尼美舒利对氯化钴诱导的人胃癌细胞HIF-1a、VEGF表达的抑制作用和机制研究
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摘要
肿瘤微环境低氧是体内肿瘤的常见现象,肿瘤组织检测表明肿瘤组织内存在低氧区。其产生与肿瘤生长迅速、肿瘤血管结构异常等原因有关。肿瘤低氧通过诱导包括血管内皮生长因子(vascular endothelial growth factor,VEGF)在内的多种促血管生长因子促进肿瘤的血管生成。低氧诱导的血管内皮生长因子表达上调是导致肿瘤血管生成的关键事件。低氧调节VEGF是由转录因子低氧诱导因子-1(HIF-1,hypoxia-inducible factor-1,HIF-1)介导的,HIF-1是由HIF-1α和HIF-1β两个亚单位组成的异源性二聚体蛋白,其中HIF-1β在mRNA和蛋白水平均呈组成性表达,不受氧分压的影响,而HIF-1α虽在mRNA水平呈组成性表达但在蛋白水平则是受细胞内氧分压严格调控的:HIF-1α蛋白在常氧下被泛素蛋白酶体途径(VHL-ubiquitin-proteasome system)快速降解,而在低氧下稳定并与HIF-1β结合为完整的HIF-1,转位至胞核,从而具有DNA结合活性,结合于低氧反应基因调控区的低氧反应元件启动其转录。已发现,多种肿瘤HIF-1α表达升高,并与肿瘤血管密度呈正相关。环氧合酶(cyclooxygenase,COX)是催化花生四烯酸转化为前列腺素类代谢中的限速酶,有两种同工型,其中,环氧合酶-1为组成性表达,而环氧合酶-2(COX-2)为诱导性表达,COX-2在多种肿瘤组织中表达升高。实验室和流行病学资料表明,COX-2抑制剂降低肿瘤发生的危险性和肿瘤的生长。COX-2抑制剂抑制肿瘤生长的机制与抑制肿瘤血管生成有关,已发现多种肿瘤组织中COX-2常与VEGF共表达,并与肿瘤血管密度呈正相关,抑制COX-2可降低VEGF表达及肿瘤血管密度。而多项细胞水平的研究表明低氧可以刺激多种细胞表达COX-2,还可以诱导多种细胞COX-2、VEGF共表达;因此肿瘤微环境低氧可
Tumor microenvironmental hypoxia is a common phenomenon in vivo, hypoxia region is present within tumor interior. Tumor tissue hypoxia can be caused by a number of factors, such as rapid growth severe structural abnormalities of tumor microvessels, and disturbed microcirculation. Tumor hypoxia promotes tumour angiogenesis by inducing the expression of many pro-angiogenic factors including vascular endothelial growth factor (VEGF). Hypoxia-induced VEGF expression upregulation is a key event in tumor angiogenesis. Under hypoxic conditions, transcriptional activation of the VEGF gene is mediated by hypoxia-inducible factor-l(HIF-l). HIF-1 is a heterodimeric protein consisting of a inductively expressed HIF-1 α subunit and a constitutively expressed HIF-1 β subunit. HIF-1 β is constitutively expressed both in mRNA and protein level and not affected by the cellular 02 concentration. In contrast to HIF-1 β , the expression of HIF-1 α is constitutive in mRNA level, but its expression is strictly regulated by the cellular 02 concentration in protein level. During normoxia, the HIF-1 α is rapidly degraded by the VHL-ubiquitin-proteasome system, while hypoxia inhibites this process and then increases the stability of the HIF-1 α protein. These events result in HIF-1 α accumulation and association with the HIF-1 β protein and the subsequent translocation of the heterodimeric complex to the
nuclear compartment where HIF-1 complex binds to the hypoxia response elements of targeted gene and promotes its transcription. HIF-1 a overexpression has been demonstrated in a variety of human cancer biopsies, and overexpression of HIF-1 a in human cells increases tumor growth and angiogenesis.Cyclooxygenase (COX) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. Two COX isoforms have been cloned, of which COX-1 is constitutively expressed, while COX-2 is an inducible, immediate-early gene, and its role has been related to carcinogenesis. Expression of COX-2 is elevated in a variety of human malignancies and in their precursor lesions. Results from epidemiological and laboratory studies indicate that regular intake of selective COX-2 inhibitors reduces the risk of several forms of human malignancies and suppresses tumor growth. The mechanism by which COX-2 inhibitor inhibiting tumor growth is involved in the inhibition of tumor angiogenesis. It has been found that expression of COX-2 is correlated with VEGF expression, angiogenesis, and that inhibiting COX-2 activity can decrease VEGF expression and tumor vascular density. As several in vitro studies have demonstrated that hypoxia can stimulate COX-2 expression or COX-2 and VEGF co-expression in many cell types, tumor microenvironmental hypoxia may be one of the important causes by which COX-2 and VEGF co-expression exist in vivo. Prostaglandin E2 (PGE2), one of the main end products of C0X-2-catalyzed reaction and a stimulating factor of angiogennesis, has been demonstrated to increase VEGF expression in many cell types. We supposed that hypoxia-induced upregulation of COX-2 expression and PGE2 level may play an important role in hypoxia-induced angiogenesis. The underlying molecular mechanisms of COX-2 inhibitor inhibiting VEGF expression and angiogenesis and the exact molecular mechanisms of COX-2-generated PGE2 inducing VEGF expression have not been completely understood. Recent studies in a human prostate cancer cell line and a human colon carcinoma cell line have demonstrated that COX-2-generated PGE2 could upregulate HIF-1 a protein through relevant signal transduction pathway and then promoted expression of VEGF accordingly,
suggesting that COX-2 inhibitor inhibits HIF-1 a ,VEGF expression and angiogenesis by reducing PGE2 production under the tumor microenvironmental hypoxia conditions in vivo. However, some studies have found that there was no correlation between the pathway of COX-2-PGE2 and HIF-1 a -VEGF in some other cell lines, suggestting that the signal transduction network is complicated and it exists diversities among the different cell types. On the basis of the fact that there are hypoxia regions in tumor tissue in vivo and hypoxia-induced HIF-1 a and VEGF expression is the vital cause of tumor angiogenesis, as well as the fact that the COX-2 and COX-2-generated PGE2 also plays an important role in tumor angiogenesis, it is required to further explore the relation between the signal pathway of COX-2-PGE2 and HIF-1 a -VEGF under hypoxia conditions. The current study was designed to investigate the influence of a selective COX-2 inhibitor, nimesulide, on the hypoxia mimic cobalt-induced expression of HIF-1 a and VEGF mRNA and protein in a human gastric low differentiation adenocarcinoma cell line BGC-823 cells in vitro, to elucidate the role of COX-2 and COX-2-catalyzed PGE2 in this effect, and finally to explore the signal transduction pathway through which PGE2 induces HIF-1 a and VEGF expression in this cell line.The studies were divided into three parts listed below:The first part: The effects of hypoxia-inducer cobalt chloride-simulated hypoxia on the expression of COX-2, HIF-1 a ,VEGF mRNA and protein in human BGC-823 cells Methods1. Cell culture, hypoxia inducion and experimental groups BGC-823 cells were grown to about 60 %-~70 % confluence and were serum starved (0.1% FCS)for 24 h and treated with 150 ^M cobalt for 0-24 h, and after incubation for a given duration, the cells were harvested. 2.Total RNA isolation and RT-PCR assay After incubation for a given duration, total RNA was extracted from BGC-823 cells
