The Improved Antigen Uptake and Presentation of Dendritic Cells Using Cell-Penetrating D-octaarginine-Linked PNVA-co-AA as a Novel Dendritic Cell-Based Vaccine.

Cancer immunotherapy using antigen-pulsed dendritic cells can induce strong cellular immune responses by priming cytotoxic T lymphocytes. In this study, we pulsed tumor cell lysates with VP-R8, a cell-penetrating D-octaarginine-linked co-polymer of N-vinylacetamide and acrylic acid (PNVA-co-AA), into the DC2.4 murine dendritic cell line to improve antigen uptake and then determined the anti-tumor effect in tumor-bearing mice. DC2.4 cells were pulsed with the cell lysate of EL4, a murine lymphoma cell line, and VP-R8 to generate the DC2.4 vaccine. For the in vivo study, DC2.4 cells pulsed with EL4 lysate and VP-R8 were subcutaneously injected into the inguinal lymph node to investigate the anti-tumor effect against EL4 and EL4-specific T cell immune responses. VP-R8 significantly improved antigen uptake into DC2.4 compared to conventional keyhole limpet hemocyanin (p < 0.05). The expression of MHC class I, MHC class II, and CD86 in DC2.4 cells significantly increased after pulsing tumor lysates with VP-R8 compared to other treatments (p < 0.05). The intra-lymph node injection of DC2.4 pulsed with both VP-R8 and EL4 lysate significantly decreased tumor growth compared to DC2.4 pulsed with KLH and lysates (p < 0.05) and induced tumor-infiltrating CD8T cells. The DC2.4 vaccine also remarkably increased the population of IFN-gamma-producing T cells and CTL activity against EL4 cells. In conclusion, we demonstrated that VP-R8 markedly enhances the efficiency of dendritic cell-based vaccines in priming robust anti-tumor immunity, suggesting its potential as a beneficial additive for dendritic cell-based immunotherapy.

Validation of the Absorption-Enhancing Ability of Oligoarginines Grafted onto a Backbone of Hyaluronic Acid through Animal Studies from Rodents to Primates.

The purpose of our research is to develop functional additives that enhance mucosal absorption of biologics, such as peptide/protein and antibody drugs, to provide their non-to-poor invasive dosage forms self-managed by patients. Our previous in vivo and in vitro studies demonstrated that the intranasal absorption of biologics in mice was significantly improved when coadministered with oligoarginines anchored chemically to hyaluronic acid via a glycine spacer, presumably through syndecan-4-mediated macropinocytosis under activation by oligoarginines. The present mouse experiments first revealed that diglycine-L-tetraarginine-linked hyaluronic acid significantly enhanced the intranasal absorption of sulpiride, which is a poor-absorptive organic compound with a low molecular weight. However, similar enhancement was not observed for levofloxacin, which has a similarly low molecular weight but is a well-absorptive organic compound, probably because its absorption was mostly dominated by passive diffusion. The subsequent monkey experiments revealed that there was no species difference in the absorption-enhancing ability of diglycine-L-tetraarginine-linked hyaluronic acid for not only organic compounds but also biologics. This was presumably because the expression levels of endocytosis-associated membrane proteins on the nasal mucosa in monkeys were almost equivalent to those in mice, and poorly membrane-permeable/membrane-impermeable drugs were mainly absorbed via syndecan-4-mediated macropinocytosis, regardless of animal species. Drug concentrations in the brain assessed in mice and monkeys and those in the cerebral spinal fluids (CSFs) assessed in monkeys indicated that drugs would be delivered from the systemic circulation to the central nervous system by crossing the blood-brain and the blood-CSF barriers under coadministration with the hyaluronic acid derivative. In line with our original hypothesis, this new set of data supported that our oligoarginine-linked hyaluronic acid would locally perform on the mucosal surface and enhance the membrane permeation of drugs under its colocalization.

Mucosal absorption of antibody drugs enhanced by cell-penetrating peptides anchored to a platform of polysaccharides.

