CDK5RAP2 is a Wnt target gene and promotes stemness and progression of oral squamous cell carcinoma.

In oral squamous cell carcinoma (OSCC), a highly aggressive and frequently lethal malignancy, the role and action mechanism of the microtubule regulatory protein CDK5RAP2 have not been fully understood. Here, we show that CDK5RAP2 is highly expressed in OSCC and its expression correlates with clinical stage and lymph node metastasis of the disease. The expression of CDK5RAP2 is regulated by the Wnt signaling pathway. Depletion of CDK5RAP2 inhibits the tumorigenesis and migration of OSCC cells and alters the OSCC cancer stem (-like) cell (CSC) signature. Notably, suppression of CDK5RAP2 expression disrupts spindle orientation during mitosis. Collectively, these results identify CDK5RAP2 as a potential CSC marker and reveal a mechanism that controls the CSC population in OSCC.

EBV latent membrane protein 1 augments γδ T cell cytotoxicity against nasopharyngeal carcinoma by induction of butyrophilin molecules.

Nasopharyngeal carcinoma (NPC) is a diverse cancer with no well-defined tumor antigen, associated with oncogenic Epstein-Barr Virus (EBV), and with usually late-stage diagnosis and survival <40%. Current radiotherapy and chemotherapy have low effectiveness and cause adverse effects, which calls for the need of new therapy. In this regard, adoptive immunotherapy using γδ T cells has potential, but needs to be coupled with butyrophilin 2A1 and 3A1 protein expression to achieve tumoricidal effect. Methods: Human γδ T cells were expanded (with Zol or PTA) and used for cytotoxicity assay against NPC cells, which were treated with the EBV EBNA1-targeting peptide (L2)P4. Effect of (L2)P4 on BTN2A1/BTN3A1 expression in NPC cells was examined by flow cytometry and Western blot. An NPC-bearing NSG mice model was established to test the effectiveness of P4 and adoptive γδ T cells. Immunofluorescence was performed on NPC tissue sections to examine the presence of γδ T cells and expression of BTN2A1 and BTN3A1. EBV gene expression post-(L2)P4 treatment was assessed by qRT-PCR, and the relationship of LMP1, NLRC5 and BTN2A1/BTN3A1 was examined by transfection, reporter assay, Western blot, and inhibition experiments. Results: Zol- or PTA-expanded the Vδ2 subset of γδ T cells that exerted killing against certain NPC cells. (L2)P4 reactivates latent EBV, which increased BTN2A1 and BTN3A1 expression and conferred higher susceptibility towards Vδ2 T cells cytotoxicity in vitro, as well as enhanced tumor regression in vivo by adoptive transfer of Vδ2 T cells. Mechanistically, (L2)P4 induced EBV LMP1, leading to IFN-γ/p-JNK and NLRC5 activation, and subsequently stimulated the expression of BTN2A1 and BTN3A1. Conclusions: This study demonstrated the effectiveness of using the EBV-targeting probe (L2)P4 and adoptive γδ T cells as a promising combinatorial immunotherapy against NPC. The identification of the LMP1-IFN-γ/p-JNK-NLRC5-BTN2A1/BTN3A1 axis may lead to new insight and therapeutic targets against NPC and other EBV+ tumors.