with Trizol. Aliquots (5 /ug) of RNA were reversely transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase system for RT-PCR analysis of COX-2, HIF-1 a and VEGF mRNA according to the manufacturer's instructions. 4 /il of cDNA were used as template for PCR of HIF-1 a , VEGF and COX-2 sequences. 3 -actin was used as an internal control. The amplified products were visualized on 1.5% agarose gels with ethidium bromide, and band intensity was quantified by photo image analyzer. The ratio of band intensity of the sample to the internal standard was calculated in the four reactions which stood for the amount of expression of COX-2, HIF-1 a and VEGF mRNA. 3.Western blot analysisAfter incubation for a given duration, cell lysates were prepared for analysis of COX-2, HIF-1 a , VEGF production by Western blot. The protein concentrations of cell extracts were determined by the ultraviolet absorpotion spectra method. Cell extracts containing an equivalent amount of protein (30 P g of protein) were separated by 10% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane by standard procedures. Membranes were blocked with 10% nonfat dry milk and were incubated with either anti-VEGF or anti-HIF-1 a antibodies. Secondary horseradish peroxidase-linked donkey anti-mouse IgG was used. Finally, the samples were visualized by incubated in DAB solution.4.1mmunocytochemistryCells were fixed in cool acetone and stained for COX-2, HIF-1 a , VEGF based on streptavidin-biotin-horseradish peroxidase complex formation which was visualized by DAB as a brown-yellow color. The results were analyzed by a cellular image analysis system. Expression levels of protein were quantified using the mean optical density value of positive signals.5.Statistical analysis All results were expressed as mean ± standard deviation (SD). Student's t test was used to assess differences between the two groups. ANOVA was employed for the statistical comparisons among groups. Pearson test
was used for correlation analysis. P<0.05 was selected as the statistically significant value. Statistical analysis was carried out using SPSS 11.0 software. Results1. RT-PCR analysis of the effects of cobalt on the expression of COX-2, HIF-1 a and VEGF mRNA in BGC-823 cellsRT-PCR analysis revealed that BGC-823 cells expressed a low level of COX-2 and VEGF mRNA but constitutively expressed HIF-1 a mRNA under normoxic condition, the density value of bands of COX-2, VEGF and HIF-1 a mRNA were 0.1934 + 0.0136, 0.2713 + 0.0164, 0.9546 + 0.0151 respectively. HIF-1 a mRNA expression was not induced by cobalt stimulation. Cobalt stimulated the expression of COX-2, VEGF mRNA in BGC-823 cells in a time-dependant manner. After stimulated by cobalt for 3, 12, 24 h, the density value of bands of COX-2 were 0.3581 ±0.0178, 0.4520 + 0.0223, 0.8232 + 0.0163 respectively(P<0.01), the density value of bands of VEGF were 0.4318 + 0.0131, 0.5221 ±0.0159, 0.9482 ±0.0203 respectively(P<0.01) and the density value of bands of HIF-1 a were 0.9743 ±0.0132, 0.9834 ± 0.0167, 0.9651 ± 0.0145 respectively (P>0.05).There was a positive correlation between the mRNA expression of COX-2 and VEGF, the correlation factor was 0.733 (P<0.01).2.Western blot analysis of the effects of cobalt on the expression of COX-2, HIF-1 a and VEGF protein in BGC-823 cellsWestern blot analysis revealed that HIF-1 a protein was weakly expressed while basal COX-2 and VEGF protein levels were found in BGC-823 cells under normoxic condition, the density value of bands of HIF-1 a ,COX-2 and VEGF protein were 5 + 1.12, 18.67±1.65, 23.16±1.43 respectively. Cobalt strongly stimulated the expression of HIF-1 a protein and induced the expression of COX-2 and VEGF protein in a time-dependant manner. After stimulated by cobalt for 3, 12, 24 h, the density value of bands of COX-2 protein were 40.33 ±0.46, 58.22 ±2.44, 5.67±2.83 respectively, the density value of bands of VEGF were 45.38±2.65, 63.31 ±1.48, 88.51 ±2.19 respectively and the density value of bands of HIF-1 a
were 91.52+1.98, 93.36 + 2.47, 94.41 + 2.81 respectively. There was a positive correlation between the protein expression of COX-2 and HIF-1 a , of COX-2 and VEGF, and of HIF-1 a and VEGF, the correlation factor was 0.755, 0.768 and 0.813 respectively (P<0.01).3. Immunocytochemistry analysis of the effects of cobalt on the expression of COX-2, HIF-1 a and VEGF protein in BGC-823 cellsImage analysis revealed that HIF-1 a protein was weakly expressed while basal COX-2 and VEGF protein levels were found in BGC-823 cells under normoxic condition, the density value of bands of HIF-1 a ,COX-2 and VEGF protein were 0.045 + 0.011, 0.119 + 0.011, 0.143 + 0.013 respectively. Cobalt markedly induced the expression of HIF-1 a , COX-2 and VEGF protein. After stimulated by cobalt for 24 h, the optical density value of COX-2, HIF-1 a and VEGF protein were 0.811+0.026, 0.963 + 0.023 and 0.852±0.018 respectively(P<0.01). The second part: The influence of nimesulide on the expression of HIF-1 a and VEGF induced by cobalt in human BGC-823 cells Methodsl.Cell culture, hypoxia inducion and experimental groupsBGC-823 cells were grown to about 60 %~70 % confluence and were serum starved (0.1% FCS)for 24 h before intervention. The experimental samples were divided into six groups:(T)no stimulation group(2)150 \i mol/L cobalt group(3)150 u mol/L cobalt plus 10 u M nimesulide group? 150 U mol/L cobalt plus 40 H M nimesulide group(5)150u mol/L plus 80 u M nimesulide group(6)150u mol/L plus 80 u M plus 100 u M PGE2 nimesulide group. After incubation for a given duration, the cells were harvested.2. Total RNA isolation and RT-PCR assay The materials and methods was the same as the first part.3.Western blot analysis The materials and methods was the same as the first part. 4.Immunocytochemistry The materials and methods was the same as the first part.
