67-kDa Laminin Receptor Research Articles

APP Maturation and Intracellular Localization Are Controlled by a Specific Inhibitor of 37/67 kDa Laminin-1 Receptor in Neuronal Cells.

Amyloid precursor protein (APP) is processed along both the nonamyloidogenic pathway preventing amyloid beta peptide (Aβ) production and the amyloidogenic pathway, generating Aβ, whose accumulation characterizes Alzheimer’s disease. Items of evidence report that the intracellular trafficking plays a key role in the generation of Aβ and that the 37/67 kDa LR (laminin receptor), acting as a receptor for Aβ, may mediate Aβ-pathogenicity. Moreover, findings indicating interaction between the receptor and the key enzymes involved in the amyloidogenic pathway suggest a strong link between 37/67 kDa LR and APP processing. We show herein that the specific 37/67 kDa LR inhibitor, NSC48478, is able to reversibly affect the maturation of APP in a pH-dependent manner, resulting in the partial accumulation of the immature APP isoforms (unglycosylated/acetylated forms) in the endoplasmic reticulum (ER) and in transferrin-positive recycling endosomes, indicating alteration of the APP intracellular trafficking. These effects reveal NSC48478 inhibitor as a novel small molecule to be tested in disease conditions, mediated by the 37/67 kDa LR and accompanied by inactivation of ERK1/2 (extracellular signal-regulated kinases) signalling and activation of Akt (serine/threonine protein kinase) with consequent inhibition of GSK3β.

A77 FURTHER CARACTERIZATION OF THE 67 KDA LAMININ RECEPTOR (67LR) IN COLORECTAL CANCER CELLS

Abstract Background The 67 kDa laminin receptor (67LR) was the first non-integrin cell surface receptor for laminin isolated on laminin affinity columns from cancer cells in the 1980’s. Initially, 67LR is found as a cytoplasmic precursor of a 37 kDa protein, named 37LR, associated with the small ribosomal subunit. In human cells, 37LR allows the formation of the polyribosome complex and plays a key role in the initiation of translation. The mechanism by which the ribosomal protein becomes the 67LR membrane receptor is still unclear. It is presumed that the process involves post-translational modifications combined with homo or hetero-dimerization with non-associated ribosomal proteins. It has been shown that 37/67LR regulates adhesion and proliferation of normal human intestinal epithelial cells. Interestingly, overexpression of 37/67LR is correlated with aggressiveness and a poor prognosis in a wide variety of cancers. Aims The aim of this study was to confirm the overexpression of 37/67LR at the membrane of colorectal cancer cells and to identify its homo or heterodimerization partners. Methods To detect the expression of 37/67LR in colorectal cancer we performed an indirect immunofluorescence on tissues from normal and diseased colons. To confirm the presence of 67LR at the membrane of Caco-2 cells we used a cellular fractionation extraction protocol combined with ultracentrifugation and detergent treatment to separate ribosome-containing fractions from the membranes and isolate the membrane associated 67LR. Mass spectrometry analysis to study the molecular identity of 67LR was performed on immunoreactive bands corresponding to 37LR and 67LR. Results Immunolocalization of 37/67LR revealed an overexpression in colorectal cancer tissues. Following analysis by western blotting, immunoreactive 67LR protein was found in the soluble fraction after ultracentrifugation at 210,000 x g while 37LR was detected in the insoluble counterpart which was solubilized after treatment with detergent, suggesting that 37LR is associated with the membrane. Mass spectrometry analysis of these fractions indicated that 37LR was not identified in the immunoreactive bands of 67LR in the soluble fraction but identified the 67 kDa elastin binding protein, another 67 kDa cell surface laminin receptor. Conclusions These results indicate that 37LR is overexpressed in colorectal adenocarcinoma cells. Further characterization of the receptor by cell fractionation and mass spectrometry indicated that the 67 kDa immunoreactive form is not related to 37LR. Supported by CIHR. Funding Agencies CIHR

Tea polyphenols and their chemopreventive and therapeutic effects on colorectal cancer.