The CBP/ß-Catenin Antagonist, ICG-001, Inhibits Tumor Metastasis via Blocking of the miR-134/ITGB1 Axis-Mediated Cell Adhesion in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy ranking as the 23rd most common cancer globally, while its incidence rate ranked the 9th in southeast Asia. Tumor metastasis is the dominant cause for treatment failure in NPC and metastatic NPC is yet incurable. The Wnt/ß-catenin signaling pathway plays an important role in many processes such as cell proliferation, differentiation, epithelial-mesenchymal transition (EMT), and self-renewal of stem cells and cancer stem cells (CSCs). Both the EMT process and CSCs are believed to play a critical role in cancer metastasis. We here investigated whether the specific CBP/ß-catenin Wnt antagonist, IGC-001, affects the metastasis of NPC cells. We found that ICG-001 treatment could reduce the adhesion capability of NPC cells to extracellular matrix and to capillary endothelial cells and reduce the tumor cell migration and invasion, events which are closely associated with distant metastasis. Through a screening of EMT and CSC-related microRNAs, it was found that miR-134 was consistently upregulated by ICG-001 treatment in NPC cells. Very few reports have mentioned the functional role of miR-134 in NPC, except that the expression was found to be downregulated in NPC. Transient transfection of miR-134 into NPC cells reduced their cell adhesion, migration, and invasion capability, but did not affect the growth of CSC-enriched tumor spheres. Subsequently, we found that the ICG-001-induced miR-134 expression resulting in downregulation of integrin ß1 (ITGB1). Such downregulation reduced cell adhesion and migration capability, as demonstrated by siRNA-mediated knockdown of ITGB1. Direct targeting of ITGB1 by miR-134 was confirmed by the 3'-UTR luciferase assay. Lastly, using an in vivo lung metastasis assay, we showed that ICG-001 transient overexpression of miR-134 or stable overexpression of miR-134 could significantly reduce the lung metastasis of NPC cells. Taken together, we present here evidence that modulation of Wnt/ß-catenin signaling pathway could inhibit the metastasis of NPC through the miR-134/ITGB1 axis.

Lanthanide-Based Peptide-Directed Visible/Near-Infrared Imaging and Inhibition of LMP1.

A lanthanide-based peptide-directed bioprobe LnP19 (Ln = Eu or Yb) is designed as an impressive example of a small molecule-based dual-functional probe for the EBV oncoprotein LMP1. The peptide P19 (Pra-KAhx-K-LDLALK-FWLY-K-IVMSDKW-K-RrRK) is designed to selectively bind to LMP1 by mimicking its TM1 region during oligomerization in lipid rafts while signal transduction is significantly suppressed. Immunofluorescence imaging and Western blotting results reveal that P19 can effectively inactivate the oncogenic cellular pathway nuclear factor κB (NF-κB) and contribute to a selective cytotoxic effect on LMP1-positive cells. By conjugation with cyclen-based europium(III) and ytterbium(III) complexes, EuP19 and YbP19 were constructed to offer visible and near-infrared LMP1-targeted imaging and cancer monitoring. In addition to the ability to target and inhibit LMP1 and to selective inhibit LMP1-positive cells, selective growth inhibition toward the LMP1-positive tumor by LnP19 is also demonstrated.

A red-light-activated sulfonamide porphycene for highly efficient photodynamic therapy against hypoxic tumor.

Photodynamic therapy (PDT) is an emerging alternative cancer treatment modality that utilizes photo-sensitivity to cause cell death upon photo-irradiation. However, PDT efficiency has been hampered by tumor hypoxia, blue-shifted excitation wavelengths, and the high dark toxicity of photo-sensitizers. We designed and synthesized two novel porphycene-based photosensitizers (TBPoS-OH and TBPoS-2OH) with potent photo-cytotoxicity and a LD50 in the nM range under both normoxic and hypoxic conditions in a variety of cell types after photo-irradiation (λ = 640 ± 15 nm). Further studies showed fast-cellular uptake for TBPoS-OH that localized lysosomes and subsequently induced cell apoptosis via the lysosomal-mitochondrial pathway. Moreover, TBPoS-OH significantly reduced tumor growth in two xenografted mouse models bearing melanoma A375 and B16 cells. Finally, TBPoS-OH exhibited no obvious immunogenicity and toxicity to blood cells and major organs in mice. These data demonstrated that these two porphycene-based photosensitizers, especially TBPoS-OH, could be developed as a potential PDT modality.

Role of miR-96/EVI1/miR-449a Axis in the Nasopharyngeal Carcinoma Cell Migration and Tumor Sphere Formation.

