PRAP1 is a novel lipid-binding protein that promotes lipid absorption by facilitating MTTP-mediated lipid transport.

Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers lipids to lipid acceptors and whether there are other biomolecules involved in MTTP-mediated lipid transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially colocalized in the endoplasmic reticulum. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated lipid transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to lipid acceptors. Both PRAP1-deficient and E85V knock-in mutant mice fed a chow diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, both genetically modified mice gained significantly less body weight and fat mass when on high-fat diets compared with littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated lipid transport and lipid absorption.

Cetyltrimethylammonium Bromide Attenuates the Mesenchymal Characteristics of Hypopharyngeal Squamous Cell Carcinoma Through Inhibiting the EGFR/PI3K/AKT Signaling Pathway.

BACKGROUND/AIM: Cetyltrimethylammonium bromide (CTAB), a quaternary ammonium surfactant, was shown to have antitumor effects in a cellular mode of head and neck squamous cell carcinoma (HNSCC), modulating apoptotic and cytotoxic processes. However, the mechanisms by which CTAB exerts its effects against the epithelial- mesenchymal transition in HNSCC remain poorly understood. In the present study, we investigated whether CTAB inhibits cellular mobility and invasiveness of hypopharyngeal squamous cell carcinoma (HPSCC) cells. MATERIALS AND METHODS: WST-1, cell-cycle phase distribution, and wound healing, as well as transwell assays were conducted. Changes in protein expression patterns and related signaling pathways involved in effects of CTAB on HPSCC cell lines were evaluated by western blotting. RESULTS: Treatment of human HPSCC cell lines with CTAB significantly altered their morphology from spindle-like to cobblestone-like by diminishing mesenchymal-like phenotypic characteristics. CTAB also hindered cell functional properties, including migration and invasion, independently of cell viability. In addition, western blot results demonstrated that treatment with CTAB reduced expression of mesenchymal markers. Further investigation showed that CTAB treatment suppressed the phosphorylation of extracellular-regulated kinase 1/2, mechanistic target of rapamycin kinase and AKT serine/threonine kinase 1. CTAB also repressed the expression and phosphorylation levels of epidermal growth factor receptor (EGFR) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), and the partial restoration of mesenchymal phenotype by EGF addition confirmed that CTAB inhibited migration and invasion in HPSCC cells by blocking the EGFR signaling pathway. CONCLUSION: Our results suggest that CTAB is involved in the suppression of EGFR-mediated mesenchymal phenotype and the molecular mechanism by which CTAB obstructs HPSCC cell metastasis may represent a promising strategy for use in HPSCC treatment.

Cetyltrimethylammonium Bromide Disrupts Mesenchymal Characteristics of Human Tongue Squamous Cell Carcinoma SCC4 Cells Through Modulating Canonical TGF-ß/Smad/miR-181b/TIMP3 Signaling Pathway.

BACKGROUND/AIM: This study investigated the anti-metastatic effects of cetyltrimethylammonium bromide (CTAB) on tongue squamous cell carcinoma (TSCC) SCC4 cells. MATERIALS AND METHODS: Cell morphology, viability, cell cycle distribution, adhesion, migration, invasion and the expression levels of associated proteins were examined using microscopy, WST-1, wound-healing, Boyden chamber assays, and western blotting, respectively. RESULTS: CTAB significantly affected SCC4 cell morphology from spindle-shaped to cobblestone-shaped and resulted in loss of adherence. CTAB significantly inhibited cell adhesion, migration, and invasion of SCC4 cells, independent of cell viability. CTAB reduced expression of matrix metalloproteinases (MMPs) such as MMP3, MMP7, and MMP14 in a concentration-dependent manner, while it increased expression of tissue inhibitors of metalloproteinase 3 (TIMP3). In addition, CTAB reduced the phosphorylation of mothers against decapentaplegic homolog 2/3 (Smad2/3) proteins, which mediated CTAB-inhibited migration and invasion in SCC4 cells. These effects were reversed by TGF-ß1. CONCLUSION: CTAB attenuates the mesenchymal characteristics through upregulation of TIMP3 by inhibiting the canonical TGF-ß/Smad/miR-181b/TIMP3 signaling involved in extracellular matrix remodeling in SCC4 cells and might be a promising anti-metastatic therapeutic agent for TSCC.