Colorectal cancer (CRC), a multifactorial disease, is usually induced and developed through complex mechanisms, including impact of diet and lifestyle, genomic abnormalities, change of signaling pathways, inflammatory response, oxidation stress, dysbiosis, and so on. As natural polyphenolic phytochemicals that exist primarily in tea, tea polyphenols (TPs) have been shown to have many clinical applications, especially as anticancer agents. Most animal studies and epidemiological studies have demonstrated that TPs can prevent and treat CRC. TPs can inhibit the growth and metastasis of CRC by exerting the anti-inflammatory, anti-oxidative or pro-oxidative, and pro-apoptotic effects, which are achieved by modulations at multiple levels. Many experiments have demonstrated that TPs can modulate several signaling pathways in cancer cells, including the mitogen-activated protein kinase pathway, phosphatidylinositol-3 kinase/Akt pathway, Wnt/β-catenin pathway, and 67 kDa laminin receptor pathway, to inhibit proliferation and promote cell apoptosis. In addition, novel studies have also suggested that TPs can prevent the growth and metastasis of CRC by modulating the composition of gut microbiota to improve immune system and decrease inflammatory responses. Molecular pathological epidemiology, a novel multidisciplinary investigation, has made great progress on CRC, and the further molecular pathological epidemiology research should be developed in the field of TPs and CRC. This review summarizes the existing in vitro and in vivo animal and human studies and potential mechanisms to examine the effects of tea polyphenols on CRC.

Epigallocatechin Gallate Induces Upregulation of LDL Receptor via the 67kDa Laminin Receptor-Independent Pathway in HepG2 Cells.

Epigallocatechin gallate (EGCG), an active polyphenol in green tea, exhibits various physiological effects, including activation of low-density lipoprotein receptors (LDLR). The previous studies have suggested that EGCG activates LDLR via extracellular signal-regulated kinase (ERK) pathway in HepG2 cells. However, the detailed molecular mechanism remains unclear. Recently, 67kDa laminin receptor (67LR) is identified as a receptor for EGCG. Therefore, this study aims to determine whether 67LR is involved in the mechanism of LDLR activation by EGCG. EGCG induces upregulation of LDLR when 67LR is knocked down in HepG2 cells. Similar effect is observed after the cells are treated with 67LR monoclonal antibody. The loss of antiallergic effect following 67LR siRNA knockdown and 67LR antibody treatment confirms the results since the antiallergic effect of EGCG is known to be mediated by 67LR. EGCG activates LDLR expression via 67LR-independent pathway in HepG2 cells.

Improvement of Cell Penetrating Peptide for Efficient siRNA Targeting of Tumor Xenografts in Zebrafish Embryos

AbstractCell‐penetrating peptides (CPPs) are one of the most efficient vectors for achieving cellular uptake of different cargos. However, their application in cancer therapy is greatly limited due to the lack of tissue selectivity, as they are widely distributed in most tissues following in vivo administration. To introduce specificity and improve tumor targeting, a combination of a cell‐penetrating peptide (CADY) and a targeting ligand, the laminin receptor binding peptide (YIGSR) to target the 37/67 kDa laminin receptor, known to be upregulated in most cancer cells and to be correlated with the invasiveness and metastatic potential, is formulated. The targeting CPP named “Y‐CADY” does not induce any change in the physico‐chemical properties (secondary structure, small interference RNA (siRNA) complexation, size, and charge) of CADY. Interestingly, Y‐CADY is more efficient than CADY for gene silencing in cell lines, and more potent for targeting and for inducing apoptosis of tumor xenografts in zebrafish embryos. The present study demonstrates that the novel targeting CPP “Y‐CADY” is efficient for gene silencing and can serve as a vector to target cancer cells in vivo.