The Wnt signaling pathway is one of the major signaling pathways used by cancer stem cells (CSC). Ecotropic Viral Integration Site 1 (EVI1) has recently been shown to regulate oncogenic development of tumor cells by interacting with multiple signaling pathways, including the Wnt signaling. In the present study, we found that the Wnt modulator ICG-001 could inhibit the expression of EVI1 in nasopharyngeal carcinoma (NPC) cells. Results from loss-of-function and gain-of-function studies revealed that EVI1 expression positively regulated both NPC cell migration and growth of CSC-enriched tumor spheres. Subsequent studies indicated ICG-001 inhibited EVI1 expression via upregulated expression of miR-96. Results from EVI1 3'UTR luciferase reporter assay confirmed that EVI1 is a direct target of miR-96. Further mechanistic studies revealed that ICG-001, overexpression of miR-96, or knockdown of EVI1 expression could restore the expression of miR-449a. The suppressive effect of miR-449a on the cell migration and tumor sphere formation was confirmed in NPC cells. Taken together, the miR-96/EVI1/miR-449a axis is a novel pathway involved in ICG-001-mediated inhibition of NPC cell migration and growth of the tumor spheres.

Targeting Epstein-Barr Virus in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection in regions in which it is endemic, including Southern China and Southeast Asia. The high mortality rates of NPC patients with advanced and recurrent disease highlight the urgent need for effective treatments. While recent genomic studies have revealed few druggable targets, the unique interaction between the EBV infection and host cells in NPC strongly implies that targeting EBV may be an efficient approach to cure this virus-associated cancer. Key features of EBV-associated NPC are the persistence of an episomal EBV genome and the requirement for multiple viral latent gene products to enable malignant transformation. Many translational studies have been conducted to exploit these unique features to develop pharmaceutical agents and therapeutic strategies that target EBV latent proteins and induce lytic reactivation in NPC. In particular, inhibitors of the EBV latent protein EBNA1 have been intensively explored, because of this protein's essential roles in maintaining EBV latency and viral genome replication in NPC cells. In addition, recent advances in chemical bioengineering are driving the development of therapeutic agents targeting the critical functional regions of EBNA1. Promising therapeutic effects of the resulting EBNA1-specific inhibitors have been shown in EBV-positive NPC tumors. The efficacy of multiple classes of EBV lytic inducers for NPC cytolytic therapy has also been long investigated. However, the lytic-induction efficiency of these compounds varies among different EBV-positive NPC models in a cell-context-dependent manner. In each tumor, NPC cells can evolve and acquire somatic changes to maintain EBV latency during cancer progression. Unfortunately, the poor understanding of the cellular mechanisms regulating EBV latency-to-lytic switching in NPC cells limits the clinical application of EBV cytolytic treatment. In this review, we discuss the potential approaches for improvement of the above-mentioned EBV-targeting strategies.

Reactivation of Epstein-Barr virus by a dual-responsive fluorescent EBNA1-targeting agent with Zn2+-chelating function.

Epstein-Barr nuclear antigen 1 (EBNA1) plays a vital role in the maintenance of the viral genome and is the only viral protein expressed in nearly all forms of Epstein-Barr virus (EBV) latency and EBV-associated diseases, including numerous cancer types. To our knowledge, no specific agent against EBV genes or proteins has been established to target EBV lytic reactivation. Here we report an EBNA1- and Zn2+-responsive probe (ZRL5P4) which alone could reactivate the EBV lytic cycle through specific disruption of EBNA1. We have utilized the Zn2+ chelator to further interfere with the higher order of EBNA1 self-association. The bioprobe ZRL5P4 can respond independently to its interactions with Zn2+ and EBNA1 with different fluorescence changes. It can selectively enter the nuclei of EBV-positive cells and disrupt the oligomerization and oriP-enhanced transactivation of EBNA1. ZRL5P4 can also specifically enhance Dicer1 and PML expression, molecular events which had been reported to occur after the depletion of EBNA1 expression. Importantly, we found that treatment with ZRL5P4 alone could reactivate EBV lytic induction by expressing the early and late EBV lytic genes/proteins. Lytic induction is likely mediated by disruption of EBNA1 oligomerization and the subsequent change of Dicer1 expression. Our probe ZRL5P4 is an EBV protein-specific agent that potently reactivates EBV from latency, leading to the shrinkage of EBV-positive tumors, and our study also suggests the association of EBNA1 oligomerization with the maintenance of EBV latency.