5.Statistical analysis The methods was the same as the first part. Resultsl.RT-PCR analysis of the effects of nimesulide on the expression of HIF-1 a ,VEGF protein in BGC-823 cells induced by cobalt and the reversed effects of PGE2RT-PCR analysis revealed that BGC-823 cells expressed a low level of VEGF mRNA but constitutively expressed HIF-1 a mRNA under normoxic conditions, the density value of bands of VEGF, HIF-1 a mRNA were 0.2656 + 0.0171, 0.9447 ±0.0144 respectively. Cobalt stimulated the expression of VEGF mRNA but did not induce HIF-1 a mRNA expression in BGC-823 cells at 24h, the density value of bands of VEGF and HIF-1 a were 0.9535 ±0.0143, 0.9628 ±0.0212 respectively. Nimesulide did not change the expression of HIF-1 a mRNA but prevented cobalt-simulated up-regulation of VEGF mRNA in a dose-dependent manner. When BGC-823 cells was treated by 150 u mol/L cobalt combined with different concentrtion of nimesulide(10, 40 and 80 n M, respectively) for 24 h, the density value of bands of HIF-1 a were 0.9736±0.0161, 0.9842±0.0153, 0.9815±0.0156 respectively, the density value of bands of VEGF were 0.6318 + 0.0131, 0.4221 ±0.0159, 0.2782 ± 0.0203 respectively(P<0.01) while extrinsic PGE2 (100 u M) almost completely reversed such inhibitory effects of 80 u M nimesulide(the density value of bands of VEGF were 0.9426 + 0.0121). There was a negative correlation between the concentrtion of nimesulide and the mRNA expression of VEGF, the correlation factor was — 0.728 (P<0.01).2.Western blot analysis of the effects of nimesulide on the expression of HIF-1 a ,VEGF protein in BGC-823 cells induced by cobalt and the reversed effects of PGE2Western blot analysis revealed that HIF-1 a protein was weakly expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a , VEGF protein were 5 ±1.14, 22.46 + 1.35 respectively. Cobalt strongly stimulated the expression of HIF-1 a and
VEGF protein at 24h ,the density value of bands of HIF-1 a and VEGF protein were 95.24 + 2.65 and 87.59 + 2.48 respectively. Nimesulide prevented cobalt-simulated up-regulation of HIF-1 a and VEGF protein in a dose-dependent manner. When BGC-823 cells was treated with 150 u mol/L cobalt combined with different concentrtion of nimesulide(10. 40 and 80 U M, respectively) for 24 h, the density value of bands of HIF-1 a were 71.45 + 1.78, 42.27 ±1.72, 7.35+1.15 respectively and the density value of bands of VEGF were 67.33 + 1.73, 43.51 + 1.18, 24.71 + 1.17 respectively while extrinsic PGE2 (100 u M) reversed such inhibitory effects of 80 U M nimesulide (the density value of bands of HIF-1 a and VEGF were 90.37 + 2.55, 82.99 + 2.25 respectively ). There was a negative correlation between the concentrtion of nimesulide and the protein expression of HIF-1 a and VEGF, the correlation factor was —0.773 and —0.729 respectively (P<0.01). 3.1mmunocytochemical analysis of the effects of nimesulide on the expression of HIF-1 a , VEGF protein in BGC-823 cells induced by cobalt and the reversed effects of PGE2Image analysis revealed that HIF-1 a protein was weakly expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a and VEGF protein were 0.045 + 0.011, 0.143 + 0.013 respectively. Cobalt markedly induced the expression of HIF-1 a and VEGF protein. After stimulated by cobalt for 24 h, the optical density value of HIF-1 a and VEGF protein were 0.963 + 0.023 and 0.852±0.018 respectively. Nimesulide prevented cobalt-simulated up-regulation of HIF-1 a and VEGF protein in a dose-dependent manner. When BGC-823 cells was treated with 150 V mol/L cobalt combined with different concentrtion of nimesulide(10, 40 and 80 U M, respectively) for 24 h, the optical density value of HIF-1 a protein were 0.713 + 0.012, 0.442±0.014 and 0.126±0.011 respectively( P < 0.01) and the optical density value of VEGF protein were 0.725 ±0.021, 0.425 + 0.019 and 0.187 ± 0.025 respectively (P < 0.01) while extrinsic PGE2 (100 u M) reversed such inhibitory effects of nimesulide, the density value of bands of HIF-1 a and VEGF
were 0.132 + 0.018, 0.211 + 0.023 respectively.The third part: The effects of extrinsic PGE2 on the expression of HIF-1 a and VEGF in BGC-823 cells and its signal transduction pathway Methodsl.Cell culture and experimental groupsBGC-823 cells were grown to about 60 %~70 % confluence and were serum starved (0.1% FCS) for 24 h before PGE2 was added. Experimental groups were divided according to experimental purposes.(DTo observe the effects of PGE2 on the expression of HIF-1 a , VEGF mRNA and protein in BGC-823 cells, the experimental samples were divided into four groups: ?no stimulation group?l u M PGE2 group? 10 u M PGE2 group? 100 u M PGE2 group. After incubation for a given duration, the cells were harvested. (2)To investigate the effects of MEK inhibitor PD98059 on the PGE2-induced expression of HIF-1 a , VEGF in BGC-823 cells, the experimental samples were divided into four groups: ?no stimulation group(2)100u M PGE2 group(3)100n M PGE2 plus 10 u M PD98059 group?50 u M PGE2 plus 50 u M PD98059 group (5) 100 u M PGE2 plus 100 u MPD98059 group. Kinase inhibitors PD98059 stimulated the cells 30 min before PGE2 treatment. After incubation for a given duration, the cells were harvested.(3)To investigate the effects of PI3K inhibitor Wortmannin on the PGE2-inducied expression of HIF-1 a and VEGF in BGC-823 cells, the experimental samples were divided into four groups: ?no stimulation group(l)100n M PGE2 group(3)100y M PGE2 plus 50 nM Wortmannin group? 100 u M PGE2 plus u M 100 nM Wortmannin group? 100 u M PGE2 plus 200 nM Wortmannin group. Kinase inhibitors Wortmannin was added 30 min before exposure to PGE2. After incubation for a given duration, the cells were harvested.2. Total RNA isolation and RT-PCR assay The materials and methods was the same as the first part. 3.Western blot analysis The materials and methods was the same as the first part.