Transduction pattern of adeno‐associated viruses in the trabecular meshwork and anterior‐segment structures in a rat model of ocular hypertension

AbstractPurposeTo investigate transduction patterns of self‐complimentary adeno‐associated virus (scAAV) serotype 2, 5, and 8 in trabecular meshwork (TM) and anterior segment structures of ocular hypertensive (OHT) rat models.MethodsWe assessed transduction efficiencies of scAAV 2, 5, and 8 in the anterior‐segment structures of the eyes of SD rats with OHT by circumlimbal suturing, followed by intracameral injection of scAAV2, 5, or 8 packaged with EGFP, and compared with the control. Expression level of receptors for scAAV2 (perlecan and syndecan), scAAV5 (2, 3‐linked sialic acid), and scAAV8 (67kDa laminin receptor) was analyzed by using immunohistochemistry and quantitative PCR.ResultsThe transduction of scAAV2 and 5 in the TM of OHT rats was markedly enhanced after 1 month, and transduction of scAAV5 was more efficient than that of scAAV2; transduction of scAAV8 into the TM did not occur. The transduction of scAAV2, 5, and 8 was enhanced in the ciliary body, iris, and corneal endothelium of the OHT eyes for 3 months. The expression levels of receptors for scAAV2 and scAAV5 were significantly increased in the OHT compared with control eyes.ConclusionsThe results showed that scAAV 2 and 5 effectively target the ciliary body and TM in OHT eyes, and OHT related changes in anterior segment structures enhance scAAV transduction.

The Regional Specific Alterations in BBB Permeability are Relevant to the Differential Responses of 67-kDa LR Expression in Endothelial Cells and Astrocytes Following Status Epilepticus.

Status epilepticus (a prolonged seizure activity, SE) differently affects vasogenic edema formation and dystrophin-aquaporin 4 (AQP4) expressions between the rat hippocampus and the piriform cortex (PC). In the present study, we explored whether the 67-kDa laminin receptor (LR) expression was relevant to the regional specific susceptibility of vasogenic edema at 3 days after SE. In spite of no difference in expression levels of 67-kDa LR, dystrophin, and AQP4 under physiological conditions, SE-induced serum extravasation was more severe in the PC than the hippocampus. Western blots demonstrated that SE reduced expression levels of 67-kDa LR, dystrophin, and AQP4 in the PC, but not in the hippocampus proper. Immunofluorescent studies revealed that SE increased 67-kDa LR expression in reactive CA1 astrocyte, but reduced it in the PC and the molecular layer of the dentate gyrus due to massive astroglial loss. Furthermore, SE decreased expressions of endothelial 67-kDa LR and SMI-71 (endothelial brain barrier antigen) in these regions. The 67-kDa LR neutralization evoked serum extravasation in these regions of normal animals without astroglial loss. Similar to SE, 67-kDa LR neutralization also reduced dystrophin-AQP4 expressions in the PC more than the total hippocampus. Furthermore, 67-kDa LR IgG infusion increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not c-Jun N-terminal kinase, independent of phosphoprotein enriched in astrocytes of 15 kDa (PEA15) activity. Co-treatment of U0126 (an ERK1/2 inhibitor) alleviated vasogenic edema formation and the reduced dystrophin-AQP4 expressions induced by 67-kDa LR neutralization. The 67-kDa LR IgG infusion also increased the susceptibility to SE induction. Therefore, our findings suggested that the cellular specific alterations in 67-kDa LR expression might be involved in the severity of SE-induced vasogenic edema formation in regional specific manners, which might affect the susceptibility to SE induction.

Peptide‐Modified Dendrimer Nanoparticles for Targeted Therapy of Colorectal Cancer