Crucifera sulforaphane (SFN) inhibits the growth of nasopharyngeal carcinoma through DNA methyltransferase 1 (DNMT1)/Wnt inhibitory factor 1 (WIF1) axis.

BACKGROUND: Sulforaphane (SFN), a natural compound present in cruciferous vegetable, has been shown to possess anti-cancer activities. Cancer stem cell (CSC) in bulk tumor is generally considered as treatment resistant cell and involved in cancer recurrence. The effects of SFN on nasopharyngeal carcinoma (NPC) CSCs have not yet been explored. PURPOSE: The present study aims to examine the anti-tumor activities of SFN on NPC cells with CSC-like properties and the underlying mechanisms. METHODS: NPC cells growing in monolayer culture, CSCs-enriched NPC tumor spheres, and also the NPC nude mice xenograft were used to study the anti-tumor activities of SFN on NPC. The population of cells expressing CSC-associated markers was evaluated using flow cytometry and aldehyde dehydrogenase (ALDH) activity assay. The effect of DNA methyltransferase 1 (DNMT1) on the growth of NPC cells was analyzed by using small interfering RNA (siRNA)-mediated silencing method. RESULTS: SFN was found to inhibit the formation of CSC-enriched NPC tumor spheres and reduce the population of cells with CSC-associated properties (SRY (Sex determining Region Y)-box 2 (SOX2) and ALDH). In the functional study, SFN was found to restore the expression of Wnt inhibitory factor 1 (WIF1) and the effect was accompanied with the downregulation of DNMT1. The functional activities of WIF1 and DNMT1 were confirmed using exogenously added recombinant WIF1 and siRNA knockdown of DNMT1. Moreover, SFN was found to inhibit the in vivo growth of C666-1 cells and enhance the anti-tumor effects of cisplatin. CONCLUSION: Taken together, we demonstrated that SFN could suppress the growth of NPC cells via the DNMT1/WIF1 axis.

The anti-tumor function of the IKK inhibitor PS1145 and high levels of p65 and KLF4 are associated with the drug resistance in nasopharyngeal carcinoma cells.

We and others have previously shown that the canonical nuclear factor kappa-B (NF-κB) pathway is essential to nasopharyngeal carcinoma (NPC) tumor development and angiogenesis, suggesting that the NF-κB pathway, including its upstream modulators and downstream effectors, are potential therapeutic targets for NPC. The inhibitor of upstream IκB kinase (IKK), PS1145, is a small molecule which can specifically inhibit the IκB phosphorylation and degradation and the subsequent nuclear translocation of NF-κB. The present study aims to determine the anti-tumor activity of PS1145 on NPC. Our results showed that PS1145 significantly inhibited the growth of tumorigenic NPC cell lines, but not in the normal nasopharyngeal epithelial cell line. Results in the in vivo study showed that low concentration of PS1145 (3 mg/kg) could significantly suppress the subcutaneous tumor formation in the nude mice bearing NPC xenografts. Apparent adverse effects were not observed in the animal study. Drug resistance against PS1145 seems to be associated with the increased levels of active NF-kB p65 and change of expression levels of kruppel-like factor 4. As can be seen, PS1145 appears to be a safe agent for animal experiments and its effects are tumor-specific, and the proteins associated with the drug resistance of PS1145 are implied.

A physiologically-based pharmacokinetic model of oseltamivir phosphate and its carboxylate metabolite for rats and humans.