4.1mmunocytochemistry The materials and methods was the same as the firstpart.5.Statistical analysis The methods was the same as the first part.Results1. RT-PCR analysis of the effects of PGE2 on the expression of HIF-1 a , VEGF mRNA in BGC-823 cellsRT-PCR analysis revealed that BGC-823 cells expressed a low level of VEGF mRNA but constitutively expressed HIF-1 a mRNA under normoxic condition, the brightness value of bands of VEGF, HIF-1 a mRNA were 0.2683 + 0.0159, 0.9489 + 0.0121 respectively. HIF-1 a mRNA expression was not induced by PGE2 stimulation. PGE2 stimulated the expression of VEGF mRNA in BGC-823 cells in a dose-dependant manner. After stimulated by PGE2 for 24 h, the brightness value of bands of HIF-1 a were 0.9523 + 0.0167, 0.9486 ±0.0193, 0.9353 + 0.0178 respectively and the brightness value of bands of VEGF were 0.3816 + 0.0122, 0.5342 + 0.0137, 0.6875 + 0.0211. There was a positive correlation between the concentrtion of PGE2 and the mRNA expression of VEGF, the correlation factor was 0.782 (P<0.01).2. Western blot analysis of the effects of PGE2 on the expression of HIF-1 a ? VEGF protein in BGC-823 cellsWestern blot analysis revealed that HIF-1 a protein was weakly or negatively expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a , VEGF protein were 5 ±1.14, 23.13 + 1.33 respectively. PGE2 significantly induced the expression of HIF-1 a and VEGF protein in normoxic cells in a dose-dependant manner at 24h. After stimulated by PGE2 for 24 h, the density value of bands of VEGF were 35.32 + 1.47, 54.41 ±1.39, 69.48 + 2.21 respectively (P < 0.01)and the density value of bands of HIF-1 a were 42.43±1.67, 67.33±2.35, 81.37±2.59 respectively(P < 0.01). There was a positive correlation between the protein expression of HIF-1 a and VEGF, the correlation factor was 0.811 (P<0.01). There was a negative
correlation between the concentrtion of PGE2 and the protein expression of HIF-1 a and VEGF, the correlation factor was 0.746 and 0.758 respectively (P<0.01).3. Immunocytochemical analysis of the effects of PGE2 on the expression of HIF-1 a and VEGF protein in BGC-823 cellsImage analysis revealed that that HIF-1 a was expressed in the cytoplasm of tumor cells and VEGF was predominantly expressed in the cytoplasm of tumor cells. HIF-1 a protein was weakly or negatively expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic condition, the density value of bands of HIF-1 a , VEGF protein were 0.0426 ± 0.0038, 0.1231 ± 0.0033 respectively. PGE2 significantly induced the expression of HIF-1 a and VEGF protein in normoxic cells in a dose-dependant manner at 24h. After stimulated by PGE2 for 24 h, the Optical density value of VEGF were 0.385 + 0.017, 0.665 + 0.014, 0.732 + 0.019 respectively and the density value of HIF-1 a were 0.476± 0.022, 0.760 + 0.013, 0.897±0.015 respectively(P < 0.01).4. Western blot analysis of the effects of PD98059 on HIF-1 a protein expression stimulated by PGE2 in BGC-823 cellsWestern blot analysis demonstrated that HIF-1 a protein was weakly or negatively expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a , VEGF protein were 4.9+1.06, 24.18+1.32 respectively. 100 y M PGE2 significantly induced the expression of HIF-1 a and VEGF protein in normoxic cells at 24h, the density value of bands of HIF-1 a and VEGF were 83.26 ± 2.61 and 72.30 + 2.31 respectively. PD98059, a inhibitor of MAPK inhibiting the activity of MEK, significantly suppressed PGE2-induced up-regulation of HIF-1 a protein expression in a dose-dependent fashion. When BGC-823 cells was treated with 100 u M PGE2 combined with different concentrtion of PD98059(10, 50 and 100 H M, respectively) for 24 h, the density value of bands of HIF-1 a were 73.36 + 1.87, 39.24+1.58, 6.67+1.09 respectively (P<0.01) and the density value of bands of VEGF were 64.52+1.43, 46.43 + 1.15, 28.31 + 1.33 respectively (P<0.01). There was a
negative correlation between the concentrtion of PD98059 and the protein expression of HIF-1 a and VEGF, the correlation factor was —0.791 and —0.772 respectively (P<0.01).5. Western blot analysis of the effects of Wortmannin on HIF-1 a protein expression stimulated by PGE2 in BGC-823 cellsWestern blot analysis demonstrated that HIF-1 a protein was weakly or negatively expressed, while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a , VEGF protein were 5.1 + 1.13, 22.17+1.27 respectively. 100 u M PGE2 significantly induced the expression of HIF-1 a and VEGF protein in normoxic cells at 24h, the density value of bands of HIF-1 a and VEGF were 80.51 + 2.81 and 68.88 ± 2.07 respectively. Wortmannin, a inhibitors of PI3K, significantly but only partially inhibited PGE2-induced up-regulation of HIF-1 a protein expression in a dose-dependent fashion. When BGC-823 cells was treated with 100 u M PGE2 combined with different concentrtion of Wortmannin (50, 100 and 200 nM, respectively) for 24 h, the density value of bands of HIF-1 a were 75.42+1.45, 58.63±1.54, 27.58+1.31 respectively (P<0.05) and the density value of bands of VEGF were 64.79 + 1.71, 51.16 + 1.13, 40.43 + 1.42 respectively (P<0.05).There was a negative correlation between the concentrtion of Wortmannin and the protein expression of HIF-1 a and VEGF, the correlation factor was —0.602 and —0.584 respectively (P<0.05). Conclusionsl.Hypoxia-inducer cobalt induced the expression of COX-2 mRNA and protein, HIF-1 a protein, VEGF mRNA and protein in human gastric cancer cell line BGC-823.2.Nimesulide had inhibitory effects on cobalt-simulated up-regulation of HIF-1 a protein, VEGF mRNA and protein in BGC-823 cells in a dose-dependent manner which may be one of the antiangiogenesis mechanisms of nimesulide. COX-2 inhibitor blocking the hypoxia-inductcd VEGF mRNA expression may act in such
a way by inhibition of HIF-1 u expression protein.3.PGE2 induced the expression of HIF-1 a protein, VEGF mRNA and protein inBGC-823 cells in a dose-dependent manner, but had no effect on HIF-1 a mRNAexpression. PGE2 induces the expression of VEGF mRNA in BGC-823 cells thatmay be mediated by the transcriptional activator HIF-1 a .4.Pharmacologic inhibition of ERK phosphorylation blocked the induction ofVEGF and HIF-1 a protein expression in response to PGE2 stimulation. Inhibitionof PI3K activity also partly blocked PGE2-induced HIF-1 a and VEGF proteinexpression.5.These evidence indicate that COX-2 plays a role in the induction of HIF-1 aexpression in hypoxia. The events demonstrated in the cultured BGC-823 cells mayalso occur in vivo, which maybe have important pathophysiological and therapeuticimplications. This observation implies that the inhibitory effects of nimesulide onhypoxic responses can be developed to suppress tumor angiogenesis.
