High Levels of Hyaluronic Acid Synthase-2 Mediate NRF2-Driven Chemoresistance in Breast Cancer Cells.

Hyaluronic acid (HA), a ligand of CD44, accumulates in some types of tumors and is responsible for tumor progression. The nuclear factor erythroid 2-like 2 (NRF2) regulates cytoprotective genes and drug transporters, which promotes therapy resistance in tumors. Previously, we showed that high levels of CD44 are associated with NRF2 activation in cancer stem like-cells. Herein, we demonstrate that HA production was increased in doxorubicin-resistant breast cancer MCF7 cells (MCF7-DR) via the upregulation of HA synthase-2 (HAS2). HA incubation increased NRF2, aldo-keto reductase 1C1 (AKR1C1), and multidrug resistance gene 1 (MDR1) levels. Silencing of HAS2 or CD44 suppressed NRF2 signaling in MCF7-DR, which was accompanied by increased doxorubicin sensitivity. The treatment with a HAS2 inhibitor, 4-methylumbelliferone (4-MU), decreased NRF2, AKR1C1, and MDR1 levels in MCF7-DR. Subsequently, 4-MU treatment inhibited sphere formation and doxorubicin resistance in MCF7-DR. The Cancer Genome Atlas (TCGA) data analysis across 32 types of tumors indicates the amplification of HAS2 gene is a common genetic alteration and is negatively correlated with the overall survival rate. In addition, high HAS2 mRNA levels are associated with increased NRF2 signaling and poor clinical outcome in breast cancer patients. Collectively, these indicate that HAS2 elevation contributes to chemoresistance and sphere formation capacity of drug-resistant MCF7 cells by activating CD44/NRF2 signaling, suggesting a potential benefit of HAS2 inhibition.

Organelle Engineering in Yeast: Enhanced Production of Protopanaxadiol through Manipulation of Peroxisome Proliferation in Saccharomyces cerevisiae.

Isoprenoids, which are natural compounds with diverse structures, possess several biological activities that are beneficial to humans. A major consideration in isoprenoid production in microbial hosts is that the accumulation of biosynthesized isoprenoid within intracellular membranes may impede balanced cell growth, which may consequently reduce the desired yield of the target isoprenoid. As a strategy to overcome this suggested limitation, we selected peroxisome membranes as depots for the additional storage of biosynthesized isoprenoids to facilitate increased isoprenoid production in Saccharomyces cerevisiae. To maximize the peroxisome membrane storage capacity of S.cerevisiae, the copy number and size of peroxisomes were increased through genetic engineering of the expression of three peroxisome biogenesis-related peroxins (Pex11p, Pex34p, and Atg36p). The genetically enlarged and high copied peroxisomes in S.cerevisiae were stably maintained under a bioreactor fermentation condition. The peroxisome-engineered S.cerevisiae strains were then utilized as host strains for metabolic engineering of heterologous protopanaxadiol pathway. The yields of protopanaxadiol from the engineered peroxisome strains were ca 78% higher than those of the parent strain, which strongly supports the rationale for harnessing the storage capacity of the peroxisome membrane to accommodate the biosynthesized compounds. Consequently, this study presents in-depth knowledge on peroxisome biogenesis engineering in S.cerevisiae and could serve as basic information for improvement in ginsenosides production and as a potential platform to be utilized for other isoprenoids.

Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors.

A major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.

The multifaceted role of NRF2 in cancer progression and cancer stem cells maintenance.

The transcription factor nuclear factor erythroid 2-like 2 (NEF2L2; NRF2) plays crucial roles in the defense system against electrophilic or oxidative stress by upregulating an array of genes encoding antioxidant proteins, electrophile/reactive oxygen species (ROS) detoxifying enzymes, and drug efflux transporters. In contrast to the protective roles in normal cells, the multifaceted role of NRF2 in tumor growth and progression, resistance to therapy and intratumoral stress, and metabolic adaptation is rapidly expanding, and the complex association of NRF2 with cancer signaling networks is being unveiled. In particular, the implication of NRF2 signaling in cancer stem cells (CSCs), a small population of tumor cells responsible for therapy resistance and tumor relapse, is emerging. Here, we described the dark side of NRF2 signaling in cancers discovered so far. A particular focus was put on the role of NRF2 in CSCs maintenance and therapy resistance, showing that low ROS levels and refractory drug response of CSCs are mediated by the activation of NRF2 signaling. A better understanding of the roles of the NRF2 pathway in CSCs will allow us to develop a novel therapeutic approach to control tumor relapse after therapy.

