Effects of Red Sorghum-Derived Deoxyanthocyanidins and Their O-ß-D-Glucosides on E-Cadherin Promoter Activity in PC-3 Prostate Cancer Cells.

Although much less common than anthocyanins, 3-Deoxyanthocyanidins (3-DAs) and their glucosides can be found in cereals such as red sorghum. It is speculated that their bioavailability is higher than that of anthocyanins. Thus far, little is known regarding the therapeutic effects of 3-DAs and their O-ß-D-glucosides on cancer, including prostate cancer. Thus, we evaluated their potential to decrease cell viability, to modulate the activity of transcription factors such as NFκB, CREB, and SOX, and to regulate the expression of the gene CDH1, encoding E-Cadherin. We found that 4',7-dihydroxyflavylium chloride (P7) and the natural apigeninidin can reduce cell viability, whereas 4',7-dihydroxyflavylium chloride (P7) and 4'-hydroxy-7-O-ß-D-glucopyranosyloxyflavylium chloride (P3) increase the activities of NFkB, CREB, and SOX transcription factors, leading to the upregulation of CDH1 promoter activity in PC-3 prostate cancer cells. Thus, these compounds may contribute to the inhibition of the epithelial-to-mesenchymal transition in cancer cells and prevent the metastatic activity of more aggressive forms of androgen-resistant prostate cancer.

Low-dose cisplatin exposure and SNAIL, Vimentin, E-cadherin expression in HEPG2 cell line / Exposición a dosis bajas de cisplatino y expresión de SNAIL, Vimentina, E-cadherina en línea celular HEPG2

Cisplatin, the first platinum compound approved for cancer treatment, is widely used in the treatment of various cancers including hepatocellular carcinoma (HCC). HCC incidence rates rise globally. Epithelial mesenchymal transition (EMT) is implicated in cancer invasion and metastasis, which are associated with increased mortality. Cisplatin dose might influence cancer invasion and metastatic behavior of the cells. The aim of the study was to investigate the effect of low-dose cisplatin treatment on EMT- related changes in HepG2 cells. Following treatment with 4 µM cisplatin, HepG2 cells were evaluated morphologically. Gene expression of E-cadherin, Vimentin, Snail1 was assessed by quantitative PCR. Immunofluorescence analyses of NA-K ATPase were performed. Although the low-dose cisplatin treated cells exhibited a more stretched morphology, no statistical difference was detected in gene expression of E-cadherin, Vimentin, Snail1 and immunofluorescence of NA-K ATPase. Findings on low-dose cisplatin effects in HepG2 might contribute to the knowledge of antineoplastic inefficacy by further understanding the molecular mechanisms of drug action.

El cisplatino, el primer compuesto de platino aprobado para el tratamiento del cáncer, es ampliamente utilizado en el tratamiento de varios tipos de cáncer, incluido el carcinoma hepatocelular (CHC). Las tasas de incidencia de CHC aumentan a nivel mundial. La transición mesenquimal epitelial (EMT) está implicada en la invasión del cáncer y la metástasis, que se asocian con un aumento de la mortalidad. La dosis de cisplatino podría influir en la invasión del cáncer y el comportamiento metastásico de las células. El objetivo del estudio fue investigar el efecto del tratamiento con dosis bajas de cisplatino en los cambios relacionados con la EMT en las células HepG2. Tras el tratamiento con cisplatino de 4 µM, se evaluaron morfológicamente las células HepG2. La expresión génica de E-cadherina, vimentina, caracol1 se evaluó mediante PCR cuantitativa. Se realizaron análisis de inmunofluorescencia de NA-K ATPasa . Aunque las células tratadas con cisplatino en dosis bajas exhibieron una morfología más estirada, no se detectaron diferencias estadísticas en la expresión génica de E-cadherina, vimentina, Snail1 e inmunofluorescencia de NA-K ATPasa. Los hallazgos sobre los efectos del cisplatino en dosis bajas en HepG2 podrían contribuir al conocimiento de la ineficacia antineoplásica al comprender mejor los mecanismos moleculares de la acción del fármaco.

Stachydrine inhibits TGF-ß1-induced epithelial-mesenchymal transition in hepatocellular carcinoma cells through the TGF-ß/Smad and PI3K/Akt/mTOR signaling pathways.