AbstractPeptides have recently emerged as a promising class of targeting ligands for specific drug delivery in cancer treatment, which avoid undesirable side effects of the systemic administration of chemotherapeutics. Their conjugation with nanoparticles has been demonstrated to improve the functionality of peptides resulting in a versatile platform for biomedical applications. In this work, the development of carboxymethylchitosan/poly(amidoamine) (CMCht/PAMAM) dendrimer nanoparticles functionalized with YIGSR laminin receptor binding peptide for the active targeting and specific delivery of therapeutic agents into colorectal cancer cells is described. The successful functionalization is confirmed by several physico‐chemical characterization techniques. The selectivity of the YIGSR‐CMCht/PAMAM dendrimer nanoparticles is first validated in vitro using a micropatterned array of 67 kDa laminin receptor. Next, the specificity of YIGSR‐CMCht/PAMAM dendrimers nanoparticles toward laminin receptor is further confirmed both in 2D and 3D settings using HCT‐116 colorectal cancer cells and L929 fibroblasts in co‐culture. Finally, gemcitabine‐loaded YIGSR‐CMCht/PAMAM dendrimer nanoparticles induce a targeted mortality on HCT‐116 cancer cells in a co‐culture scenario. Overall, the study shows solid evidence that YIGSR laminin receptor binding peptide coupled to CMCht/PAMAM dendrimer nanoparticles may be employed as an anticancerous target for the specific and intracellular delivery of chemotherapeutic agents.

LRP::FLAG Rescues Cells from Amyloid-β-Mediated Cytotoxicity Through Increased TERT Levels and Telomerase Activity

Alzheimer's disease (AD) represents the most common form of neurodegenerative disorders with only palliative treatments currently available. Amyloid plaque formation caused by amyloid-β (Aβ) aggregation and neurofibrillary tangle formation caused by hyperphosphorylated tau are hallmarks for the development of AD. The 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in AD and tools blocking or downregulating LRP/LR impede amyloid plaque formation in vitro and in vivo. We have recently shown that LRP::FLAG enhances telomerase activity with a concomitant reduction of senescent markers. Here, we overexpressed LRP::FLAG in HEK293 and SH-SY5Y cells, which resulted in an increase in hTERT levels as well as increased telomerase activity and increased cell viability in the presence of cytotoxic levels of exogenous Aβ. LRP::FLAG overexpression decreased Aβ shedding and intracellular Aβ levels in HEK293 cells. This suggests that LRP::FLAG rescues cells from Aβ-induced cytotoxicity through increased telomerase activity. This study recommends LRP::FLAG as a novel alternative therapeutic for AD treatment through activation of telomerase activity.

Dysfunction of 67-kDa Laminin Receptor Disrupts BBB Integrity via Impaired Dystrophin/AQP4 Complex and p38 MAPK/VEGF Activation Following Status Epilepticus.

Status epilepticus (SE, a prolonged seizure activity) impairs brain-blood barrier (BBB) integrity, which results in secondary complications following SE. The non-integrin 67-kDa laminin receptor (67-kDa LR) plays a role in cell adherence to laminin (a major glycoprotein component in basement membrane), and participates laminin-mediated signaling pathways including p38 mitogen-activated protein kinase (p38 MAPK). Thus, we investigated the role of 67-kDa LR in SE-induced vasogenic edema formation in the rat piriform cortex (PC). SE diminished 67-kDa LR expression, but increased laminin expression, in endothelial cells accompanied by the reduced SMI-71 (a rat BBB barrier marker) expression. Astroglial 67-kDa LR expression was also reduced in the PC due to massive astroglial loss. 67-kDa LR neutralization led to serum extravasation in the PC concomitant with the reduced SMI-71 expression. 67-kDa LR neutralization also decreased expressions of dystrophin and aquaporin-4 (AQP4). In addition, it increased p38 MAPK phosphorylation and expressions of vascular endothelial growth factor (VEGF), laminin and endothelial nitric oxide synthase (eNOS), which were abrogated by SB202190, a p38 MAPK inhibitor. Therefore, our findings indicate that 67-kDa LR dysfunction may disrupt dystrophin-AQP4 complex, which would evoke vasogenic edema formation and subsequent laminin over-expression via activating p38 MAPK/VEGF axis.

Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models.

Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.

Cell signaling associated with internalization of 67 kDa laminin receptor (67LR) by soluble laminin and its implication for protection against neurodegenerative diseases.

Cell signaling associated with internalization of 67 kDa laminin receptor (67LR) by soluble laminin and its implication for protection against neurodegenerative diseases.