Negative correlation of urinary miR-199a-3p level with ameliorating effects of sarpogrelate and cilostazol in hypertensive diabetic nephropathy.

The prevalence of chronic kidney disease is increasing globally; however, effective therapeutic options are limited. In this study, we aimed to identify urinary miRNAs reflecting the effect of therapeutic intervention in rats with comorbid hypertension and diabetes. Additionally, the potential beneficial effects of anti-platelet sarpogrelate and cilostazol were investigated. Nephropathy progression in streptozotocin (STZ)-treated spontaneously hypertensive rats (SHRs), including albuminuria, collagen deposition, and histopathological changes, was alleviated by sarpogrelate and antihypertensive agent telmisartan. Global analysis of urinary miRNAs identified that miR-199a-3p was commonly reduced by sarpogrelate and telmisartan treatment. In vitro analysis suggested CD151 as a target gene of miR-199a-3p: miR-199a-3p overexpression repressed CD151 levels and miR-199a-3p interacted with the 3'-untranslated region of the CD151 gene. In addition, we demonstrated that the miR-199a-3p/CD151 axis is associated with the transforming growth factor-ß1 (TGF-ß1)-induced fibrogenic pathway. TGF-ß1 treatment led to miR-199a-3p elevation and CD151 suppression, and miR-199a-3p overexpression or CD151-silencing enhanced TGF-ß1-inducible collagen IV and α-smooth muscle actin (α-SMA) levels. In vivo analysis showed that the decrease in CD151 and the increase in collagen IV and α-SMA in the kidney from STZ-treated SHR were restored by sarpogrelate and telmisartan administration. In an additional animal experiment using cilostazol and telmisartan, there was a correlation between urinary miR-199a-3p reduction and the ameliorating effects of cilostazol or combination with telmisartan. Collectively, these results indicate that urinary miR-199a-3p might be utilized as a marker for nephropathy treatment. We also provide evidence of the benefits of antiplatelet sarpogrelate and cilostazol in nephropathy progression.

Role of CD133/NRF2 Axis in the Development of Colon Cancer Stem Cell-Like Properties.

Cancer stem cells (CSCs) exhibit intrinsic therapy/stress resistance, which often cause cancer recurrence after therapy. In this study, we investigated the potential relationship between the cluster of differentiation (CD)-133, a CSC marker of colon cancer, and nuclear factor erythroid 2-like 2 (NFE2L2; NRF2), a master transcription factor for the regulation of multiple antioxidant genes. In the first model of CSC, a sphere culture of the colorectal cell line HCT116, showed increased levels of CD133 and NRF2. Silencing of CD133 reduced the levels of CSC markers, such as Kruppel-like factor 4 (KLF4) and ATP-binding cassette subfamily G member 2 (ABCG2), and further suppressed the expression levels of NRF2 and its target genes. As a potential molecular link, CD133-mediated activation of phosphoinositide 3-kinase/serine-threonine kinase (PI3K/AKT) signaling appears to increase the NRF2 protein levels via phosphorylation and the consequent inhibition of glycogen synthase kinase (GSK)-3ß. Additionally, NRF2-silenced HCT116 cells showed attenuated sphere formation capacity and reduced CSC markers expression, indicating the critical role of the NRF2 pathway in the development of CSC-like properties. As a second model of CSC, the CD133high cell population was isolated from HCT116 cells. CSC-like properties, including sphere formation, motility, migration, colony formation, and anticancer resistance, were enhanced in the CD133high population compared to CD133low HCT116 cells. Levels of NRF2, which were elevated in CD133high HCT116, were suppressed by CD133-silencing. In line with these, the analysis of The Cancer Genome Atlas (TCGA) database showed that high levels of CD133 expression are correlated with increased NRF2 signaling, and alterations in CD133 gene or expression are associated with unfavorable clinical outcome in colorectal carcinoma patients. These results indicate that the CD133/NRF2 axis contributes to the development of CSC-like properties in colon cancer cells, and that PI3K/AKT signaling activation is involved in CD133-mediated NRF2 activation.

Microbial Production of Retinyl Palmitate and Its Application as a Cosmeceutical.

