Expression Levels Of Claudin-1 Research Articles

Claudin-1 upregulation is associated with favorable tumor features and a reduced risk for biochemical recurrence in ERG-positive prostate cancer.

Claudin-1 is a membrane-tight junction protein and important for the sealing of the paracellular cleft in epithelial and endothelial cells. Differential expression of Claudin-1 is linked to disease outcome in various cancers. To evaluate the potential relevance of Claudin-1 expression in prostate cancer, a tissue microarray containing samples of 17,747 tumors with annotated clinico-pathological and molecular data was immunohistochemically analyzed for Claudin-1 expression. In normal prostate, glandular cells were always Claudin-1-negative while there was a strong staining of gland-surrounding basal cells. In contrast to normal prostatic glands, a positive Claudin-1 immunostaining, was found, however, in 38.7% of 12,441 interpretable cancers and was considered weak in 12.7%, moderate in 13.2%, and strong in 12.8% of cases. Positive Claudin-1 immunostaining was associated with favorable tumor features like low pT (p = 0.0032), low Gleason grade (p< 0.0001), and a reduced risk of PSA recurrence (p = 0.0005). A positive Claudin-1 staining was markedly more frequent in ERG-positive (63%) than in ERG-negative cancers (23%; p < 0.0001). Subset analyses revealed that all associations of Claudin-1 expression and favorable phenotype and prognosis were driven by ERG-positive cancers. Multivariate analyses revealed, however, that even in ERG-positive cancers, the prognostic impact of high Claudin-1 expression was not independent of established clinico-pathological parameters. Comparison with 12 previously analyzed chromosomal deletions identified conspicuous associations with PTEN and 12p13 deletions potentially indicating functional interactions. These data identify a peculiar role for Claudin-1 in prostate cancer. The protein is overexpressed in a fraction of prostate cancers and increased Claudin-1 expression levels predict a favorable prognosis in ERG-positive cancer.

Claudin-1 silencing increases sensitivity of liver cancer HepG2 cells to 5-fluorouracil by inhibiting autophagy.

Liver cancer is one of the most common cancer types globally. However, the acquisition of drug resistance limits the effectiveness of chemotherapy and commonly results in metastasis. Therefore, an effective therapeutic approach to target chemoresistance-associated cellular molecules is imperative. Claudin-1 (CLDN1) has previously been reported to be associated with the development of drug resistance. The present study investigated the effect of CLDN1 on the sensitivity of 5-fluorouracil (5-FU)-resistant liver cancer cells. Firstly, a 5-FU-resistant HepG2 liver cancer cell line (Hep/5FU) was developed by continuous 5-FU treatment. MTT proliferation, Transwell and Matrigel assays indicated that Hep/5FU cells were significantly resistant to 5-FU, and demonstrated increased migration and invasion abilities, compared with parental HepG2 cells. Furthermore, reverse transcription-quantitative polymerase chain reaction and western blot analysis indicated that mRNA and protein expression levels of CLDN1 were significantly increased in Hep/5FU cells, compared with HepG2 cells. CLDN1 was knocked down by transfection with small interference RNA. MTT and Annexin V-fluorescein isothiocyanate/propidium iodide assays demonstrated that CLDN1 silencing significantly inhibits proliferation and enhances apoptosis induced by 5-FU treatment in Hep/5FU cells, compared with non-silenced Hep/5FU cells. Additionally, CLDN1 silencing attenuated the migration and invasion capabilities of Hep/5FU cells. In addition, it was identified that CLDN1 silencing decreased drug resistance by inhibiting autophagy, which was associated with a decrease in the ratio of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I and upregulation of P62. A cell proliferation assay revealed that the addition of autophagy inhibitor 3-methyladenine decreased drug resistance of Hep/5FU cells. By contrast, incubation with the autophagy agonist Rapamycin elevated drug resistance of CLDN1-silenced Hep/5FU cells. In summary, these data indicate that CLDN1 may be a potential target for resensitizing resistant liver cancer HepG2 cells to 5-FU by regulating cell autophagy.

Mesenchymal stem cells decrease blood\u2013brain barrier permeability in rats with severe acute pancreatitis

BackgroundImpairment of the blood–brain barrier (BBB) could result in secondary cerebral edema and life-threatening pancreatic encephalopathy in patients with severe acute pancreatitis (SAP). Mesenchymal stem cells (MSCs) have been widely adopted in clinical research because of their pleiotropic functions. The aim of this study was to investigate the impact of MSCs on BBB permeability in SAP and the potential mechanisms driving these effects.MethodsSprague-Dawley rats were randomly assigned to the control, SAP and SAP+MSCs groups. Pancreatic impairment was assessed. The serum levels of amylase, TNF-α and IL-10, expression levels of claudin-5, Bax, Bcl-2 and MMP-9, and the BBB permeability were measured. Endothelial cell apoptosis was evaluated.ResultsSAP rats showed BBB impairment with increased permeability and secondary cerebral edema, which was confirmed using the Evans blue assay and the calculation of the brain dry/wet ratio. Treatment with MSCs decreased the serum levels of amylase and TNF-α, increased the serum levels of IL-10, attenuated the apoptosis of brain microvascular endothelial cells, upregulated claudin-5 expression and downregulated MMP-9 expression. This treatment attenuated the increased BBB permeability in SAP rats.ConclusionsMSCs attenuated the impairment of the BBB and decreased its permeability, producing protective effects in SAP rats.