Our previous studies demonstrated that L-octaarginine grafted onto hyaluronic acid via a tetraglycine spacer significantly enhanced intranasal absorption of protein drugs with a molecular weight (Mw) of 22 kDa or less. The present study focused on its potential as an absorption enhancer for antibody drugs with a larger Mw and the enhancement mechanism. When ranibizumab (48 kDa) alone was intranasally administered in mice, its absolute bioavailability was 0.67% on average. The mean bioavailability elevated to 6.2% under coadministration with tetraglycine-L-octaarginine-linked hyaluronic acid. A similar result was observed under substitution of ranibizumab with certolizumab pegol (91 kDa), although bioavailability itself decreased with the Mw increase, irrespective of coadministration with the hyaluronic acid derivative. Rat experiments also revealed that coadministration with the polysaccharide derivative resulted in significant enhancement of intranasal absorption of trastuzumab (148 kDa). In vitro studies using gene-knocked down cells indicated that syndecan-4-induced macropinocytosis played a crucial role on acceleration of antibody uptake into epithelial cells on the nasal mucosa, irrespective of their Mw. It appeared that neither clathrin heavy chain nor caveolin-1 involved in cellular uptake of antibodies. Tetraglycine-L-octaarginine-linked hyaluronic acid was concluded to be a promising delivery tool that possessed universal absorption-enhancing abilities independent to Mw of biologics.

Performance of Cell-Penetrating Peptides Anchored to Polysaccharide Platforms Applied via Various Mucosal Routes as an Absorption Enhancer.

We have been investigating the potential of cell-penetrating peptides anchored to polymeric platforms as a novel absorption enhancer which delivers biologics into systemic circulation via mucosal routes. Our previous mouse experiments demonstrated that hyaluronic acid modified with l-octaarginine, a typical cell-penetrating peptide, via a tetraglycine spacer significantly enhanced the mucosal absorption of protein drugs applied into the nasal cavities, irrespective of the molecular weights (Mw) of the drugs. The present study evaluated the performance of tetraglycine-l-octaarginine-linked hyaluronic acid applied via various mucosal routes. Somatropin (Mw: ca. 22.1 kDa) was moderately absorbed from the lung mucosa, and the mean absolute bioavailability (BA) reached 19% under enhancer-free conditions; nevertheless, its BA under intranasal administration was approximately 1% or less. Its BA significantly elevated to 46% on average through intrapulmonary coadministration with tetraglycine-l-octaarginine-linked hyaluronic acid. When the administration site was replaced with the oral cavities, an extreme reduction in somatropin absorption was observed with a mean BA of 0.056% under enhancer-free conditions. Intraoral coadministration with tetraglycine-l-octaarginine-linked hyaluronic acid resulted in a 6.3-fold elevation of somatropin absorption with statistical significance. A similar enhancement was observed under intrarectal administration with a further reduction in BA. On the other hand, the hyaluronic acid derivative did not exhibit the absorption-enhancing ability under intragastric administration, probably due to the lack of stabilization effects against enzyme-susceptible biologics. The results indicated that the intrapulmonary route was suitable for maximizing the mucosal absorption of biologics, and that there was a likelihood of the intraoral route with user convenience. When somatropin was substituted with fluorescein isothiocyanate-conjugated dextran with an average Mw range of 4-70 kDa, similar phenomena were observed under intrapulmonary and intranasal administration. BA decreased with an increase in the Mw of dextran; however, the ratio of BA under enhancer-present conditions to that under enhancer-free conditions was consistently around 3, indicating that the performance of the hyaluronic acid derivative was Mw-independent, irrespective of the administration route.

Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines.

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Acquisition of absorption-enhancing abilities of cationic oligopeptides with short chain arginine residues through conjugation to hyaluronic acid.

Cell-penetrating peptides such as oligoarginines are one of promising tools that improve mucosal absorption of poorly membrane-permeable biologics. We have already demonstrated that conjugation of L-octaarginine to hyaluronic acid via a tetraglycine spacer resulted in a 3-fold enhancement of nasal absorption of somatropin (Mw: ca. 22.1 kDa) in mice when compared with the unmodified peptide. Here, we evaluated absorption-enhancing abilities and safety profiles of oligopeptides with short chain arginine residues conjugated to hyaluronic acid. Somatropin absorption was hardly ever enhanced by diglycine-L-tetraarginine. The peptide acquired the absorption-enhancing ability through the conjugation; however, it disappeared when arginine residues were halved. In vivo data were consistent to in vitro cellular uptake of somatropin. When somatropin was substituted with exendin-4 (Mw: ca. 4.2 kDa), cellular uptake was significantly enhanced by diglycine-L-diarginine conjugated to hyaluronic acid under comparison with the unmodified peptide. The conjugate also exhibited the enhancement ability in mice, as observed for hyaluronic acid derivatives with four and more arginine residues. Another cell studies revealed that oligoarginine-linked hyaluronic acid tended to be less toxic as arginine residues were reduced. Results indicated that diglycine-L-tetraarginine-linked hyaluronic acid was the most suitable candidate as an absorption enhancer whose Mw-independent enhancement ability and safety were well-balanced.