Chemically synthesized retinyl palmitate has been widely used in the cosmetic and biotechnology industry. In this study, we aimed to demonstrate the microbial production of retinyl palmitate and the benefits of microbial retinyl palmitate in skin physiology. A heterologous retinyl palmitate biosynthesis pathway was reconstructed in metabolically engineered Escherichia coli using synthetic expression modules from Pantoea agglomerans, Salinibacter ruber, and Homo sapiens. High production of retinyl palmitate (69.96 ± 2.64 mg/L) was obtained using a fed-batch fermentation process. Moreover, application of purified microbial retinyl palmitate to human foreskin HS68 fibroblasts led to increased cellular retinoic acid-binding protein 2 (CRABP2) mRNA level [1.7-fold (p = 0.001) at 100 µg/mL], acceleration of cell proliferation, and enhancement of procollagen synthesis [111% (p < 0.05) at 100 µg/mL], strongly indicating an anti-ageing-related effect of this substance. These results would pave the way for large-scale production of retinyl palmitate in microbial systems and represent the first evidence for the application of microbial retinyl palmitate as a cosmeceutical.

PHGDH supports liver ceramide synthesis and sustains lipid homeostasis.

BACKGROUND: d-3-phosphoglycerate dehydrogenase (PHGDH), which encodes the first enzyme in serine biosynthesis, is overexpressed in human cancers and has been proposed as a drug target. However, whether PHGDH is critical for the proliferation or homeostasis of tissues following the postnatal period is unknown. METHODS: To study PHGDH inhibition in adult animals, we developed a knock-in mouse model harboring a PHGDH shRNA under the control of a doxycycline-inducible promoter. With this model, PHGDH depletion can be globally induced in adult animals, while sparing the brain due to poor doxycycline delivery. RESULTS: We found that PHGDH depletion is well tolerated, and no overt phenotypes were observed in multiple highly proliferative cell compartments. Further, despite detectable knockdown and impaired serine synthesis, liver and pancreatic functions were normal. Interestingly, diminished PHGDH expression reduced liver serine and ceramide levels without increasing the levels of deoxysphingolipids. Further, liver triacylglycerol profiles were altered, with an accumulation of longer chain, polyunsaturated tails upon PHGDH knockdown. CONCLUSIONS: These results suggest that dietary serine is adequate to support the function of healthy, adult murine tissues, but PHGDH-derived serine supports liver ceramide synthesis and sustains general lipid homeostasis.

The Diverse Functions of Non-Essential Amino Acids in Cancer.

Far beyond simply being 11 of the 20 amino acids needed for protein synthesis, non-essential amino acids play numerous important roles in tumor metabolism. These diverse functions include providing precursors for the biosynthesis of macromolecules, controlling redox status and antioxidant systems, and serving as substrates for post-translational and epigenetic modifications. This functional diversity has sparked great interest in targeting non-essential amino acid metabolism for cancer therapy and has motivated the development of several therapies that are either already used in the clinic or are currently in clinical trials. In this review, we will discuss the important roles that each of the 11 non-essential amino acids play in cancer, how their metabolic pathways are linked, and how researchers are working to overcome the unique challenges of targeting non-essential amino acid metabolism for cancer therapy.

High NRF2 level mediates cancer stem cell-like properties of aldehyde dehydrogenase (ALDH)-high ovarian cancer cells: inhibitory role of all-trans retinoic acid in ALDH/NRF2 signaling.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is one of cancer stem cell (CSC) markers, and high ALDH1 expression has been related to drug resistance and facilitated tumor growth. In this study, we investigated the potential involvement of nuclear factor erythroid 2-like 2 (NFE2L2/NRF2) in CSC-like properties of ALDH-high ovarian CSCs. Our experimental system, ALDH1A1-high (ALDH-H) subpopulation, was isolated and stabilized using doxorubicin-resistant ovarian cancer A2780 cells. ALDH-H exerted CSC-like properties such as drug resistance, colony/sphere formation, and enhanced tumor growth along with high levels of CSCs markers compared to ALDH1A1-low (ALDH-L). Levels of NRF2 and subsequent target genes substantially increased in ALDH-H cells, and the increase in ALDH1A1 and p62 was associated with NRF2 upregulation. ALDH1A1-silencing blocked increases in NRF2, drug efflux transporters, and p62, along with CSC markers in ALDH-H cells. The inhibition of p62, which was elevated in ALDH-H, suppressed NRF2 activation. High NRF2 level was confirmed in the ALDH1-high subpopulation from colon cancer HCT116 cells. The functional implication of NRF2 activation in ovarian CSCs was verified by two experimental approaches. First, CSC-like properties such as high CSC markers, chemoresistance, colony/sphere formation, and tumor growth were significantly inhibited by NRF2-silencing in ALDH-H cells. Second, all-trans retinoic acid (ATRA) suppressed ALDH1 expression, inhibiting NRF2 activation, which led to the attenuation of CSC-like properties in ALDH-H cells but not in ALDH-L cells. These results provide insight into the molecular basis of the ALDH1A1-mediated development of CSC-like properties such as stress/treatment resistance, and further suggest the therapeutic potential of ATRA in ALDH-high ovarian CSCs.

