NADPH Oxidase Promotes Pancreatic Cancer Cell Survival via Inhibiting JAK2 Dephosphorylation by Tyrosine Phosphatases
2007; Elsevier BV; Volume: 133; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.08.022
ISSN1528-0012
AutoresJong Kyun Lee, Mouad Edderkaoui, Patrick A. Truong, Izumi Ohno, Kee‐Taek Jang, Andrea Berti, Stephen J. Pandol, Anna S. Gukovskaya,
Tópico(s)Immune cells in cancer
ResumoBackground & Aims: Growth factors, such as insulin-like growth factor-1 (IGF-I), protect pancreatic cancer (PaCa) cells from death. We recently showed that reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase Nox4 mediate the antiapoptotic effect of growth factors. Here, we examine the mechanisms of the antiapoptotic role of NADPH oxidase. We hypothesized that ROSs produced by NADPH oxidase inhibit key protein tyrosine phosphatases (PTPs) and thus sustain the activation of kinases mediating antiapoptotic pathways in PaCa cells. Methods: Transfections and pharmacologic inhibition were used to assess the effects of NADPH oxidase on Janus kinase 2 (JAK2) kinase, the low molecular weight–protein tyrosine phosphatase (LMW-PTP), and apoptosis. Results: We found that 1 target of ROSs is JAK2, an important antiapoptotic kinase in PaCa cells. Both serum-induced and IGF-I biphasic JAK2 phosphorylation, with a rapid (minutes) and transient first phase, and a slow and sustained (24–72 hours) second phase. Nox4 mediated the sustained phase of JAK2 phosphorylation, which was required for the antiapoptotic effects of IGF-I and serum. Transfection experiments identified the LMW-PTP as a negative regulator of sustained JAK2 phosphorylation. Growth factors inhibited LMW-PTP through its oxidation by NADPH oxidase. LMW-PTP colocalizes with Nox4 both in PaCa cells and in human pancreatic adenocarcinoma. Conclusions: The results suggest a novel signaling pathway, in which NADPH oxidase activation results in inhibition of PTPs, such as LMW-PTP, leading, in turn, to enhanced and sustained phosphorylation of kinases, such as JAK2, and suppression of apoptosis. This pathway mediates the prosurvival effect of ROSs and suggests new targets for pancreatic cancer treatment. Background & Aims: Growth factors, such as insulin-like growth factor-1 (IGF-I), protect pancreatic cancer (PaCa) cells from death. We recently showed that reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase Nox4 mediate the antiapoptotic effect of growth factors. Here, we examine the mechanisms of the antiapoptotic role of NADPH oxidase. We hypothesized that ROSs produced by NADPH oxidase inhibit key protein tyrosine phosphatases (PTPs) and thus sustain the activation of kinases mediating antiapoptotic pathways in PaCa cells. Methods: Transfections and pharmacologic inhibition were used to assess the effects of NADPH oxidase on Janus kinase 2 (JAK2) kinase, the low molecular weight–protein tyrosine phosphatase (LMW-PTP), and apoptosis. Results: We found that 1 target of ROSs is JAK2, an important antiapoptotic kinase in PaCa cells. Both serum-induced and IGF-I biphasic JAK2 phosphorylation, with a rapid (minutes) and transient first phase, and a slow and sustained (24–72 hours) second phase. Nox4 mediated the sustained phase of JAK2 phosphorylation, which was required for the antiapoptotic effects of IGF-I and serum. Transfection experiments identified the LMW-PTP as a negative regulator of sustained JAK2 phosphorylation. Growth factors inhibited LMW-PTP through its oxidation by NADPH oxidase. LMW-PTP colocalizes with Nox4 both in PaCa cells and in human pancreatic adenocarcinoma. Conclusions: The results suggest a novel signaling pathway, in which NADPH oxidase activation results in inhibition of PTPs, such as LMW-PTP, leading, in turn, to enhanced and sustained phosphorylation of kinases, such as JAK2, and suppression of apoptosis. This pathway mediates the prosurvival effect of ROSs and suggests new targets for pancreatic cancer treatment. One reason why pancreatic adenocarcinoma is so aggressive and unresponsive to chemotherapy and radiotherapy is its resistance to apoptosis. Growth factors (GFs) and extracellular matrix (ECM) are the 2 major "environmental factors" that protect pancreatic cancer (PaCa) cells from death.1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar, 3Vaquero E.C. Edderkaoui M. Nam K.J. Gukovsky I. Pandol S.J. Gukovskaya A.S. Extracellular matrix proteins protect pancreatic cancer cells from death via mitochondrial and nonmitochondrial pathways.Gastroenterology. 