Oseltamivir phosphate (OP, Tamiflu®) is a widely used prodrug for the treatment of influenza viral infections. Orally administered OP is rapidly hydrolyzed by the carboxylesterases in animals to oseltamivir carboxylate (OC), a potent influenza virus neuraminidase inhibitor. The goals of this study were to develop and validate a physiologically-based pharmacokinetic (PBPK) model of OP/OC in rats and humans, and to predict the internal tissue doses for OP and OC in humans after receiving OP orally. To this end, a PBPK model of OP/OC was first developed in the rat, which was then scaled up to humans by replacing the physiological and biochemical parameters with human-specific values. The proposed PBPK model consisted of an OP and an OC sub-models each containing nine first-order, flow-limited tissue/organ compartments. OP metabolism to OC was assumed to carry out mainly by hepatic carboxylesterases although extra-hepatic metabolism also occurred especially in the plasma. The PBPK model was developed and validated by experimental data from our laboratories and from the literature. The proposed PBPK model accurately predicted the pharmacokinetic behavior of OP and OC in humans and rats after receiving a single or multiple doses of OP orally or an OC dose i.v. The PBPK model was used to predict the internal tissue doses of OP and OC in a hypothetical human after receiving the recommended dose of 75 mg/kg OP b.i.d. for 6 days. Steady-state OC concentrations in the plasma and major organs such as the lung and the brain were higher than the minimum in vitro IC50 reported for H1N1 influenza virus neuraminidase, confirming OP is an effective, anti-viral agent. OP side-effects in the gastrointestinal tract and brain of humans were explainable by the tissue doses found in these organs. The PBPK model provides a quantitative tool to evaluate the relationship between an externally applied dose of OP and the internal tissue doses in humans. As such the model can be used to adjust the dose regimens for adult patients in disease states e.g., renal failure and liver damage.

The Wnt modulator ICG­001 mediates the inhibition of nasopharyngeal carcinoma cell migration in vitro via the miR­150/CD44 axis.

The Wnt signaling pathway is known to serve an important role in the control of cell migration. The present study analyzed the mechanisms underlying the in vitro modulation of the migration of nasopharyngeal carcinoma (NPC) cells by the CREB­binding protein/catenin antagonist and Wnt modulator ICG­001. The results revealed that ICG­001­mediated inhibition of tumor cell migration involved downregulated mRNA and protein expression of the Wnt target gene cluster of differentiation (CD)44. It was also demonstrated that ICG­001 downregulated the expression of CD44, and this effect was accompanied by restored expression of microRNA (miRNA)­150 in various NPC cell lines. Using a CD44 3'­untranslated region luciferase reporter assay, miR­150 was confirmed to be a novel CD44­targeting miRNA, which could directly target CD44 and subsequently regulate the migration of NPC cells. The present study provides further insight into the inhibition of tumor cell migration through the modulation of miRNA expression by the Wnt modulator ICG­001.

EBNA1-targeted inhibitors: Novel approaches for the treatment of Epstein-Barr virus-associated cancers.

Epstein-Barr virus (EBV) infects more than 90% of humans worldwide and establishes lifelong latent infection in the hosts. It is closely associated with endemic forms of a wide range of human cancers and directly contributes to the formation of some. Despite its critical role in cancer development, no EBV- or EBV latent protein-targeted therapy is available. The EBV-encoded latent protein, Epstein-Barr nuclear antigen 1 (EBNA1), is expressed in all EBV-associated tumors and acts as the only latent protein in some of these tumors. This versatile protein functions in the maintenance, replication, and segregation of the EBV genome and can therefore serve as an attractive therapeutic target to treat EBV-associated cancers. In the last decades, efforts have been made for designing specific EBNA1 inhibitors to decrease EBNA1 expression or interfere with EBNA1-dependent functions. In this review, we will briefly introduce the salient features of EBNA1, summarize its functional domains, and focus on the recent developments in the identification and design of EBNA1 inhibitors related to various EBNA1 domains as well as discuss their comparative merits.