Stachydrine is a bioactive alkaloid that has been found to exert tumor-suppressive potential. However, the effect of stachydrine on hepatocellular carcinoma (HCC) has not been previously investigated. In the present study, we investigated the effect of transforming growth factor-ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) in HepG2 cells. Our results showed that stachydrine significantly suppressed TGF-ß1-induced HepG2 cell migration and invasion in a dose-dependent manner. Stachydrine prevented TGF-ß1-induced EMT in HepG2 cells, as proved by the increased expression level of E-cadherin and decreased expression levels of N-cadherin and vimentin. In addition, stachydrine attenuated TGF-ß1-induced upregulation of TGF-ß receptor I (TßRI) in both protein and mRNA levels. Further mechanism investigations proved that stachydrine prevented TGF-ß1-induced activation of Smad2/3 and phosphoinositol-3-kinase (PI3K)/Akt/mTOR signaling pathways in HepG2 cells. In conclusion, these findings demonstrated that stachydrine prevented TGF-ß1-induced EMT in HCC cells through Smad2/3 and PI3K/Akt/mTOR signaling pathways. Thus, stachydrine might be a potential therapeutic agent for the treatment of HCC.

Overexpression of heat shock protein 70 inhibits epithelial-mesenchymal transition and cell migration induced by transforming growth factor-ß in A549 cells.

Heat shock protein 70 (HSP70) is a key member of the HSP family that contributes to a pre-cancerous environment; however, its role in lung cancer remains poorly understood. The present study used geranylgeranylacetone (GGA) to induce HSP70 expression, and transforming growth factor-ß (TGF-ß) was used to construct an epithelial-mesenchymal transition (EMT) model by stimulating A549 cells in vitro. Western Blot was performed to detect protein levels of NADPH oxidase 4 (NOX4) and the EMT-associated proteins E-cadherin and vimentin both before and after HSP70 expression. Cell morphological changes were observed, and the effect of HSP70 on cell migration ability was detected via the wound healing. The results demonstrated that GGA at 50 and 200 µmol/L could significantly induce HSP70 expression in A549 cells (P < 0.05). Furthermore, HSP70 induced by 200 µmol/L GGA significantly inhibited the changes of E-cadherin, vimentin, and cell morphology induced by TGF-ß (P < 0.05), while HSP70 induced by 50 µmol/L GGA did not. The results of the wound healing assay indicated that 200 µmol/L GGA significantly inhibited A549 cell migration induced by TGF-ß. Taken together, the results of the present study demonstrated that overexpression of HSP70 inhibited the TGF-ß induced EMT process and changed the cell morphology and migratory ability induced by TGF-ß in A549 cells.

Evodiamine inhibits vasculogenic mimicry in HCT116 cells by suppressing hypoxia-inducible factor 1-alpha-mediated angiogenesis.

Evodiamine (Evo), a quinazoline alkaloid and one of the most typical polycyclic heterocycles, is mainly isolated from Evodia rugulosa. Vasculogenic mimicry (VM) is a newly identified way of angiogenesis during tumor neovascularization, which is prevalent in a variety of highly invasive tumors. The purpose of this study was to investigate the effect and mechanism of Evo on VM in human colorectal cancer (CRC) cells. The number of VM structures was calculated by the three-dimensional culture of human CRC cells. Wound-healing was used to detect the migration of HCT116 cells. Gene expression was detected by reverse transcription-quantitative PCR assay. CD31/PAS staining was used to identify VM. Western blotting and immunofluorescence were used to detect protein levels. The results showed that Evo inhibited the migration of HCT116 cells, as well as the formation of VM. Furthermore, Evo reduced the expression of hypoxia-inducible factor 1-alpha (HIF-1α), VE-cadherin, VEGF, MMP2, and MMP9. In a model of subcutaneous xenotransplantation, Evo also inhibited tumor growth and VM formation. Our study demonstrates that Evo could inhibit VM in CRC cells HCT116 and reduce the expression of HIF-1α, VE-cadherin, VEGF, MMP2, and MMP9.

Modifying gap junction communication in cancer therapy.

