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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

100%

Bobarykina A. Y., Minchenko D. O., Opentanova I. L., Moenner M., Caro J., Esumi H., Minchenko O. H.

2006

vol. 53

issue 4

789-799

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-1α, HIF-2α, 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-α 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-1α protein are increased in both gastric and pancreatic cancer cell lines under hypoxic conditions. However, the basal level of HIF-1α as well as HIF-2α mRNA expression and their hypoxia responsiveness are different in the MKN45 and NUGC3 cancer cells. Thus, the expression of HIF-1α mRNA is decreased in both gastric cancer cell lines treated by hypoxia or dimethyloxalylglycine, but HIF-2α 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-1α 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.

Inhibition of kinase and endoribonuclease activity of ERN1/IRE1α affects expression of proliferation related genes in U87 glioma cells

100%

Minchenko O. H., Tsymbal D. O., Minchenko D. O., Moenner M., Kovalevska O. V., Lypova N. M.

Endoplasmic Reticulum Stress in Diseases

2015

vol. 2

issue 1

Inhibition of ERN1/IRE1α (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme-1α), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth. We have studied the expression of transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F1), and HOXC6 (homeobox C6) in U87 glioma cells overexpressing dominant-negative ERN1/IRE1α defective in endoribonuclease (dnr-ERN1) as well as defective in both kinase and endonuclease (dn-ERN1) activity of ERN1/IRE1α. We have demonstrated that the expression of all studied genes is decreased at the mRNA level in cells with modified ERN1/IRE1α; TBX3, however, is increased in these cells as compared to control glioma cells. Changes in protein levels of E2F8, HOXC6, ATF3, and TBX3 corresponded to changes in mRNAs levels. We also found that two mutated ERN1/IRE1α have differential effects on the expression of studied transcripts. The presence of kinase and endonuclease deficient ERN1/IRE1α in glioma cells had a less profound effect on the expression of E2F8, HOXC6, and TBX3 genes than the blockade of the endoribonuclease activity of ERN1/IRE1α alone. Kinase and endonuclease deficient ERN1/IRE1α suppresses ATF3 and FOXF1 gene expressions, while inhibition of only endoribonuclease of ERN1/IRE1α leads to the up-regulation of these gene transcripts. The present study demonstrates that fine-tuning of the expression of proliferation related genes is regulated by ERN1/IRE1α an effector of endoplasmic reticulum stress. Inhibition of ERN1/IRE1α, especially its endoribonuclease activity, correlates with deregulation of proliferation related genes and thus slower tumor growth.

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1 Acta Biochimica Polonica

1 Endoplasmic Reticulum Stress in Diseases

1 2015

1 2006

2 Minchenko D. O.

2 Minchenko O. H.

2 Moenner M.

1 Bobarykina A. Y.

1 Caro J.

1 Esumi H.

1 Kovalevska O. V.

1 Lypova N. M.

1 Opentanova I. L.

1 Tsymbal D. O.

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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

Inhibition of kinase and endoribonuclease activity of ERN1/IRE1α affects expression of proliferation related genes in U87 glioma cells