2003; 125: 1188-1202Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Among GFs, insulin-like growth factor-1 (IGF-I) is believed to play a key role in pancreatic tumorigenesis. It is overexpressed in PaCa cells, suppressing apoptosis and stimulating growth both in cell lines and animal models of pancreatic cancer.4Korc M. Role of growth factors in pancreatic cancer.Surg Oncol Clin N Am. 1998; 7: 25-41Abstract Full Text PDF PubMed Google Scholar, 5Min Y. Adachi Y. Yamamoto H. et al.Genetic blockade of the insulin-like growth factor-I receptor: a promising strategy for human pancreatic cancer.Cancer Res. 2003; 63: 6432-6441PubMed Google Scholar We recently showed that the antiapoptotic effects of GFs in PaCa cells are mediated by reactive oxygen species (ROS).1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar We found that GFs activate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, in particular its Nox4 isoform, and that ROSs produced by NADPH oxidase inhibit apoptosis in PaCa cells. Recently, these results were confirmed.6Mochizuki T. Furuta S. Mitsushita J. et al.Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells.Oncogene. 2006; 25: 3699-3707Crossref PubMed Scopus (210) Google Scholar Several prosurvival signaling mechanisms operate in PaCa cells. One important mechanism involves Janus kinases (JAKs), a family of nonreceptor tyrosine kinases that mediate signal transduction from various GFs and cytokines.7Rane S.G. Reddy E.P. Janus kinases: components of multiple signaling pathways.Oncogene. 2000; 19: 5662-5679Crossref PubMed Scopus (399) Google Scholar On activation, JAKs phosphorylate signal transducers and activators of transcription (STATs), the transcription factors that play key roles in cellular proliferation, differentiation, and survival. STATs are overexpressed and persistently activated in a large number of human cancers,8Greten F.R. Weber C.K. Greten T.F. et al.Stat3 and NF-kappa B activation prevents apoptosis in pancreatic carcinogenesis.Gastroenterology. 2002; 123: 2052-2063Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 9Yu H. Jove R. The stats of cancer: new molecular targets come of age.Nat Rev Cancer. 2004; 4: 97-105Crossref PubMed Scopus (2014) Google Scholar resulting in up-regulation of antiapoptotic proteins such as survivin and XIAP (X-linked inhibitor of apoptosis protein), resulting in suppression of apoptosis.8Greten F.R. Weber C.K. Greten T.F. et al.Stat3 and NF-kappa B activation prevents apoptosis in pancreatic carcinogenesis.Gastroenterology. 2002; 123: 2052-2063Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 9Yu H. Jove R. The stats of cancer: new molecular targets come of age.Nat Rev Cancer. 2004; 4: 97-105Crossref PubMed Scopus (2014) Google Scholar JAK2 is constitutively activated in PaCa cells and its inhibition stimulates apoptosis.10Scholz A. Heinze S. Detjen K.M. et al.Activated signal transducer and activator of transcription 3 (STAT3) supports the malignant phenotype of human pancreatic cancer.Gastroenterology. 