AIM: Drug delivery is crucial for therapeutic efficacy and gap junction communication channels (GJIC) facilitate movement within the tumour. Pro-drug activation, a modality of cancer therapy leads to Ganciclovir triphosphate (GCV-TP) incorporation into newly synthesized DNA resulting in cell death. The objective was to enhance, with Histone deacetylase inhibitors (HDACi) and All Trans Retinoic Acid (ATRA), GJIC, crucial for drug delivery, and with combination, abrogate the observed detrimental effect of Dexamethasone (DXM). METHODS: Cell lines (NT8E, and HeLa) were pre-treated with Valproic Acid (VPA) (1 mM), 4 Phenyl Butyrate (4PB) (2 mM), ATRA (10 µM) and Dexamethasone (1 µM). Protein quantitated with the Bicinchoninic (BCA) assay for cell lysates, membrane and soluble fractions was assessed with Western blotting for Connexins (43, 26 and 32) and E-Cadherin. A qRT-PCR was done for CX 43-GJA1, CX 26-GJB2, CX 32-GJB1 and E-Cadherin, and normalized with Glyceraldehyde Phosphate dehydrogenase (GAPDH). Further, localization of Connexins (CX) and E-Cadherin, GJIC competence, pre-clinical in-vitro studies and the mechanism of cell death were evaluated. RESULTS: There was no toxicity or change in growth patterns observed with the drugs. In both the cell lines CX 43 localized to the membrane whereas CX 32 and CX 26 were present but not membrane bound. E-Cadherin was present on the membrane in NT8E and completely absent in HeLa cells. Effects of HDACi, DXM and ATRA were seen on the expression of Connexins and E-Cadherin in both the cell lines. NT8E and HeLa cell lines showed enhanced GJIC with 4PB [30 %], VPA [36 %] and ATRA [54 %] with a 60 % increase in cytotoxicity and an abrogation of Dexamethasone inhibition on combination with VPA or ATRA. CONCLUSION: An enhancement of GJIC function by HDACi and ATRA increased cytotoxicity and could be effective in the presence of Dexamethasone, when combined with ATRA or VPA.

Effect of Platelet-Activating Factor on Barrier Function of ARPE-19 Cells.

AIM: To examine the effects of platelet-activating factor (PAF) on the barrier functions of cultured retinal pigment epithelial (RPE) cells. METHODS: A human RPE cell line (ARPE-19) was cultured on microporous filter supports and treated with PAF and WEB 2086, a specific PAF-receptor (PAF-R) antagonist. The permeability of the RPE monolayer was measured using transepithelial electrical resistance (TER) and sodium fluorescein flux. The expression of the tight junction protein zonula occludens (ZO)-1 and the adherens junction protein N-cadherin was assessed using immunohistochemistry and Western blotting. We also measured the vascular endothelial growth factor (VEGF) concentrations in PAF-treated cultures and re-measured RPE monolayer permeability in the presence of VEGF-neutralizing antibodies. RESULTS: PAF significantly decreased the TER and enhanced the sodium fluorescein flux of the RPE monolayer and downregulated the expression of ZO-1 and N-cadherin. These effects were abolished by WEB 2086-mediated blockage of the PAF-R. PAF stimulation increased VEGF expression in RPE cells, and the antibody-mediated neutralization of VEGF caused a partial recovery of the barrier properties. CONCLUSION: The barrier functions of ARPE-19 cells were altered by PAF, and these effects were partly mediated by an upregulation of VEGF expression in these cells. Our results contribute to the growing body of evidence supporting the role of PAF in choroidal neovascularization. Our findings suggest that PAF is a novel target in the development of therapies for increased permeability of the RPE monolayer.

Blocking TG2 attenuates bleomycin-induced pulmonary fibrosis in mice through inhibiting EMT.

BACKGROUND: Epithelial-mesenchymal transformation (EMT) is a central mechanism for the occurrence and development of pulmonary fibrosis. Therefore, to identify the key target molecules regulating the EMT process is considered as an important direction for the prevention and treatment of pulmonary fibrosis. Transglutaminase 2 (TG2) has been recently found to play an important role in the regulation of inflammation and the generation of extracellular matrix. Here, our study focuses on the roles of TG2 in pulmonary fibrosis and EMT. METHODS: at first, the expression of TG2 and the EMT-related markers like E-cadherin, Vimentin, and α-SMA were detected with Western Blotting, immunohistochemistry and other methods in the mice with pulmonary fibrosis induced by bleomycin. Further, MLE 12 cells were used to study the effects on EMT of the inhibition of TG2 in vitro. Finally, GK921, an inhibitor against TG2, was used to show its function in both prevention and treatment of pulmonary fibrosis induced by bleomycin in mice. RESULTS: bleomycin succeeded to induce pulmonary fibrosis in mice, with increased TG2 expression, EMT and Akt activation. Knock-down of TG2 by siRNA technique in MLE 12 cell (a mouse alveolar epithelial cell line) and GK921 (an inhibitor of TG2) all inhibited the EMT process, however SC79, an activator of Akt rescued above inhibition. Finally, GK921 alleviated pulmonary fibrosis in mice induced by bleomycin. CONCLUSION: Blocking TG2 reduces bleomycin-induced pulmonary fibrosis in mice via inhibiting EMT.