Expression and significance of PTEN and Claudin-3 in prostate cancer.

Expression and significance of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Claudin-3 in the blood of patients with prostate cancer [prostate cancer (PCa)] were investigated. Retrospective analysis of 84 cases of PCa patients confirmed by pathological diagnosis were studied, as the experiment group. Moreover, the physical examination data of 84 healthy volunteers examined in the Affiliated Hospital of Beihua University were the control group. The expression levels of blood in the PTEN and Claudin-3 of both the experiment group and the control group were determined by enzyme-linked immunosorbent assay. According to the blood expression in PTEN and Claudin-3 between both the experiment group and the control group, the test value of the ROC curve in PTEN and Claudin-3 were detected by both single detection and joint detection. The expression levels of PTEN in the experiment group were significantly lower than the control group (P<0.05). The expression levels of Claudin-3 were higher in the experiment group than the control group (P<0.01). The expression levels of PTEN and Claudin-3 in the experiment group were significantly associated with the distant metastasis of cancer cells, preoperative prostate-specific antigen levels, tumor diameter and pathological stages (P<0.01). The expression levels of PTEN in the pathological stage of T1-T2 group was lower than that of the T3-T4 group (P<0.01). The expression levels of PTEN and Claudin-3 are closely related to the distant metastasis of cancer cells, preoperative prostate-specific antigen level, tumor diameter and pathological stage. Combined detection of both PTEN and Claudin-3 can improve the specificity levels of PCa for diagnosis and has an important diagnostic value for PCa. It can be used as a biological indicator for PCa diagnosis, disease severity analysis and efficacy evaluation.

Epidermal growth factor treatment has protective effects on the integrity of the blood-brain barrier against cerebral ischemia injury in bEnd3 cells.

Tight junctions (TJs) serve an important role in maintaining the integrity of the blood-brain barrier (BBB), while neurological disorders, including ischemic stroke, induce TJ disruption and increase BBB permeability; results include edema formation and hemorrhage transformation. Cerebral endothelium protection presents a promising approach in ischemic stroke therapy. In the current study, protective effects of the epidermal growth factor (EGF) on ischemia-induced disruption of BBB integrity were examined using an oxygen-glucose deprivation (OGD) model in bEnd3 cells. Expression levels of claudin-5 and TJ protein-1 (ZO-1) were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Cell viability was evaluated by cell counting kit-8 assay and the endothelial permeability of Lucifer yellow (LY) was assessed using Transwell assays. The results revealed that post-ischemia administration of EGF (250 ng/ml) significantly attenuated the decrease in mRNA (P<0.05) and protein (P<0.01) expression levels of claudin-5 and ZO-1, and the increase in endothelial permeability of LY (P<0.05) induced by 4 h OGD exposure followed by 24 h reoxygenation. In addition, EGF did not significant affect cell viability. The current study suggested a potential of EGF to improve BBB integrity against ischemic injury by upregulating the expression of TJ proteins and reducing endothelial permeability.

Vascular endothelial growth factor A promotes platelet adhesion to collagen IV and causes early brain injury after subarachnoid hemorrhage.

The role of vascular endothelial growth factor A in platelet adhesion in cerebral microvessels in the early stage of subarachnoid hemorrhage remains unclear. In this study, the endovascular puncture method was used to produce a rat model of subarachnoid hemorrhage. Then, 30 minutes later, vascular endothelial growth factor A antagonist anti-vascular endothelial growth factor receptor 2 antibody, 10 μg, was injected into the right ventricle. Immunohistochemistry and western blot assay were used to assess expression of vascular endothelial growth factor A, occludin and claudin-5. Immunohistochemical double labeling was conducted to examine co-expression of GP Ia-II integrin and type IV collagen. TUNEL was used to detect apoptosis in the hippocampus. Neurological score was used to assess behavioral performance. After subarachnoid hemorrhage, the expression of vascular endothelial growth factor A increased in the hippocampus, while occludin and claudin-5 expression levels decreased. Co-expression of GP Ia-II integrin and type IV collagen and the number of apoptotic cells increased, whereas behavioral performance was markedly impaired. After treatment with anti-vascular endothelial growth factor receptor 2 antibody, occludin and claudin-5 expression recovered, while co-expression of GP Ia-II integrin and type IV collagen and the number of apoptotic cells decreased. Furthermore, behavioral performance improved notably. Our findings suggest that increased vascular endothelial growth factor A levels promote platelet adhesion and contribute to early brain injury after subarachnoid hemorrhage. This study was approved by the Biomedical Ethics Committee, Medical College of Xi'an Jiaotong University, China in December 2015.