MicroRNA profiling study reveals miR-150 in association with metastasis in nasopharyngeal carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in pathogenesis of human cancers. Several miRNAs have been shown to involve in nasopharyngeal carcinoma (NPC) pathogenesis through alteration of gene networks. A global view of the miRNA expression profile of clinical specimens would be the best way to screen out the possible miRNA candidates that may be involved in disease pathogenesis. In this study, we investigated the expression profiles of miRNA in formalin-fixed paraffin-embedded tissues from patients with undifferentiated NPC versus non-NPC controls using a miRNA real-time PCR platform, which covered a total of 95 cancer-related miRNAs. Hierarchical cluster analysis revealed that NPC and non-NPC controls were clearly segregated. Promisingly, 10 miRNA candidates were differentially expressed. Among them, 9 miRNAs were significantly up-regulated of which miR-205 and miR-196a showed the most up-regulated in NPC with the highest incidence percentage of 94.1% and 88.2%, respectively, while the unique down-regulated miR-150 was further validated in patient sera. Finally, the in vitro gain-of-function and loss-of-function assays revealed that miR-150 can modulate the epithelial-mesenchymal-transition property in NPC/HK-1 cells and led to the cell motility and invasion. miR-150 may be a potential biomarker for NPC and plays a critical role in NPC tumourigenesis.

Role of G3BP1 in glucocorticoid receptor-mediated microRNA-15b and microRNA-23a biogenesis in endothelial cells.

MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control of miRNA biogenesis by androgen and estrogen has been demonstrated, but the direct effects of the glucocorticoid receptor (GR) on miRNA biogenesis are unknown. This study revealed the role of GR in miRNA maturation. We showed that two GR agonists, dexamethasone and ginsenoside-Rg1 rapidly suppressed the expression of mature miR-15b, miR-23a, and miR-214 in human endothelial cells. RNA pulldown coupled with proteomic analysis identified GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) as one of the RNA-binding proteins mediating GR-regulated miRNA maturation. Activated GR induced phosphorylation of v-AKT Murine Thymoma Viral Oncogene Homologue (AKT) kinase, which in turn phosphorylated and promoted nuclear translocation of G3BP1. The nuclear G3BP1 bound to the G3BP1 consensus sequence located on primary miR-15b~16-2 and miR-23a~27a~24-2 to inhibit their maturation. The findings from this study have advanced our understanding of the non-genomic effects of GR in the vascular system.

Anti-inflammatory effects of indirubin derivatives on influenza A virus-infected human pulmonary microvascular endothelial cells.

Influenza A virus (IAV) poses global threats to human health. Acute respiratory distress syndrome and multi-organ dysfunction are major complications in patients with severe influenza infection. This may be explained by the recent studies which highlighted the role of the pulmonary endothelium as the center of innate immune cells recruitment and excessive pro-inflammatory cytokines production. In this report, we examined the potential immunomodulatory effects of two indirubin derivatives, indirubin-3'-(2,3-dihydroxypropyl)-oximether (E804) and indirubin-3'-oxime (E231), on IAV (H9N2) infected-human pulmonary microvascular endothelial cells (HPMECs). Infection of H9N2 on HPMECs induced a high level of chemokines and cytokines production including IP-10, RANTES, IL-6, IFN-ß and IFN-γ1. Post-treatment of E804 or E231 could significantly suppress the production of these cytokines. H9N2 infection rapidly triggered the activation of innate immunity through phosphorylation of signaling molecules including mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STAT) proteins. Using specific inhibitors or small-interfering RNA, we confirmed that indirubin derivatives can suppress H9N2-induced cytokines production through MAPKs and STAT3 signaling pathways. These results underscore the immunomodulatory effects of indirubin derivatives on pulmonary endothelium and its therapeutic potential on IAV-infection.

Therapeutic targeting of CBP/ß-catenin signaling reduces cancer stem-like population and synergistically suppresses growth of EBV-positive nasopharyngeal carcinoma cells with cisplatin.