High CD44 expression mediates p62-associated NFE2L2/NRF2 activation in breast cancer stem cell-like cells: Implications for cancer stem cell resistance.

Cluster of differentiation 44 (CD44) is the most common cancer stem cell (CSC) marker and high CD44 expression has been associated with anticancer drug resistance, tumor recurrence, and metastasis. In this study, we aimed to investigate the molecular mechanism by which CD44 and nuclear factor erythroid 2-like 2 (NFE2L2; NRF2), a key regulator of antioxidant genes, are linked to CSC resistance using CD44high breast CSC-like cells. NRF2 expression was higher in CD44high cell populations isolated from doxorubicin-resistant MCF7 (ADR), as well as MCF7, MDA-MB231, and A549 cells, than in corresponding CD44low cells. High NRF2 expression in the CD44highCD24low CSC population (ADR44P) established from ADR cells depended on standard isoform of CD44. Silencing of CD44 or overexpression of CD44 resulted in the reduction or elevation of NRF2, respectively, and treatment with hyaluronic acid, a CD44 ligand, augmented NRF2 activation. As functional implications, NRF2 silencing rendered ADR44P cells to retain higher levels of reactive oxygen species and to be sensitive to anticancer drug toxicity. Moreover, NRF2-silenced ADR44P cells displayed tumor growth retardation and reduced colony/sphere formation and invasion capacity. In line with these, CD44 significantly colocalized with NRF2 in breast tumor clinical samples. The molecular mechanism of CD44-mediated NRF2 activation was found to involve high p62 expression. CD44 elevation led to an increase in p62, and inhibition of p62 resulted in NRF2 suppression in ADR44P. Collectively, our results showed that high CD44 led to p62-associated NRF2 activation in CD44high breast CSC-like cells. NRF2 activation contributed to the aggressive phenotype, tumor growth, and anticancer drug resistance of CD44high CSCs. Therefore, the CD44-NRF2 axis might be a promising therapeutic target for the control of stress resistance and survival of CD44high CSC population within breast tumors.

Complete genome sequence of Planococcus faecalis AJ003T, the type species of the genus Planococcus and a microbial C30 carotenoid producer.

A novel type strain, Planococcus faecalis AJ003T, isolated from the feces of Antarctic penguins, synthesizes a rare C30 carotenoid, glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid. The complete genome of P. faecalis AJ003T comprises a single circular chromosome (3,495,892 bp; 40.9% G + C content). Annotation analysis has revealed 3511 coding DNA sequences and 99 RNAs; seven genes associated with the MEP pathway and five genes involved in the carotenoid pathway have been identified. The functionality and complementation of 4,4'-diapophytoene synthase (CrtM) and two copies of heterologous 4,4'-diapophytoene desaturase (CrtN) involved in carotenoid biosynthesis were analyzed in Escherichia coli.

Amelioration of high fat diet-induced nephropathy by cilostazol and rosuvastatin.

Multiple comorbidities of metabolic disorders are associated with facilitated chronic kidney disease progression. Anti-platelet cilostazol is used for the treatment of peripheral artery disease. In this study, we investigated the potential beneficial effects of cilostazol and rosuvastatin on metabolic disorder-induced renal dysfunctions. C57BL/6 mice that received high fat diet (HFD) for 22 weeks and a low dose of streptozotocin (STZ, 40 mg/kg) developed albuminuria and had increased urinary cystatin C excretion, and cilostazol treatment (13 weeks) improved these markers. Histopathological changes, including glomerular mesangial expansion, tubular vacuolization, apoptosis, and lipid accumulation were ameliorated by cilostazol treatment. Tubulointerstitial fibrosis that was indicated by the increases in collagen and transforming growth factor-ß1 subsided by cilostazol. Renoprotective effects were also observed in rosuvastatin-treated mice, and combinatorial treatment with cilostazol and rosuvastatin demonstrated enhanced ameliorative effects in histopathological evaluations. Notably, repressed renal heme oxygenase-1 (Ho-1) level in HFD/STZ mice was restored in cilostazol group. Further, we demonstrated that cilostazol enhanced Nrf2/Ho-1 signaling in cultured proximal tubular epithelial cells. Collectively, these results suggest the potential advantageous use of cilostazol as an adjunctive therapy with statins for the amelioration of metabolic disorder-associated renal injury.