Activation of the α7 nicotinic acetylcholine receptor upregulates blood-brain barrier function through increased claudin-5 and occludin expression in rat brain endothelial cells

The blood-brain barrier (BBB) is formed by brain endothelial cells (BECs) and regulates brain homeostasis by restricting the entry of blood-borne substances into the brain. Recent in vivo studies have shown that administration of nicotinic acetylcholine receptor (nAChR) agonists protects against BBB disruption and neuroinflammation induced by stroke and traumatic brain injury through the systemic cholinergic anti-inflammatory pathway. In the present study, we focused on the nAChRs expressed on BECs rather than those widely expressed in the central nervous system and peripheral tissues, and examined whether activation of the nAChRs on BECs facilitates BBB function. We used primary cultures of rat brain endothelial cells to evaluate brain endothelial permeability and tight junction (TJ)-related protein expression after a 24-h exposure to PHA543613 (a selective α7 nAChR agonist) or 5-iodo-A-85380 (a selective α4β2 nAChR agonist). We found that PHA543613 decreased sodium fluorescein permeability and increased the expression levels of claudin-5 and occludin, key TJ components. In contrast, 5-iodo-A-85380 had no effect on brain endothelial permeability or TJ protein expression. These findings suggest that the selective activation of α7 nAChRs on BECs has a specific role in upregulating BBB properties through increased claudin-5 and occludin expression.

Protective effect of catalpolon destruction of tight junctions of high glucose induced BMECs

This paper aimed to observe the protective effect of catalpol on the high glucose induced destruction of tight junctions of rat primary brain microvascular endothelial cells (BMECs). Catalpol co-administrated with high glucose increased BMECs survival, decreased its ET-1 secretion, and improved transmembrane electrical resistance in a time-dependent manner. Furthermore, transmission electron microscopy was used to observe catalpol's protective effect on tight junction. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and up-regulated the tight junction proteins claudin-5 and ZO-1, which were further demonstrated by the mRNA expression levels of claudin-5, occludin, ZO-1, ZO-2, ZO-3, α-actintin, vinculin and cateinins. This study indicated that catalpol reverses the disaggregation of cytoskeleton actin in BMECs and up-regulates the expression of tight junction proteins, such as claudin-5, occludin, and ZO-1, and finally alleviates the increase in high glucose-induced BMECs injury.

Clostridium butyricum protects the epithelial barrier by maintaining tight junction protein expression and regulating microflora in a murine model of dextran sodium sulfate-induced colitis

Objective: To investigate the protective effects of Clostridium butyricum on the epithelial barrier in dextran sodium sulfate (DSS)-induced colitis.Methods: Eight-week-old BALB/c mice were divided into a healthy control group and DSS-induced groups, including negative control (normal saline), 5-aminosalicylic acid (5-ASA), C. butyricum group, and 5-ASA + C. butyricum groups. Colitis was induced by 5% DSS ad libitum for 7 d. We assessed the disease activity index (DAI), histological grading, and ultrastructural changes by transmission electron microscopy. Stools were collected for bacterial microflora analysis. Tight junction (TJ) proteins, including claudin-1, claudin-2, occluding, and zonula occludens-1, were detected by immunohistochemical staining and western blot. We also assessed NF-κB and cytokines, including IL-1β, IL-13, and IL-10, by western blot.Results: C. butyricum decreased DAI and histological injury scores in DSS-induced mice, and repaired the damaged structure of TJs. Moreover, C. butyricum exerted its regulatory effect on fecal microflora by increasing and decreasing the growth of Lactobacillus spp. and Enterococcus spp., respectively, in the colon lumen. Expression levels of claudin-1, occludin and zonula occludens-1 were also elevated by the administration of C. butyricum. In addition, C. butyricum increased the expression of the anti-inflammatory cytokine, IL-10, and decreased levels of pro-inflammatory cytokines, including IL-1β, TNF, and IL-13, as well as NF-κB. Moreover, the beneficial effects of C. butyricum combined with 5-ASA were superior to treatment with C. butyricum only.Conclusion: In a mouse model of ulcerative colitis (UC), C. butyricum exerted a protective effect on the epithelial barrier by regulating microflora, maintaining the expression of TJ proteins and exerting immunoregulatory effects.