2003; 125: 891-905Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 11Toyonaga T. Nakano K. Nagano M. et al.Blockade of constitutively activated Janus kinase/signal transducer and activator of transcription-3 pathway inhibits growth of human pancreatic cancer.Cancer Lett. 2003; 201: 107-116Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar NADPH oxidase mediates activation of the JAK2/STAT pathway induced by angiotensin in smooth muscle cells12Schieffer B. Luchtefeld M. Braun S. Hilfiker A. Hilfiker-Kleiner D. Drexler H. Role of NAD(P)H oxidase in angiotensin II-induced JAK/STAT signaling and cytokine induction.Circ Res. 2000; 87: 1195-1201Crossref PubMed Scopus (244) Google Scholar and by a respiratory virus in alveolar epithelial cells.13Liu T.S. Castro S. Brasier A.R. Jamaluddin M. Garofalo R.P. Casola A. Reactive oxygen species mediate virus-induced STAT activation: role of tyrosine phosphatases.J Biol Chem. 2004; 279: 2461-2469Crossref PubMed Scopus (133) Google Scholar These data suggest that activation of the JAK2/STAT pathway could be 1 mechanism through which NADPH oxidase inhibits apoptosis in PaCa cells. JAK activity is determined by the level of its phosphorylation. ROSs could activate JAK2 by stimulating its receptor-induced phosphorylation7Rane S.G. Reddy E.P. Janus kinases: components of multiple signaling pathways.Oncogene. 2000; 19: 5662-5679Crossref PubMed Scopus (399) Google Scholar or by inhibiting protein tyrosine phosphatases (PTPs) that dephosphorylate JAK2.14Myers M.P. Andersen J.N. Cheng A. et al.TYK2 and JAR2 are substrates of protein-tyrosine phosphatase 1B.J Biol Chem. 2001; 276: 47771-47774Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar Evidence indicates that PTPs are more sensitive to ROSs than are tyrosine kinases.15Chiarugi P. Cirri P. Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction.Trends Biochem Sci. 2003; 28: 509-514Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar All PTPs contain an essential cysteine residue in their catalytic pocket, which has an unusually low pKa (negative logarithm of acid ionization constant) and is thus present predominantly as a thiolate anion, rendering it highly susceptible to oxidation.15Chiarugi P. Cirri P. Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction.Trends Biochem Sci. 2003; 28: 509-514Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 16Tonks N.K. Redox redux: revisiting PTPs and the control of cell signaling.Cell. 2005; 121: 667-670Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar As a result, PTPs are reversibly oxidized and inactivated by low levels of ROSs.15Chiarugi P. Cirri P. Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction.Trends Biochem Sci. 2003; 28: 509-514Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 16Tonks N.K. Redox redux: revisiting PTPs and the control of cell signaling.Cell. 2005; 121: 667-670Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar Low molecular weight (LMW)–PTP is highly expressed in a number of cancer cell types.17Malentacchi F. Marzocchini R. Gelmini S. et al.Up-regulated expression of low molecular weight protein tyrosine phosphatases in different human cancers.Biochem Biophys Res Commun. 2005; 334: 875-883Crossref PubMed Scopus (72) Google Scholar LMW-PTP regulates signaling from platelet-derived GF and insulin receptors,18Taddei M.L. Chiarugi P. Cirri P. et al.LMW-PTP exerts a differential regulation on PDGF- and insulin-mediated signaling.Biochem Biophys Res Commun. 2000; 270: 564-569Crossref PubMed Scopus (32) Google Scholar and phosphorylation of Ephrin receptors19Stein E. Lane A.A. Cerretti D.P. et al.Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses.Genes Dev. 1998; 12: 667-678Crossref PubMed Scopus (372) Google Scholar and focal adhesion kinases.20Rigacci S. Rovida E. Dello Sbarba P. Berti A. Low M-r phosphotyrosine protein phosphatase associates and dephosphorylates p125 focal adhesion kinase, interfering with cell motility and spreading.J Biol Chem. 2002; 277: 41631-41636Crossref PubMed Scopus (42) Google Scholar In fibroblasts, exogenous H2O2 oxidized and inactivated LMW-PTP.21Chiarugi P. Fiaschi T. Taddei M.L. et al.Two vicinal cysteines confer a peculiar redox regulation to low molecular weight protein tyrosine phosphatase in response to platelet-derived growth factor receptor stimulation.J Biol Chem. 2001; 276: 33478-33487Crossref PubMed Scopus (170) Google Scholar Here, we examined the roles of NADPH oxidase, JAK2 kinase, and LMW-PTP in the prosurvival effects of IGF-I and serum in PaCa cells. We found that ROSs, which are generated