Lysyl Oxidase Protein Expression Research Articles

Transcriptome analysis of PPARγ target genes reveals the involvement of lysyl oxidase in human placental cytotrophoblast invasion.

Human placental development is characterized by invasion of extravillous cytotrophoblasts (EVCTs) into the uterine wall during the first trimester of pregnancy. Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in placental development, and activation of PPARγ by its agonists results in inhibition of EVCT invasion in vitro. To identify PPARγ target genes, microarray analysis was performed using GeneChip technology on EVCT primary cultures obtained from first-trimester human placentas. Gene expression was compared in EVCTs treated with the PPARγ agonist rosiglitazone versus control. A total of 139 differentially regulated genes were identified, and changes in the expression of the following 8 genes were confirmed by reverse transcription-quantitative polymerase chain reaction: a disintegrin and metalloproteinase domain12 (ADAM12), connexin 43 (CX43), deleted in liver cancer 1 (DLC1), dipeptidyl peptidase 4 (DPP4), heme oxygenase 1 (HMOX-1), lysyl oxidase (LOX), plasminogen activator inhibitor 1 (PAI-1) and PPARγ. Among the upregulated genes, lysyl oxidase (LOX) was further analyzed. In the LOX family, only LOX, LOXL1 and LOXL2 mRNA expression was significantly upregulated in rosiglitazone-treated EVCTs. RNA and protein expression of the subfamily members LOX, LOXL1 and LOXL2 were analyzed by absolute RT-qPCR and western blotting, and localized by immunohistochemistry and immunofluorescence-confocal microscopy. LOX protein was immunodetected in the EVCT cytoplasm, while LOXL1 was found in the nucleus and nucleolus. No signal was detected for LOXL2 protein. Specific inhibition of LOX activity by β-aminopropionitrile in cell invasion assays led to an increase in EVCT invasiveness. These results suggest that LOX, LOXL1 and LOXL2 are downstream PPARγ targets and that LOX activity is a negative regulator of trophoblastic cell invasion.

Expression of lysyl oxidase and vascular endothelial growth factor in gastric cancer and their effects on tumor invasion and metastasis

Objective To study the expression of lysyl oxidase (LOX) and vascular endothelial growth factor (VEGF) in gastric cancer and paracancerous tissues,and their relationshipa with genesis of gastric cancer.Methods Sixty-five samples of freshly surgically resected gastric cancer tissues and the paracancerous tissues 5 cm away from the gastric cancer tissues each were collected in Wuhan Puren Hospital from Sep.2009 to Jan.2013.By using the method of Western blotting,the protein expression of LOX and VEGF in gastric cancer and paracancerous tissues was detected.The Spearman rank correlation analysis was done to analyze the correlation between the LOX and VEGF expression in gastric cancer.Results LOX relative expression in 65 cases of gastric cancer tissues and their paracancerous tissues was 0.4178 ± 0.1135 and 0.2174 ± 0.1524 respectively (P ＜ 0.05).VEGF in 65 cases of gastric cancer tissues and their paracancerous tissues was 0.4793 ± 0.1019 and 0.2116 ± 0.1146 respectively (P ＜ 0.05).Conclusion The expression levels of LOX and VEGF are obviously higher in the gastric cancer tissues than in the paracancerous tissues.LOX and VEGF may play an important role in the pathogenesis of gastric cancer. Key words: Gastric cancer; Lysyl oxidase; Vascular endothelial growth factor

Expression and Significance of Hypoxia Inducible Factor-1α and Lysyl Oxidase in Non-small Cell Lung Cancer

To detect expression of hypoxia inducible factor-1α (HIF-1α) and lysyl oxidase (LOX) in non-small cell lung cancer (NSCLC) and explore their roles in prognosis. The mRNA levels of HIF-1α and LOX were investigated by real-time reverse-transcriptase polymerase chain reaction in 40 cases of tumour and paired normal tissues. In addition, protein expression of HIF-1α and LOX was examined by immunohistochemistry in 82 cases of tumour and 45 paired normal tissues. The relationship between HIF-1α or LOX and clinicopathologic characteristics, as well as the correlation between HIF-1α and LOX, were also examined. Kaplan-Meier survival curves and the log-rank test were used to analyze progression-free survival. HIF-1α or LOX mRNA levels in tumor tissues was significantly higher than those in paired normal tissues (p<0.01). Positive HIF-1α or LOX protein expression in tumor tissues was noted in 46/82 (56.1%) and 49/82 (59.8%) of the cases, respectively, being significantly higher than those in paired normal tissues (p<0.05). There was significant correlation between the expression of HIF-1α or LOX and tumor size, lymph node metastasis and pathological stage (p<0.05). The expression of HIF-1α and LOX had a significant inverse impact on survival of patients with NSCLC. HIF-1α and LOX may play a pivotal role in the development of NSCLC, and may act in synergy to promote the progression of NSCLC.

