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Dual oxidase expression is epigenetically silenced in pancreatic ductal adenocarcinoma
A. Newman / Free Radical Biology and Medicine 128 (2018) S61–S78
cancer cell lines SCC-40 (Squamous Cell Carcinoma) and Papillary Cystadenoma Lymphomatosum (PCL) by using western blots and the Agilent Seahorse XF Cell Mito Stress Test. We also studied the migratory capacity of PCL cells using wound-healing assays. A wound-healing assay mimics cell migration during wound healing in-vivo. The results we obtained from this project indicate an increase in the amount of oxidative phosphorylation taking place in the mitochondria of PCL and SCC-40 cancer cell lines, as well as a decrease in the rate of glycolysis. As a result, fusarochromanone is shown to cause an increase in the amount of Reactive Oxygen Species (ROS) in the mitochondria of these cancer cell lines and thus causes an increase in mitochondrial bioenergetics. The data serves to strengthen the potential of FC-101 as an anti-cancer drug.
S71
148
Pharmacological ascorbate enhances expression of dual oxidase which mediates sustained toxicity via alterations in cellular metabolism Adrienne Gibson, Brianne O'Leary*, Matthew Alexander, Juan Du, Brett Wagner, Prabhat Goswami, Garry Buettner, Joseph Cullen The University of Iowa, USA
https://doi.org/10.1016/j.freeradbiomed.2018.10.150
147
Glyoxalase 2 correlates with redox signaling and enhances survival outcome in patients with hepatocellular carcinoma Dinh-Truong Nguyen 1,*, Quynh Hoa Tran 2, Xuan The Hoang 2, Ngoc Huyen Vu 2, Kim Van Thi Le 3, Van Thai Than 4, Minh Nam Nguyen 5 1
Tan Tao University, Viet Nam Ho Chi Minh University of Food Industry, Viet Nam 3 National Institute of Medicinal Materials, Viet Nam 4 Nguyen Tat Thanh University, Viet Nam 5 Ton Duc Thang University, Viet Nam 2
Glyoxalase 2 (Glo2), the second of two enzymes in the glyoxalase system, protects against cellular damage by detoxification of methylglyoxal which is involved in oxidative stress and mitochondrial dysfunction. However, the role of Glo2 in Hepatocellular carcinoma (HCC) progression and survival outcome remains unclear. Here, we investigated the expression of Glo2 in HCC and normal tissues, as well as identified the correlation of Glo2 with the clinicopathological, recurrence risk, patient survival, and other genes related to redox signaling and oxidative stress. Clinical information and gene expression data were retrieved from The Cancer Genome Atlas and Gene Expression Omnibus, comprising of 648 patients. We found that Glo2 was significantly decreased in primary HCC samples relative to pair-matched normal tissues (p-value o 2.2e-16). In addition, Glo2 was significantly associated with serum alpha-fetoprotein level, nodularity, tumor size, and TNM staging. Importantly, patients with high Glo2 expression had longer relapse-free survival (p ¼ 0.012), disease-free survival (p ¼ 0.002), and overall survival (p ¼ 5.91e 04). Multivariate analysis showed that low Glo2 expression was an independent predictor for survival outcome [HR ¼ 2.00, 95% CI ¼ 1.22– 3.29; p ¼ 0.006] and tumor recurrence [HR ¼ 1.63, 95% CI ¼ 1.08–2.46; p ¼ 0.019]. Furthermore, Glo2 expression was strongly correlated with antioxidant, redox signaling, oxidative stress genes, such as catalase, glutaredoxin, epoxide hydrolase 2, nicotinamide nucleotide transhydrogenase, arylamine N-acetyltransferase 1, Paraoxonase-1, Paraoxonase3, hydroxyacid oxidase 1, and hydroxyacid oxidase 2. These findings reveal the critical role of Glo2 in HCC progression. High expression of Glo2 in HCC is associated with better clinical outcomes, suggesting that this gene is a promise prognostic marker in the era of precision medicine and that it also may be a potential therapeutic target for treating HCC.