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy prevalent in southern China. Presence of treatment-resistant cancer stem cells (CSC) may associate with tumor relapse and metastasis in NPC. ICG-001 is a specific CBP/ß-catenin antagonist that can block CBP/ß-catenin-mediated transcription of stem cell associated genes and enhance p300/ß-catenin-mediated transcription, thereby reducing the CSC-like population via forced differentiation. In this study, we aimed to evaluate the effect of ICG-001 on the CSC-like population, and the combination effect of ICG-001 with cisplatin in the C666-1 EBV-positive NPC cells. Results showed that ICG-001 inhibited C666-1 cell growth and reduced expression of CSC-associated proteins with altered expression of epithelial-mesenchymal transition (EMT) markers. ICG-001 also inhibited C666-1 tumor sphere formation, accompanied with reduced SOX2(hi)/CD44(hi) CSC-like population. ICG-001 was also found to restore the expression of a tumor suppressive microRNA-145 (miR-145). Ectopic expression of miR-145 effectively repressed SOX2 protein expression and inhibited tumor sphere formation. Combination of ICG-001 with cisplatin synergistically suppressed in vitro growth of C666-1 cells and significantly suppressed growth of NPC xenografts. These results suggested that therapeutically targeting of the CBP/ß-catenin signaling pathway with ICG-001 can effectively reduce the CSC-like population and combination with cisplatin can effectively suppress the growth of NPC.

Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins.

Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 µg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future.

Monitoring and inhibition of Plk1: amphiphilic porphyrin conjugated Plk1 specific peptides for its imaging and anti-tumor function.

Polo-like kinase 1 (Plk1) is well-known for taking part in cell cycle progression and regulation. Using small molecules for Plk inhibition has been well documented in the literature. However, there are several intrinsic and intractable problems associated with this approach. For example monitoring small molecule Plk inhibitors as anti-tumor agents in vitro/in vivo is often ineffective, they can have poor cell internalization and be susceptible to enzymatic degradation. Herein, we report the synthesis of cell-permeable, water-soluble amphiphilic porphyrin ­ Plk1 specific peptide bioconjugates, Por-P1 and Por-P2. In addition to resolving the aforementioned problems of the small molecule inhibitors Por-P2 manifests responsive emission enhancement upon binding with Plk1 in aqueous medium and in vitro, while potently triggering G2-M phase arrest and then apoptosis selectively in the cancer cells tested. In combination our findings make Por-P2 a promising candidate for the preparation of a new generation of smart chemotherapeutic targeting agents (imaging and inhibition) for Plk1 in particular cancer cell lines.

Ginsenoside compound K induces apoptosis in nasopharyngeal carcinoma cells via activation of apoptosis-inducing factor.

BACKGROUND: Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southern China. Although there are conventional therapies, the side effects and toxicities are not always tolerable for patients. Recently, the tumoricidal effect of ginsenosides on different cancer cells has been studied. This study aims to investigate the anti-cancer effect of ginsenosides on NPC cells and their underlying mechanism. METHODS: The cytotoxicity of ginsenosides on NPC cell line HK-1 was measured by MTT assay. Apoptosis was detected by propidium iodide staining followed by flow cytometry. A xenograft tumor model was established by injecting nude mice with HK-1 cells. The activation of caspases and apoptosis-inducing factor (AIF) were evaluated by Western blot analysis. Nuclear translocation of AIF was also studied by immunofluorescence staining. Mitochondrial membrane potential was measured by JC-1 dye using flow cytometry. RESULTS: Four ginsenosides, 20 (S)-Rh2, compound K (CK), panaxadiol (PD) and protopanaxadiol (PPD), induced apoptotic cell death in HK-1 cells in a concentration-dependent manner. CK inhibited HK-1 xenograft tumor growth most extensively and depleted mitochondrial membrane potential depolarization and induced translocation of AIF from cytoplasm to nucleus in HK-1 cells. In addition, depletion of AIF by siRNA abolished CK-induced HK-1 cell death. CONCLUSION: Ginsenoside CK-induced apoptosis of HK-1 cells was mediated by the mitochondrial pathway and could significantly inhibit tumor growth in vivo.