Cetyltrimethylammonium Bromide Suppresses the Migration and Invasion of Hepatic Mahlavu Cells by Modulating Fibroblast Growth Factor Signaling.

BACKGROUND/AIM: Liver cancer is the fourth leading cause of cancer-related mortality globally, of which hepatocellular carcinoma (HCC) accounts for 85-90% of total primary liver cancer. A drug shortage for HCC therapy triggered us to screen the small-molecule database with a high-throughput cellular screening system. Herein, we examined whether cetyltrimethylammonium bromide (CTAB) inhibits cellular mobility and invasiveness of Mahlavu HCC cells. MATERIALS AND METHODS: The effects of CTAB on cell viability were assessed using WST-1 assay, cell-cycle distribution using flow cytometric analysis, migration/invasion using woundhealing and transwell assays, and associated protein levels using western blotting. RESULTS: Treatment of Mahlavu cells with CTAB transformed its mesenchymal spindle-like morphology. In addition, CTAB exerted inhibitory effects on the migration and invasion of Mahlavu cells dose-dependently. CTAB also reduced the protein levels of matrix metalloproteinase-2 (MMP2), MMP9, RAC family small GTPase 1, SNAIL family transcriptional repressor 1 (SNAI1), SNAI2, TWIST family basic helix-loop-helix transcription factor 1 (TWIST1), vimentin, N-cadherin, phospho-fibroblast growth factor (FGF) receptor, phospho-phosphoinositide 3-kinase, phospho-v-Akt murine thymoma viral oncogene and phospho-signal transducer and activator of transcription 3 but increased the protein levels of tissue inhibitor of metalloproteinases-1/2 and E-cadherin. Rescue experiments proved that CTAB induced mesenchymal-epithelial transition in Mahlavu cells and this was significantly dose-dependently mitigated by basic FGF. CONCLUSION: CTAB suppressed the migration and invasion of Mahlavu cells through inhibition of the FGF signaling pathway. CTAB seems to be a potential agent for preventing metastasis of hepatic cancer.

Cetyltrimethylammonium Bromide Disrupts the Mesenchymal Characteristics of HA22T/VGH Cells Via Inactivation of c-Met/PI3K/Akt/mTOR Pathway.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) arises from hepatocytes, and is the most frequently occurring malignancy of primary liver cancer. In this study, we investigated the anti-metastatic effects of the quaternary ammonium compound, cetyltrimethylammonium bromide (CTAB), on HA22T/VGH HCC cells. MATERIALS AND METHODS: According to our preliminary data, the effect of CTAB on cell cycle distribution, migration, invasion and the associated protein levels was examined using flow cytometry, wound-healing migration, Matrigel transwell invasion assay and western blotting under sub-lethal concentrations. RESULTS: CTAB treatment of HA22T/VGH cells casued dose-dependent mesenchymal-epithelial transition (MET)-like changes and impaired migration and invasion capabilities. In addition, CTAB reduced the levels of metastasis-related proteins including c-Met, phosphoinositide 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), Twist, N-cadherin, and Vimentin. Moreover, pretreatment with hepatocyte growth factor (HGF) rescued CTAB-mediated effects. CONCLUSION: CTAB exhibited potent anti-EMT and anti-metastatic activities through the inhibition of migration and invasion of HA22T/VGH cells. CTAB interrupted the mesenchymal characteristics of HA22T/VGH cells, which were significantly alleviated by HGF in a dose-dependent manner. CTAB has the potential to evolve as a therapeutic agent for HCC.

Myeloid Zinc Finger 1 (MZF1) Maintains the Mesenchymal Phenotype by Down-regulating IGF1R/p38 MAPK/ERα Signaling Pathway in High-level MZF1-expressing TNBC cells.