Gremlin utilizes canonical and non-canonical TGFβ signaling to induce lysyl oxidase (LOX) genes in human trabecular meshwork cells

The TGFβ/BMP signaling pathways are involved in glaucomatous damage to the trabecular meshwork (TM) leading to elevated intraocular pressure (IOP), which is a major risk factor for the development and progression of glaucoma. The BMP antagonist gremlin is elevated in glaucomatous TM cells and tissues and can directly elevate IOP. Gremlin utilizes the TGFβ2/SMAD pathway to induce TM extracellular matrix (ECM) proteins. The purpose of this study is to determine whether expression of the ECM cross-linking lysyl oxidase (LOX) genes is regulated by gremlin in cultured human TM cells. Human TM cells were treated with recombinant gremlin, and expression of the LOX genes was examined by quantitative RT-PCR and western immunoblotting. TM cells were pretreated with TGFBR inhibitors (LY364947 or SB431542), an inhibitor of the SMAD signaling pathway (SIS3), or with JNK (SP600125) and p38 MAPK (SB203580) inhibitors to identify the signaling pathway(s) involved in gremlin induction of LOX protein expression. All five LOX genes (LOX and LOXL1–4) were induced by gremlin. Gremlin induction of LOX genes and protein expression was blocked by TGFBR inhibitors as well as by inhibitors of the SMAD3, JNK and p38 MAPK signaling pathways. We conclude that gremlin employs both canonical TGFβ/SMAD and the non-canonical JNK and p38 MAPK signaling pathways to induce LOX genes and proteins in cultured human TM cells. Increased LOX levels may be at least partially responsible for gremlin-mediated IOP elevation and increased aqueous humor outflow resistance leading to glaucoma.

Long-Term Ethanol Exposure-Induced Hepatocellular Carcinoma Cell Migration and Invasion through Lysyl Oxidase Activation Are Attenuated by Combined Treatment with Pterostilbene and Curcumin Analogues

Ethanol consumption induces hepatocellular carcinoma (HCC) cell metastasis by changing the extracellular matrix (ECM). Lysyl oxidase (LOX) catalyzes the cross-linkage of collagen or elastin in the ECM. LOX protein and mRNA overexpression (>21-fold compared with controls, n = 6) was detected in cirrhotic HCC patients with a history of alcoholism. LOX protein expression was induced in HCC cells after long-term treatment with ethanol (10 mM) for 20-40 passages (denoted E20-E40 cells). Pterostilbene (PSB, 1 μM) displayed significant potency to reduce LOX-mediated activity in E40 cells when combined with curcumin and its analogues. The ability of E40 cells to form colonies in soft agar was reduced by both genetic depletion of LOX and by chemical inhibitors of LOX expression. This study suggests that targeting LOX expression with food components such as PSB and curcumin may be a novel strategy to overcome ethanol-induced HCC cell metastasis in liver cancer patients.

Lysyl oxidase inhibition in the volume overloaded heart prevents adverse collagen remodeling, apoptosis, and cardiac dysfunction

Heart failure is the most prevalent and costly disease in the U.S. Our goal was to determine if the over‐activation of lysyl oxidase (LOX), a collagen cross‐linking enzyme, accelerates cardiac disease and failure. LOX is elevated in human failing hearts, but it is not known if LOX plays a causative role in disease. Using the aortocaval fistula (ACF) rat surgical model of volume overload, we assessed the role of LOX activity in progression of heart failure over 14 wks. LOX activity was inhibited by BAPN (100 mg/kg/d) at 2 wks post‐surgery. Echocardiography was used to evaluate cardiac function and progression of LV remodeling. LOX expression and activity, collagen content, cross‐linking, and typing were determined in LV homogenates. Fixed sections of mid‐LV were assessed for apoptosis by TUNEL. ACF surgery caused significant ventricular dilatation (43% increase) and dysfunction (26% decreased, %FS). LOX protein expression was increased (65%) with concomitant increases in LOX activity. These increases in LOX were associated with significantly elevated collagen concentration, cross‐linking, and type I/III. LOX inhibition prevented ACF‐induced changes in LV collagen, and led to maintenance of systolic function. LOX inhibition also attenuated LV dilatation and prevented apoptosis, but did not reduce LV hypertrophy. These data indicate that LOX inhibition is cardioprotective in the volume overloaded heart.