Lysines residing in putative Small Ubiquitin-like MOdifier (SUMO) motifs regulate fate and function of 37 KDa laminin receptor

There is a putative precursor to mature receptor relationship between 37 Laminin Receptor (LR) and 67 LR. As such, the pair are frequently referred to as a single entity, the 37/67 kDa Laminin Receptor (37/67 LR) and 67 LR was identified as a laminin binding entity. 37/67 LR has been of clinical interest for many years, as 37/67 LR is a prognostic indicator for many cancers including breast, lung, colon, and prostate. However, the genesis of 67 LR is controversial, and confounded by its stability under SDS-PAGE conditions, a lack of splice variants, and the existence of post-translational modifications that cannot account for the mass discrepancy between 37 and 67 LR. In the present work, we mutated potential SUMO motif sites (Lysine residues) in 37 LR and generated a series of 37 LR-expressing plasmids with a C-terminal histidine tag. We report an inability to detect 67 LR formation, suggesting that SUMOylation does not appear to directly occur at the lysine residues proposed. However, the work revealed that these lysine mutations still appear to be important and can impact the fate and function of 37 LR, by impairing half-life and steady state pre-mRNA levels. These results suggest that the Lys residues within putative SUMO motifs of 37 LR are important for 37 LR function.

Abstract 2678: Antitumor effect against 67 kDa laminin receptor expressed on high-grade tumor cells

Abstract [Purpose] In chemotherapy for cancer, liposomes as drug delivery system (DDS) carriers are expected to have a useful effect. The aim of this study was, therefore to evaluate the antitumor effects of, and elucidate the mechanisms underlying, (-)-epigallocatechin-3-O-gallate (EGCG) and polyethyleneglycol (PEG)-modified liposomes. EGCG functions as a target ligand of the 67 kDa laminin receptor (67LR), which is expressed on high-grade tumor cells. An EGCG derivative was synthesized for binding to the end site of PEG structure. In this study, the antitumor effects of EGCG-modified liposomes as active-targeting liposome were evaluated. [Methods] Liposome was loaded with doxorubicin (DOX) as an antitumor agent. C57BL/6 mice were subcutaneously inoculated with B16F10 mouse melanoma cells (5x105 cells/animal). Each sample was administered intravenously with 2.5 mg/kg DOX. EGCG solution (EGCG sol.) was also administered intravenously at 11.3 mg/kg, based on the amount of EGCG modification in DOX-loaded EGCG-PEG-modified liposomes (EPL). Caspase-3 was evaluated as 7-amino-4-trifluoromethyl coumarin (Ex: 400 nm, Em: 505 nm), following a reaction with the fluorescent substrate DEVD-AFC. Caspase-8 was determined reacted with DEVD-pNA and detected as chromophore p-nitroanilin (λ: 400nm). [Results and Discussion] EPL significantly decreased tumor size against B16F10 mouse melanoma cells, in mice bearing 67LR-high-expression tumors. The tumor weight of the EPL group was significantly lower than that of the control (p<0.01). Typical passive-targeting liposomes (PL) accumulated into the tumors due to improved blood circulation and an enhanced permeability and retention (EPR) effect, and decreased tumor weight. On the other hand, EPL disappeared from the blood immediately, compared with PL or PL + EGCG sol. DOX concentration of liver and spleen in EPL group did not increase, indicating that the rapid disappearance of EPL from the blood was not due to reticuloendothelial system (RES) trapping. Caspase-3 activity, which indicates apoptosis induction, was also elevated only in the EPL group, and the caspase-3 activity of the PL + EGCG sol. group was the same as that of the PL group. Hence, it was important that EGCG was included to modify the liposomes via PEG, as soluble EGCG did not accumulate at a high enough concentration to exert an apoptotic effect. Moreover, EPL significantly increased caspase-8 activity. It was suggested that EPL-induced apoptosis occurred due to caspase-8 activity induced following the binding of EGCG to 67LR as a cell-death ligand. In conclusion, it was suggested that EPL was a superior antitumor agent in high 67LR-expressing tumor cells as the liposomes had dual effects, namely antitumor effects due to the loaded DOX and apoptosis induced by the bound EGCG. Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. Antitumor effect against 67 kDa laminin receptor expressed on high-grade tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2678.