NFE2L2/NRF2 silencing-inducible miR-206 targets c-MET/EGFR and suppresses BCRP/ABCG2 in cancer cells.

The nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2) plays a critical role in the expression of multiple antioxidant and detoxifying enzymes. Herein, we provide evidence of the molecular links between NRF2 and oncogenic signaling hepatocyte growth factor receptor (HGFR/c-MET) and epidermal growth factor receptor (EGFR). Interfering RNA-induced stable inhibition of NRF2 in ovarian carcinoma SKOV3 and renal carcinoma A498 reduced the levels of c-MET and EGFR. MicroRNA-206 (miR-206) that was increased in both NRF2-silenced cells was predicted as a dual regulator of c-MET and EGFR. As experimental evidence, miR-206 decreased c-MET and EGFR levels through a direct binding to the 3'-untranslated region of the c-MET and EGFR genes. The treatment of NRF2-knockdown cells with the miR-206 inhibitor could restore c-MET and EGFR levels. The miR-206-mediated c-MET/EGFR repression resulted in two outcomes. First, presumably through the inhibition of c-MET/EGFR-dependent cell proliferation, overexpression of miR-206 inhibited tumor growth in SKOV3-inoculated nude mice. Second, reduced c-MET/EGFR in NRF2-silenced cells affected breast cancer resistance protein (BCRP/ABCG2) levels. The pharmacological and genetic inhibition of c-MET or EGFR, as well as the miR-206 mimic treatment, repressed BCRP levels and increased cellular accumulation of doxorubicin. In line with these, treatment of NRF2-silenced SKOV3 with the miR-206 inhibitor elevated BCRP levels and consequently made these cells more resistant to doxorubicin treatment. Collectively, our results demonstrated that the NRF2 silencing-inducible miR-206 targeted both c-MET and EGFR, and subsequently suppressed the BCRP level in cancer cells.

Involvement of NRF2 Signaling in Doxorubicin Resistance of Cancer Stem Cell-Enriched Colonospheres.

Cancer stem cells (CSCs) are a subset of tumor cells, which are characterized by resistance against chemotherapy and environmental stress, and are known to cause tumor relapse after therapy. A number of molecular mechanisms underlie the chemoresistance of CSCs, including high expression levels of drug efflux transporters. We investigated the role of the antioxidant transcription factor NF-E2-related factor 2 (NRF2) in chemoresistance development, using a CSC-enriched colonosphere system. HCT116 colonospheres were more resistant to doxorubicin-induced cell death and expressed higher levels of drug efflux transporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) compared to HCT116 monolayers. Notably, levels of NRF2 and expression of its target genes were substantially elevated in colonospheres, and these increases were linked to doxorubicin resistance. When NRF2 expression was silenced in colonospheres, Pgp and BCRP expression was downregulated, and doxorubicin resistance was diminished. Collectively, these results indicate that NRF2 activation contributes to chemoresistance acquisition in CSC-enriched colonospheres through the upregulation of drug efflux transporters.

Beneficial Effects of Sarpogrelate and Rosuvastatin in High Fat Diet/Streptozotocin-Induced Nephropathy in Mice.