Correlations between claudin-1 and PIGF expressions in retinoblastoma.

To investigate the expressions of claudin-1 and placental growth factor (PlGF) proteins in retinoblastoma (RB) and their relationships with the differentiation of RB, the infiltration of optic nerve and choroid and clinical stages. Immunohistochemical (IHC) method was used to detect the expressions of claudin-1 and PlGF proteins in 56 cases of RB paraffin-embedded tissue samples. The x2-test and Fisher exact test were used to compare the qualitative variables. The Pearson correlation coefficient was used to detect the correlation of the expression of claudin-1 with that of PlGF in RB tissues. 1) Among RB tissues, the positive expression rates of claudin-1 in clinical stage I tumors and clinical stage III tumors were 69.2% and 38.9%, respectively, and claudin-1 was not expressed in all clinical stage II tumors (p=0.002). In case of optic nerve invasion, the lowly positive expression of claudin-1 was detected, and the difference was significant (p=0.001). 2) The positive expression rate of PlGF proteins in RB was 73.8%, which was higher in tumors with optic nerve invasion than in tumors without the invasion; the expression was significantly different (p=0.001). In addition, the positive expression rate of PlGF in tumors with choroidal invasion was 74.1%. 3) The expression of claudin-1 in RB was negatively correlated with the presence of choroidal invasion (r=0.52, p≤0.0001) and optic nerve infiltration (r=0.49, p=0.0003). There was a significant positive correlation between the expression of PlGF and the presence of optic nerve invasion (r=0.30, p=0.009). In addition, there was a significant positive correlation between the expression of claudin-1 and that of PlGF (r=0.41, p=0.006). The expression level of claudin-1 is negatively correlated with the differentiation of RB cells, optic nerve infiltration and clinical stages, while the expression of PlGF was positively correlated with the optic nerve infiltration and clinical stages of RB. The role of claudin-1 may be opposite to that of matrix metalloproteinase-2 (MMP-2) in the development of RB.

Leptin alleviates intestinal mucosal barrier injury and inflammation in obese mice with acute pancreatitis.

Obesity is an independent risk factor for severe acute pancreatitis (AP). Leptin plays an important role in energy homeostasis. It has been reported that leptin might also participate in the regulation of the intestinal mucosal barrier and inflammatory response. This study aimed to evaluate the effects of leptin on the intestinal mucosal barrier and inflammatory injury in obese mice with AP. AP was induced in leptin-deficient (ob/ob) or wild type (WT) mice by peritoneal injection of caerulein. The animals were divided into 4 groups: WT mice with or without exogenous leptin injection and ob/ob mice with or without leptin treatment. The inflammatory scoring of the pancreas and intestine were evaluated. Intestinal permeability, ileal interleukin (IL)-6 and IL-1β, proliferation, apoptosis and intestinal expression levels of claudin-1 and occludin were measured. Pancreatic pathologic scores (8.50 ± 0.96 vs. 3.78 ± 1.35, p < 0.001), pancreatic levels of IL-6 (8.34 ± 3.21 ng/mg vs. 4.99 ± 0.53 ng/mg, p = 0.022), intestinal oedema scores (2.25 ± 0.46 vs. 1.14 ± 0.69, p = 0.001) and intestinal permeability to FD4 (0.78 ± 0.06 μg/ml vs. 0.53 ± 0.11 μg/ml, p < 0.001) were significantly higher in ob/ob mice than those in WT mice. Leptin replacement in ob/ob mice greatly improved the intestinal permeability (FD4 0.66 ± 0.03 μg/ml, vs. 0.78 ± 0.06 μg/ml, p = 0.012), increased the ileal expression of claudin-1(1.07 ± 0.08 vs. 0.83 ± 0.07 relative densitometry, p = 0.001) and reduced intestinal IL-6 and IL-1β to levels comparable to those in WT mice. The pancreatic level of IL-6 in ob/ob mice treated with leptin was also significantly decreased relative to that of untreated ob/ob mice (4.45 ± 1.71 ng/mg vs. 8.34 ± 3.21 ng/mg, p = 0.010). Obesity may aggravate intestinal inflammation and increase intestinal permeability under the condition of acute pancreatitis. Exogenous leptin supplementation was in favour of anti-inflammation and improvement of intestinal mucosal barrier.