LRP/LR specific antibody IgG1-iS18 impedes neurodegeneration in Alzheimer's disease mice.

Alzheimer’s disease (AD) is a neurodegenerative disease caused by accumulation of amyloid beta (Aβ) plaque and neurofibrillary tangle formation. We have shown in vitro, that knock-down and blockade of the 37 kDa/67 kDa Laminin Receptor (LRP/LR) resulted in reduced Aβ induced cytotoxicity and Aβ accumulation. In order to test the effect of blocking LRP/LR on Aβ formation and AD associated symptoms, AD transgenic mice received the anti-LRP/LR specific antibody, IgG1-iS18 through intranasal administration. We show that this treatment resulted in an improvement in memory, and decreased Aβ plaque formation. Moreover, a significant decrease in Aβ42 protein expression with a concomitant increase in amyloid precursor protein (APP) and telomerase reverse transcriptase (mTERT) levels was observed. These data recommend IgG1-iS18 as a potentially powerful therapeutic antibody for AD treatment.

Coptisine Induces Apoptosis in Human Hepatoma Cells Through Activating 67-kDa Laminin Receptor/cGMP Signaling.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. Hence, new anti-liver cancer treatment strategies need to be urgently developed. Coptisine is a natural alkaloid extracted from rhizoma coptidis which exhibits anticancer activity in various preclinical models, including liver cancer. However, the molecular mechanisms underlying the anti-liver cancer effects of coptisine remains unclear. We used flow cytometry to assess the binding of coptisine to 67LR expressed on the surface of SMMC7721, HepG2, LO2 and H9 cells. Then SMMC7721, HepG2 and BEL7402 cells, belonging to the HCC cell lines, were treated with coptisine. The cell viability was detected using a cell counting kit-8 assay. Apoptosis was evaluated using flow cytometry and transferase-mediated dUTP nick-end labeling (TUNEL) assay. Apoptotic-related proteins and tumor death receptor 67-kDa laminin receptor (67LR) were detected using Western blot analysis. The cyclic guanosine 3′,5′-monophosphate (cGMP) concentration was determined using enzyme-linked immunosorbent assay. sh67LR lentivirus, anti67LR antibody, and cGMP inhibitor NS2028 were used to determine how a 67LR/cGMP signaling pathway regulated coptisine-induced apoptosis. Tumor growth inhibited by coptisine was confirmed in a SMMC7721 cell xenograft mouse model. Coptisine selectively exhibited cell viability in human hepatoma cells but not in normal human hepatocyte cell line LO2 cells. Coptisine promoted SMMC7721 and HepG2 cell apoptosis by increasing 67LR activity. Both 67LR antibody and sh67LR treatment blocked coptisine-induced apoptosis and inhibition of cell viability. Coptisine upregulated the expression of cGMP. Moreover, cGMP inhibitor NS2028 significantly decreased coptisine-induced apoptosis and inhibition of cell viability. In vivo experiments confirmed that coptisine could significantly suppress the tumor growth and induce apoptosis in SMMC7721 xenografts through a 67LR/cGMP pathway. Coptisine-mediated 67LR activation may be a new therapeutic strategy for treating hepatic malignancy.

SiRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases

The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and −9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway.Implications: siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis.

Dual-effect liposomes with increased antitumor effects against 67-kDa laminin receptor-overexpressing tumor cells

This study sought to evaluate the antitumor effects of and elucidate the mechanisms underlying (−)-epigallocatechin-3-O-gallate (EGCG) and polyethyleneglycol (PEG)-modified liposomes. EGCG functions as a target ligand of the 67-kDa laminin receptor (67LR), which is expressed on high-grade tumor cells. An EGCG derivative was synthesized for binding to the end of PEG. Doxorubicin (DOX)-loaded EGCG-PEG-modified liposome (EPL) significantly decreased tumor size in mice bearing high 67LR-high-expressing tumors. Caspase-3 activity, which indicates induction of apoptosis, was also elevated only in the EPL group. The importance of PEG for the antitumor effects of EGCG was noted, as soluble EGCG did not accumulate at a sufficient concentration to exert an apoptotic effect. Moreover, EPL significantly increased caspase-8 activity, suggesting that EPL-induced apoptosis occurred due to caspase-8 activity induced following the binding of EGCG to 67LR as a cell-death ligand. In conclusion, EPL appear to have superior antitumor activity against high 67LR-expressing tumor cells, as the liposomes had dual effects, namely antitumor effects due to the loaded DOX and apoptosis induced by the bound EGCG.