Combination of celecoxib and calyculin-A inhibits epithelial-mesenchymal transition in human oral cancer cells.

Expression of cyclo-oxygenase-2 (COX-2) and protein phosphatase 2A (PP2A) deactivation occurs frequently in oral squamous cell carcinoma (OSCC). We initially assessed COX-2 and PP2A protein expression in OSCC specimens using immunohistochemical (IHC) staining and western blot analysis. We found strong COX-2 and phosphorylated PP2A (p-PP2A) expression in OSCC samples. No significant difference in total PP2A expression was observed between cancer and nontumor tissues. The effect of combining COX-2 inhibitor and celecoxib (CXB) with the PP2A inhibitor, calyculin-A (CLA) on the OSCC cell line, HSC3, was evaluated in vitro. We found that a combination of 1 nM CLA and 50 µM CXB significantly inhibited cell viability, and migration and invasion of HSC3 cells. Western blots for AKT, p-AKT, ERK, p-ERK, E-cadherin, vimentin and ß-catenin were conducted after treatment with CXB and/or CLA. Increased E-cadherin and decreased ß-catenin expression were found in CXB or CLA treated hsc-3 cells, whereas the combined CXB and CLA treatment showed no difference in E-cadherin or ß-catenin expression. Our findings suggest that CLA alone was more effective than CXB alone, but not in the combined drug treatment.

Rikkunshito attenuates induction of epithelial-mesenchymal switch via activation of Sirtuin1 in ovarian cancer cells.

Rikkunshito, a traditional Japanese herbal medicine, improves appetite via activation of gastrointestinal hormone ghrelin pathway. The function of ghrelin is mediated by growth hormone secretagogue receptor (GHSR1a), and ghrelin has been known to possess diverse physiological functions including growth suppression of some cancer cells. Considering that increased ghrelin signaling by Rikkunshito could enhance sirtuin1 (SIRT1) activity in nervous system, we aimed to investigate the effect of Rikkunshito in ovarian cancer cells. Ovarian cancer cell lines were treated with Rikkunshito, and cellular viability, gene expressions and epithelial-mesenchymal transition (EMT) status were investigated. To investigate the involvement of SIRT1 by Rikkunshito in SKOV3 cancer cells, endogenous expression of SIRT1 was depleted using small interfering RNA (siRNA). Treatment with Rikkunshito elevated ghrelin, GHSR1a and SIRT1, while cellular viability was decreased. The treatment of Rikkunshito also inhibited cellular migration and invasion status in a dose-dependent manner, and these effects were translated to the enhanced EMT status, although the role of SIRT1 was not determined. Our study revealed a novel function of Rikkunshito in enhancing EMT status of ovarian cancer cells. Therefore, we would like to propose that Rikkunshito may be used as a novel adjunctive therapy in chemotherapy of ovarian cancer because platinum-based chemotherapy frequently used for the treatment of ovarian cancer inevitably impairs appetite.

Combining Ellagic Acid with Temozolomide Mediates the Cadherin Switch and Angiogenesis in a Glioblastoma Model.

OBJECTIVE: We aimed to evaluate the combined effect of ellagic acid (EA) and temozolomide (TEM) on the cadherin switch and angiogenesis in the C6 glioma cell line. METHODS: A total of 100 µM EA and 100 µM TEM were applied to rat C6 glioma cells for 24, 48, and 72 hours. Cell proliferation was detected by 5-bromo-2'-deoxyuridine immunohistochemistry. The messenger RNA and protein levels of E-cadherin, N-cadherin, and vascular endothelial growth factor (VEGF) were determined by real-time polymerase chain reaction and their immunohistochemistry, respectively, subsequent to EA treatment combined with TEM. RESULTS: EA in combination with TEM conspicuously reduced the viability of C6 glioma cells at all incubation times (P < 0.001). EA upregulated the expression of E-cadherin at the gene and protein levels in a time-independent manner (P < 0.05 and P < 0.001, respectively). By the presence of TEM, the increase was exaggerated at 24-hour incubation (P < 0.01). Conversely, EA reduced N-cadherin expression and immunoreactivity in a time-independent manner (P < 0.05 and P < 0.001, respectively), and combination with TEM enhanced this effect at the 24th hour (P < 0.001). Combination also downregulated the gene expression (P < 0.001) and immunoreactivity of VEGF only at 72 hours (P < 0.001). CONCLUSIONS: A successful therapeutic efficacy of EA combined with TEM is suggested probably by inhibiting the cadherin switch and angiogenesis.