Differential regulation of collagen, lysyl oxidase and MMP‐2 in human periodontal ligament cells by low‐ and high‐level mechanical stretching

Mechanical stretching modulates extracellular matrix (ECM) protein synthesis by periodontal ligament (PDL) cells. However, the mechanoregulation of lysyl oxidase (LOX), a key enzyme for collagen cross-linking, is not fully understood. In the present study, we hypothesized that low-level and high-level mechanical stretching differentially regulates collagen deposition and the expression of LOX and the enzymes responsible for ECM degradation, such as MMP-2 in PDL cells. Human PDL cells were cultured on flexible-bottom culture plates and subjected to cyclic mechanical stretching (3% and 10% elongation at 0.1 Hz) for 24 and 48 h in a Flexercell FX-4000 strain unit. The levels of expression of type I collagen alpha 1 (COL1A1), type III collagen alpha 1 (COL3A1), lysyl oxidase (LOX), MMP2 and TIMP2 mRNAs were analyzed using an RT-PCR technique. The cell layer and the culture medium were separately collected and processed for detection of the following ECM-related molecules: (i) total collagen content using a Sircol dye-binding method; (ii) LOX protein expression by western blotting; (iii) LOX activity using a fluorometric assay; and (iv) MMP-2 enzyme activity by gelatin zymography. Low-level (3%) mechanical stretching of PDL cells upregulated the expression of COL1A1, COL3A1 and LOX mRNAs, enhanced the production of collagen and increased the LOX activity but did not change the level of expression of MMP2 or TIMP2 mRNA. The collagen content and LOX activity showed obvious elevation in the medium, but not in the cell layer. High-level (10%) mechanical stretching downregulated COL1A1 mRNA but upregulated COL3A1 mRNA; however, the effect on COL3A1 was smaller, and occurred earlier, compared with the effect on the COL1A1 gene. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs but did not change collagen production or LOX activity. Moreover, high-level mechanical stretching increased the level of pro-MMP-2, especially in the cell layer. This study substantiates the mechanoregulation of the expression of ECM-related molecules in PDL cells. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs, but did not affect collagen production or LOX activity. In addition to increasing the transcription of COL1A1, COL3A1 and LOX genes, low-level mechanical stretching enhanced total collagen production and LOX activity, which should favor ECM stabilization. As an effective regulator of ECM remodeling, mechanical stretching can be exploited in periodontal regeneration and ligament tissue engineering via application of appropriate mechanical stimulation.

Effects of lysyl oxidase down-regulation on invasion, migration and E-cadherin protein expression of hypoxic lung cancer cells

To observe the effects of lysyl oxidase (LOX) down-regulation on invasion, migration and epithelial-mesenchymal transition phenotype molecule E-cadherin protein expression, induced by hypoxia in lung cancer NCI-H460 cells. Small interfering RNA against human LOX gene (LOX siRNA) was used to transfect lung cancer cells under normoxia (19%O2). After a 24 h incubation, the cells were plated for 24 h in hypoxic incubator (0.5%O2). Real-time polymerase chain reaction (PCR) was performed to detect the LOX mRNA expression. The protein levels of LOX and E-cadherin were determined by Western blot. And invasion and migration capacities were detected by transwell chamber. Compared with NCI-H460 cells under normoxia (set to 1), hypoxia increased to the levels of LOX mRNA and protein expression up to 26.04 ± 1.78 and 5.57 ± 1.27 respectively (both P < 0.05). Compared with control siRNA group (set to 1), LOX mRNA and protein expression after LOX siRNA transfection were 0.24 ± 0.03 and 0.29 ± 0.03 respectively, cellular invasive and migratory capacities were 0.57 ± 0.03 and 0.49 ± 0.02 respectively, the protein expression of E-cadherin was 2.17 ± 0.21 (all P < 0.05). LOX down-regulation reduces invasion and migration potentials of hypoxic human lung cancer cell and potentiates the protein expression of E-cadherin.