Epigallocatechin-3-Gallate Regulates Anti-Inflammatory Action Through 67-kDa Laminin Receptor-Mediated Tollip Signaling Induction in Lipopolysaccharide-Stimulated Human Intestinal Epithelial Cells

Background/Aims: Inflammatory bowel disease (IBD) is a condition that involves chronic inflammation in all or part of the digestive tract. Often painful and debilitating, IBD can lead to life-threatening complications and increase the risk for colon cancer. In this study, we investigated the epigallocatechin-3-gallate (EGCG) mediated anti-inflammation response in lipopolysaccharide (LPS)-stimulated human colorectal cells through the negative regulator of Toll-like receptor (TLR) signaling. Methods: human intestinal epithelial cells (HT-29) were used in all experiments. Cell cytotoxicity and nitric oxide (NO) were evaluated by WST-1 and the Griess reagent. Western blot analysis and ELISA were used to determine inflammatory mediators and 67-kDa laminin receptor (67LR)-mediated Tollip signaling pathways. Results: Treatment of EGCG and LPS did not affect the cytotoxicity in HT-29 cells. LPS treatment dose-dependently increased the pro-inflammatory cytokine, such as interleukin (IL)-8, whereas EGCG significantly reduced the LPS-stimulated IL-8 production. Additionally, EGCG treatment markedly increased the Toll-interacting protein (Tollip) expression, which negatively regulates the TLR signaling in a dose and time-dependent manner. In particular, in the result from an RNA interference-mediated assay, our finding showed that silencing of Tollip resulted in abrogation of the inhibitory action of EGCG on LPS-induced production of pro-inflammatory mediators (inducible nitric oxide synthase-mediated NO/COX2, and IL-8) and activation of MAPKs and NF-κB signaling pathways. Interestingly, we also found that Tollip expression induced by EGCG could be modulated through 67LR expressed on the surface of HT-29 cells. Conclusions: Our novel finding indicates that 67LR and Tollip signaling activated by EGCG treatment is essential for inhibition of inflammation in human intestinal epithelial cells.

Epigallocatechin gallate induces GLUT4 translocation in skeletal muscle through both PI3K- and AMPK-dependent pathways.

Our previous report demonstrated that epigallocatechin gallate (EGCg) promotes translocation of glucose transporter 4 (GLUT4) in skeletal muscle. In this study, we investigated the molecular mechanism of GLUT4 translocation by EGCg at the physiological concentration range. In L6 cells, EGCg induced phosphorylation of phosphatidylinositide 3'-kinase (PI3K) and downstream protein kinase C (PKC) λ/ξ without affecting the phosphorylation of insulin receptor and Akt. EGCg-induced GLUT4 translocation was suppressed by RNA interference-mediated knockdown of PI3K and treatment with PKC inhibitor Go6983. Moreover, EGCg increased Rac1 activity and actin remodelling as downstream events of PKCλ/ξ. These results indicate that EGCg induced GLUT4 translocation through a PI3K-dependent pathway, but its mode of action differed from that of insulin. EGCg also induced GLUT4 translocation through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent pathway. 67 kDa laminin receptor, which is a target molecule of EGCg, was not involved in EGCg-induced glucose uptake in L6 cells. The oral administration of EGCg suppressed postprandial hyperglycaemia accompanied by GLUT4 translocation through both PI3K- and AMPK-dependent pathways, and promoted glycogen accumulation in skeletal muscle of ICR mice. EGCg promotes GLUT4 translocation through both PI3K- and AMPK-dependent pathways and glycogen accumulation in skeletal muscle.