lincRNA-p21 Mediates the Anti-Cancer Effect of Ginkgo Biloba Extract EGb 761 by Stabilizing E-Cadherin Protein in Colon Cancer.

BACKGROUND Ginkgo biloba extract EGb 761 is a putative antioxidant and has been used for thousands of years to treat a variety of ailments, including cancer. While it is known that cell behavior can be modulated by long non-coding RNAs (lncRNAs), the contributions of lncRNAs in EGb 761-induced anti-cancer effects are largely unknown. MATERIAL AND METHODS Colon cancer cell lines HT29 and HCT116 were used in this study. RT-qPCR was used to detect the relative expression of lincRNA-p21 in colon cancer cells. Wound-healing assay and Matrigel Transwell assay were performed to investigate the migration and invasion of colon cancer cells. Immunoprecipitation and Western blot experiments were used to verify ubiquitination and the interaction between lincRNA-p21 and E-cadherin, or E-cadherin and b-transducin repeat containing (BTRC) E3 ubiquitin protein ligase. RESULTS Cell function assay verified that treatment with EGb 761 suppressed the migratory and invasive abilities of colon cancer cells in a dose-dependent manner via the suppression of E-cadherin expression level. lincRNA-p21 was upregulated in colon cancer cells after treatment with EGb 761, and knockdown of lincRNA-p21 reversed the EGb 761-induced anti-metastatic effect. Furthermore, lincRNA-p21 was localized in cytoplasm of colon cells and regulated E-cadherin expression at a post-transcriptional level. Specifically, lincRNA-p21 promotes E-cadherin stability by preventing the interaction between BTRC and E-cadherin, which leads to the inhibition of E-cadherin ubiquitination. CONCLUSIONS We demonstrated that lincRNA-p21 mediates the anti-cancer effect of Ginkgo biloba extract EGb 761 by stabilizing E-cadherin protein in colon cancer, which may help define the functional role of EGb 761 in cancer treatment.

Angioedema and Hemorrhage After 4.5-Hour tPA (Tissue-Type Plasminogen Activator) Thrombolysis Ameliorated by T541 via Restoring Brain Microvascular Integrity.

Background and Purpose- tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time window. Methods- Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage, cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then. An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate effect of T541 on tight junctions and F-actin in the presence of tPA. Results- tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate, whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1, occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile, ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and IV declined, whereas malondialdehyde and 8-Oxo-2'-deoxyguanosine increased and F-actin arrangement disordered. All the insults after tPA treatment were attenuated by addition of T541 dose dependently. Conclusions- The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-brain barrier is likely attributable to its effects observed.

Anti-Tumor Effects of Paeoniflorin on Epithelial-To-Mesenchymal Transition in Human Colorectal Cancer Cells.

BACKGROUND Colorectal cancer is one of the leading causes of death in China, and the development of effective drugs is urgently needed. Here, we report on Paeoniflorin (PF), a product isolated from the roots of the peony plant, as a possible candidate because of its anti-tumor effects on epithelial-to-mesenchymal transition (EMT) of PF in human colorectal cancer (CRC). MATERIAL AND METHODS Cell proliferation, wound healing, and Transwell assays were used to analyze the effects of PF on in vitro cell migration and invasion of HCT116 and SW480, 2 colorectal cancer cell lines. The tumor xenograft model was used to verify the anti-metastasis effects of PF in vivo. The RNA and protein levels of epithelia-cadherin (E-cadherin), Vimentin, and histone deacetylase2 (HDAC2) were measured by qPCR and Western blot analysis to explore the mechanism involved. RESULTS Our results showed that PF inhibited colorectal cancer cell migration and invasion and suppressed the metastatic potential of the cancer cells in vivo. Moreover, PF significantly decreased the expression of HDAC2 and Vimentin, while increasing the expression of E-cadherin. CONCLUSIONS These results suggest that PF inhibits colorectal cancer cell migration and invasion ability and reverses the EMT process, through inhibiting the expression of HDAC2, and then affects the expression level of E-cadherin and Vimentin at the cell level. Our results were also verified in the tumor xenograft model. This indicates that PF may be a candidate for colorectal cancer treatment.

