Hypoxic regulation of PFKFB-3 and PFKFB-4 gene expression in gastric and pancreatic cancer cell lines and expression of PFKFB genes in gastric cancers.

Previously we have shown that hypoxia strongly induces the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) genes in several cancer cell lines via a HIF-dependent mechanism. In this paper we studied the expression and hypoxic regulation of PFKFB-4 and PFKFB-3 mRNA as well as its correlation with HIF-1alpha, HIF-2alpha, VEGF and Glut1 mRNA expression in the pancreatic cancer cell line Panc1 and two gastric cancer cell lines MKN45 and NUGC3. This study clearly demonstrated that PFKFB-3 and PFKFB-4 mRNA are expresses in MKN45, NUGC3 and Panc1 cancers cells and that both genes are responsive to hypoxia in vitro. However, their basal level of expression and hypoxia responsiveness vary in the different cells studied. Particularly, PFKFB-3 mRNA is highly expressed in MKN45 and NUGC3 cancer cells, with the highest response to hypoxia in the NUGC3 cell line. The PFKFB-4 mRNA has a variable low basal level of expression in both gastric and pancreatic cancer cell lines. However, the highest hypoxia response of PFKFB-4 mRNA is found in the pancreatic cancer cell line Panc1. The basal level of PFKFB-4 protein expression is the highest in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with the highest response to hypoxia in the pancreatic cancer cell line. Further studies showed that PFKFB-3 and PFKFB-4 gene expression was highly responsive to the hypoxia mimic dimethyloxalylglycine, a specific inhibitor of HIF-alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of PFKFB-3 and PFKFB-4 genes in these cell lines is regulated by the HIF transcription complex. The expression of VEGF and Glut1, which are known HIF-dependent genes, is also strongly induced under hypoxic conditions in gastric and pancreatic cancer cell lines. The levels of HIF-1alpha protein are increased in both gastric and pancreatic cancer cell lines under hypoxic conditions. However, the basal level of HIF-1alpha as well as HIF-2alpha mRNA expression and their hypoxia responsiveness are different in the MKN45 and NUGC3 cancer cells. Thus, the expression of HIF-1alpha mRNA is decreased in both gastric cancer cell lines treated by hypoxia or dimethyloxalylglycine, but HIF-2alpha mRNA expression is not changed significantly in NUGC3 and slightly increased in MKN45 cells. Expression of PFKFB-4 and PFKFB-3 was also studied in gastric cancers and corresponding nonmalignant tissue counterparts from the same patients on both the mRNA and protein levels. The expression of PFKFB-3 and PFKFB-4 mRNA as well as PFKFB-1 and PFKFB-2 mRNA was observed in normal human gastric tissue and was increased in malignant gastric tumors. The basal level of PFKFB-4 protein expression in gastric cancers was much higher as compared to the PFKFB-3 isoenzyme. In conclusion, this study provides evidence that PFKFB-4 and PFKFB-3 genes are also expressed in gastric and pancreatic cancer cells, they strongly respond to hypoxia via a HIF-1alpha dependent mechanism and, together with the expression of PFKFB-1 and PFKFB-2 genes, possibly have a significant role in the Warburg effect which is found in malignant cells.

Bobarykina AY ，Minchenko DO ，Opentanova IL ，Moenner M ，Caro J ，Esumi H ，Minchenko OH ... -  《ACTA BIOCHIMICA POLONICA》

Hypoxia induces transcription of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 gene via hypoxia-inducible factor-1alpha activation.

The PFKFB4 gene encodes isoenzyme of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB or PFK-2/FBPase-2) which originally was found in the testes. We have studied hypoxic regulation of PFKFB4 gene in prostate cancer cell line, PC-3, and several other cancer cell lines. It was shown that hypoxia significantly induced PFKFB4 mRNA levels in PC-3 as well as in HeLa, Hep3B and HepG2 cell lines. Hypoxia increased PFKFB4 protein levels also. Moreover, desferrioxamine and cobalt chloride, which are known to mimic hypoxia, also had a stimulatory effect on the expression of PFKFB4 mRNA. In order to investigate the mechanisms of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, which has the ability to mimic effect of hypoxia by significant induction of hypoxia-inducible factor (HIF-1alpha) protein levels. Our studies showed that PFKFB4 mRNA expression in PC-3, HeLa, Hep3B and HepG2 cell lines was highly responsive to dimethyloxalylglycine, an inhibitor of HIF-1alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of this gene is regulated by HIF proteins. To better understand the hypoxic regulation of PFKFB4 gene expression, we isolated genomic DNA, which includes the promoter region of PFKFB4. Cell transfection, deletion and site-specific mutagenesis of the PFKFB4 promoter region indicates that hypoxic induction of PFKFB4 gene expression is mediated by the hypoxia-responsive element (HRE). These experiments identified a HRE 422-429 bp upstream from the translation start site. Thus, our results indicate that testis-specific form of PFKFB or PFK-2/FBPase-2 is also expressed in several cancer cell lines and that hypoxia induces transcription of PFKFB4 gene in these cell lines by HIF-1alpha dependent mechanism. HRE in 5'-promoter region of PFKFB4 gene mediates hypoxic induction of PFKFB4 gene transcription.