https://doi.org/10.1016/j.freeradbiomed.2018.10.151
Pancreatic Ductal Adenocarcinoma is a leading cause of cancer related death in the United States that is in critical need of adjuvant therapies to enhance patient survival. One such adjuvant is pharmacological ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C). P-AscH- treatment results in a short-term increased flux of H2O2 that is preferentially cytotoxic to cancer cells vs. normal cells. Treatment with P-AscH- results in sustained increases in oxygen consumption (25-30 attomoles cell-1 sec-1) and reactive oxygen species production (1.5 fold as DCFH-DA oxidation), with no change in glucose metabolism in tumor cells compared to nontumorigenic pancreatic ductal epithelial cells. These results lead to the hypothesis that there is a sustained effect ( 4 24 h) of P-AscH- that contributes to cytotoxicity. Seahorse XF96 mito stress test (48 h) demonstrated that P-AscH- results in a significant and sustained increase in nonmitochondrial OCR ( 15 attomoles cell-1 sec-1). A potential source of this sustained non-mitochondrial OCR is the NADPH oxidase family of enzymes Dual Oxidase 1 and 2 (DUOX) which are up-regulated after P-AscH-. P-AscH- treatment of PDAC cell lines resulted in a dose (1-10 mM; 4-6 fold) and duration (24-72 h; 5-20 fold) dependent increase in DUOX. Catalase pretreatment reverses the P-AscH--induced increases in DUOX, while DUOX inhibition partially rescues P-AscH- toxicity. Additionally, nutritional ascorbate (100 mm; Ascorbate-2-Phosphate) is unable to mediate an increase in DUOX expression. P-AscH- also induces DUOX protein expression in pancreatic tumor xenografts. These results suggest that P-AscH--induced toxicity may be enhanced by a sustained metabolic shift in tumor cells that is related to the H2O2 produced from P-AscH-. We suggest that P-AscH- results in a feed-forward mechanism of H2O2 generation and induction of metabolic stress via enhanced DUOX expression and OCR.
https://doi.org/10.1016/j.freeradbiomed.2018.10.152
149
Dual oxidase expression is epigenetically silenced in pancreatic ductal adenocarcinoma Adrienne Gibson, Brianne O'Leary*, Juan Du, Carlos Chan, Joseph Cullen The University of Iowa, USA
Recent reports have suggested that Dual Oxidases (DUOX1 & DUOX2) are epigenetically silenced in non-small cell lung, hepatocellular, and breast cancers and may act as tumor suppressor genes. These NADPH Oxidase (NOX) family members are responsible for the production of hydrogen peroxide (H2O2) on the extracellular surface of cells. However, epigenetic silencing of DUOX in pancreatic ductal adenocarcinoma has never been studied. In vitro, we observed that DUOX1 and DUOX2 mRNA and protein expression was significantly reduced in all PDAC cell lines tested when compared to a non-tumorigenic pancreatic ductal epithelial cell line. In addition, 5-azacitidine, an inhibitor of DNA
S72
A. Newman / Free Radical Biology and Medicine 128 (2018) S61–S78
methyltransferases, induced the expression of DUOX1 and DUOX2 (5-20 fold, P o 0.05). To determine if there is a correlation with DUOX promoter methylation and patient survival, the Xena Genome Browser was utilized. Our analyses determined that high DUOX1 promoter methylation is correlated with poor patient outcome (p o 0.05 vs low methylation). Furthermore, immunofluorescent staining of DUOX1 and DUOX2 in pancreatic ductal adenocarcinoma resections demonstrated significant reductions ( 450%) in expression of DUOX 1 and 2 when compared to adjacent, normal pancreas. Overall, these results suggest that DUOX expression is epigenetically silenced and may present as a novel therapeutic target for the treatment of pancreatic ductal adenocarcinoma.
https://doi.org/10.1016/j.freeradbiomed.2018.10.153
150
Use of phenylarsine oxide-affinity chromatography to identify common cellular targets of cancer-active electrophiles Stefan Olsen*, Timothy Foley, Giancarlo Montovano, Connor Lynch University of Scranton, USA
Trivalent arsenicals, such as arsenic trioxide, and 3-bromopyruvate (3BP) are thiol-reactive electrophiles that have shown remarkable ability to selectively impair the viability of cancer cells both in vitro and in vivo. However, the broad reactivity of these substances, potentially forming adducts with many different proteins and altering numerous cellular pathways, has precluded a clear identification of the pivotal mechanisms underlying their preferential anticancer activities. Furthermore, the possibility that these chemically-distinctive substances may act in part by targeting the same protein(s) is unknown. The study described here employed Saccharomyces cerevisiae, which reproduces several metabolic characteristics of cancer cells, as a model to examine the cytotoxicity and protein binding partners of phenylarsine oxide (PAO), a representative trivalent arsenical, and 3BP. Both PAO and 3BP abolished cellular growth. In addition, up to 10% of the yeast proteome bound immobilized PAO in a dithiothreitol-sensitive manner. Among the most abundant PAO-binding proteins were glycolytic enzymes and the vacuolarATPase which play key roles in tumorigenesis. Moreover, binding of the yeast proteins to the immobilized PAO was inhibited by about 50% by prior incubation with 3BP demonstrating that a substantial fraction of protein can bind both PAO and 3BP. The proteins binding uniquely to PAO and those binding both PAO and 3BP, identified by LC-MS/MS, will be reported and discussed in the contexts of cellular viability and cancer.
