Inhibition of HIV-1 entry by the tricyclic coumarin GUT-70 through the modification of membrane fluidity.

Membrane fusion between host cells and HIV-1 is the initial step in HIV-1 infection, and plasma membrane fluidity strongly influences infectivity. In the present study, we demonstrated that GUT-70, a natural product derived from Calophyllum brasiliense, stabilized plasma membrane fluidity, inhibited HIV-1 entry, and down-regulated the expression of CD4, CCR5, and CXCR4. Since GUT-70 also had an inhibitory effect on viral replication through the inhibition of NF-κB, it is expected to be used as a dual functional and viral mutation resistant reagent. Thus, these unique properties of GUT-70 enable the development of novel therapeutic agents against HIV-1 infection.

Therapeutic effects of hybrid liposomes with downregulation of inflammatory cytokine for model mice of rheumatoid arthritis in vivo.

Therapeutic effects of HL for a collagen-induced arthritis (CIA) mouse models of HL-23 composed of 95mol% l-α-dimyristoylphosphatidylcholine (DMPC) and 5mol% polyoxyethylenedodecylether (C12(EO)23) in vivo were examined. Remarkably high therapeutic effects of HL-23 for CIA mouse models were obtained on the basis of clinical assessment of arthritis. The reduction of hyperplastic synovial membrane (pannus tissue) and destruction of the cartilage and bone by HL-23 was revealed on the basis of hematoxylin and eosin (HE) and safranin O staining. Furthermore, the downregulation of inflammatory cytokines such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 for CIA mouse models treated with HL-23 were investigated. Remarkably high therapeutic effects without joint swelling were obtained in CIA mouse models treated with HL-23.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes.

Cepharanthine inhibited HIV-1 cell-cell transmission and cell-free infection via modification of cell membrane fluidity.

The anti-HIV-1 activity of cepharanthine (CEP), a natural product derived from Stephania cepharantha Hayata, was evaluated. CEP stabilized plasma membrane fluidity and inhibited HIV-1 envelope-dependent cell-to-cell fusion of HIV-1-infected cells as well as cell-free infection. It is suggested that CEP inhibited the HIV-1 entry process by reducing plasma membrane fluidity, and the plasma membrane is therefore an identical target to prevent viral infection.

Therapeutic effects of cationic hybrid liposomes on the hepatic metastasis of colon carcinoma along with apoptosis in vivo.

Therapeutic effects of cationic hybrid liposomes (HL) composed of 87 mol% dimyristoyl-phosphatidylcholine (DMPC), 5 mol% polyoxyethylene (21) dodecyl ether (C12(EO)21) and 8 mol% O,O'-ditetradecanoyl-N-(α-trimethyl-ammonioacetyl) diethanolamine chloride (2C14ECl) on the metastasis of human colon carcinoma (HCT116) cells were examined in vivo. Cationic HL having a hydrodynamic diameter less than 150 nm were preserved for one month. Therapeutic effects were obtained in the hepatic metastasis mouse models of HCT116 cells after the intravenous injection of cationic HL. The histological analysis indicated the induction of apoptosis in the liver section of the hepatic metastasis mouse models treated with cationic HL in vivo. Therapeutic effects of cationic HL without any drugs on the hepatic metastasis were revealed for the first time on the basis of histological analyses in vivo.

Novel liposomes composed of dimyristoylphosphatidylcholine and trehalose surfactants inhibit the growth of tumor cells along with apoptosis.

Novel liposomes composed of L-α-dimyristoylphosphatidylcholine (DMPC) and trehalose surfactant (DMTre) were produced and inhibitory effects of DMTre on the growth of human colon carcinoma (HCT116) and gastric carcinoma (MKN45) in vitro were examined. The remarkably high inhibitory effects of DMTre on the growth of HCT116 and MKN45 cells were obtained without affecting the growth of normal cells. The thickness of fixed aqueous layer of DMTre was larger than that of DMPC liposomes and increased in a dose-dependent manner. The induction of apoptosis by DMTre was revealed on the basis of flow cytometric analysis. DMTre induced apoptosis through the activation of caspases and mitochondria via Bax. It is noteworthy that remarkable inhibitory effects of DMTre on the growth of human colon and gastric carcinoma cells leading to apoptosis were obtained for the first time.

