Tomoka Takatani-Nakase

Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

CXCR4 Stimulates Macropinocytosis: Implications for Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and HIV

CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

High Glucose Level Promotes Migration Behavior of Breast Cancer Cells through Zinc and Its Transporters

BACKGROUND The diabetes patients have been associated with an increased risk of mortality by breast cancer and there are difference between the breast cancer patients with diabetes, and their nondiabetic counterparts in the regimen choice and effects of breast cancer treatment. However, the pathophysiological relationships of diabetes and breast cancer have not yet been elucidated in detail. In this study, we investigate the breast cancer cell line, MCF-7 motility, which linked to invasion and metastasis, in high glucose level corresponding to hyperglycemia and the role of Zn and its transporter. METHODOLOGY/PRINCIPAL FINDINGS We demonstrated the significant motility of MCF-7 cultured in hyperglycemic level (25 mM glucose) in comparison to normal physiological glucose level (5.5 mM glucose). The other hand, the osmotic control medium, 5.5 mM glucose with 19.5 mM mannitol or fructose had no effect on migratory, suggesting that high glucose level promotes the migration of MCF-7. Moreover, the activity of intracellular Zn(2+) uptake significantly increased in high glucose-treated cells in comparison to 5.5 mM glucose, and the mRNA expression of zinc transporters, ZIP6 and ZIP10, was upregulated in 25 mM glucose-treated cells. The deficiency of ZIP6 or ZIP10 and intracellular Zn(2+) significantly inhibited the high migration activity in 25 mM glucose medium, indicating that Zn(2+) transported via ZIP6 and ZIP10 play an essential role in the promotion of cell motility by high glucose stimulation. CONCLUSION/SIGNIFICANCE Zinc and its transporters, ZIP6 and ZIP10, are required for the motility stimulated with high glucose level. These findings provide the first evidence proposing the novel strategies for the diagnosis and therapy of breast cancer with hyperglycemia.

Curdione Plays an Important Role in the Inhibitory Effect of Curcuma aromatica on CYP3A4 in Caco-2 Cells

Curcuma aromatica is a plant belonging to genus Curcuma of family Zingiberaceae and is widely used as supplements in Japan. Rhizomes of C. aromatica have curcumin as a major yellow pigment and curdione as a main ingredient of essential oils. In this study, we investigated the affect of C. aromatica on CYP3A4 using 1α,25-(OH)2-D3-treated Caco-2 clone cells. Caco-2 cells were treated with methanol extract (0.1 mg ml−1), its hexane soluble fraction (0.1 mg ml−1), curcumin (4 μM) and curdione (20 μM) for 72 hours. Nifedipine was used as a substrate of CYP3A4. Methanol extract, hexane fraction and curdione inhibited the formation of oxidized nifedipine by 50–70%, and curcumin showed no effect. The IC50s of methanol extract, hexane fraction and curdione to oxidized nifedipine formation were 21, 14 and 3.9 μg ml−1 (16.9 μM), respectively. The content of curdione in methanol extract was 11.4%. Moreover, all of methanol extract, hexane fraction and curdione decreased CYP3A4 protein expression but had no affect on CYP3A4 mRNA expression. Our results showed that these drugs further decreased the CYP3A4 protein expression level after the protein synthesis was inhibited by cychroheximide. These findings suggest that curdione plays an important role in the CYP3A4 inhibitory activity of C. aromatica and curdione might inhibit the activity by accelerating the degradation of CYP3A4.

Application of hydrotropy to transdermal formulations: hydrotropic solubilization of polyol fatty acid monoesters in water and enhancement effect on skin permeation of 5‐FU

Objectives  A hydrotropic formulation containing a percutaneous enhancer was developed for the transdermal formulation of a water‐soluble drug and the solubilizing mechanisms of a percutaneous enhancer in water by a hydrotropic agent were investigated. The enhancement effect was also compared with the hydrotropic formulation and the other formulations using ethanol, propylene glycol or mixed micelles.

Cardioprotective activity of urocortin by preventing caspase-independent, non-apoptotic death in cultured neonatal rat cardiomyocytes exposed to ischemia

Caspase-independent, non-apoptotic cell death in ischemic heart disease is considered to be one of the important therapeutic targets, however, the detailed mechanisms of this cell death process are not clear. In this study, we investigated the mechanisms of non-apoptotic cell death in cultured neonatal rat cardiomyocytes during ischemia, and the cardioprotection by preventing the mechanisms. We found that ischemia caused elevation of the phospholipase A2 (iPLA2) expression in the myocytes, leading to distinctive non-apoptotic nuclear shrinkage, and cell death. Moreover, we investigated whether the potent cardioprotective corticotropin-releasing hormone (CRH), urocortin, which had been less focused on non-apoptotic cell death, inhibits the ischemic myocyte death. Ischemia-augmented nuclear shrinkage of the myocytes was suppressed by the pretreatment of ∼10 nM urocortin before the cells were exposed to ischemia. Urocortin could significantly suppress the expression and activity of iPLA2, resulting in preventing the ischemia-induced cell death. The survival-promoting effect of urocortin was abrogated by the CRH receptor antagonist astressin. These findings provide the first evidence linking the targets of the urocortin-mediated cardioprotection to the suppression of the caspase-independent, non-apoptotic death in cardiac myocytes exposed to ischemia.

Zinc and its transporter ZIP6 are key mediators of breast cancer cell survival under high glucose conditions

Recent studies have shown that hyperglycaemia is related to breast cancer progression; however, the mechanisms underlying the relationship between hyperglycaemia and breast cancer cell survival remain unknown. Here, we demonstrate that as compared to physiological glucose conditions, high glucose conditions promote a significant increase in MCF‐7 cell survival under hypoxia. High glucose levels inhibit apoptosis and induce epithelial‐to‐mesenchymal transition, resulting in increased cell viability under hypoxic conditions. Moreover, high glucose‐treated cells display significant increases in intracellular Zn2+ levels and reduction in mRNA expression of the zinc (Zn) transporter Zrt‐ and Irt‐like protein 6 (ZIP6) in hypoxia. ZIP6 deficiency disturbs intracellular Zn2+ homeostasis, leading to increased cell survival in hypoxia and reduced E‐cadherin expression, indicating that decreased ZIP6 expression is strongly associated with resistance to hypoxia.

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

Arginine-rich cell-penetrating peptide-modified extracellular vesicles for active macropinocytosis induction and efficient intracellular delivery

Extracellular vesicles (EVs) including exosomes have been shown to play crucial roles in cell-to-cell communication because of their ability to carry biofunctional molecules (e.g., microRNAs and enzymes). EVs also have pharmaceutical advantages and are highly anticipated to be a next-generation intracellular delivery tool. Here, we demonstrate an experimental technique that uses arginine-rich cell-penetrating peptide (CPP)-modified EVs to induce active macropinocytosis for effective cellular EV uptake. Modification of arginine-rich CPPs on the EV membrane resulted in the activation of the macropinocytosis pathway, and the number of arginine residues in the peptide sequences affected the cellular EV uptake efficiency. Consequently, the ribosome-inactivating protein saporin-encapsulated EVs modified with hexadeca-arginine (R16) peptide effectively attained anti-cancer activity.