Cerebral cavernous malformation 3 gene deficiency promotes early changes in Alzheimer disease-like lesions induced by low lead exposure

Objective: To investigate the effects of cerebral cavernous malformation 3 (CCM3) gene knockout on the lead exposure-induced blood-brain barrier malfunction in mice brain, and the relationship between CCM3 knockout and the Alzheimer's disease (AD). Methods: Wide type (WT) mice and CCM3(+)/- mice were divided into 4 groups, control group and lead exposed group in WT as well as CCM3(+/-) mice. Lead exposed groups were treated with 0.05% lead acetate in drinking water for 12 weeks, while control group drink deionized water freely. Blood lead and brain lead levels in each group were detected by graphite furnace atomic absorption spectrometry. The brain tissue of each group was made into paraffin sections, whose morphology were observed by HE staining. The expression of Aβ(1-42) in brain tissue was detected by immunohistochemistry and the brain capillaries were labeled by VRGFR2. The protein expression of Claudin-5, ZO-1, and p-Tau was detected by Western blot. The brain tissue RNA was extracted and the relative expression of LRP-1 mRNA was detected by qRT-PCR. Results: The levels of blood lead WT (216.07±84.16) and CCM3(+/-) (189.64±101.86) μg/L in lead exposed group were higher than those in control group WT (19.52±11.46) and CCM3(+/-) (11.79±8.20) μg/L, the difference was statistically significant (t=4.18, P=0.006; t=3.79, P=0.016). The levels of brain lead WT (1.78±0.69) and CCM3(+/-) (1.74±0.66) μg/L were higher than those in control group WT (1.06±0.87) and CCM3(+/-) (0.97±0.64) μg/L, the difference was statistically significant (t=3.67, P=0.018; t=3.88, P=0.015). The HE staining showed no obvious lesions in the brain of each group of mice. The results of immunohistochemistry showed that there was no Aβ(1)-42 deposition in the brain of mice in each group. The numbers of microvessels in the brain of CCM3(+/-) mice in the lead exposed group were decreased. Compared with the relative expression levels of Claudin-5 (WT: 1.30±0.03, CCM3(+/-): 1.07±0.08) in control group mice brain, the relative expression of Claudin-5 (WT: 0.96±0.04, CCM3(+/-): 0.59±0.01) was decreased with statistical significance (F=199.27, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of lead exposed group (WT: 0.32±0.10, CCM3(+/-): 0.06±0.01) was higher than that of unexposed group (WT:1.00±0.06, CCM3(+/-):2.12±0.18), the difference was statistically significant (F=288.29, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of CCM3(+)/- mice exposed to lead was lower than that of WT mice ((0.06±0.01)vs(0.32±0.10), t=26.90, P<0.001). Conclusion: The mice did not show significant AD-like lesions under low-does lead exposure, but resulted in early damage of brain blood-brain barrier and early changes of AD-like lesions in mice, with CCM3(+/-) mice being sensitive to lead exposure stronger than that of WT mice, suggesting that deletion of CCM3 gene may be one of the potential risk factors for accelerating the development of AD in mice exposed to lead.

The protective roles of L-borneolum, D-borneolum and synthetic borneol in cerebral ischaemia via modulation of the neurovascular unit

ObjectiveBorneol has been used to treat stroke in China since ancient times. In our previous research, we demonstrated the effect of borneol on cerebral ischaemia injury via meta-analysis. The neurovascular unit (NVU) is the structural basis of the preservation of the brain microenvironment and is believed to be a promising target in treating stroke. In this research, we explored the roles of three kinds of borneol, namely, L-borneolum (B1), D-borneolum (B2) and synthetic borneol (B3), in the NVU with permanent middle cerebral artery occluded (pMCAO) rats. MethodsThe Longa scoring method was used to evaluate nerve function deficits in the pMCAO rats. Awakening time, brain water content, brain index and brain edema rate were also measured. TTC staining was used to calculate the cerebral infarction rate. The morphology of the ischaemia penumbra brain tissue was observed via HE staining, and the neuronal denatured cell index (DCI) was calculated. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of vascular endothelial growth factor VEGF and TNF-α in the serum. Moreover, the ultrastructures of the neurons and of the blood-brain barrier (BBB) were observed using transmission electron microscopy. The expression levels of Claudin-5, Bcl-2 and Bax in the ischaemia penumbra of pMCAO rats were detected using real-time PCR and immunohistochemistry. ResultsPretreatment with B1, B2 and B3 delayed the recovery time (P < 0.01). B1 remarkably ameliorated neurological deficits 24 h after cerebral ischaemia (P < 0.05). Moreover, B1 and B3 were both able to ameliorate brain edema and the area of cerebral infarction. In addition, B1, B2 and B3 all increased serum VEGF levels and decreased serum TNF-α levels (P < 0.01). For the ultrastructure determination, the BBB and the nerve centre were significantly improved by B1, B2 and B3. The mechanistic exploration revealed that B2 and B3 protected the brain by reducing the Bax/Bcl-2 ratio (P < 0.05, P < 0.01, respectively). Immunohistochemistry suggested that B1, B2 and B3 could also enhance the expression of Claudin-5 (P < 0.01). ConclusionThe three kinds of borneol demonstrated different protective effects on cerebral ischaemia injury. L-Borneolum displayed the most prominent anti-cerebral ischaemia effect among them. The mechanism was most likely executed via anti-apoptosis and anti-inflammation effects and maintenance of the stability of the BBB and TJs to comprehensively improve NVU function.

Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells

Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.

Protective effect and mechanism of p-hydroxybenzaldehyde on blood-brain barrier

The disruption of blood-brain barrier(BBB) induced by oxidative stress is an important pathological reaction which results in secondary brain injury during the cerebral ischemia-reperfusion. This study was designed to investigate the protective effect and mechanism of p-hydroxybenzaldehyde (p-HBA) from Gastrodia elata on BBB. The BBB is mainly consisted of vascular endothelial cells and astrocytes, so brain microvascular endothelial cell line (bEnd.3) and astrocytes (Ast) in mice were used in this study to establish BBB model. H₂O₂-induced oxidative stress was employed to induct the BBB damage. The bEnd.3 cells or astrocytes were exposed to different concentrations of H₂O₂ (0.125, 0.25, 0.5, 0.75 mmol·L⁻¹) for 4 h, then exposed to 0.5 mmol·L⁻¹ H₂O₂ for different duration (1, 2, 4, 6 h) to detect the reasonable condition of oxidative injury. After intervention by different concentrations of p-HBA(12.5, 25, and 50 mg·L⁻¹), LDH leakage rate was detected for bEnd.3 and Ast cells; the expression levels of tight junction protein claudin-5 and occludin in bEnd.3 cells were determined by Western blot and immunofluorescence. Nrf2, HO-1 and NQO1 in normal bEnd.3 cells and astrocytes as well as H₂O₂-induced damaged in astrocytes were detected by western blot after treatment with p-HBA. The results showed that the optimal condition of H₂O₂ induced damage in bEnd.3 cells and astrocytes was set up as exposure the cells to 0.5 mmol·L⁻¹ H₂O₂ for 4 h. Different concentrations of p-HBA could decrease LDH leakage rate after bEnd.3 and Ast injury was induced by H₂O₂; increase the protein expression levels of claudin-5, occludin, Nrf2, HO-1 and NQO1; and increase the expression levels of Nrf2, HO-1 and NQO1 in normal and H₂O₂-induced damaged astrocytes. These findings indicate that the p-HBA has protective effect on the BBB, and the related mechanism seems to involve up-regulating tight junction protein of the bEnd.3 cells and enhancing endogenous antioxidant capacity by activating the Nrf2/ARE pathway in both of bEnd.3 cells and astrocytes.

Interleukin-10 attenuates impairment of the blood-brain barrier in a severe acute pancreatitis rat model

BackgroundImpairment of the blood-brain barrier (BBB) in severe acute pancreatitis (SAP) could result in life-threatening pancreatic encephalopathy. Interleukin-10 (IL-10) is a classical cytokine that is well-known for its strong immunoregulatory and anti-inflammatory abilities. However, whether and how IL-10 protects the BBB in SAP are still unclear.MethodsThis study includes in vivo experiments using a SAP rat model and in vitro experiments using an in vitro BBB model consisting of a monolayer of brain microvascular endothelial cells (BMECs). The study groups are divided into the control, SAP (in vivo)/TNF-α (in vitro), IL-10 treatment, IL-10 + signal transducer and activator of transcription 3 (STAT3) inhibitor S3I-201 treatment groups. Pancreatic pathological scores, serum amylase, serum TNF-α levels and BBB permeability by Evan’s blue assay in SAP rat models were evaluated. BMEC apoptosis in SAP rats or induced by TNF-αin vitro was detected by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and flow cytometry, separately. Expression levels of claudin-5 and proteins involved in the STAT3 signaling pathway were measured by Western blotting. Location and changes of junctional structure of claudin-5 on BMECs were assessed by immunohistochemistry and immunofluorescence.ResultsIn vivo, IL-10 alleviated the severity of inflammation, attenuated the increased BBB permeability in SAP rat models by reducing BMEC apoptosis via the STAT3 pathway and ameliorated the down-regulation of claudin-5 expression in BMECs; in vitro, IL-10 improved BBB integrity against TNF-α by attenuating BMEC apoptosis via the STAT3 pathway, the impairment of tight junction structure and the down-regulation of claudin-5 expression in BMECs.ConclusionsIL-10 improves BBB properties in SAP by attenuating the down-regulation of claudin-5 expression and the impairment of tight junctions and by STAT3 pathway-mediated anti-apoptotic effects on BMECs.

Blood-brain Barrier Disruption Leads to Postoperative Cognitive Dysfunction.