Therapeutic effects of autologous lymphocytes activated with trastuzumab for xenograft mouse models of human breast cancer.

Trastuzumab (TTZ) is molecular targeted drug used for metastatic breast cancer patients overexpressing human epidermal growth factor receptor 2 (HER2). Therapeutic effects of lymphocytes activated with TTZ (TTZ-LAK) using xenograft mouse models of human breast cancer (MDA-MB-453) cells were examined in vivo. Remarkable reduction of tumor volume in a xenograft mouse models intravenously treated with TTZ-LAK cells after the subcutaneously inoculated of MDA-MB-453 cells was verified in vivo. The migration of TTZ-LAK cells in tumor of mouse models subcutaneously inoculated MDA-MB-453 cells was observed on the basis of histological analysis using immunostaining with CD-3. Induction of apoptosis in tumor of xenograft mice treated with TTZ-LAK cells was observed in micrographs using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method. It was noteworthy that the therapeutic effects of TTZ-LAK cells along with apoptosis were obtained for xenograft mouse models of human breast tumor in vivo.

Selective accumulation and growth inhibition of hybrid liposomes to human hepatocellular carcinoma cells in relation to fluidity of plasma membranes.

Hybrid liposomes (HLs), composed of l-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) dodecyl ether, have selectively inhibited the growth of human hepatocellular carcinoma (HCC) cells without affecting normal hepatocytes to trigger apoptosis via caspase-3 activation. Furthermore, HLs distinguished between the HCC and normal cells which had higher and lower membrane fluidities respectively, then fused and accumulated preferentially into the membranes of HCC cells. It is noteworthy that the anti-cancer activity of HLs correlated well with the fluidity of cell membranes for HCC and other cancer cells. These results suggest that HLs could target cancer cell-membranes in relation to their lipid fluidity that provide the possibility of novel nanotherapy for intractable cancer.

Hybrid liposomes inhibit growth and invasion of human osteosarcoma cells leading to apoptosis.

Marked inhibitory effects of hybrid liposomes (HL-n; n=21, 23, 25) composed of 90 mol% l-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene(n) dodecyl ethers on the growth of two human osteosarcoma cell lines (MG-63 and U-2 OS) were obtained. Furthermore, fluorescence microscopic and flow cytometric analyses revealed the induction of apoptosis by HL-n in both cells. It is noteworthy that HL-23 could inhibit the invasion and migration of U-2 OS cells on the basis of matrigel invasion assay and scratch wound assay, respectively.

Hybrid liposomes affect cellular lipid constituents and caveolae structures.

We examined alterations of lipid constituents induced by hybrid liposomes (HLs) in cancer cells. As early as 1h after HL treatment, amounts of the raft/caveolae lipids sphingomyelin, ceramide, and ether-type PC were altered. In addition, the structures of caveolae on the cytoplasmic surface of the cell membrane were significantly changed. Our results suggest that alterations of lipid composition in caveolae mediate HL signaling for apoptosis.

Anti-tumor effects of cationic hybrid liposomes against colon carcinoma along with apoptosis in vitro.

New-type three-component cationic hybrid liposomes (HLs) composed of dimyristoylphosphatidylcholine (DMPC), polyoxyethylene(21)dodecyl ether (C(12)(EO)(21)) and O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl) diethanolamine chloride (2C(14)ECl) were produced. Cationic HLs were smaller and more stable than pure DMPC liposomes. It is noteworthy that cationic HLs could remarkably inhibit the growth of human colon cancer (HCT116) cells along with apoptosis in vitro for the first time in this study.

Anticancer effects of residual powder from Barley-Shochu distillation remnants against the orthotopic xenograft mouse models of hepatocellular carcinoma in vivo.