Tumor microenvironmental hypoxia is a common phenomenon in vivo, hypoxia region is present within tumor interior. Tumor tissue hypoxia can be caused by a number of factors, such as rapid growth severe structural abnormalities of tumor microvessels, and disturbed microcirculation. Tumor hypoxia promotes tumour angiogenesis by inducing the expression of many pro-angiogenic factors including vascular endothelial growth factor (VEGF). Hypoxia-induced VEGF expression upregulation is a key event in tumor angiogenesis. Under hypoxic conditions, transcriptional activation of the VEGF gene is mediated by hypoxia-inducible factor-l(HIF-l). HIF-1 is a heterodimeric protein consisting of a inductively expressed HIF-1 α subunit and a constitutively expressed HIF-1 β subunit. HIF-1 β is constitutively expressed both in mRNA and protein level and not affected by the cellular 02 concentration. In contrast to HIF-1 β , the expression of HIF-1 α is constitutive in mRNA level, but its expression is strictly regulated by the cellular 02 concentration in protein level. During normoxia, the HIF-1 α is rapidly degraded by the VHL-ubiquitin-proteasome system, while hypoxia inhibites this process and then increases the stability of the HIF-1 α protein. These events result in HIF-1 α accumulation and association with the HIF-1 β protein and the subsequent translocation of the heterodimeric complex to the
nuclear compartment where HIF-1 complex binds to the hypoxia response elements of targeted gene and promotes its transcription. HIF-1 a overexpression has been demonstrated in a variety of human cancer biopsies, and overexpression of HIF-1 a in human cells increases tumor growth and angiogenesis.Cyclooxygenase (COX) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. Two COX isoforms have been cloned, of which COX-1 is constitutively expressed, while COX-2 is an inducible, immediate-early gene, and its role has been related to carcinogenesis. Expression of COX-2 is elevated in a variety of human malignancies and in their precursor lesions. Results from epidemiological and laboratory studies indicate that regular intake of selective COX-2 inhibitors reduces the risk of several forms of human malignancies and suppresses tumor growth. The mechanism by which COX-2 inhibitor inhibiting tumor growth is involved in the inhibition of tumor angiogenesis. It has been found that expression of COX-2 is correlated with VEGF expression, angiogenesis, and that inhibiting COX-2 activity can decrease VEGF expression and tumor vascular density. As several in vitro studies have demonstrated that hypoxia can stimulate COX-2 expression or COX-2 and VEGF co-expression in many cell types, tumor microenvironmental hypoxia may be one of the important causes by which COX-2 and VEGF co-expression exist in vivo. Prostaglandin E2 (PGE2), one of the main end products of C0X-2-catalyzed reaction and a stimulating factor of angiogennesis, has been demonstrated to increase VEGF expression in many cell types. We supposed that hypoxia-induced upregulation of COX-2 expression and PGE2 level may play an important role in hypoxia-induced angiogenesis. The underlying molecular mechanisms of COX-2 inhibitor inhibiting VEGF expression and angiogenesis and the exact molecular mechanisms of COX-2-generated PGE2 inducing VEGF expression have not been completely understood. Recent studies in a human prostate cancer cell line and a human colon carcinoma cell line have demonstrated that COX-2-generated PGE2 could upregulate HIF-1 a protein through relevant signal transduction pathway and then promoted expression of VEGF accordingly,
suggesting that COX-2 inhibitor inhibits HIF-1 a ,VEGF expression and angiogenesis by reducing PGE2 production under the tumor microenvironmental hypoxia conditions in vivo. However, some studies have found that there was no correlation between the pathway of COX-2-PGE2 and HIF-1 a -VEGF in some other cell lines, suggestting that the signal transduction network is complicated and it exists diversities among the different cell types. On the basis of the fact that there are hypoxia regions in tumor tissue in vivo and hypoxia-induced HIF-1 a and VEGF expression is the vital cause of tumor angiogenesis, as well as the fact that the COX-2 and COX-2-generated PGE2 also plays an important role in tumor angiogenesis, it is required to further explore the relation between the signal pathway of COX-2-PGE2 and HIF-1 a -VEGF under hypoxia conditions. The current study was designed to investigate the influence of a selective COX-2 inhibitor, nimesulide, on the hypoxia mimic cobalt-induced expression of HIF-1 a and VEGF mRNA and protein in a human gastric low differentiation adenocarcinoma cell line BGC-823 cells in vitro, to elucidate the role of COX-2 and COX-2-catalyzed PGE2 in this effect, and finally to explore the signal transduction pathway through which PGE2 induces HIF-1 a and VEGF expression in this cell line.The studies were divided into three parts listed below:The first part: The effects of hypoxia-inducer cobalt chloride-simulated hypoxia on the expression of COX-2, HIF-1 a ,VEGF mRNA and protein in human BGC-823 cells Methods1. Cell culture, hypoxia inducion and experimental groups BGC-823 cells were grown to about 60 %-~70 % confluence and were serum starved (0.1% FCS)for 24 h and treated with 150 ^M cobalt for 0-24 h, and after incubation for a given duration, the cells were harvested. 2.Total RNA isolation and RT-PCR assay After incubation for a given duration, total RNA was extracted from BGC-823 cells