Lysyl Oxidase May Play a Critical Role in Hypoxia-Induced NSCLC Cells Invasion and Migration

Lysyl oxidase (LOX), a copper-dependent amine oxidase known to function both intracellularly and extracellularly, is implicated in promoting tumor progression and hypoxic metastasis in certain malignancies. Nonsmall cell lung cancer (NSCLC) is a highly aggressive cancer with poor prognosis worldwide. However, the role and molecular mechanism by which LOX involving in hypoxic NSCLC invasion and migration are poorly understood. This study explores the effect of LOX on invasion and migration of NSCLC cells under hypoxic conditions. Small interfering RNA (siRNA) targeting LOX was used to silence LOX expression of hypoxic NSCLC cells, SPCA1 and A549. Cellular invasive and migratory potentials were determined by matrigel invasion and migration assays. Expression of LOX, Src, Src activation (Tyr418 phosphorylation of Src), and Snail were evaluated by real-time PCR and western blot, respectively. The results showed that LOX mRNA and protein expression were upregulated under hypoxic conditions in NSCLC cells. Knockdown of LOX led to inhibition of hypoxia-induced invasion and migration. Phosphorylated Src (Tyr418) and Snail proteins were decreased along with LOX downregulation. Our data provide molecular evidences that LOX is mechanistically linked to increased invasion and migration of hypoxic NSCLC cells, and may serve as an antimetastasis target of human NSCLC.

Mechanism of lysyl oxidase (LOX) in breast cancer invasion and metastasis

To investigate possible mechanism of silencing lysyl oxidase (LOX) gene by RNA interference affecting on invasion and metastasis of breast cancer cells. LOX-RNAi-LV was designed and synthesized, which was transfected into breast cancer cell line MDA-MB-231. The expressions of LOX, MMP-2 and MMP-9 were determined by Real-time PCR in MDA-MB-231 cells, and the protein expression of LOX was determined by Western blot. The cells migration and invasion abilities were measured by cell migration and invasion test. 111 cases of breast cancer tissue and cancer-adjacent breast tissues and 20 cases of benign lesion tissues of LOX, MMP-2 and MMP-9 were detected by immunohistochemistry, and the relationship of LOX and clinicopathological characteristics was analyzed. The expression levels of LOX mRNA and protein were down-regulated obviously after transfecting LOX-RNAi-LV, with the inhibition rate 89.2% ± 1.3% and 84.4% ± 0.4% respectively. The relative expressions of MMP-2 and MMP-9 mRNA were 0.496 ± 0.021 and 0.571 ± 0.099 in RNAi group, which was significantly lower than that in negative control group (0.846 ± 0.047, 0.786 ± 0.042) and blank control group (1.000 ± 0.000, 1.000 ± 0.000) (both P < 0.05). Cell migration and invasion test showed the average cell numbers per field in the group RNAi were 47 ± 2 and 63 ± 2, was significantly lower than that in negative control group (100 ± 1, 118 ± 2) and blank control group (100 ± 1, 118 ± 2) (both P < 0.05). The expression of LOX protein in breast cancer, cancer-adjacent breast tissues and benign breast tumor were 48.6% (54/111), 26.1% (29/111), 20.0% (4/20), the expression of LOX protein in breast cancer was significantly higher than that in cancer-adjacent breast tissues and benign lesion tissues (P = 0.019). The expression of LOX protein was associated with clinical stage, lymph node metastasis, tumor size. Correlation analysis showed that LOX protein expression was significantly positive correlation with MMP-2 (r = 0.262, P = 0.005) and MMP-9 (r = 0.424, P = 0.000). LOX can promote invasion and metastasis of breast cancer; LOX and MMP-2, MMP-9 may have a synergistic role in promoting invasion and metastasis of breast cancer.