Barely-Shochu is a traditional Japanese liquor distilled from fermented barley with Saccharomyces cerevisiae. Barely-Shochu distillation remnants (SDR) are by-products in the manufacturing process of barley-Shochu. We have already reported on valuable powder from Shochu distillation remnants (PSDR) including antioxidative compounds such as polyphenols. In this study, we investigated the therapeutic effects of barely-PSDR against orthotopic xenograft mouse models of hepatocellular carcinoma (HCC) in vivo. We constructed a mouse model of HCC by orthotopical inoculation of HepG2 cells into the liver of SCID mice. Barely-PSDR (2250 mg/kg) was orally treated once each day for 21 d after the inoculation of HepG2 cells. The livers were removed from anaesthetized mice after the treatment with barely-PSDR and fixed in formalin. The liver sections were analyzed by hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) methods. Remarkably high reduction of tumorigenesis was obtained in the mouse models of HCC after the oral administration of barely-PSDR in vivo. Induction of apoptosis in the liver section on the mouse models treated with barely-PSDR was observed. Furthermore, prolonged survival was obtained. Thus, therapeutic effects of barely-PSDR without side effects on the orthotopic xenograft mouse models were revealed for the first time.

New cancer immunotherapy using autologous lymphocytes activated with trastuzumab.

It is well known that trastuzumab (TTZ) is molecular target drug for breast cancer overexpressing human epidermal growth factor receptor 2 (HER2). Novel immunotherapy by human peripheral blood mononuclear cells (PBMCs) activated with TTZ were examined. Proliferation of lymphocytes after adding of TTZ was obtained. Furthermore, lymphocytes activated with TTZ inhibited growth of breast cancer cells in vitro. It is noteworthy that remarkably high cellular cytotoxicity in lymphocytes activated with TTZ compared with that of CD3- and lymphokine (interleukin (IL)2)-activated killer (CD3-LAK) cells commonly used in immunotherapy were revealed.

Inhibitory effects of hybrid liposomes on the growth of synoviocyte causing rheumatoid arthritis.

Inhibitory effects of HL-n composed of 95 mol% L-α-dimyristoylphosphatidylcholin (DMPC) and 5 mol% polyoxyethylenedodecylether (C(12)(EO)(n), n = 21, 23, or 25) on the growth of human rheumatoid arthritis (RA) fibroblast-like synoviocytes (HFLS-RA) in vitro were examined. Remarkably high inhibitory effects of HL-n on the growth of HFLS-RA cells were obtained. The induction of apoptosis by HL-n was revealed on the basis of TUNEL method. Furthermore, the therapeutic effects of HL-23 using mouse models of arthritis were investigated. Therapeutic effects without joint swelling were obtained in mouse models of RA treated with HL.

Selective accumulation of hybrid liposomes into adult T-cell leukemia cells along with induction of apoptosis.

Markedly inhibitory effects of hybrid liposomes (HL-n) composed of 90 mol% L-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene(n) dodecyl ethers on the growth of adult T-cell leukemia cells were obtained for the first time. It is noteworthy that HL-n could selectively accumulate into the adult T-cell leukemia cells and induce apoptosis via caspase-3 activation.

Specific interaction between Ca2+ and anionic lipids in cancer cells along with apoptosis.

Markedly inhibitory effects of Ca(2+) on the growth of human tumor cells were attained through the induction of apoptosis in vitro. On the other hand, a good correlation between the growth inhibition effects of Ca(2+) and the amounts of phosphatidylserines (PS) in the cell membranes (plasma membranes) was obtained. Furthermore, the decrease of membrane fluidity and the localization of lipid microdomains "lipid rafts" in the cell membranes were observed in the presence of Ca(2+). The findings in this study suggest that Ca(2+) could induce apoptosis toward tumor cells through the localization of lipid rafts in plasma membranes by the specific interactions between extracellular Ca(2+) and PS.

Therapeutic effects of hybrid liposomes composed of phosphatidylcholine and docosahexaenoic acid on the hepatic metastasis of colon carcinoma along with apoptosis in vivo.