Postoperative Cognitive Dysfunction (POCD) has received considerable attention as one of the main postoperative complications. The underlying mechanism of POCD in elderly subjects has not been fully elucidated to date. The Central Nervous System (CNS) is isolated from the bloodstream by the Blood Brain Barrier (BBB) that consists of endothelial cells, capillary blood vessels and tight junctions. The tight junctions carry out significant biological functions that are associated with the CNS and blood circulation. In this review, I present a hypothesis that blood-brain barrier disruption leads to postoperative cognitive dysfunction. A total of 81 healthy male Wistar rats were used for the present study. All the experimental animals were randomly divided into 3 groups: normal control group, isoflurane group and splenectomy group. The control group was not subjected to any form of treatment. The rats in isoflurane group were given 1.5-2% isoflurane under intubation and mechanical ventilation. The rats in splenectomy group underwent splenectomy under the same anesthesia as the isoflurane group. The Morris water maze was used to examine the learning and memory ability of the animals. The expression of the Tight Junctions Proteins (TJPs) in the hippocampus was analyzed using Western blotting. The concentration of Evans Blue (EB) in the supernatant was analyzed using UV spectroscopy. Ultrastructure changes in the basal laminas, the Tight Junctions (TJs), mitochondria and the endoplasmic reticulum surrounding the capillaries were assessed by Transmission Electron Microscopy (TEM). Following splenectomy, the rats displayed concomitant significant cognitive deficits in the Morris water maze test. Taken together, the results indicate that the expression levels of occludin (65KD) following splenectomy were reduced on days one and three in aged rats. No significant difference was noted in the expression levels of claudin-5, except for a reduction after surgery on day one. The leakage of EB was higher following splenectomy than control group and isoflurane group. The ultrastructure of the neurovascular unit was monitored on the day prior to surgery and on the 1st, 3rd and 7th day following surgery using a transmission electronmicroscope. The alterations in the levels of tight junction proteins following splenectomy may contribute to the BBB permeability increase, which in turn will induce postoperative cognitive dysfunction.

Vitamin A and Retinoic Acid Exhibit Protective Effects on Necrotizing Enterocolitis by Regulating Intestinal Flora and Enhancing the Intestinal Epithelial Barrier

Exaggerated inflammation that characterizes necrotizing enterocolitis (NEC) is caused by the invasion of pathogens through an immature intestinal barrier. Vitamin A (VA) and retinoic acid (RA) play important roles in the growth of epithelial tissue and in modulating immune function. To investigate the roles of VA and RA in the development of NEC. Levels of serum retinol in patients and in a NEC mouse model were detected with high-performance liquid chromatography. Bacterial communities of NEC mice treated with VA or PBS were detected by high-throughput sequencing. Invitro and invivo, levels of inflammatory factors were measured by ELISA and RT-PCR, and expression levels of claudin-1, occludin, and ZO-1 were detected by Western blotting. Transepithelial electrical resistance (TEER) was measured in Caco-2cell monolayers. The level of VA in the NEC patients was lower than in the control patients. In the NEC mice that were treated with VA versus PBS, the proportion of Escherichia-Shigella was lower, while the abundance of Bacteroides was markedly higher. Both invivo and invitro, the levels of inflammatory factors were significantly reduced, while the expression levels of claudin-1, occludin, and ZO-1 were increased, after the VA and RA treatments. Meanwhile, TEER was increased and lipopolysaccharide-induced damage was reduced in Caco-2cell monolayers after RA treatment. These results suggest that VA may regulate intestinal flora, alleviate inflammatory reactions, and enhance the intestinal epithelial barrier in NEC. Thus, VA may be an effective drug for providing protection against NEC in newborns.

Abstract A80: Differential expression of claudin-3 during colorectal tumorigenesis and its role in modulation and interaction with other tight junction proteins