Minchenko O ，Opentanova I ，Minchenko D ，Ogura T ，Esumi H ... -  《FEBS LETTERS》

Mechanisms of regulation of PFKFB expression in pancreatic and gastric cancer cells.

Minchenko OH ，Tsuchihara K ，Minchenko DO ，Bikfalvi A ，Esumi H ... -  《-》

Effects of YC-1 on hypoxia-inducible factor 1-driven transcription activity, cell proliferative vitality, and apoptosis in hypoxic human pancreatic cancer cells.

To investigate the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) on HIF-1-driven transcription activity, cell proliferative vitality, and apoptosis in hypoxic human pancreatic cancer cells. Human pancreatic cancer PC-3 cells were incubated under normoxic or hypoxic conditions. YC-1 was added to the media with different concentrations. The HIF-1alpha protein expression was detected by means of immunocytochemical staining and Western blotting. Semiquantitative reverse transcriptase polymerase chain reaction was used to determine the mRNA expression of HIF-1alpha, vascular endothelial growth factor (VEGF), and glucose phosphate isomerase (GPI). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry were used to detect the cells' proliferative vitality and apoptosis. Hypoxic PC-3 cells expressed a higher level of HIF-alpha protein in nucleus compared with the normoxic controls. When the dose of YC-1 was at 100 micromol/L, the expression location of HIF-alpha shifted from nucleus to cytoplasm. Western blotting revealed that YC-1 reduced the level of HIF-1alpha protein expression, and the inhibitory effect was dose dependent. Moreover, YC-1 dose dependently inhibited mRNA expression levels of VEGF and GPI in hypoxic cells. YC-1 inhibited proliferative vitality and induced apoptosis of hypoxic PC-3 cells in a dose-dependent manner. YC-1 inhibits HIF-1alpha expression in hypoxic pancreatic cancer cells, which is accompanied by the translocation of HIF-1alpha from nucleus to cytoplasm, decreased mRNA expression of VEGF and GPI, reduced cell proliferative vitality, and increased apoptosis. These results suggest that HIF-1 is a potential therapeutic target for pancreatic cancer.

Zhao Q ，Du J ，Gu H ，Teng X ，Zhang Q ，Qin H ，Liu N ... -  《-》

Constitutive expression of hypoxia-inducible factor-1alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and nutrient deprivation.

Hypovasculature is an outstanding characteristic of pancreatic cancers in imaging diagnosis, suggesting that blood supply is poor in pancreatic cancer tissues. Despite poor blood supply, pancreatic cancer cells survive and proliferate in severe hypoxia and nutrient deprivation. To demonstrate how pancreatic cancer cells adapt themselves to hypoxia and nutrient deprivation, we investigated the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) protein and HIF-1-inducible genes in human pancreatic cancer cell lines in comparison with other cancer cell lines. We found that HIF-1alpha protein was constitutively expressed in 15 of 20 pancreatic cancer cell lines (75%) but in none of other cancer cell lines tested in this study. The cells with constitutive expression of HIF-1alpha were more resistant to apoptosis induced by hypoxia and glucose deprivation than those without constitutive expression of HIF-1alpha. Transfection with HIF-1alpha transformed the latter cells resistant to apoptosis and increased in vivo tumorigenicity. Furthermore, anaerobic metabolism-associated genes, Glut1 and aldolase A, were more highly expressed in the cells with constitutive expression of HIF-1alpha than in the cells without it. These results suggest that constitutive expression of HIF-1alpha contributes to the survival and proliferation of pancreatic cancer cells in hypoxia and glucose deprivation through the activation of anaerobic metabolism.

Akakura N ，Kobayashi M ，Horiuchi I ，Suzuki A ，Wang J ，Chen J ，Niizeki H ，Kawamura Ki ，Hosokawa M ，Asaka M ... -  《CANCER RESEARCH》