Estrogen receptor dependence of lysyl oxidase expression and activity in cardiac fibroblasts

Lysyl oxidase (LOX) is a collagen crosslinking enzyme essential for the deposition of collagen. We investigated the mechanism of estrogen mediated increases of LOX expression in rat cardiac fibroblasts and the role of estrogen receptors (ER). Adult rat cardiac fibroblasts were treated with ER‐α (MPP) and ‐β (PHTPP) specific antagonists, and a general ER inhibitor (ICI 181780), then treated with estrogen for 48 hrs. LOX protein expression was analyzed by Western blot. LOX catalytic activity was measured by fluorometric assay of cell‐conditioned media. Estrogen (1 to 50 ng/ml) increased LOX protein expression (150–290% for active form and 210–310% for proLOX vs control; P&lt;0.05). Estrogen also increased LOX activity in media (120–140% vs media from untreated). LOX activation by estrogen was prevented by ER‐α and ‐β blockade; however ER‐β antagonism had a greater effect, reducing LOX activity below that of control (75% versus control and ER‐α block; P&lt;0.05). ER‐β blockade also had a greater effect on LOX protein expression (58% decrease for active LOX vs estrogen treated) than ER‐α inhibitor (25%). ICI also significantly decreased LOX protein expression. These data indicate that both ER‐α and ‐β are involved in estrogen induced upregulation of LOX expression and activity in rat cardiac fibroblasts, with ER‐β playing a more significant role (Louisiana Board of Regents LEQSF2009‐12‐RDA10 and AHA #11GRNT7700002).

Induction of cardiac fibroblast lysyl oxidase by TGF-β1 requires PI3K/Akt, Smad3, and MAPK signaling

Lysyl oxidase (LOX) is a key extracellular enzyme responsible for the post-translational modification of collagens I and III to form mature fibrillar collagen. Increased expression of LOX is associated with fibrosis and cardiac dysfunction, yet little is known about the regulation of LOX in the heart. In this study, the cell signaling pathways responsible for the regulation of LOX expression by transforming growth factor (TGF)-β1 were assessed. Adult cardiac fibroblasts were isolated from male Sprague–Dawley rat hearts by enzymatic digestion. Fibroblasts were grown in DMEM with 10% FBS until approximately 80% confluent, growth arrested for 24 h, and then treated with TGF-β1 (0–10 ng/ml), in the absence or presence of inhibitors of (1) PI3K (wortmannin), (2) Smad3 (SIS3), (3) p38-MAPK (PD169316), (4) JNK (SP600125) and (5) ERK1/2 (PD98059). TGF-β1 treatment significantly upregulated LOX mRNA and protein expression in cardiac fibroblasts, as well as activity in the cell-conditioned media. Concomitant increases in collagen types I and III, and bone morphogenic protein (BMP-1) expression were found in response to TGF-β1. The increase of LOX protein in response to TGF-β1 was prevented by inhibitors of PI3K, Smad3, p38-MAPK, JNK and ERK1/2. Blockade of PI3K also decreased TGF-β1 induced phosphorylation of Smad3, suggesting that the PI3K/Akt and Smad pathways may be integrated in TGF-β1 signaling. Further studies are warranted to address the regulation of LOX in the normal and diseased heart, and how this critical extracellular enzyme may be targeted for clinical benefit.

Compensatory response of cardiac lysyl oxidase to chronic volume overload

Volume overload causes extensive remodeling of the ventricle, with structural and functional alterations. These alterations include disruption of extracellular collagen, ventricular hypertrophy and dilatation, and eventual heart failure. The collagen crosslinking enzyme, lysyl oxidase (LOX), is necessary for collagen maturation, but little is known about its role in the remodeling heart. We hypothesized that chronic volume overload induced remodeling and dysfunction are accompanied by elevations of cardiac LOX protein expression and activity. Surgically induced volume overload and sham‐operated rats were studied at 4, 14 and 21 wks post‐surgery. Ultrasound was used to evaluate cardiac function. Left ventricular (LV) LOX expression and activity were determined by Western blot and Amplex red assay, respectively. Collagen content was determined by picrosirius red assay. Volume overload caused progressive ventricular dilatation and dysfunction. LV LOX protein expression was elevated after 4, 14 and 21 wks of volume overload (30%, 65% and 92% increases vs. sham, p&lt;0.05). This elevation correlated with increased LOX activity at 4 and 14 wks, but not at 21 wks. Despite increased LOX expression and activity, a progressive loss of collagen was observed. These data indicate that compensatory increases in LOX occur in response to volume overload, but are insufficient to prevent collagen loss and dilatation.