BACKGROUND/AIM: Signaling regulation of myeloid zinc finger 1 (MZF1) has been implicated in the progression of many human malignancies; however, the mechanistic action of MZF1 in triple-negative breast cancer (TNBC) progression remains elusive. In this study, the aim was to investigate the molecular mechanisms of MZF1 and its functional role in TNBC cellular migration and invasion. MATERIALS AND METHODS: Hs578T and MDA-MB-231 cells were transfected to stably express the acidic domain of MZF1 (MZF160-72), or were transfected with MZF1-specific or ELK1-specific short hairpin RNA (shRNA). Changes in cell morphology and distributions of cellular proteins were observed and subsequently migration and invasion were measured by wound healing and transwell assays. Expression levels of epithelial-mesenchymal transition (EMT)-related genes were carried out using immunoblotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Data of transcriptional regulation were obtained from promoter-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. RESULTS: Herein, we found that MZF1 in high-level MZF1-expressing TNBC cells is associated with cell migration, invasion, and mesenchymal phenotype. MZF1 interacted with the promoter region of insulin-like growth factor 1 receptor (IGF1R) to drive invasion and metastasis of high-level MZF1-expressing TNBC cells. Exogenous expression of the acidic domain of MZF1 repressed the binding of endogenous MZF1 to IGF1R promoter via blocking the interaction with ETS-like gene 1 (ELK1). This blockage not only caused MZF1 protein degradation, but also restrained ELK1 nuclear localization in high-level MZF1-expressing TNBC cells. MZF1, but not ELK1, was necessary for the retention of mesenchymal phenotype by repressing IGF1R promoter activity in TNBC cells expressing high levels of MZF1. Activation of the IGF1R-driven p38MAPK-ERα-slug-E-cadherin signaling axis mediated the conversion of mesenchymal cell to epithelial phenotype, caused by MZF1 destabilization. These results suggest that MZF1 is an oncogenic inducer. CONCLUSION: Blocking of the MZF1/ELK1 interaction to reduce MZF1 protein stability by saturating the endogenous MZF1/ELK1 binding domains might be a promising therapeutic strategy for the treatment of high-level MZF1-expressing TNBC.

Cetrimonium Bromide Inhibits Cell Migration and Invasion of Human Hepatic SK-HEP-1 Cells Through Modulating the Canonical and Non-canonical TGF-ß Signaling Pathways.

BACKGROUND/AIM: Cetrimonium bromide (CTAB), a quaternary ammonium surfactant, is an antiseptic agent against bacteria and fungi. However, the mechanisms by which its pharmacological actions affect epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells, such as adenocarcinoma in SK-HEP-1 cells, have not been investigated. We, thereby, investigated whether CTAB inhibits cellular mobility and invasiveness of human hepatic adenocarcinoma in SK-HEP-1 cells. MATERIALS AND METHODS: SK-HEP-1 cells were treated with CTAB, and subsequent migration and invasion were measured by wound healing and transwell assays. Protein expression was detected by immunoblotting analysis. RESULTS: Our data revealed that treatment of SK-HEP-1 cells with CTAB altered their mesenchymal spindle-like morphology. CTAB exerted inhibitory effects on the migration and invasion of SK-HEP-1 cells dose-dependently, and reduced protein levels of matrix metalloproteinase-2 (MMP-2), MMP-9, snail, slug, twist, vimentin, fibronectin, N-cadherin, Smad2, Smad3, Smad4, phosphoinositide-3-kinase (PI3K), p-PI3K, Akt, p-Akt, ß-catenin, mammalian target of rapamycin (mTOR), p-mTOR, p-p70S6K, p-extracellular signal-regulated kinases (ERK)1/2, p-p38 mitogen-activated protein kinase (MAPK) and p-c-Jun N-terminal kinase (JNK), but increased protein levels of tissue inhibitor matrix metalloproteinase-1 (TIMP-1), TIMP-2, claudin-1 and p-GSK3ß. Based on these observations, we suggest that CTAB not only inhibits the canonical transforming growth factor-ß (TGF-ß) signaling pathway though reducing SMADs (an acronym from the fusion of Caenorhabditis elegans Sma genes and the Drosophila Mad, Mothers against decapentaplegic proteins), but also restrains the non-canonical TGF-ß signaling including MAPK pathways like ERK1/2, p38 MAPK, JNK and PI3K. CONCLUSION: CTAB is involved in the suppression of TGF-ß-mediated mesenchymal phenotype and could be a potent medical agent for use in controlling the migration and invasion of hepatic adenocarcinoma.

Isochlorogenic Acid C Reverses Epithelial-Mesenchymal Transition via Down-regulation of EGFR Pathway in MDA-MB-231 cells.