with Trizol. Aliquots (5 /ug) of RNA were reversely transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase system for RT-PCR analysis of COX-2, HIF-1 a and VEGF mRNA according to the manufacturer's instructions. 4 /il of cDNA were used as template for PCR of HIF-1 a , VEGF and COX-2 sequences. 3 -actin was used as an internal control. The amplified products were visualized on 1.5% agarose gels with ethidium bromide, and band intensity was quantified by photo image analyzer. The ratio of band intensity of the sample to the internal standard was calculated in the four reactions which stood for the amount of expression of COX-2, HIF-1 a and VEGF mRNA. 3.Western blot analysisAfter incubation for a given duration, cell lysates were prepared for analysis of COX-2, HIF-1 a , VEGF production by Western blot. The protein concentrations of cell extracts were determined by the ultraviolet absorpotion spectra method. Cell extracts containing an equivalent amount of protein (30 P g of protein) were separated by 10% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane by standard procedures. Membranes were blocked with 10% nonfat dry milk and were incubated with either anti-VEGF or anti-HIF-1 a antibodies. Secondary horseradish peroxidase-linked donkey anti-mouse IgG was used. Finally, the samples were visualized by incubated in DAB solution.4.1mmunocytochemistryCells were fixed in cool acetone and stained for COX-2, HIF-1 a , VEGF based on streptavidin-biotin-horseradish peroxidase complex formation which was visualized by DAB as a brown-yellow color. The results were analyzed by a cellular image analysis system. Expression levels of protein were quantified using the mean optical density value of positive signals.5.Statistical analysis All results were expressed as mean ± standard deviation (SD). Student's t test was used to assess differences between the two groups. ANOVA was employed for the statistical comparisons among groups. Pearson test
was used for correlation analysis. P<0.05 was selected as the statistically significant value. Statistical analysis was carried out using SPSS 11.0 software. Results1. RT-PCR analysis of the effects of cobalt on the expression of COX-2, HIF-1 a and VEGF mRNA in BGC-823 cellsRT-PCR analysis revealed that BGC-823 cells expressed a low level of COX-2 and VEGF mRNA but constitutively expressed HIF-1 a mRNA under normoxic condition, the density value of bands of COX-2, VEGF and HIF-1 a mRNA were 0.1934 + 0.0136, 0.2713 + 0.0164, 0.9546 + 0.0151 respectively. HIF-1 a mRNA expression was not induced by cobalt stimulation. Cobalt stimulated the expression of COX-2, VEGF mRNA in BGC-823 cells in a time-dependant manner. After stimulated by cobalt for 3, 12, 24 h, the density value of bands of COX-2 were 0.3581 ±0.0178, 0.4520 + 0.0223, 0.8232 + 0.0163 respectively(P<0.01), the density value of bands of VEGF were 0.4318 + 0.0131, 0.5221 ±0.0159, 0.9482 ±0.0203 respectively(P<0.01) and the density value of bands of HIF-1 a were 0.9743 ±0.0132, 0.9834 ± 0.0167, 0.9651 ± 0.0145 respectively (P>0.05).There was a positive correlation between the mRNA expression of COX-2 and VEGF, the correlation factor was 0.733 (P<0.01).2.Western blot analysis of the effects of cobalt on the expression of COX-2, HIF-1 a and VEGF protein in BGC-823 cellsWestern blot analysis revealed that HIF-1 a protein was weakly expressed while basal COX-2 and VEGF protein levels were found in BGC-823 cells under normoxic condition, the density value of bands of HIF-1 a ,COX-2 and VEGF protein were 5 + 1.12, 18.67±1.65, 23.16±1.43 respectively. Cobalt strongly stimulated the expression of HIF-1 a protein and induced the expression of COX-2 and VEGF protein in a time-dependant manner. After stimulated by cobalt for 3, 12, 24 h, the density value of bands of COX-2 protein were 40.33 ±0.46, 58.22 ±2.44, 5.67±2.83 respectively, the density value of bands of VEGF were 45.38±2.65, 63.31 ±1.48, 88.51 ±2.19 respectively and the density value of bands of HIF-1 a
were 91.52+1.98, 93.36 + 2.47, 94.41 + 2.81 respectively. There was a positive correlation between the protein expression of COX-2 and HIF-1 a , of COX-2 and VEGF, and of HIF-1 a and VEGF, the correlation factor was 0.755, 0.768 and 0.813 respectively (P<0.01).3. Immunocytochemistry analysis of the effects of cobalt on the expression of COX-2, HIF-1 a and VEGF protein in BGC-823 cellsImage analysis revealed that HIF-1 a protein was weakly expressed while basal COX-2 and VEGF protein levels were found in BGC-823 cells under normoxic condition, the density value of bands of HIF-1 a ,COX-2 and VEGF protein were 0.045 + 0.011, 0.119 + 0.011, 0.143 + 0.013 respectively. Cobalt markedly induced the expression of HIF-1 a , COX-2 and VEGF protein. After stimulated by cobalt for 24 h, the optical density value of COX-2, HIF-1 a and VEGF protein were 0.811+0.026, 0.963 + 0.023 and 0.852±0.018 respectively(P<0.01). The second part: The influence of nimesulide on the expression of HIF-1 a and VEGF induced by cobalt in human BGC-823 cells Methodsl.Cell culture, hypoxia inducion and experimental groupsBGC-823 cells were grown to about 60 %~70 % confluence and were serum starved (0.1% FCS)for 24 h before intervention. The experimental samples were divided into six groups:(T)no stimulation group(2)150 \i mol/L cobalt group(3)150 u mol/L cobalt plus 10 u M nimesulide group? 150 U mol/L cobalt plus 40 H M nimesulide group(5)150u mol/L plus 80 u M nimesulide group(6)150u mol/L plus 80 u M plus 100 u M PGE2 nimesulide group. After incubation for a given duration, the cells were harvested.2. Total RNA isolation and RT-PCR assay The materials and methods was the same as the first part.3.Western blot analysis The materials and methods was the same as the first part. 4.Immunocytochemistry The materials and methods was the same as the first part.