Mechanism on antigen delivery under mucosal vaccination using cell-penetrating peptides immobilized at multiple points on polymeric platforms.

We have developed an aggregate of D-octaarginine immobilized at multiple points on a co-polymer of N-vinylacetamide and acrylic acid. Previous studies revealed that immunoglobulin G and A were induced when mice were inoculated with influenza virus antigens under coadministration with the D-octaarginine-immobilized polymers as a mucosal vaccine adjuvant. Infection experiments demonstrated that mice vaccinated with a mixture of inactivated influenza viruses and the polymers were protected from infection with mouse-adapted infectious viruses. In the present study, we investigated the mechanism on antigen delivery under mucosal vaccination using the polymers. Two-hour retention of fluorescein-labeled ovalbumin (F-OVA) on the nasal mucosa was observed when applied with the polymers; nevertheless F-OVA was eliminated less than 10 min under polymer-free conditions. F-OVA mixed with the polymers was vigorously taken up into murine dendritic cells. Electrophoresis and dynamic light scattering analysis indicated that OVA interacted with the polymers. The uptake of F-OVA was hardly ever inhibited by the addition of an excess amount of intact OVA. The results suggested that viral antigens were accumulated on the mucosa and delivered into dendritic cells under basolateral membranes via dendrites extending to the mucosal surface and/or subsequent to their permeation through epithelial cells, when they were coadministered with D-octaarginine-immobilized polymers.

Drug resistance via radixin-mediated increase of P-glycoprotein membrane expression during SNAI1-induced epithelial-mesenchymal transition in HepG2 cells.

OBJECTIVES: Epithelial-mesenchymal transition (EMT) plays a role in cancer metastasis as well as in drug resistance through various mechanisms, including increased drug efflux mediated by P-glycoprotein (P-gp). In this study, we investigated the activation mechanism of P-gp, including its regulatory factors, during EMT in hepatoblastoma-derived HepG2 cells. METHODS: HepG2 cells were transfected with SNAI1 using human adenovirus serotype 5 vector. We quantified mRNA and protein expression levels using qRT-PCR and western blot analysis, respectively. P-gp activity was evaluated by uptake assay, and cell viability was assessed by an MTT assay. KEY FINDINGS: P-gp protein expression on plasma membrane was higher in SNAI1-transfected cells than in Mock cells, although there was no difference in P-gp protein level in whole cells. Among the scaffold proteins such as ezrin, radixin and moesin (ERM), only radixin was increased in SNAI1-transfected cells. Uptake of both Rho123 and paclitaxel was decreased in SNAI1-transfected cells, and this decrease was blocked by verapamil, a P-gp inhibitor. The reduced susceptibility of SNAI1-transfected cells to paclitaxel was reversed by elacridar, another P-gp inhibitor. CONCLUSIONS: Increased expression of radixin during SNAI1-induced EMT leads to increased P-gp membrane expression in HepG2 cells, enhancing P-gp function and thereby increasing drug resistance.

Functional Alterations of Multidrug Resistance-Associated Proteins 2 and 5, and Breast Cancer Resistance Protein upon Snail-Induced Epithelial-Mesenchymal Transition in HCC827 Cells.