https://doi.org/10.1016/j.freeradbiomed.2018.10.154
151
Rac1-induced ROS is implicated in the sustained activation of anti-apoptotic protein Bcl-2 in cancer cells Shazib Pervaiz*, Stephen Jun Fei Chong National University of Singapore, Singapore
The anti-apoptotic protein Bcl-2 is strongly associated with the development of therapy resistance in a host of hematopoietic and other cancers. To
that end, the post-translational modification of Bcl-2, in particular its mono- or multi-site phosphorylation, have been reported to impact its apoptosis inhibitory activity. While multi-site phosphorylation compromises its death inhibitory activity, our recent work has highlighted the critical involvement of serine 70 phosphorylation (S70pBcl-2) in stabilizing its anti-apoptotic activity. S70pBcl-2 is a function of either an amplification of the activities of various kinases involved in this post-translational modification or a loss of function of the phosphatase regulating this modification. The pleiotropic phosphatase, PP2A, is specifically involved in the dephosphorylation of Bcl-2 at S70. In our search to identify redox dependent pathways impacting the sustained activation of Bcl-2 via S70P, we provided evidence that redox modification of the B56δ sub-unit of PP2A resulted in the sustained S70P of Bcl-2, thereby promoting its death inhibitory activity in cancer cells. Interestingly, here we present substantial evidence for the existence of a similar redox-dependent regulation of Bcl-2 phosphorylation in cells expressing GTP-bound active Rac1. We show that active Rac1 promotes sustained S70pBcl2 levels in cancer cells via physical interaction with Bcl-2, thereby permitting Rac1-induced production of superoxide (O2–) in close proximity to inactivate PP2A holoenzyme assembly and specific activity. Corroborating this, pharmacological inhibition of the interaction with BH3 mimetics such as HA14-1 or ABT199 as well as scavengers of O2– such as Tiron reduced S70pBcl2. Importantly, the interaction between Rac1 and Bcl-2 to promote S70pBcl2 as well as the oxidative inhibition of PP2A are re-capitulated in primary cells derived from lymphoma patients. Collectively, our findings demonstrate a novel redox mechanism of active Rac1 in sustaining S70pBcl2 in cancer cells.
https://doi.org/10.1016/j.freeradbiomed.2018.10.155
152
Pulmonary and systemic nitric oxide deficiency in the early phase of lung metastasis in 4T1 metastatic breast cancer murine model Bartosz Proniewski 1,*, Agnieszka Kij 1, Marta Smeda 1, Elzbieta Buczek 1, Anna Kieronska 1, Kamil Kus 1, Kamil Przyborowski 1, Katarzyna Derszniak 1, Karolina Matyjaszczyk 1, Agnieszka Jasztal 1, Marta Stojak 1, Maria Walczak 1, Joanna Wietrzyk 2, Stefan Chlopicki 1 1 2
Jagiellonian University, Poland Hirszfeld Institute of Immunology and Experimental Therapy, Poland
The role of nitric oxide (NO) in metastatic cancer progression has not been fully explained. In the present work we characterized the development of NO deficiency in the lung and examined changes in NO and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in Balb/c mice. Furthermore, orthotopic and intravenous models of 4T1 metastatic cancer in mice were used to study the differential effects of L-NAME-induced NO deficiency on the primary tumor and the metastatic burden. NO was assessed in isolated aorta and lungs using EPR spin-trapping and chemiluminescence, while NOx in blood were measured using either EPR, chemiluminescence or Griess reaction. Early pulmonary metastasis was correlated with lung inflammation. Pulmonary endothelial function and systemic NO availability in 4T1 breast cancer-bearing mice were compromised prior to the development of pulmonary metastasis,preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT). Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway. L-NAME-induced NO-deficiency showed that NO plays a major role in the primary tumor development, but it is not the key mediator responsible for the regulation of cancer cell extravasation to the
cancer cell lines SCC-40 (Squamous Cell Carcinoma) and Papillary Cystadenoma Lymphomatosum (PCL) by using western blots and the Agilent Seahorse XF Cell Mito Stress Test. We also studied the migratory capacity of PCL cells using wound-healing assays. A wound-healing assay mimics cell migration during wound healing in-vivo. The results we obtained from this project indicate an increase in the amount of oxidative phosphorylation taking place in the mitochondria of PCL and SCC-40 cancer cell lines, as well as a decrease in the rate of glycolysis. As a result, fusarochromanone is shown to cause an increase in the amount of Reactive Oxygen Species (ROS) in the mitochondria of these cancer cell lines and thus causes an increase in mitochondrial bioenergetics. The data serves to strengthen the potential of FC-101 as an anti-cancer drug.