Fucosyltransferase 2 induced epithelial-mesenchymal transition via TGF-ß/Smad signaling pathway in lung adenocarcinaoma.

Fucosyltransferase 2 (FUT2), the enzyme catalyzing α-1,2-fucosylation in mammals, has been implicated in cancer. The up-regulation of FUT2 has been observed in lung adenocarcinoma (LUAD), and FUT2 can enhance the cell migration and invasion of LUAD cell lines. However, the underlying mechanism of FUT2 in LUAD remains largely unknown. Abundant studies have revealed that epithelial-mesenchymal transition (EMT) played a pivotal role during lung cancer metastasis and progression. In the present study, we showed that knocking down FUT2 in LUAD cell lines increased the expression of E-cadherin and reduced the expression of Vimentin, N-cadherin, TßRII, p-Smad2, p-Smad3 and Snail, which were the makers of EMT. Meanwhile, the expression of E-cadherin was decreased, and the expression of Vimentin was increased by restoring the expression of FUT2 in RNA interference FUT2 (RNAi-FUT2) cells, suggesting that FUT2 enhanced the EMT process in LUAD. Additionally, silencing FUT2 expression can up-regulate E-cadherin and down-regulate Vimentin, significantly attenuated EMT in vivo. Treated with the SIS3, a new-type inhibitor of p-Smad3 of TGF-ß signaling, the expression of E-cadherin, Vimentin and Snail were not affected by RNAi-FUT2 cells, indicating that the effect of FUT2 on EMT depended on TGF-ß/Smad signaling. Overall, the current results indicated that FUT2 might promote LUAD metastasis through the EMT initiated by TGF-ß/Smad signaling. Therefore, FUT2 might be a prognostic factor and therapeutic target for LUAD.

Saquinavir plus methylprednisolone ameliorates experimental acute lung injury

Glucocorticoid insensitivity is an important barrier to the treatment of several inflammatory diseases, including acute lung injury (ALI). Saquinavir (SQV) is an inhibitor of the human immunodeficiency virus protease, and the therapeutic effects of SQV in ALI accompanied with glucocorticoid insensitivity have not been previously investigated. In this study, the effects of SQV on lipopolysaccharide (LPS)-mediated injury in human pulmonary microvascular endothelial cells (HPMECs), human type I alveolar epithelial cells (AT I), and alveolar macrophages were determined. In addition, the effects of SQV on an LPS-induced ALI model with or without methylprednisolone (MPS) were studied. In LPS-stimulated HPMECs, SQV treatment resulted in a decrease of high mobility group box 1 (HMGB1), phospho-NF-κB (p-NF-κB), and toll-like receptor 4 (TLR4), and an increase of VE-cadherin. Compared to MPS alone, MPS plus SQV attenuated the decrease of glucocorticoid receptor alpha (GRα) and IκBα in LPS-stimulated HPMECs. HMGB1, TLR4, and p-NF-κB expression were also lessened in LPS-stimulated alveolar macrophages with SQV treatment. In addition, SQV reduced the injury in human AT I with a decrease of HMGB1 and p-NF-κB, and with an increase of aquaporin 5 (AQP 5). SQV ameliorated the lung injury caused by LPS in rats with reductions in vascular permeability, myeloperoxidase activity (MPO) and histopathological scores, and with lowered HMGB1, TLR4, and p-NF-κB expression, but with enhanced VE-cadherin expression. By comparison, SQV plus MPS increased GRα and IκBα in lung tissues of rats with ALI. This study demonstrated that SQV prevented experimental ALI and improved glucocorticoid insensitivity by modulating the HMGB1/TLR4 pathway.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

Inhibition of cervical cancer cell metastasis by benzothiazole through up-regulation of E-cadherin expression.