Our previous report indicated that Snail-induced epithelial-mesenchymal transition (EMT) enhanced P-glycoprotein (P-gp) function and drug resistance to P-gp substrate anticancer drug in a human non-small cell lung cancer (NSCLC) cell line, HCC827. Our objective is to evaluate the changes in the mRNA and protein expression levels and the functions of multidrug resistance-associated protein (MRP) 2, MRP5 and breast cancer resistance protein (BCRP). Snail-expressing HCC827 cells showed increased mRNA levels of Snail and a mesenchymal marker vimentin, and decreased mRNA levels of an epithelial marker E-cadherin after transduction, indicating that Snail had induced EMT consistent with our previous reports. The mRNA level of MRP2 was significantly decreased, while that of MRP5 remained unchanged, in Snail-expressing cells. The expression levels of MRP2 and MRP5 proteins in whole-cell homogenate were unchanged in Snail-expressing cells, but MRP5 protein showed significantly increased membrane localization. Snail-transduction increased the efflux transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDCF), a substrate of MRP2, 3 and 5. This increase was blocked by MK571, which inhibits MRP1, 2, and 5. Toxicity of cisplatin, a substrate of MRP2 and 5, was significantly decreased in Snail-expressing cells. BCRP mRNA and protein levels were both decreased in Snail-expressing cells, which showed an increase in the intracellular accumulation of 7-ethyl-10-hydroxycamptothecin (SN-38), a BCRP substrate, resulting in reduced viability. These results suggested that MRP5 function appears to be increased via an increase in membrane localization, whereas the BCRP function is decreased via a decrease in the expression level in HCC827 cells with Snail-induced EMT.

Nasal absorption enhancement of protein drugs independent to their chemical properties in the presence of hyaluronic acid modified with tetraglycine-L-octaarginine.

Our previous mouse studies demonstrated that mean bioavailability of exendin-4, which is an injectable glucagon-like peptide-1 (GLP-1) analogue whose molecular weight (Mw) and isoelectric point (pI) are ca. 4.2 kDa and 4.5, respectively, administered nasally with poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing D-octaarginine, which is a typical cell-penetrating peptide, was 20% relative to subcutaneous administration even though it was less than 1% when exendin-4 alone was given nasally. The studies also revealed that the absorption-enhancing ability of D-octaarginine-linked PNVA-co-AA for exendin-4 was statistically equivalent to that of sodium salcaprozate (SNAC), which is an absorption enhancer formulated in tablets of semaglutide approved recently as an orally available GLP-1 analogue. From a perspective of clinical application of our technology, we have separately developed hyaluronic acid modified with L-octaarginine via a tetraglycine spacer which would be degraded in biological conditions. The present study revealed that tetraglycine-L-octaarginine-linked hyaluronic acid enhanced nasal absorption of exendin-4 in mice, as did D-octaarginine-linked PNVA-co-AA. There was no significant difference in absorption-enhancing abilities between the hyaluronic acid derivative and SNAC when octreotide (Mw: ca. 1.0 kDa, pI: 8.3) and lixisenatide (Mw: ca. 4.9 kDa, pI: 9.5) were used as a model protein drug. On the other hand, SNAC did not significantly enhance nasal absorption of somatropin (Mw: ca. 22.1 kDa, pI: 5.3) when compared with absorption enhancer-free conditions. Substitution of SNAC with tetraglycine-L-octaarginine-linked hyaluronic acid resulted in a 5-fold increase in absolute bioavailability of somatropin with statistical significance. It appeared that pI hardly ever influenced absorption-enhancing abilities of both enhancers. Results indicated that our polysaccharide derivative would be a promising absorption enhancer which delivers biologics applied on the nasal mucosa into systemic circulation and was of greater advantage than SNAC for enhancing nasal absorption of protein drugs with a larger Mw.

Moesin-Mediated P-Glycoprotein Activation During Snail-Induced Epithelial-Mesenchymal Transition in Lung Cancer Cells.

Epithelial-mesenchymal transition (EMT) plays a role in not only cancer metastasis, but also drug resistance, which is associated with increased levels of efflux transporters such as P-glycoprotein (P-gp). Here, we examined whether P-gp activation during Snail-induced EMT of lung cancer cells is mediated by ezrin, radixin, and moesin (ERM), which regulate transporter localization. HCC827 lung cancer cells overexpressing the transcription factor Snail showed increased Rhodamine123 efflux and increased paclitaxel resistance, reflecting increased P-gp activity. Concomitantly, the expression level of moesin, but not ezrin or radixin, was significantly increased. The increase of P-gp activity was suppressed by knockdown of moesin. Thus, the increase of P-gp activity associated with Snail-induced EMT may be mediated mainly by moesin in HCC827 cells. On the other hand, the Snail mRNA expression level was correlated with the expression level of each ERM in 4 non-small-cell lung cancer cell lines (HCC827, A549, H441, H1975) and in tumor tissues, but not normal tissues, of patients with lung cancer. These results suggest that P-gp activation during EMT is at least partially due to increased expression of moesin. Coadministration of moesin inhibitors with anticancer drugs might block P-gp-mediated drug efflux organ-specifically, improving treatment efficacy and minimizing side effects on other organs.