S71
148
Pharmacological ascorbate enhances expression of dual oxidase which mediates sustained toxicity via alterations in cellular metabolism Adrienne Gibson, Brianne O'Leary*, Matthew Alexander, Juan Du, Brett Wagner, Prabhat Goswami, Garry Buettner, Joseph Cullen The University of Iowa, USA
https://doi.org/10.1016/j.freeradbiomed.2018.10.150
147
Glyoxalase 2 correlates with redox signaling and enhances survival outcome in patients with hepatocellular carcinoma Dinh-Truong Nguyen 1,*, Quynh Hoa Tran 2, Xuan The Hoang 2, Ngoc Huyen Vu 2, Kim Van Thi Le 3, Van Thai Than 4, Minh Nam Nguyen 5 1
Tan Tao University, Viet Nam Ho Chi Minh University of Food Industry, Viet Nam 3 National Institute of Medicinal Materials, Viet Nam 4 Nguyen Tat Thanh University, Viet Nam 5 Ton Duc Thang University, Viet Nam 2
Glyoxalase 2 (Glo2), the second of two enzymes in the glyoxalase system, protects against cellular damage by detoxification of methylglyoxal which is involved in oxidative stress and mitochondrial dysfunction. However, the role of Glo2 in Hepatocellular carcinoma (HCC) progression and survival outcome remains unclear. Here, we investigated the expression of Glo2 in HCC and normal tissues, as well as identified the correlation of Glo2 with the clinicopathological, recurrence risk, patient survival, and other genes related to redox signaling and oxidative stress. Clinical information and gene expression data were retrieved from The Cancer Genome Atlas and Gene Expression Omnibus, comprising of 648 patients. We found that Glo2 was significantly decreased in primary HCC samples relative to pair-matched normal tissues (p-value o 2.2e-16). In addition, Glo2 was significantly associated with serum alpha-fetoprotein level, nodularity, tumor size, and TNM staging. Importantly, patients with high Glo2 expression had longer relapse-free survival (p ¼ 0.012), disease-free survival (p ¼ 0.002), and overall survival (p ¼ 5.91e 04). Multivariate analysis showed that low Glo2 expression was an independent predictor for survival outcome [HR ¼ 2.00, 95% CI ¼ 1.22– 3.29; p ¼ 0.006] and tumor recurrence [HR ¼ 1.63, 95% CI ¼ 1.08–2.46; p ¼ 0.019]. Furthermore, Glo2 expression was strongly correlated with antioxidant, redox signaling, oxidative stress genes, such as catalase, glutaredoxin, epoxide hydrolase 2, nicotinamide nucleotide transhydrogenase, arylamine N-acetyltransferase 1, Paraoxonase-1, Paraoxonase3, hydroxyacid oxidase 1, and hydroxyacid oxidase 2. These findings reveal the critical role of Glo2 in HCC progression. High expression of Glo2 in HCC is associated with better clinical outcomes, suggesting that this gene is a promise prognostic marker in the era of precision medicine and that it also may be a potential therapeutic target for treating HCC.