Chronic kidney disease (CKD) is a major complication of metabolic disorders such as diabetes mellitus, obesity, and hypertension. Comorbidity of these diseases is the factor exacerbating CKD progression. Statins are commonly used in patients with metabolic disorders to decrease the risk of cardiovascular complications. Sarpogrelate, a selective antagonist of 5-hydroxytryptamine (5-HT) 2A receptor, inhibits platelet aggregation and is used to improve peripheral circulation in diabetic patients. Here, we investigated the effects of sarpogrelate and rosuvastatin on CKD in mice that were subjected to a high fat diet (HFD) for 22 weeks and a single low dose of streptozotocin (STZ, 40 mg/kg). When mice were administrated sarpogrelate (50 mg/kg, p.o.) for 13 weeks, albuminuria and urinary cystatin C excretion were normalized and histopathological changes such as glomerular mesangial expansion, tubular damage, and accumulations in lipid droplets and collagen were significantly improved. Sarpogrelate treatment repressed the HFD/STZ-induced CD31 and vascular endothelial growth factor receptor-2 expressions, indicating the attenuation of glomerular endothelial proliferation. Additionally, sarpogrelate inhibited interstitial fibrosis by suppressing the increases in transforming growth factor-ß1 (TGF-ß1) and plasminogen activator inhibitor-1 (PAI-1). All of these functional and histological improvements were also seen in rosuvastatin (20 mg/kg) group and, notably, the combinatorial treatment with sarpogrelate and rosuvastatin showed additive beneficial effects on histopathological changes by HFD/STZ. Moreover, sarpogrelate reduced circulating levels of PAI-1 that were elevated in the HFD/STZ group. As supportive in vitro evidence, sarpogrelate incubation blocked TGF-ß1/5-HT-inducible PAI-1 expression in murine glomerular mesangial cells. Taken together, sarpogrelate and rosuvastatin may be advantageous to control the progression of CKD in patients with comorbid metabolic disorders, and particularly, the use of sarpogrelate as adjunctive therapy with statins may provide additional benefits on CKD.

NRF2 Signaling Negatively Regulates Phorbol-12-Myristate-13-Acetate (PMA)-Induced Differentiation of Human Monocytic U937 Cells into Pro-Inflammatory Macrophages.

Blood monocytes are recruited to injured tissue sites and differentiate into macrophages, which protect against pathogens and repair damaged tissues. Reactive oxygen species (ROS) are known to be an important contributor to monocytes' differentiation and macrophages' function. NF-E2-related factor 2 (NRF2), a transcription factor regulating cellular redox homeostasis, is known to be a critical modulator of inflammatory responses. We herein investigated the role of NRF2 in macrophage differentiation using the human monocytic U937 cell line and phorbol-12-myristate-13-acetate (PMA). In U937 cells with NRF2 silencing, PMA-stimulated cell adherence was significantly facilitated when compared to control U937 cells. Both transcript and protein levels for pro-inflammatory cytokines, including interleukine-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNFα) were highly elevated in PMA-stimulated NRF2-silenced U937 compared to the control. In addition, PMA-inducible secretion of monocyte chemotactic protein 1 (MCP-1) was significantly high in NRF2-silenced U937. As an underlying mechanism, we showed that NRF2-knockdown U937 retained high levels of cellular ROS and endoplasmic reticulum (ER) stress markers expression; and subsequently, PMA-stimulated levels of Ca2+ and PKCα were greater in NRF2-knockdown U937 cells, which caused enhanced nuclear accumulation of nuclear factor-Ò¡B (NFÒ¡B) p50 and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. Whereas the treatment of NRF2-silenced U937 cells with pharmacological inhibitors of NFÒ¡B or ERK1/2 largely blocked PMA-induced IL-1ß and IL-6 expression, indicating that these pathways are associated with cell differentiation. Taken together, our results suggest that the NRF2 system functions to suppress PMA-stimulated U937 cell differentiation into pro-inflammatory macrophages and provide evidence that the ROS-PKCα-ERK-NFÒ¡B axis is involved in PMA-facilitated differentiation of NRF2-silenced U937 cells.

Activation of NRF2 by p62 and proteasome reduction in sphere-forming breast carcinoma cells.

Cancer stem cells (CSCs) express high levels of drug efflux transporters and antioxidant genes, and are therefore believed to be responsible for cancer recurrence following chemo/radiotherapy intervention. In this study, we investigated the role of NF-E2-related factor 2 (NRF2), a master regulator of antioxidant gene expression, in the growth and stress resistance of CSC-enriched mammosphere. The MCF7 mammospheres expressed significantly higher levels of the NRF2 protein and target gene expression compared to the monolayer. As underlying mechanisms, we observed that proteolytic activity and expression of the proteasome catalytic subunits were decreased in the mammospheres. Additionally, mammospheres retained a high level of p62 and the silencing of p62 was observed to attenuate NRF2 activation. NRF2 increase was confirmed in sphere-cultures of the colon and ovarian cancer cells. The functional implication of NRF2 was demonstrated in NRF2-knockdown mammospheres. NRF2-silenced mammospheres demonstrated increased cell death and retarded sphere growth as a result of target gene repression. Moreover, unlike the control mammospheres, NRF2-knockdown mammospheres did not develop anticancer drug resistance. Collectively, these results indicated that altered proteasome function and p62 expression caused NRF2 activation in CSC-enriched mammospheres. In addition, NRF2 appeared to play a role in CSC survival and anticancer drug resistance.