IL‐1β and TNF‐α Differentially Regulate Lysyl Oxidase Expression and Activity in Adult Cardiac Fibroblasts

Cardiac fibroblasts constitutively express lysyl oxidase (LOX), an enzyme that catalyzes the crosslinking of collagen and modulates extracellular matrix maturation. Pro‐inflammatory cytokines, IL‐1β and TNF‐α, are elevated during cardiac disease and are known to modulate LOX expression in non‐cardiac cells. However, little is known about the regulation of LOX expression or activity in the heart. We tested the hypothesis that IL‐1β and TNF‐α decrease LOX expression and activity in isolated adult rat cardiac fibroblasts. After incubation in minimal serum media, fibroblasts were treated with TNF‐α (20 ng/ml) or IL‐1β (10 ng/ml) for 48 hrs. Cells were harvested and LOX expression assayed by Western blot. LOX activity was measured in cell culture media using Amplex red assay. Treatment with TNF‐α led to a significant reduction in LOX protein expression. However, IL‐1β did not affect LOX expression. Despite these differences in expression, both TNF‐α and IL‐1β significantly decreased LOX activity (64% and 60% vs. control, respectively). In addition, the TNF‐α regulation of LOX protein occurs in part through a Rho‐associated protein kinase (ROCK) pathway, as incubation of fibroblasts with the ROCK inhibitor, Y‐27632, attenuated the reduction of LOX expression. These findings indicate that the expression and activation of LOX in cardiac fibroblasts is differentially regulated by TNF‐α and IL‐1β.

Myocardial lysyl oxidase regulation of cardiac remodeling in a murine model of diet-induced metabolic syndrome

Metabolic syndrome (MetS) represents an increased risk of cardiovascular disease. Although its individual components adversely affect cardiac structure and function, the extent to which multiple components of MetS affect the cardiac extracellular matrix (ECM) has not been well characterized. Lysyl oxidase (LOX) is one of the cardiac ECM-modifying enzymes that catalyze the formation of collagen cross-linking. Our objective was to define the effect of diet-induced MetS on the LOX enzyme. MetS was induced in male C57BL/6 mice by administrating a high-fat, high-simple carbohydrate diet for 6 mo. Gene expression was determined by real-time PCR. The cardiac protein expression and enzymatic activity of LOX were measured. The severity of fibrosis was assessed by histology and hydroxylproline assay. Cardiac diastolic function was assessed by in vivo analysis of the pressure-volume relationship. LOX, matrix metalloproteinases, and their tissue inhibitors were analyzed, and of these three, LOX was most significantly changed in the MetS mice. Despite the blunted gene expression of LOX isoforms, MetS mice demonstrated a significant upregulation of bone morphogenetic protein-1. Correspondingly, there was an increase in the ratio of protein expression of mature to proenzyme LOX by 25.9%, enhanced LOX activity by 50.0%, and increased cardiac cross-linked collagen compared with the controls. This fibrotic response coincided with a marked increase in end-diastolic pressure, increased left ventricular stiffness, and impaired diastolic filling pattern. Our data signify that diet-induced MetS alters the remodeling enzymes, mainly LOX, thereby altering ECM structure by increasing the amount of cross-linking and inducing diastolic dysfunction.

Expression of Lysyl Oxidase Is Correlated with Lymph Node Metastasis and Poor Prognosis in Esophageal Squamous Cell Carcinoma

Lysyl oxidase (LOX), an extracellular matrix-remodeling enzyme, has been reported to regulate tumor metastasis. We investigated the clinical significance of LOX expression in esophageal squamous cell carcinoma (ESCC). We examined LOX expression in ESCC cell lines by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. We also examined LOX expression by real-time RT-PCR in 39 surgically resected ESCC and by immunohistochemistry in 122 surgically resected ESCC. LOX messenger RNA (mRNA) was expressed at a high level in TTn (originating from an ESCC metastatic lesion); at a moderate level in TE-2 and TE-15; and at a low level in TE-1, TE-8, and TE-13. In Western blotting, all cell lines expressed the catalytically inactive 50-kDa LOX at approximately the same levels, but catalytically active 32-kDa LOX was overexpressed only in TTn. LOX mRNA levels in ESCC tissues were significantly higher than those observed in normal esophageal tissues (P < 0.001) and had no significant correlation with tumor-node-metastasis (TNM) factors. High LOX protein expression had a significant correlation with presence of lymph node metastasis (P = 0.009) and number of lymph node metastases (P = 0.047). Overall and cancer-specific survival rates of patients with ESCC with high LOX expression were significantly lower than those of patients with ESCC with low LOX expression (P = 0.024 and P = 0.012). Univariate and multivariate analyses revealed that high LOX protein expression was an independent prognostic factor for ESCC. Our findings suggest that LOX can serve as a predictive marker of lymph node metastasis and prognosis in ESCC.