Abstract Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths in the world. According to the International Agency for Research on Cancer data, Brazil belongs to the group of countries that presented an increase in both incidence and mortality average of the CRC in the last decade. The loss of the apical-basal polarity can lead to cancer progression, since neoplastic cells shows deficiency in the apical junctional complexes (AJC), exhibiting a malign potential. Tight junction (TJ) is one of the junctions found in the AJC. It is a structure that helps to maintain the polarity and also presents a barrier function, which regulates the transit of important molecules through the paracellular flow. Claudin protein family are the main components of the TJ and are composed of transmembrane proteins. These proteins can interact with other TJ proteins such as occludins or scaffold proteins, essentially ZO-1/2. Our group has already showed that patients with CRC overexpress claudin-3 in the tumor tissue. Our goal was to evaluate the importance of the expression and interaction among claudin-3, ZO-1, and occludin proteins during the progression of CRC. For the molecular analysis, samples were obtained from biopsy after patients were submitted to surgery at the Brazilian National Cancer Institute. Adenocarcinoma tissue and normal adjacent region were obtained through colectomy and classified by the TNM staging of tumor. The Immunoblotting essay was performed to quantify the expression of claudin-3 and occludin proteins in those patients in different stages of the disease. Moreover, the interaction between these proteins was identified through immunoprecipitation essay. In vitro assay was performed using transfected HT-29 human adenocarcinoma cells overexpressing claudin-3. In these cells we evaluated the expression levels of claudin-3 and ZO-1, and we also analyzed the subcellular distribution of claudin-3, ZO-1, and occludin by immunofluorescence. This study is being carried out with approval of the INCA Research Ethics Committee (Protocol 84/04). The analysis by immunoblotting of claudin-3 expression showed that the patients can be segregated in two groups: one where the level of this protein in the tumor is low and other group where its levels are high. No alteration in the levels of occludin was observed when comparing the tumor and the normal adjacent tissue. However, the patients in whom the tumor presented high levels of claudin-3 showed less interaction with occludin. In vitro assay using transfected HT-29 cells that overexpress claudin-3 allowed us to observe that there was a reduction in the protein levels of ZO-1 as well as a reduced colocalization between these in the regions of cell-cell contact. Associated with these results, we also observed claudin-3 and occludin colocalization in an intermittent fashion in the regions of cell-cell contact, being more evident in the region of claudin-3 accumulation in HT-29 cells. Altogether, our data suggest that in CRC there are groups of patients that show elevated level of claudin-3 in the tumor and other group that shows low level. Further, it was observed that the increase of claudin-3 expression can be related to the decrease of the ZO-1 expression and a decrease in the interaction between claudin-3 and both, occludin and ZO-1. Our results can help to improve the understanding of the molecular mechanisms involved in the colorectal tumorigenesis, and point out potential prognostic markers aiming a therapeutic design to specific targets. Financial support: FAPERJ, CAPES and MS. Citation Format: Waldemir Fernandes de Souza, Perôny da Silva Nogueira, Maria Teresa dos Santos Guedes, Bruno Kaufmann Robbs, Joao Paulo de Biaso Viola, José Andrés Morgado-Díaz. Differential expression of claudin-3 during colorectal tumorigenesis and its role in modulation and interaction with other tight junction proteins [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A80.

Abstract 843: CLDN1 mediates tumor invasion in HNSCC by regulation of EMT through AMPK-TGF-β signalling

Abstract Background: Claudin-1 (CLDN1), a major component of tight junction complexes in epithelium, maintains cellular polarity and plays critical roles in cell-to-cell communication and epithelial cell homeostasis. Although, the role of CLDN1 in cancer has been generally studied in cancers, the exact regulatory mechanisms and downstream pathways in cancer have been controversial and are not completely understood. Materials and Methods: After, analysed mutational profiling of tumor/non-tumor paired tissue in the patients with head and neck squamous cell carcinoma (HNSCC) by next-generation sequencing (NGS), CLDN1 expression levels were measured in human HNSCC cell lines and tissues from HNSCC patients using next-generation sequencing. For functional in vitro analysis, cell proliferation testing using a WST-1 assay, an invasion assay, and Western blotting was performed after CLDN1 expression regulation. In vivo validations were performed using nude mouse and patient-derived tumor xenograft (PDTX) model. Results: Mutational profiling of tumor/non-tumor paired HNSCC tissue by NGS revealed that HNSCC tumor tissues and cell lines had relatively high levels of CLDN1 expression. Although, CLDN1 regulation did not affect proliferation of HNSCC cell lines, knock down of CLDN1 inhibited invasive characteristics (migration/invasion) with down-regulation of EMT associated proteins (vimentin and slug). We also found that CLDN1 negatively correlated with AMPK phosphorylation and AMPK activator (AICAR) showed the same effect with CLDN1 suppression on HNSCC cells without change in CLDN1 expression which lead to inhibition of TGF-β-induced Smad phosphorylation and their transcriptional activity, suggesting that CLDN1/AMPK/TGF-β cascade. Then, we confirmed antitumorigenic effects of suppression of CLDN1 in HNSCC in a nude mouse and PDTX model. Conclusions: Our findings suggest that CLDN1 acts as oncogenic driver in HNSCC via regulating EMT through AMPK/TGF-β signalling and if validated in further studies, a CLDN1-targeting agent partnered with an existing anti-TGF-β modality could be a novel tactic to overcome HNSCC. Citation Format: Jae Won Chang, Seung-Nam Jung, Lihua Liua, Jin Man Kim, Myung Jin Ban, Yeon Soo Kim, Bok-Soon Lee, Yang Eun Kim, Ho Ryun Won, Chul-Ho Kim, Bon Seok Koo. CLDN1 mediates tumor invasion in HNSCC by regulation of EMT through AMPK-TGF-β signalling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2017-843