5.Statistical analysis The methods was the same as the first part. Resultsl.RT-PCR analysis of the effects of nimesulide on the expression of HIF-1 a ,VEGF protein in BGC-823 cells induced by cobalt and the reversed effects of PGE2RT-PCR analysis revealed that BGC-823 cells expressed a low level of VEGF mRNA but constitutively expressed HIF-1 a mRNA under normoxic conditions, the density value of bands of VEGF, HIF-1 a mRNA were 0.2656 + 0.0171, 0.9447 ±0.0144 respectively. Cobalt stimulated the expression of VEGF mRNA but did not induce HIF-1 a mRNA expression in BGC-823 cells at 24h, the density value of bands of VEGF and HIF-1 a were 0.9535 ±0.0143, 0.9628 ±0.0212 respectively. Nimesulide did not change the expression of HIF-1 a mRNA but prevented cobalt-simulated up-regulation of VEGF mRNA in a dose-dependent manner. When BGC-823 cells was treated by 150 u mol/L cobalt combined with different concentrtion of nimesulide(10, 40 and 80 n M, respectively) for 24 h, the density value of bands of HIF-1 a were 0.9736±0.0161, 0.9842±0.0153, 0.9815±0.0156 respectively, the density value of bands of VEGF were 0.6318 + 0.0131, 0.4221 ±0.0159, 0.2782 ± 0.0203 respectively(P<0.01) while extrinsic PGE2 (100 u M) almost completely reversed such inhibitory effects of 80 u M nimesulide(the density value of bands of VEGF were 0.9426 + 0.0121). There was a negative correlation between the concentrtion of nimesulide and the mRNA expression of VEGF, the correlation factor was — 0.728 (P<0.01).2.Western blot analysis of the effects of nimesulide on the expression of HIF-1 a ,VEGF protein in BGC-823 cells induced by cobalt and the reversed effects of PGE2Western blot analysis revealed that HIF-1 a protein was weakly expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a , VEGF protein were 5 ±1.14, 22.46 + 1.35 respectively. Cobalt strongly stimulated the expression of HIF-1 a and
VEGF protein at 24h ,the density value of bands of HIF-1 a and VEGF protein were 95.24 + 2.65 and 87.59 + 2.48 respectively. Nimesulide prevented cobalt-simulated up-regulation of HIF-1 a and VEGF protein in a dose-dependent manner. When BGC-823 cells was treated with 150 u mol/L cobalt combined with different concentrtion of nimesulide(10. 40 and 80 U M, respectively) for 24 h, the density value of bands of HIF-1 a were 71.45 + 1.78, 42.27 ±1.72, 7.35+1.15 respectively and the density value of bands of VEGF were 67.33 + 1.73, 43.51 + 1.18, 24.71 + 1.17 respectively while extrinsic PGE2 (100 u M) reversed such inhibitory effects of 80 U M nimesulide (the density value of bands of HIF-1 a and VEGF were 90.37 + 2.55, 82.99 + 2.25 respectively ). There was a negative correlation between the concentrtion of nimesulide and the protein expression of HIF-1 a and VEGF, the correlation factor was —0.773 and —0.729 respectively (P<0.01). 3.1mmunocytochemical analysis of the effects of nimesulide on the expression of HIF-1 a , VEGF protein in BGC-823 cells induced by cobalt and the reversed effects of PGE2Image analysis revealed that HIF-1 a protein was weakly expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a and VEGF protein were 0.045 + 0.011, 0.143 + 0.013 respectively. Cobalt markedly induced the expression of HIF-1 a and VEGF protein. After stimulated by cobalt for 24 h, the optical density value of HIF-1 a and VEGF protein were 0.963 + 0.023 and 0.852±0.018 respectively. Nimesulide prevented cobalt-simulated up-regulation of HIF-1 a and VEGF protein in a dose-dependent manner. When BGC-823 cells was treated with 150 V mol/L cobalt combined with different concentrtion of nimesulide(10, 40 and 80 U M, respectively) for 24 h, the optical density value of HIF-1 a protein were 0.713 + 0.012, 0.442±0.014 and 0.126±0.011 respectively( P < 0.01) and the optical density value of VEGF protein were 0.725 ±0.021, 0.425 + 0.019 and 0.187 ± 0.025 respectively (P < 0.01) while extrinsic PGE2 (100 u M) reversed such inhibitory effects of nimesulide, the density value of bands of HIF-1 a and VEGF
were 0.132 + 0.018, 0.211 + 0.023 respectively.The third part: The effects of extrinsic PGE2 on the expression of HIF-1 a and VEGF in BGC-823 cells and its signal transduction pathway Methodsl.Cell culture and experimental groupsBGC-823 cells were grown to about 60 %~70 % confluence and were serum starved (0.1% FCS) for 24 h before PGE2 was added. Experimental groups were divided according to experimental purposes.(DTo observe the effects of PGE2 on the expression of HIF-1 a , VEGF mRNA and protein in BGC-823 cells, the experimental samples were divided into four groups: ?no stimulation group?l u M PGE2 group? 10 u M PGE2 group? 100 u M PGE2 group. After incubation for a given duration, the cells were harvested. (2)To investigate the effects of MEK inhibitor PD98059 on the PGE2-induced expression of HIF-1 a , VEGF in BGC-823 cells, the experimental samples were divided into four groups: ?no stimulation group(2)100u M PGE2 group(3)100n M PGE2 plus 10 u M PD98059 group?50 u M PGE2 plus 50 u M PD98059 group (5) 100 u M PGE2 plus 100 u MPD98059 group. Kinase inhibitors PD98059 stimulated the cells 30 min before PGE2 treatment. After incubation for a given duration, the cells were harvested.(3)To investigate the effects of PI3K inhibitor Wortmannin on the PGE2-inducied expression of HIF-1 a and VEGF in BGC-823 cells, the experimental samples were divided into four groups: ?no stimulation group(l)100n M PGE2 group(3)100y M PGE2 plus 50 nM Wortmannin group? 100 u M PGE2 plus u M 100 nM Wortmannin group? 100 u M PGE2 plus 200 nM Wortmannin group. Kinase inhibitors Wortmannin was added 30 min before exposure to PGE2. After incubation for a given duration, the cells were harvested.2. Total RNA isolation and RT-PCR assay The materials and methods was the same as the first part. 3.Western blot analysis The materials and methods was the same as the first part.