The present study was aimed to investigate the effect of benzothiazole on the invasive and metastasis potential of HeLa DH cervical cancer cells and the underlying mechanism. HeLa DH cervical cells were cultured with 5, 10, 15, 20, 25 and 30 µM concentrations of benzothiazole for 48 h. Benzothiazole treatment did not induce any cytotoxic effect on HeLa DH cells after 48 h of incubation. The results from wound healing assay revealed that migration potential of HeLa DH cells was reduced to 4% on treatment with 20 µM concentration of benzithiazole compared to 99% in the control cells. The invasion potential of HeLa DH cells was reduced to 13% on treatment with 20 µM concentration of benzithiazole. Inhibition of HeLa DH cell migration was also significantly (p < 0.002) higher in the benzithiazole treated cell cultures compared to the control cells. HeLa DH cervical cancer cells on treatment with various concentrations of benzithiazole for 48 h showed a significant (p < 0.05) increase in the expression of E-cadherin in a dose dependent manner. Among the various concentrations of benzithiazole used, western blot assay revealed that the increase in E-cadherin was maximum at 20 µM. Analysis of the levels of mRNA corresponding to E-cadherin by RT-qPCR showed significant increase in HeLa DH cervical cancer cells on treatment with 20 µM concentration of benzithiazole. Thus benzithiazole treatment suppresses the invasive and metastasis potential of HeLa DH cervical cells through upregulation of E-cadherin expression. Therefore, benzithiazole has a potential to be used for the treatment of cervical cancer.

FOXA1 Induces E-Cadherin Expression at the Protein Level via Suppression of Slug in Epithelial Breast Cancer Cells.

Epithelial-to-mesenchymal transition (EMT) is an important process during embryonic development and tumor progression by which adherent epithelial cells acquire mesenchymal properties. Forkhead box protein A1 (FOXA1) is a transcriptional regulator preferentially expressed in epithelial breast cancer cells, and its expression is lost in mesenchymal breast cancer cells. However, the implication of this biased expression of FOXA1 in breast cancer is not fully understood. In this study, we analyzed the involvement of FOXA1 in EMT progression in breast cancer, and found that stable expression of FOXA1 in the mesenchymal breast cancer MDA-MB-231 cells strongly induced the epithelial marker E-cadherin at the mRNA and protein levels. Furthermore, stable expression of FOXA1 was found to reduce the mRNA and protein expression of Slug, a repressor of E-cadherin expression. FOXA1 knockdown in the epithelial breast cancer MCF7 cells reduced E-cadherin protein expression without decreasing its mRNA expression. In addition, FOXA1 knockdown in MCF7 cells up-regulated Slug mRNA and protein expression. Notably, similar to FOXA1 knockdown, stable expression of Slug in MCF7 cells reduced E-cadherin protein expression without decreasing its mRNA expression. Taken together, these results suggest that although FOXA1 can induce E-cadherin mRNA expression, it preferentially promotes E-cadherin expression at the protein level by suppressing Slug expression in epithelial breast cancer, and that the balance of this FOXA1-Slug axis regulates EMT progression.

E-cadherin Mediates the Preventive Effect of Vitamin D3 in Colitis-associated Carcinogenesis.

Vitamin D3 is beneficial in ameliorating or preventing inflammation and carcinogenesis. Here, we evaluated if vitamin D3 has a preventive effect on colitis-associated carcinogenesis. Administration of azoxymethane (AOM), followed with dextran sulfate sodium (DSS), was used to simulate colitis-associated colon cancer in mice. The supplement of vitamin D3 at different dosages (15, 30, 60 IU·g·w), started before AOM or immediately after DSS treatment (post 60), was sustained to the end of the experiment. Dietary vitamin D3 significantly reduced the number of tumors and tumor burden in a dose-dependent manner. Of note, vitamin D3 in high doses showed significant preventive effects on carcinogenesis regardless of administration before or after AOM-DSS treatment. Cell proliferation decreased in vitamin D3 groups compared with the control group after inhibition of expression of ß-catenin and its downstream target gene cyclin D1 in the colon. In vitro, vitamin D3 reduced the transcriptional activity and nuclear level of ß-catenin, and it also increased E-cadherin expression and its binding affinity for ß-catenin. Moreover, repression of E-cadherin was rescued by supplemental vitamin D3 in mouse colons. Taken together, our results indicate that vitamin D3 effectively suppressed colonic carcinogenesis in the AOM-DSS mouse model. Our findings further suggest that upregulation of E-cadherin contributes to the preventive effect of vitamin D3 on ß-catenin activity.