https://doi.org/10.1016/j.freeradbiomed.2018.10.151
Pancreatic Ductal Adenocarcinoma is a leading cause of cancer related death in the United States that is in critical need of adjuvant therapies to enhance patient survival. One such adjuvant is pharmacological ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C). P-AscH- treatment results in a short-term increased flux of H2O2 that is preferentially cytotoxic to cancer cells vs. normal cells. Treatment with P-AscH- results in sustained increases in oxygen consumption (25-30 attomoles cell-1 sec-1) and reactive oxygen species production (1.5 fold as DCFH-DA oxidation), with no change in glucose metabolism in tumor cells compared to nontumorigenic pancreatic ductal epithelial cells. These results lead to the hypothesis that there is a sustained effect ( 4 24 h) of P-AscH- that contributes to cytotoxicity. Seahorse XF96 mito stress test (48 h) demonstrated that P-AscH- results in a significant and sustained increase in nonmitochondrial OCR ( 15 attomoles cell-1 sec-1). A potential source of this sustained non-mitochondrial OCR is the NADPH oxidase family of enzymes Dual Oxidase 1 and 2 (DUOX) which are up-regulated after P-AscH-. P-AscH- treatment of PDAC cell lines resulted in a dose (1-10 mM; 4-6 fold) and duration (24-72 h; 5-20 fold) dependent increase in DUOX. Catalase pretreatment reverses the P-AscH--induced increases in DUOX, while DUOX inhibition partially rescues P-AscH- toxicity. Additionally, nutritional ascorbate (100 mm; Ascorbate-2-Phosphate) is unable to mediate an increase in DUOX expression. P-AscH- also induces DUOX protein expression in pancreatic tumor xenografts. These results suggest that P-AscH--induced toxicity may be enhanced by a sustained metabolic shift in tumor cells that is related to the H2O2 produced from P-AscH-. We suggest that P-AscH- results in a feed-forward mechanism of H2O2 generation and induction of metabolic stress via enhanced DUOX expression and OCR.
https://doi.org/10.1016/j.freeradbiomed.2018.10.152
149
Dual oxidase expression is epigenetically silenced in pancreatic ductal adenocarcinoma Adrienne Gibson, Brianne O'Leary*, Juan Du, Carlos Chan, Joseph Cullen The University of Iowa, USA
Recent reports have suggested that Dual Oxidases (DUOX1 & DUOX2) are epigenetically silenced in non-small cell lung, hepatocellular, and breast cancers and may act as tumor suppressor genes. These NADPH Oxidase (NOX) family members are responsible for the production of hydrogen peroxide (H2O2) on the extracellular surface of cells. However, epigenetic silencing of DUOX in pancreatic ductal adenocarcinoma has never been studied. In vitro, we observed that DUOX1 and DUOX2 mRNA and protein expression was significantly reduced in all PDAC cell lines tested when compared to a non-tumorigenic pancreatic ductal epithelial cell line. In addition, 5-azacitidine, an inhibitor of DNA
S72
A. Newman / Free Radical Biology and Medicine 128 (2018) S61–S78
methyltransferases, induced the expression of DUOX1 and DUOX2 (5-20 fold, P o 0.05). To determine if there is a correlation with DUOX promoter methylation and patient survival, the Xena Genome Browser was utilized. Our analyses determined that high DUOX1 promoter methylation is correlated with poor patient outcome (p o 0.05 vs low methylation). Furthermore, immunofluorescent staining of DUOX1 and DUOX2 in pancreatic ductal adenocarcinoma resections demonstrated significant reductions ( 450%) in expression of DUOX 1 and 2 when compared to adjacent, normal pancreas. Overall, these results suggest that DUOX expression is epigenetically silenced and may present as a novel therapeutic target for the treatment of pancreatic ductal adenocarcinoma.
https://doi.org/10.1016/j.freeradbiomed.2018.10.153
150
Use of phenylarsine oxide-affinity chromatography to identify common cellular targets of cancer-active electrophiles Stefan Olsen*, Timothy Foley, Giancarlo Montovano, Connor Lynch University of Scranton, USA
Trivalent arsenicals, such as arsenic trioxide, and 3-bromopyruvate (3BP) are thiol-reactive electrophiles that have shown remarkable ability to selectively impair the viability of cancer cells both in vitro and in vivo. However, the broad reactivity of these substances, potentially forming adducts with many different proteins and altering numerous cellular pathways, has precluded a clear identification of the pivotal mechanisms underlying their preferential anticancer activities. Furthermore, the possibility that these chemically-distinctive substances may act in part by targeting the same protein(s) is unknown. The study described here employed Saccharomyces cerevisiae, which reproduces several metabolic characteristics of cancer cells, as a model to examine the cytotoxicity and protein binding partners of phenylarsine oxide (PAO), a representative trivalent arsenical, and 3BP. Both PAO and 3BP abolished cellular growth. In addition, up to 10% of the yeast proteome bound immobilized PAO in a dithiothreitol-sensitive manner. Among the most abundant PAO-binding proteins were glycolytic enzymes and the vacuolarATPase which play key roles in tumorigenesis. Moreover, binding of the yeast proteins to the immobilized PAO was inhibited by about 50% by prior incubation with 3BP demonstrating that a substantial fraction of protein can bind both PAO and 3BP. The proteins binding uniquely to PAO and those binding both PAO and 3BP, identified by LC-MS/MS, will be reported and discussed in the contexts of cellular viability and cancer.