4.1mmunocytochemistry The materials and methods was the same as the firstpart.5.Statistical analysis The methods was the same as the first part.Results1. RT-PCR analysis of the effects of PGE2 on the expression of HIF-1 a , VEGF mRNA in BGC-823 cellsRT-PCR analysis revealed that BGC-823 cells expressed a low level of VEGF mRNA but constitutively expressed HIF-1 a mRNA under normoxic condition, the brightness value of bands of VEGF, HIF-1 a mRNA were 0.2683 + 0.0159, 0.9489 + 0.0121 respectively. HIF-1 a mRNA expression was not induced by PGE2 stimulation. PGE2 stimulated the expression of VEGF mRNA in BGC-823 cells in a dose-dependant manner. After stimulated by PGE2 for 24 h, the brightness value of bands of HIF-1 a were 0.9523 + 0.0167, 0.9486 ±0.0193, 0.9353 + 0.0178 respectively and the brightness value of bands of VEGF were 0.3816 + 0.0122, 0.5342 + 0.0137, 0.6875 + 0.0211. There was a positive correlation between the concentrtion of PGE2 and the mRNA expression of VEGF, the correlation factor was 0.782 (P<0.01).2. Western blot analysis of the effects of PGE2 on the expression of HIF-1 a ? VEGF protein in BGC-823 cellsWestern blot analysis revealed that HIF-1 a protein was weakly or negatively expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a , VEGF protein were 5 ±1.14, 23.13 + 1.33 respectively. PGE2 significantly induced the expression of HIF-1 a and VEGF protein in normoxic cells in a dose-dependant manner at 24h. After stimulated by PGE2 for 24 h, the density value of bands of VEGF were 35.32 + 1.47, 54.41 ±1.39, 69.48 + 2.21 respectively (P < 0.01)and the density value of bands of HIF-1 a were 42.43±1.67, 67.33±2.35, 81.37±2.59 respectively(P < 0.01). There was a positive correlation between the protein expression of HIF-1 a and VEGF, the correlation factor was 0.811 (P<0.01). There was a negative
correlation between the concentrtion of PGE2 and the protein expression of HIF-1 a and VEGF, the correlation factor was 0.746 and 0.758 respectively (P<0.01).3. Immunocytochemical analysis of the effects of PGE2 on the expression of HIF-1 a and VEGF protein in BGC-823 cellsImage analysis revealed that that HIF-1 a was expressed in the cytoplasm of tumor cells and VEGF was predominantly expressed in the cytoplasm of tumor cells. HIF-1 a protein was weakly or negatively expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic condition, the density value of bands of HIF-1 a , VEGF protein were 0.0426 ± 0.0038, 0.1231 ± 0.0033 respectively. PGE2 significantly induced the expression of HIF-1 a and VEGF protein in normoxic cells in a dose-dependant manner at 24h. After stimulated by PGE2 for 24 h, the Optical density value of VEGF were 0.385 + 0.017, 0.665 + 0.014, 0.732 + 0.019 respectively and the density value of HIF-1 a were 0.476± 0.022, 0.760 + 0.013, 0.897±0.015 respectively(P < 0.01).4. Western blot analysis of the effects of PD98059 on HIF-1 a protein expression stimulated by PGE2 in BGC-823 cellsWestern blot analysis demonstrated that HIF-1 a protein was weakly or negatively expressed while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a , VEGF protein were 4.9+1.06, 24.18+1.32 respectively. 100 y M PGE2 significantly induced the expression of HIF-1 a and VEGF protein in normoxic cells at 24h, the density value of bands of HIF-1 a and VEGF were 83.26 ± 2.61 and 72.30 + 2.31 respectively. PD98059, a inhibitor of MAPK inhibiting the activity of MEK, significantly suppressed PGE2-induced up-regulation of HIF-1 a protein expression in a dose-dependent fashion. When BGC-823 cells was treated with 100 u M PGE2 combined with different concentrtion of PD98059(10, 50 and 100 H M, respectively) for 24 h, the density value of bands of HIF-1 a were 73.36 + 1.87, 39.24+1.58, 6.67+1.09 respectively (P<0.01) and the density value of bands of VEGF were 64.52+1.43, 46.43 + 1.15, 28.31 + 1.33 respectively (P<0.01). There was a
negative correlation between the concentrtion of PD98059 and the protein expression of HIF-1 a and VEGF, the correlation factor was —0.791 and —0.772 respectively (P<0.01).5. Western blot analysis of the effects of Wortmannin on HIF-1 a protein expression stimulated by PGE2 in BGC-823 cellsWestern blot analysis demonstrated that HIF-1 a protein was weakly or negatively expressed, while basal VEGF protein levels were found in BGC-823 cells under normoxic conditions, the density value of bands of HIF-1 a , VEGF protein were 5.1 + 1.13, 22.17+1.27 respectively. 100 u M PGE2 significantly induced the expression of HIF-1 a and VEGF protein in normoxic cells at 24h, the density value of bands of HIF-1 a and VEGF were 80.51 + 2.81 and 68.88 ± 2.07 respectively. Wortmannin, a inhibitors of PI3K, significantly but only partially inhibited PGE2-induced up-regulation of HIF-1 a protein expression in a dose-dependent fashion. When BGC-823 cells was treated with 100 u M PGE2 combined with different concentrtion of Wortmannin (50, 100 and 200 nM, respectively) for 24 h, the density value of bands of HIF-1 a were 75.42+1.45, 58.63±1.54, 27.58+1.31 respectively (P<0.05) and the density value of bands of VEGF were 64.79 + 1.71, 51.16 + 1.13, 40.43 + 1.42 respectively (P<0.05).There was a negative correlation between the concentrtion of Wortmannin and the protein expression of HIF-1 a and VEGF, the correlation factor was —0.602 and —0.584 respectively (P<0.05). Conclusionsl.Hypoxia-inducer cobalt induced the expression of COX-2 mRNA and protein, HIF-1 a protein, VEGF mRNA and protein in human gastric cancer cell line BGC-823.2.Nimesulide had inhibitory effects on cobalt-simulated up-regulation of HIF-1 a protein, VEGF mRNA and protein in BGC-823 cells in a dose-dependent manner which may be one of the antiangiogenesis mechanisms of nimesulide. COX-2 inhibitor blocking the hypoxia-inductcd VEGF mRNA expression may act in such
a way by inhibition of HIF-1 u expression protein.3.PGE2 induced the expression of HIF-1 a protein, VEGF mRNA and protein inBGC-823 cells in a dose-dependent manner, but had no effect on HIF-1 a mRNAexpression. PGE2 induces the expression of VEGF mRNA in BGC-823 cells thatmay be mediated by the transcriptional activator HIF-1 a .4.Pharmacologic inhibition of ERK phosphorylation blocked the induction ofVEGF and HIF-1 a protein expression in response to PGE2 stimulation. Inhibitionof PI3K activity also partly blocked PGE2-induced HIF-1 a and VEGF proteinexpression.5.These evidence indicate that COX-2 plays a role in the induction of HIF-1 aexpression in hypoxia. The events demonstrated in the cultured BGC-823 cells mayalso occur in vivo, which maybe have important pathophysiological and therapeuticimplications. This observation implies that the inhibitory effects of nimesulide onhypoxic responses can be developed to suppress tumor angiogenesis.
引文
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36.Schmedtje JF Jr, Ji YS, Liu WL, DuBois RN, Runge MSHypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. J Biol Chem. 1997 Jan 3;272(1):601-8.
37.Bonazzi A, Mastyugin V, Mieyal PA, Dunn MW, Laniado-Schwartzman M. Regulation of cyclooxygenase-2 by hypoxia and peroxisome proliferators in the corneal epithelium. J Biol Chem. 2000 Jan 28;275(4):2837-44.
38.Demasi M, Cleland LG, Cook-Johnson RJ, Caughey GE, James MJ.Effects of hypoxia on monocyte inflammatory mediator production: Dissociation between changes in cyclooxygenase-2 expression and eicosanoid synthesisJ Biol Chem. 2003 Oct 3;278(40):38607-16.
39.Liu XH, Kirschenbaum A. Yao S, et al.Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line. Clin Exp Metastasis. 1999;17(8):687-94.
40.Pichiule P, Chavez JC, LaManna JC.Hypoxic regulation of angiopoietin-2 expression in endothelial cells.J Biol Chem. 2004 Mar 26;279(13):12171-80.