Cross-Protective Abilities of Hyaluronic Acid Modified with Tetraglycine-l-octaarginine as a Mucosal Adjuvant against Infection with Heterologous Influenza Viruses.

Mucosal vaccination, which secretion of immunoglobulin A (IgA) on the mucosa is accompanied by induction of immunoglobulin G (IgG) in the blood, is one of the most effective ways to circumvent influenza epidemics caused by incorrect prediction of epidemic viral strains or viral mutation. Secreted IgA is expected to prevent hosts from being infected with heterologous viruses because this antibody cross-reacts to strains other than those used for immunization. Our previous mouse experiments revealed that intranasal IgA with cross-reactivity was induced through nasal inoculation with inactivated whole viral particles of the H1N1 A/New Caledonia/20/99 IVR116 (NCL) strain in the presence of hyaluronic acid modified with tetraglycine-l-octaarginine. In the present study, heterologous influenza virus challenge was performed to validate a potential of the hyaluronic acid derivative as a mucosal adjuvant with cross-protective abilities. Serious weight loss was observed when mice were nasally inoculated with inactivated NCL viruses alone and subsequently exposed to mouse-adapted infectious viruses of the H1N1 A/Puerto Rico/8/34 (PR8) strain. The symptom associated with virus infection was hardly ever observed for mice inoculated with a mixture of the viral antigens and tetraglycine-l-octaarginine-linked hyaluronic acid, presumably due to high induction of IgG and IgA capable of cross-reacting to PR8 viruses. Less proliferation of PR8 viruses in those mice was also supported by an insignificant elevation of antibody levels through virus exposure. Our polysaccharide derivative enabled hosts to acquire adaptive immunity with cross-protective abilities against heterologous virus infection.

Biodegradable Hyaluronic Acid Modified with Tetraglycine-l-octaarginine as a Safe Adjuvant for Mucosal Vaccination.

We have been investigating the potential use of polymers modified with cell-penetrating peptides as an adjuvant for mucosal vaccination and have already developed nondegradable poly( N-vinylacetamide- co-acrylic acid) (PNVA- co-AA) with which d-octaarginine, a typical cell-penetrating peptide, was grafted. Our previous murine infection experiments demonstrated that immunoglobulin G (IgG) and immunoglobulin A (IgA) were induced in systemic circulation and secreted on nasal mucosa, respectively, through 4-time nasal inoculations with a mixture of influenza viral antigens and d-octaarginine-linked PNVA- co-AA at 7-day intervals, and that immunized mice were perfectly protected from homologous virus infection. In the present study, we designed novel biodegradable polymers bearing cell-penetrating peptides from a perspective of clinical application. Hyaluronic acid whose glucuronic acid was modified with tetraglycine-l-octaarginine at a monosaccharide unit ratio of 30% was successfully developed. The hyaluronic acid derivative exhibited adjuvant activities identical to PNVA- co-AA bearing either d-octaarginine or tetraglycine-d-octaarginine under the above-mentioned inoculation schedule. We further found that there was no difference in humoral immunity between the 4-time inoculations at 7-day intervals and the 2-time inoculations at 28-day intervals. Intranasal IgA induced through the latter schedule with a smaller number of inoculations, which is clinically practical, exhibited cross-reactivity beyond the subtype of viral strains. In vitro toxicity studies demonstrated that the hyaluronic acid derivative was much less toxic than the corresponding PNVA- co-AA derivatives, and that both the polymers and their metabolites did not exhibit genotoxicity. Our results suggested that tetraglycine-l-octaarginine-linked hyaluronic acid would be a clinically valuable and safe adjuvant for mucosal vaccination.

Tumor recognition of peanut agglutinin-immobilized fluorescent nanospheres in biopsied human tissues.

