implantation). (E) A transplantation tumor from the NCI-H446/Ad5 group (10 d after implantation). (F) A transplantation tumor from the NCI-H446/Ad5-siRNA group (10 d after implantation). (G) Comparing to the growth curves in NCI-H446 group the tendency of the curves in NCI-H446/Ad5 group and NCI-H446/Ad5-siRNA group had no significant changes. (*p > 0.05 represents NCI-H446 group vs. NCI-H446/Ad5 group; **p > 0.01 represents NCI-H446/Ad5-siRNA group vs. NCI-H446 group).

Figure 4

Angiogenesis quantification of CAM. The entire process of angiogenesis quantification on the CAM was divided into four steps. (A) The image of one special domain in the CAM was collected for the assay. (B) The background of the image was cleaned up. (C) The profiles of the vessels for the assay were deepened. (D) The result of the MIQAS quantified system analysis for the number of vessel branch points as marked by the red points.

Figure 5

RT-PCR analysis of human and chicken angiogenic factors mRNA. Microarray analysis was performed to screen out the angiogenic factors affected by HIF-1α in SCLC cells (table 2). Afterwards, RT-PCR analysis was used to detect the expression of angiogenic factors affected by HIF-1a in the transplantation tumors of CAM in vivo. (A), Human and chicken VEGF-A, TNFAIP6, PDGFC, FN1, MMP28, MMP14, SOCS2 and IGFBP3 mRNA expression: Representative images of three independent experiments (Lane 1: control group-no human mRNA expression, Lane 2: transplantation tumor of NCI-H446 cells transduction by empty vector Ad5-NCI-H446 cells group, Lane 3: ransplantation tumor of NCI-H446 cells with transduction by HIF-1α-NCI-H446/HIF-1α group, Lane 4: transplantation tumor of NCI-H446 cells with transduction by siHIF-1α-NCI-H446/siHIF-1α group). (B and C), Relative expression levels of mRNA in NCI-H446/HIF-1α group and NCI-H446/siHIF-1α group compared with that in control group and NCI-H446 cells group (p < 0.05).

Figure 6

RT-PCR analysis of human and chicken glycolytic factors mRNA. RT-PCR analysis was used to detect the expression of glycolytic factors affected by HIF-1a in the transplantation tumors of CAM in vivo. (A), Human and chicken GLUT1 and GLUT2 mRNA expression: Representative images of three independent experiments (Lane 1: control group-no human mRNA expression, Lane 2: transplantation tumor of NCI-H446 cells transduction by empty vector Ad5-NCI-H446 cells group, Lane 3: ransplantation tumor of NCI-H446 cells with transduction by HIF-1α-NCI-H446/HIF-1α group, Lane 4: transplantation tumor of NCI-H446 cells with transduction by siHIF-1α-NCI-H446/siHIF-1α group). (B and C), Relative expression levels of mRNA in NCI-H446/HIF-1α group and NCI-H446/siHIF-1α group compared with that in control group and NCI-H446 cells group (p < 0.05).

Figure 7

Western blot analysis of the human and chicken VEGF-A protein in the CAM. In the NCI-H446/HIF-1α and NCI-H446/siHIF-1α groups, the SCLC cells were transduced with Ad-HIF-1α or Ad-siHIF-1α (MOI = 50) for 60 h before implanting onto the CAM to form transplantation tumors. Western blots were performed to detect the VEGF-A protein level in the tumors and peripheral tissues on day 17 of incubation. Data are presented as means ± SD. (A) Representative images of three independent experiments (Lane A - human VEGF-A protein expression in the tumors from the NCI-H446 group; Lane B - human VEGF-A protein expression in the tumors from the NCI-H446/HIF-1α group; and Lane C - human VEGF-A protein expression in the tumors from the NCI-H446/siHIF-1α group) (human - * p < 0.05 group C vs. group B; ** p < 0.05 group C vs. group D) (chicken - * p < 0.05 group C vs. group B; ** p < 0.05 group C vs. group D). (B) Representative images of three independent experiments (Lane A - chicken VEGF-A protein expression of control group; Lane B - chicken VEGF-A protein expression in the tumors from the NCI-H446 group; Lane C - chicken VEGF-A protein expression in the tumors from the NCI-H446/HIF-1α group; and Lane D - Chicken VEGF-A protein expression in tumors from the NCI-H446/siHIF-1α group). (C) Densitometry analysis of the relative expression of VEGF-A protein compared to the corresponding β-actin in each group (p < 0.05).