BACKGROUND/AIM: Epidermal growth factor receptor (EGFR) has been suggested to play an important role in survival, proliferation, migration, differentiation, and tumorigenesis of many cell types. Breast cancer patients with high EGFR expression have a poor prognosis. In this study, we investigated the molecular mechanism of the inhibitory effect of isochlorogenic acid c (ICAC) extracted from Lonicera japonica on elevated EGFR levels of the triple-negative breast cancer (TNBC) cell line, MDA-MB-231. MATERIALS AND METHODS: The cell viability and cell-cycle analysis were evaluated using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometry, respectively. The migration ability and invasiveness of ICAC-treated MDA-MB-231 were examined by migration and Matrigel invasion assay. The epithelial-mesenchymal-transition (EMT)-related protein expression was examined by western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ICAC led to significant morphological changes and suppressed migration and invasion capacities of highly metastatic MDA-MB-231 cells. Western blot analysis for EGFR/EMT-associated proteins suggested that ICAC attenuated the mesenchymal traits as observed by up-regulation of epithelial markers and down-regulation of mesenchymal markers as well as decreased activities of matrix metalloproteinase-9 (MMP-9). CONCLUSION: These results suggested that the inhibitory effects of ICAC against EGFR-induced EMT and MDA-MB-231 cell invasion were dependent on the EGFR/ phospholipase Cγ (PLCγ)/extracellular regulated protein kinase ½ (ERK½)/slug signaling pathway. Therefore, the obtained results could provide us clues for the next therapeutic strategy in the treatment of TNBC.

MZF-1/Elk-1/PKCα is Associated with Poor Prognosis in Patients with Hepatocellular Carcinoma.

Background: Protein kinase C alpha (PKCα) is a key signaling molecule in human cancer development. As a therapeutic strategy, targeting PKCα is difficult because the molecule is ubiquitously expressed in non-malignant cells. PKCα is regulated by the cooperative interaction of the transcription factors myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) in human cancer cells. Methods: By conducting tissue array analysis, herein, we determined the protein expression of MZF-1/Elk-1/PKCα in various cancers. Results: The data show that the expression of MZF-1/Elk-1 is correlated with that of PKCα in hepatocellular carcinoma (HCC), but not in bladder and lung cancers. In addition, the PKCα down-regulation by shRNA Elk-1 was only observed in the HCC SK-Hep-1 cells. Blocking the interaction between MZF-1 and Elk-1 through the transfection of their binding domain MZF-160-72 decreased PKCα expression. This step ultimately depressed the epithelial-mesenchymal transition potential of the HCC cells. Conclusion: These findings could be used to develop an alternative therapeutic strategy against patients with the PKCα-derived HCC.

MZF-1/Elk-1 interaction domain as therapeutic target for protein kinase Cα-based triple-negative breast cancer cells.

Recent reports demonstrate that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) correlates with decreased survival outcomes. However, off-target effects of targeting PKCα and limited understanding of the signaling mechanisms upstream of PKCα have hampered previous efforts to manipulate this ubiquitous gene. This study shows that the expression of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) correlates with PKCα expression in TNBC. We found that the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 facilitate the heterodimeric interaction between the two genes before the complex formation binds to the PKCα promoter. Blocking the formation of the heterodimer by transfection of MZF-160-72 or Elk-1145-157 peptide fragments at the MZF-1 / Elk-1 interface decreases DNA-binding activity of the MZF-1 / Elk-1 complex at the PKCα promoter. Subsequently, PKCα expression, migration, tumorigenicity, and the epithelial-mesenchymal transition potential of TNBC cells decrease. These subsequent effects are reversed by transfection with full-length PKCα, confirming that the MZF-1/Elk-1 heterodimer is a mediator of PKCα in TNBC cells. These data suggest that the next therapeutic strategy in treating PKCα-related cancer will be developed from blocking MZF-1/Elk-1 interaction through their binding domain.

Protein kinase C α is involved in the regulation of AXL receptor tyrosine kinase expression in triple-negative breast cancer cells.