We are investigating an imaging agent for early detection of colorectal cancer. The agent, named the nanobeacon, is coumarin 6-encapsulated polystyrene nanospheres whose surfaces are covered with poly(N-vinylacetamide) and peanut agglutinin that reduces non-specific interactions with the normal mucosa and exhibits high affinity for terminal sugars of the Thomsen-Friedenreich antigen, which is expressed cancer-specifically on the mucosa, respectively. We expect that cancer can be diagnosed by detecting illumination of intracolonically administered nanobeacon on the mucosal surface. In the present study, biopsied human tissues were used to evaluate the potential use of the nanobeacon in the clinic. Prior to the clinical study, diagnostic capabilities of the nanobeacon for detection of colorectal cancer were validated using 20 production batches whose characteristics were fine-tuned chemically for the purpose. Ex vivo imaging studies on 66 normal and 69 cancer tissues removed from the colons of normal and orthotopic mouse models of human colorectal cancer, respectively, demonstrated that the nanobeacon detected colorectal cancer with excellent capabilities whose rates of true and false positives were 91% and 5%, respectively. In the clinical study, normal and tumor tissues on the large intestinal mucosa were biopsied endoscopically from 11 patients with colorectal tumors. Histological evaluation revealed that 9 patients suffered from cancer and the rest had adenoma. Mean fluorescence intensities of tumor tissues treated with the nanobeacon were significantly higher than those of the corresponding normal tissues. Correlation of magnitude relation of the intensity in individuals was observed in cancer patients with a high probability (89%); however, the probability reduced to 50% in adenoma patients. There was a reasonable likelihood for diagnosis of colorectal cancer by the nanobeacon applied to the mucosa of the large intestine.

Entinostat reverses P-glycoprotein activation in snail-overexpressing adenocarcinoma HCC827 cells.

Epithelial-to-mesenchymal transition (EMT) in cancer cells facilitates tumor progression by promoting invasion and metastasis. Snail is a transcriptional factor that induces EMT, while P-glycoprotein (P-gp) is an efflux transporter involved in anticancer drug resistance, and P-gp efflux activity is stimulated in Snail-overexpressing lung cancer cells with EMT characteristics. Since the histone deacetylase (HDAC) inhibitor entinostat (Ent) reverses EMT features, our aim in this study was to determine whether Ent also suppresses P-gp activation in Snail-induced cells. First, we confirmed that Ent treatment reduced migration activity, downregulated E-cadherin and upregulated vimentin at the mRNA level in Snail-overexpressing cells, thus inhibiting EMT. Efflux and uptake assays using rhodamine123 (Rho123), a fluorescent P-gp substrate, showed that Ent also inhibited Snail-induced activation of P-gp. Moreover, P-gp activity was more strongly inhibited by Ent in Snail-overexpressing cells than in Mock cells. When we evaluated the uptakes of Rho123 by LLC-PK1 cells and P-gp-overexpressing LLC-GA5COL150 cells, Rho123 accumulation in LLC-GA5COL150 cells was significantly decreased compared with that in LLC-PK1 cells. Coincubation with Ent had no effect on Rho123 accumulation in either of the cell lines. Thus, Ent appears to be an inhibitor, but not a substrate, of P-gp at low concentration. Our results suggest that Ent treatment might suppress not only Snail-induced cancer malignant alteration, but also P-gp-mediated multidrug resistance.

Advances in Studies of P-Glycoprotein and Its Expression Regulators.

This review deals with recent advances in studies on P-glycoprotein (P-gp) and its expression regulators, focusing especially on our own research. Firstly, we describe findings demonstrating that the distribution of P-gp along the small intestine is heterogeneous, which explains why orally administered P-gp substrate drugs often show bimodal changes of plasma concentration. Secondly, we discuss the post-translational regulation of P-gp localization and function by the scaffold proteins ezrin, radixin and moesin (ERM proteins), together with recent reports indicating that tissue-specific differences in regulation by ERM proteins in normal tissues might be retained in corresponding cancerous tissues. Thirdly, we review evidence that P-gp activity is enhanced in the process of epithelial-to-mesenchymal transition (EMT), which is associated with cancer progression, without any increase in expression of P-gp mRNA. Finally, we describe two examples in which P-gp critically influences the brain distribution of drugs, i.e., oseltamivir, where low levels of P-gp associated with early development allow oseltamivir to enter the brain, potentially resulting in neuropsychiatric side effects in children, and cilnidipine, where impairment of P-gp function in ischemia allows cilnidipine to enter the ischemic brain, where it exerts a neuroprotective action.