The c-MET/PI3K signaling is associated with cancer resistance to doxorubicin and photodynamic therapy by elevating BCRP/ABCG2 expression.

Overexpression of BCRP/ABCG2, a xenobiotic efflux transporter, is associated with anticancer drug resistance in tumors. Proto-oncogene c-MET induces cancer cell proliferation, motility, and survival, and its aberrant activation was found to be a prognostic factor in advanced ovarian cancers. In the present study, we investigated the potential crossresistance of doxorubicin-resistant ovarian cancer cells to the pheophorbide a (Pba)-based photodynamic therapy (PDT), and suggest c-MET and BCRP/ABCG2 overexpression as an underlying molecular mechanism. The doxorubicin-resistant A2780 cell line (A2780DR), which was established by incubating A2780 with stepwise increasing concentrations of doxorubicin, showed low levels of cellular Pba accumulation and reactive oxygen species generation, and was more resistant to PDT cytotoxicity than A2780. In a microarray analysis, BCRP/ABCG2 was found to be the only drug transporter whose expression was upregulated in A2780DR; this increase was confirmed by Western blot and immunocytochemical analyses. As functional evidence, the treatment with a BCRP/ABCG2-specific inhibitor reversed A2780DR resistance to both doxorubicin and PDT. We identified that c-MET increase is related to BCRP/ABCG2 activation. The c-MET downstream phosphoinositide 3-kinase (PI3K)/AKT signaling was activated in A2780DR and the inhibition of PI3K/AKT or c-MET repressed resistance to doxorubicin and PDT. Finally, we showed that the pharmacological and genetic inhibition of c-MET diminished levels of BCRP/ABCG2 in A2780DR. Moreover, c-MET inhibition could repress BCRP/ABCG2 expression in breast carcinoma MDA-MB-231 and colon carcinoma HT29, resulting in sensitization to doxorubicin. Collectively, our results provide a novel link of c-MET overexpression to BCRP/ABCG2 activation, suggesting that this mechanism leads to crossresistance to both chemotherapy and PDT.

The sensitivity of cancer cells to pheophorbide a-based photodynamic therapy is enhanced by Nrf2 silencing.

Photodynamic therapy (PDT) has emerged as an effective treatment for various solid tumors. The transcription factor NRF2 is known to protect against oxidative and electrophilic stress; however, its constitutive activity in cancer confers resistance to anti-cancer drugs. In the present study, we investigated NRF2 signaling as a potential molecular determinant of pheophorbide a (Pba)-based PDT by using NRF2-knockdown breast carcinoma MDA-MB-231 cells. Cells with stable NRF2 knockdown showed enhanced cytotoxicity and apoptotic/necrotic cell death following PDT along with increased levels of singlet oxygen and reactive oxygen species (ROS). A confocal microscopic visualization of fluorogenic Pba demonstrated that NRF2-knockdown cells accumulate more Pba than control cells. A subsequent analysis of the expression of membrane drug transporters showed that the basal expression of BCRP is NRF2-dependent. Among measured drug transporters, the basal expression of breast cancer resistance protein (BCRP; ABCG2) was only diminished by NRF2-knockdown. Furthermore, after incubation with the BCRP specific inhibitor, differential cellular Pba accumulation and ROS in two cell lines were abolished. In addition, NRF2-knockdown cells express low level of peroxiredoxin 3 compared to the control, which implies that diminished mitochondrial ROS defense system can be contributing to PDT sensitization. The role of the NRF2-BCRP pathway in Pba-PDT response was further confirmed in colon carcinoma HT29 cells. Specifically, NRF2 knockdown resulted in enhanced cell death and increased singlet oxygen and ROS levels following PDT through the diminished expression of BCRP. Similarly, PDT-induced ROS generation was substantially increased by treatment with NRF2 shRNA in breast carcinoma MCF-7 cells, colon carcinoma HCT116 cells, renal carcinoma A498 cells, and glioblastoma A172 cells. Taken together, these results indicate that the manipulation of NRF2 can enhance Pba-PDT sensitivity in multiple cancer cells.