AXL receptor tyrosine kinase is overexpressed in triple-negative breast cancer (TNBC), and has a function in cancer progression and metastases. However, the mechanism underlying AXL gene regulation in TNBC remains unknown. In this study, the involvement of protein kinase C α (PKCα) in the expression of AXL was investigated in human TNBC cells. The microarray data from other studies showed that PKCα is significantly correlated with AXL expression in TNBC cell lines. Tissue array analysis also confirmed their correlation in TNBC. The PKCα inhibitor Go6976 was used to treat MDA­MB­231 and Hs578T TNBC cells, which resulted in decreased expression of AXL and epithelia-mesenchymal transition-related gene vimentin, and decreased cell proliferation. An MZF­1 acidic domain fragment (MZF-1 peptide), which was designed to downregulate PKCα expression, was transfected into the cells and resulted in inhibition of AXL expression. This effect was reversed by co­treatment with the constitutive form of PKCα. Moreover, the downregulation of PKCα was also confirmed by treatment with TAT­fused MZF­1 peptide. Thus, the current study proposes that AXL may be correlated with PKCα­dependent TNBC cells, and could be modulated by MZF­1 peptides.

Anemone altaica Induces Apoptosis in Human Osteosarcoma Cells.

In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.

Antitumor activity of acriflavine in human hepatocellular carcinoma cells.

Patients suffering from advanced hepatocellular carcinoma can generally be treated only by targeted therapy to achieve a survival rate that lasts a few months more than that achieved with conventional therapy. To develop better drugs against hepatocellular carcinoma, we screened a variety of compounds and treated four human hepatocellular carcinoma (HCC) cell lines with different drug concentrations. We then examined cell viability using the MTT assay. Results show that a new candidate drug, acriflavine (ACF), suppresses the viability of HCC cell lines in a dose-dependent manner. Flow cytometry analysis reveals that ACF significantly induces the accumulation of a Sub-G1 population of Mahlavu cells. Moreover, ACF decreases Bcl-2 expression and caspase-3 activation. The content of cleaved poly-(ADP-ribose)polymerase-1 (PARP-1) is significantly increased. These findings suggest that ACF suppresses HCC cell growth through the caspase-3 activation pathway. Compared to clinically-approved drugs, the IC50 of ACF (1 µM) is nearly ten-fold lower than that of sorafenib (13 µM). In the in vivo test, nude mice received Mahlavu cell xenografts subcutaneously and were randomly assigned into two groups: control and experimental groups. Treatment was initiated 3 days after implantation and intraperitoneal injection of 0.9 % normal saline or 2 mg/Kg of ACF was continued daily for five weeks. Tumors were palpable in vehicle-treated mice by day 3 and grew to approximately 2000 mm3 by the end of the experiment, whereas mice treated with ACF experience tumor growth to approximately 500 mm3. We, thus, suggest that ACF can inhibit cell growth in HCC cells. Our results may assist the delineation of the mechanism(s) leading to HCC cell growth inhibition and provide a new target therapy capable to prolong the survival rate of patients in advanced stage.

Antitumor activity of acriflavine in lung adenocarcinoma cell line A549.

UNLABELLED: Aim/Materials and Methods: In order to develop better drugs against non-small cell lung cancer (NSCLC), we screened a variety of compounds and treated the human lung adenocarcinoma cell line A549 with different drug concentrations. We then examined the cell viability using the MTT assay. RESULTS: Data show that a new candidate drug, acriflavine (ACF), suppresses the viability of A549 cells in a concentration- and time-dependent manner. Flow cytometry analysis revealed that ACF significantly caused cell growth arrest in the G2/M phase on A549 cells. Moreover, ACF decreased Bcl-2 expression and increased Bax expression. The content of cleaved poly(ADP-ribose)polymerase-1 (PARP-1) and caspase-3 are significantly increased. These findings suggest that ACF is cytotoxic against A549 cells and suppresses A549 cells growth through the caspase-3 activation pathway. In the in vivo test, nude mice bearing A549 cells xenografts by intravenous injection were randomly assigned into two groups: control and experimental group. Treatment was initiated 10 days after implantation and intraperitoneal injection of 0.9% normal saline or 2 mg/kg of ACF was continued daily for five weeks. ACF treatment significantly decreased tumor size and tumor spots on lung surface of tumor-bearing mice. CONCLUSION: ACF can inhibit cell growth in A549 cells. Our results may assist on the delineation of the mechanism(s) leading to NSCLC cell growth inhibition and provide a new antitumor strategy against NSCLC.