Therapeutic effects of hybrid liposomes (L-α-dimyristoylphosphatidylcholine (DMPC)/docosahexaenoic acid (DHA)) composed of 50 mol% DMPC and 50 mol% DHA on the metastasis of human colon carcinoma (HCT116) cells were examined in vivo. DMPC/DHA having a hydrodynamic diameter less than 100 nm were preserved for one month. Remarkably high therapeutic effects were obtained in the hepatic metastasis mouse models of HCT116 cells after the intravenous injection of DMPC/DHA. The histological analysis indicated the induction of apoptosis was observed in the liver section of the hepatic metastasis mouse models treated with DMPC/DHA in vivo. Furthermore, prolonged survival was obtained in the hepatic metastasis mouse models after the treatment with DMPC/DHA. Therapeutic effects of DMPC/DHA without any drugs on the hepatic metastasis were revealed on the basis of histological and biochemical analyses for the first time in vivo.

Effective drug delivery system for duchenne muscular dystrophy using hybrid liposomes including gentamicin along with reduced toxicity.

It is known that gentamicin (GM) could be a possible treatment for Duchenne Muscular Dystrophy (DMD). However, GM therapy has been hindered by several problems such as severe side effects of GM. In order to resolve these problems, we developed the drug delivery system (DDS) of GM using hybrid liposomes (HL) composed of L-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) lauryl ether (C12(EO)23). The hydrodynamic diameters of HL including GM (GM-HL) were 60-90 nm with a narrow range of the size distribution and the sizes were kept almost constant for over 4 weeks, suggesting that GM-HL could avoid the reticuloendothelial system in vivo. Furthermore, GM-HL accumulated more to the skeletal muscle cells of X chromosome-linked muscular distrophy (mdx) mice as compared to those of normal mice. Significantly, we succeeded in increasing dystrophin positive fibers in skeletal muscle cells of mdx mice using GM-HL along with the reduction of ototoxicity. It is suggested that GM should be carried more efficiently into the muscular cells of mdx mice by HL. These results indicate that HL could be an effective carrier in the DDS of GM therapy for DMD.

Inhibitory effect of drug-free hybrid liposomes on metastasis of human neuroblastoma.

PURPOSE: Hybrid liposomes composed of vesicular and micellar molecules have been used as drug-delivery systems. It has become clear that hybrid liposomes alone have an inhibitory effect against the growth of various tumor cells. The present study was designed to determine whether a drug-free hybrid liposome composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylenealkyl ether (EO) [90 mol% DMPC/10% C(12)(EO)(21) (HL21), 90 mol% DMPC/10% C(12)(EO)(23) (HL23), or 90 mol% DMPC/10% C(12)(EO)(25) (HL25)], inhibit the liver metastasis of human neuroblastoma cells and thus increases survival. METHODS: A human neuroblastoma cell, TNB9, and BALB/C-nu/nu athymic mice were used in this study. First, we determined the inhibitory effect of the hybrid liposomes on TNB9 cells in vitro. Next, to determine the inhibitory effect of the hybrid liposomes on metastasis of neuroblastoma cells to the liver, we made a murine hepatic metastasis model by implanting TNB9 cells (2 × 106) in the spleen of the mice and compared anatomic appearance, weights, and histological findings of the livers of treated mice and control mice 60 days after the beginning of a 7-day intraperitoneal injection of a hybrid liposome. We also compared survival rates using the Kaplan-Meier method. RESULTS: In mice implanted with TNB9 neuroblastoma cells and treated with HL21 or HL25, no histological evidence of metastasis was found, the weight of the liver was normal, and survival was a mean of 88 and 87.9 days, respectively. In contrast, mice treated with HL23 and control mice had countless tumor cell masses histologically, their liver weight was increased, and their survival was 73.0 and 68.6 days, respectively. CONCLUSIONS: Two kinds of hybrid liposomes, HL21 and HL25, inhibit metastasis of human neuroblastoma cells to the liver, and thus increase survival.

Highly selective fusion and accumulation of hybrid liposomes into primary effusion lymphoma cells along with induction of apoptosis.

Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by human herpes virus-8 infection, and is generally resistant to chemotherapy. Hybrid liposomes, composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (21) dodecyl ether (C12(EO)21) (HL-21), were rapidly accumulated in the membrane of PEL cells. HL-21 also increased membrane fluidity of PEL cells, and induced caspase-3 activation along with cell death. These results suggest that HL-21 should be an effective and attractive regent for PEL treatment.