by GF-activated Nox4, inhibit PTP activity in PaCa cells and, in particular, oxidize and inactivate LMW-PTP. In turn, PTP inhibition is required to maintain sustained JAK2 phosphorylation, resulting in suppression of apoptosis. Our results suggest a novel mechanism that may mediate the antiapoptotic effect of GFs in cancer cells. See supplemental material available online at www.gastrojournal.org. GFs are known to induce a rapid (over minutes) and transient activation of JAK kinases.22Rui H. Kirken R.A. Farrar W.L. Activation of receptor-associated tyrosine kinase JAK2 by prolactin.J Biol Chem. 1994; 269: 5364-5368Abstract Full Text PDF PubMed Google Scholar By contrast, we found that in PaCa cells the GF-induced increase in ROS is characterized by slow (hours and days) kinetics.1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar Therefore, to determine the role of ROS in the regulation of JAK2 activation, we first measured a detailed time course of GF-induced JAK2 phosphorylation in MIA PaCa-2 and PANC-1 cells. The level of JAK2 phosphorylation is a measure of its activation. We found that both serum and IGF-I caused a biphasic JAK2 activation in PaCa cells (Figure 1). In addition to the rapid and transient first phase, there were a second, slow (detected after 4 hours of culture with GFs) phase and sustained (up to 72 hours of observation) phase of JAK2 phosphorylation (Figure 1A and C). Densitometric analysis (Figure 1B and D) showed that the level of JAK2 phosphorylation during the second phase reached ∼70% of the first peak. Of note, in the absence of GFs there were no changes in JAK2 phosphorylation during the 72-hour culture. Similarly, serum induced a biphasic time-dependent increase in Akt phosphorylation in MIA PaCa-2 cells (Supplementary Figure 1; see supplemental material online at www.gastrojournal.org), indicating that sustained phosphorylation is not unique for JAK2. In parallel, we measured the effects of fetal bovine serum (FBS) and IGF-I on intracellular ROS levels, using cells labeled with the ROS-sensitive dye dichlorofluorescein (DCF).1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar As we described previously,1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar the GFs stimulated a slow increase in cellular ROS (Figure 1B and D). The second phase of JAK2 phosphorylation coincides with the ROS increase, whereas the first, transient phase occurs before the increase in ROS (Figure 1B and D). These data suggest that the sustained phase of GF-induced JAK2 phosphorylation could be linked with the ROS increase. To determine the role of NADPH oxidase in JAK2 phosphorylation, we first applied diphenylene iodonium (DPI), which, as we showed before, inhibited ROS increases induced by IGF-I and serum, as well as the NADPH oxidase activation. DPI prevented the second, sustained phase of JAK2 phosphorylation in both MIA PaCa-2 and PANC-1 cells (Figure 2). DPI not only inhibited JAK2 phosphorylation but also somewhat decreased JAK2 protein levels. The effects of DPI on phosphorylated JAK2 were, however, much greater than on total JAK2 (Figure 2A and B). DPI prevented the second, sustained phase of JAK2 phosphorylation, whereas it did not affect the first, transient phase (Figure 2C). Indeed, DPI inhibited the sustained JAK2 phosphorylation independently of whether it was added before or 1 hour after the GFs, that is, when the first, transient peak subsided (Figure 2D and E). These results suggest that the sustained phase of GF-induced JAK2 phosphorylation is mediated by NADPH oxidase. We next measured JAK2 phosphorylation in cells transfected with either the Nox4 antisense oligonucleotides1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar or the Nox4 small interfering RNA (siRNA) plasmid23Park H.S. Jung H.Y. Park E.Y. Kim J. Lee W.J. Bae Y.S. Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B.J Immunol. 2004; 173: 3589-3593Crossref PubMed Scopus (545) Google Scholar (as well as with the control oligonucleotide or plasmid) (Figure 3). As we showed previously,1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar Nox4 antisense oligonucleotide efficiently decreased Nox4 protein levels in both cell lines (Figure 3B and D). A similar inhibition of Nox4 was achieved by using the Nox4 siRNA (Figure 3F). We1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar and Mochizuki et al6Mochizuki T. Furuta S. Mitsushita J. et al.Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells.Oncogene. 2006; 25: 3699-3707Crossref PubMed Scopus (210) Google Scholar also showed that Nox4 inhibition with antisense oligonucleotide or siRNA decreased ROS levels in PaCa cells by 25%–60%. Inhibition of Nox4 with antisense or siRNA resulted in 30%–65% decrease of JAK2 phosphorylation in PaCa cells cultured with GFs (Figure 3A, C, and E). The decrease of JAK2 phosphorylation was similar to that in Nox4 protein levels (Figure 3B, D, and F). Nox4 inhibition with antisense or siRNA did not affect JAK2 protein level (Figure 3A, C, and E). The combined results in Figure 1, Figure 2, Figure 3 indicate that GF-induced activation of NADPH oxidase, in particular Nox4, mediates the sustained phase of JAK2 phosphorylation in PaCa cells. As we showed,1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar GFs inhibit apoptosis in MIA PaCa-2 and PANC-1 cells. This inhibition was dose dependently prevented by the specific inhibitor of JAK2, AG490; for example, 50 μmol/L AG490 increased the internucleosomal DNA fragmentation 2.5- to 3.5-fold (Figure 4A and B). There was little effect of AG490 in cells cultured without GFs (Figure 4A). We also measured the effect of AG490 on cell death using Annexin-V and propidium iodide (AnV/PI) staining to monitor phosphatidylserine externalization, an early event in apoptosis. As we previously showed, in PaCa cells both AnV+/PI− and AnV+/PI+ groups contain apoptotic cells.3Vaquero E.C. Edderkaoui M. Nam K.J. Gukovsky I. Pandol S.J. Gukovskaya A.S. Extracellular matrix proteins protect pancreatic cancer cells from death via mitochondrial and nonmitochondrial pathways.Gastroenterology. 2003; 125: 1188-1202Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar AnV+/PI− group is comprised of apoptotic cells only, whereas the AnV+/PI+ group included both primary necrotic cells and apoptotic cells associated with secondary necrosis. We measured that AG490 markedly increased the percentage of cells in both AnV+/PI− and AnV+/PI+ groups (Figure 4C), suggesting that JAK2 mediates the prosurvival effect of the GFs through suppressing both apoptosis and necrosis in PaCa cells. We inhibited JAK2 by transfecting PANC-1 cells with JAK2 antisense oligonucleotide, which resulted in a pronounced decrease in JAK2 protein (as well as the levels of phosphorylated JAK2) (Figure 4D). Transfection with JAK2 antisense markedly increased DNA fragmentation and the percentage of AnV+/PI− and AnV+/PI+ cells compared with cells transfected with control oligonucleotide (Figure 4E and F). To specifically assess the role of the sustained phase of JAK2 activation in the antiapoptotic effect of JAK2, we added AG490 either before or after 1-hour stimulation of cells with serum, ie, after the first, transient phase of JAK2 phosphorylation subsided (Figure 1). AG490 stimulated apoptosis (ie, DNA fragmentation) to the same extent independently of whether it was added before or 1 hour after serum (Figure 4G). Western blot analysis verified that in both situations AG490 similarly inhibited sustained JAK2 phosphorylation (data not shown). The effect of DPI on apoptosis was similar to that observed with AG490 (Figure 4G), namely, DPI prevented the antiapoptotic effect of serum independently of whether it was added before or 1 hour after serum. Together with Figure 2, Figure 3, these data indicate that the effect of DPI on apoptosis is through inhibition of the sustained phase of JAK2 phosphorylation, that is, the phase mediated by NADPH oxidase (ie, Nox4). We showed previously that inhibiting NADPH oxidase, (in particular, Nox4), but not other ROS-producing systems, abrogates the antiapoptotic effect of GFs in PaCa cells.1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar Indeed, DPI or Nox4 knockdown with Nox4 siRNA both stimulated apoptosis in PaCa cells1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar; similar stimulation was observed with the superoxide scavenger Tiron.1Vaquero E.C. Edderkaoui M. Pandol S.J. Gukovsky I. Gukovskaya A.S. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells.J Biol Chem. 2004; 279: 34643-34654Crossref PubMed Scopus (319) Google Scholar, 2Edderkaoui M. Hong P. Vaquero E.C. et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G1137-G1147Crossref PubMed Scopus (121) Google Scholar By contrast, buthylhydroxyanisole, which decreases cellular ROSs through inhibiting mitochondrial ROS production,24Ferreira J. Effect of butylated hydroxyanisole on electron transport in rat liver mitochondria.Biochem Pharmacol. 1990; 40: 677-684Crossref PubMed Scopus (38) Google Scholar had no effect on apoptosis in MIA PaCa-2 cells (not shown). One mechanism through which JAK2 inhibits apoptosis is by phosphorylation of the transcription factors STAT1 and STAT3.9Yu H. Jove R. The stats of cancer: new molecular targets come of age.Nat Rev Cancer. 2004; 4: 97-105Crossref PubMed Scopus (2014) Google Scholar, 25Kovacic B. Stoiber D. Moriggl R. et al.STAT1 acts as a tumor promoter for leukemia development.Cancer Cell. 2006; 10: 77-87Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar Both IGF-I and FBS stimulated STAT1 phosphorylation in MIA PaCa-2 cells (Figure 5A). JAK2 antisense but not control oligonucleotide inhibited STAT1 phosphorylation (Figure 5B and E), indicating that STAT1 is a target of JAK2 in these cells. STAT1 knockdown with siRNA markedly stimulated DNA fragmentation in both MIA PaCa-2 and PANC-1 cells, indicating a prosurvival role for STAT1 (Figure 5C and F). To determine the underlying mechanisms we measured the effect of STAT1 knockdown on endogenous caspase inhibitors XIAP and survivin, potent antiapoptotic proteins.26McDermott U. Longley D.B. Galligan L. et al.Effect of p53 status and STAT1 on chemotherapy-induced, Fas-mediated apoptosis in colorectal cancer.Cancer Res. 2005; 65: 8951-8960Crossref PubMed Scopus (60) Google Scholar STAT1 siRNA decreased the levels of XIAP and survivin in MIA PaCa-2 cells treated with IGF-I and FBS (Figure 5D), suggesting that 1 mechanism of the antiapoptotic effect of STAT1 is through up-regulation of these endogenous caspase inhibitors. JAK2 antisense oligonucleotide also inhibited STAT3 phosphorylation (Supplementary Figure 2; see supplemental material online at www.gastrojournal.org), indicating that both STAT1 (Figure 5B and E) and STAT3 (Supplementary Figure 2) are targets of JAK2 in PaCa cells. STAT3 inhibitor pyridone 6 at doses 1–4 μmol/L increased apoptosis in PaCa cells (Supplementary Figure 2; data not shown). Of note, pyridone 6 is specific against STAT327Pedranzini L. Dechow T. Berishaj M. et al.Pyridone 6, a pan-janus-activated kinase inhibitor, induces growth inhibition of multiple myeloma cells.Cancer Res. 2006; 66: 9714-9721Crossref PubMed Scopus (139) Google Scholar; however, it inhibits activation of STAT3 by all JAKs, including JAK2.27Pedranzini L. Dechow T. Berishaj M. et al.Pyridone 6, a pan-janus-activated kinase inhibitor, induces growth inhibition of multiple myeloma cells.Cancer Res. 2006; 66: 9714-9721Crossref PubMed Scopus (139) Google Scholar Differently, AG490 is more specific for JAK2, but it inhibits both STAT3 and STAT1.28McWhinney C.D. Hunt R.A. Conrad K.M. Dostal D.E. Baker K.M. The type I angiotensin II receptor couples to Stat1 and Stat3 activation through Jak2 kinase in neonatal rat cardiac myocytes.J Mol Cell Cardiol. 1997; 29: 2513-2524Abstract Full Text PDF PubMed Scopus (102) Google Scholar Pyridone 6 increased DNA fragmentation by only 50% (Supplementary Figure 2), whereas AG490 increased apoptosis in PANC-1 cells >2 times (Figure 4). These data indicate that both STAT1 and STAT3 are anti
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