Impact of Treatment on Myocardial Lysyl Oxidase Expression and Collagen Cross-Linking in Patients With Heart Failure

The aim of this study was to investigate whether torasemide modifies collagen cross-linking in the failing human heart. We analyzed the degree of cross-linking and the expression of the enzyme lysyl oxidase, which regulates cross-linking, in the myocardium of patients with chronic heart failure at baseline and after 8 months of treatment with either torasemide or furosemide in addition to their standard heart failure therapy. Whereas lysyl oxidase protein expression was very scarce in normal hearts, it was highly expressed in failing hearts. Cross-linking was increased (P<0.001) in heart failure patients compared with normal hearts. These 2 parameters decreased (P=0.021 and P=0.034) in torasemide-treated patients and remained unchanged in furosemide-treated patients. In addition, more (P=0.009) patients showed normalization of left ventricular chamber stiffness in the torasemide subgroup than in the furosemide subgroup after treatment. Lysyl oxidase expression correlated with cross-linking (r=0.661; P<0.001), and cross-linking correlated with left ventricular chamber stiffness (r=0.452; P=0.002) in all patients. These findings show for the first time that lysyl oxidase overexpression is associated with enhanced collagen cross-linking in the failing human heart. In addition, we report that the ability of torasemide to correct both lysyl oxidase overexpression and enhanced collagen cross-linking results in normalization of left ventricular chamber stiffness in patients with heart failure. Lysyl oxidase may thus represent a target for reduction of stiff collagen and improvement of left ventricular mechanical properties in heart failure patients.

Hypoxia/reoxygenation: A dynamic regulator of lysyl oxidase‐facilitated breast cancer migration

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.

Association of Expression Aberrances and Genetic Polymorphisms of Lysyl Oxidase with Areca-Associated Oral Tumorigenesis

Areca nut use is the major cause of oral squamous cell carcinoma (OSCC) in Southern Asians. Areca nut contains a high level of free copper ions. Lysyl oxidase (LOX) is a copper-activated enzyme critical for extracellular matrix organization. Contradictory evidence has been put forward to suggest that LOX may be either an oncogenic or a suppressive element. This study investigated the oncogenic significance of LOX in areca-associated OSCC. The expression assays and polymorphism analysis were done to know the clinicopathologic implications of LOX status in OSCC. Knockdown and overexpression experiments were conducted to know the phenotypic effects of LOX on OSCC cells. Up-regulation of LOX mRNA and LOX protein expression in OSCCs relative to adjacent oral mucosa was found. Precancerous lesions had the highest LOX mRNA expression. Areca nut extract up-regulated LOX expression in oral epithelial cells. Knockdown of LOX induced cellular migration and invasion, but it reduced the anchorage-independent growth and xenographic tumorigenesis of OSCC cells. The reduction of migration and invasion by LOX overexpression was partially rescued by blockage of LOX activity. The Arg158Gln polymorphism was associated with earlier clinical stage of OSCC. Wild-type LOX overexpression induced anchorage-independent growth in OSCC cells, but this was not for LOXArg158Gln overexpression. LOX exerts oncogenic roles in areca-associated OSCC. This potential could be affected by the existence of LOX propeptide domain or genetic polymorphism.

Lysyl oxidase expression in bronchogenic carcinoma

Background Lysyl oxidase catalyzes a key step in the cross-linking of collagen and elastin in the extracellular matrix. Recent studies have documented differential lysyl oxidase expression in the stromal reaction to colon, breast, prostate, and lung cancer. The present study was undertaken to test the hypothesis that lysyl oxidase mRNA and protein expression decrease with advancing tumor stage in patients with bronchogenic carcinoma. Methods Tumor specimens were obtained from 17 patients undergoing resection for bronchogenic carcinoma. Real-time polymerase chain reaction was used to determine steady-state lysyl oxidase mRNA expression, and protein expression was qualitatively assessed by immunohistochemistry. Results Real-time polymerase chain reaction studies documented a 3.4-fold graded decrease in lysyl oxidase mRNA levels as tumors progressed from stage I to IV. Similar qualitative changes in lysyl oxidase protein expression were demonstrated by immunohistochemistry. Conclusions These results support the hypothesis that variations in lysyl oxidase expression may correlate with the invasive and metastatic potential of bronchogenic carcinoma.