https://doi.org/10.1016/j.freeradbiomed.2018.10.154
151
Rac1-induced ROS is implicated in the sustained activation of anti-apoptotic protein Bcl-2 in cancer cells Shazib Pervaiz*, Stephen Jun Fei Chong National University of Singapore, Singapore
The anti-apoptotic protein Bcl-2 is strongly associated with the development of therapy resistance in a host of hematopoietic and other cancers. To
that end, the post-translational modification of Bcl-2, in particular its mono- or multi-site phosphorylation, have been reported to impact its apoptosis inhibitory activity. While multi-site phosphorylation compromises its death inhibitory activity, our recent work has highlighted the critical involvement of serine 70 phosphorylation (S70pBcl-2) in stabilizing its anti-apoptotic activity. S70pBcl-2 is a function of either an amplification of the activities of various kinases involved in this post-translational modification or a loss of function of the phosphatase regulating this modification. The pleiotropic phosphatase, PP2A, is specifically involved in the dephosphorylation of Bcl-2 at S70. In our search to identify redox dependent pathways impacting the sustained activation of Bcl-2 via S70P, we provided evidence that redox modification of the B56δ sub-unit of PP2A resulted in the sustained S70P of Bcl-2, thereby promoting its death inhibitory activity in cancer cells. Interestingly, here we present substantial evidence for the existence of a similar redox-dependent regulation of Bcl-2 phosphorylation in cells expressing GTP-bound active Rac1. We show that active Rac1 promotes sustained S70pBcl2 levels in cancer cells via physical interaction with Bcl-2, thereby permitting Rac1-induced production of superoxide (O2–) in close proximity to inactivate PP2A holoenzyme assembly and specific activity. Corroborating this, pharmacological inhibition of the interaction with BH3 mimetics such as HA14-1 or ABT199 as well as scavengers of O2– such as Tiron reduced S70pBcl2. Importantly, the interaction between Rac1 and Bcl-2 to promote S70pBcl2 as well as the oxidative inhibition of PP2A are re-capitulated in primary cells derived from lymphoma patients. Collectively, our findings demonstrate a novel redox mechanism of active Rac1 in sustaining S70pBcl2 in cancer cells.
https://doi.org/10.1016/j.freeradbiomed.2018.10.155
152
Pulmonary and systemic nitric oxide deficiency in the early phase of lung metastasis in 4T1 metastatic breast cancer murine model Bartosz Proniewski 1,*, Agnieszka Kij 1, Marta Smeda 1, Elzbieta Buczek 1, Anna Kieronska 1, Kamil Kus 1, Kamil Przyborowski 1, Katarzyna Derszniak 1, Karolina Matyjaszczyk 1, Agnieszka Jasztal 1, Marta Stojak 1, Maria Walczak 1, Joanna Wietrzyk 2, Stefan Chlopicki 1 1 2
Jagiellonian University, Poland Hirszfeld Institute of Immunology and Experimental Therapy, Poland
The role of nitric oxide (NO) in metastatic cancer progression has not been fully explained. In the present work we characterized the development of NO deficiency in the lung and examined changes in NO and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in Balb/c mice. Furthermore, orthotopic and intravenous models of 4T1 metastatic cancer in mice were used to study the differential effects of L-NAME-induced NO deficiency on the primary tumor and the metastatic burden. NO was assessed in isolated aorta and lungs using EPR spin-trapping and chemiluminescence, while NOx in blood were measured using either EPR, chemiluminescence or Griess reaction. Early pulmonary metastasis was correlated with lung inflammation. Pulmonary endothelial function and systemic NO availability in 4T1 breast cancer-bearing mice were compromised prior to the development of pulmonary metastasis,preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT). Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway. L-NAME-induced NO-deficiency showed that NO plays a major role in the primary tumor development, but it is not the key mediator responsible for the regulation of cancer cell extravasation to the