SPECIFIC MOLECULAR RECOGNITION AS A STRATEGY TO DELINEATE TUMOR MARGIN USING TOPICALLY APPLIED FLUORESCENCE EMBEDDED NANOPARTICLES.

The Thomsen-Friedenreich (TF) antigen is a tumor-associated antigen consistently expressed on the apical surface of epithelial-based cancer cells, including pancreatic cancer. In this work, we report the development of multimodal imaging probe, the tripolymer fluorescent nanospheres, whose surface was fabricated with peanut agglutinin (PNA) moieties as TF molecular recognition molecules. Here, we demonstrate that the probe is able to detect TF antigen in human pancreatic cancer tissues and differentiate from normal tissue. What is most noteworthy regarding the probe is its ability to visualize tumor margins defined by epithelial TF antigen expression. Further, in vivo preclinical studies using an orthotopic mouse model of pancreatic cancer suggest the potential use of the nanospheres for laparoscopic imaging of pancreatic cancer tumor margins to enhance surgical resection and improve clinical outcomes.

Gastrointestinal Hormone Cholecystokinin Increases P-Glycoprotein Membrane Localization and Transport Activity in Caco-2 Cells.

It was reported that stimulation of taste receptor type 2 member 38 by a bitter substance, phenylthiocarbamide (PTC), increased P-glycoprotein (P-gp) mRNA level and transport activity via release of the gastrointestinal hormone cholecystokinin-8 (CCK-8) at 9 h. Therefore, we hypothesized that CCK-8 and PTC might also regulate P-gp activity more rapidly via a different mechanism. As a result, we found that the pretreatment of human colon adenocarcinoma (Caco-2) cells with 10-mM PTC significantly decreased the intracellular accumulation of P-gp substrate rhodamine 123 (Rho123) compared with the control after 90-min incubation. Moreover, CCK-8 treatments significantly reduced the accumulation of Rho123 within 30 min, compared with the control. On the other hand, when Caco-2 cells were pretreated with PTC, the efflux ratio of Rho123 was significantly increased compared with control. The efflux ratio of Rho123 in CCK-8 treatment cells was also significantly increased compared with control. Furthermore, CCK-8 increased the phosphorylation of the scaffold proteins ezrin, radixin, and moesin, which regulate translocation of P-gp to the plasma membrane. Therefore, our results indicate that PTC induced release of CCK-8, which in turn induced the phosphorylation of ezrin, radixin, and moesin proteins, leading to upregulation of P-gp transport activity via increased membrane localization of P-gp.

Snail-Induced Epithelial-to-Mesenchymal Transition Enhances P-gp-Mediated Multidrug Resistance in HCC827 Cells.

Overexpression and activation of P-glycoprotein (P-gp), which mediates efflux transport of various anticancer drugs in cancer cells, is associated with multidrug resistance (MDR). On the other hand, malignant cancer cells frequently undergo epithelial-to-mesenchymal transition (EMT), thereby acquiring high migratory mobility and invasive ability. Snail is a transcriptional factor that represses multiple other factors, and its overexpression is a trigger of EMT. Because both P-gp and Snail are involved in malignant evolution of cancer, in this work, we evaluated whether EMT induced by overexpression of Snail influences P-gp expression and activity. Snail-overexpressing cells showed downregulation of epithelial markers, E-cadherin, occludin, and claudin-1, and upregulation of mesenchymal markers, vimentin and ZEB1. Although Western blot analysis showed that P-gp expression levels were similar in Mock and Snail-overexpressing cells, the results of P-gp functional assays with P-gp substrates rhodamine123 and paclitaxel indicated that P-gp is activated in Snail-overexpressing cells. Indeed, Snail-overexpressing cells showed greater viability than Mock cells in the presence of paclitaxel. We observed caveolin-1 dephosphorylation and decreased growth factor receptor-bound protein 2 (GRB2) expression in Snail-overexpressing cells. These findings suggest a novel pathway leading to cancer MDR, in which Snail induces EMT concomitantly with a decrease in GRB2-mediated caveolin-1 phosphorylation, resulting in activation of P-gp.

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein.

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment.