Inhibitory effect of hybrid liposomes on the growth of liver cancer stem cells.

PURPOSE: Cancer stem cells (CSCs), also known as tumor-initiating cells, are involved in tumor progression, metastasis, and drug resistance. Hybrid liposomes (HLs) are nano-sized liposomal particles that can be easily prepared by ultrasonicating a mixture of vesicular and micellar molecules in buffer solutions. In this study, we investigated the inhibitory effects of HL on the growth of CSC subpopulations in liver cancer cells (HepG2) in vitro. METHODS: HLs composed of 90 mol% L-α-dimyristoylphosphatidylcholine and 10 mol% polyoxyethylene(23) dodecyl ether were prepared by sonication. Cell viability was determined by the trypan blue exclusion assay. In liver cancer cells, CSCs were identified by the presence of the cell surface marker proteins CD133 and EpCAM by flow cytometry. A soft agar colony formation assay was performed using HepG2 cells pretreated with HLs. RESULTS: HLs selectively inhibited liver cancer cell growth without affecting normal hepatocytes. Additionally, HLs induced apoptosis of HepG2 cells by a"ctivating caspase-3. Notably, the CD133(+)/EpCAM(+) CSC sub-population of liver cancer cells treated with HLs was reduced. Furthermore, HLs markedly decreased the number of colony-forming cells. Finally, we confirmed the fusion and accumulation of HLs into the cell membranes of CSCs using a fluorescently labeled lipid (NBDPC). Significant accumulation of HL/NBDPC into the CSCs (particularly EpCAM(+) cells) occurred in a dose-dependent manner. CONCLUSION: These results suggest that HLs are a novel nanomedical therapeutic agent for targeting CSCs in liver cancer therapy.

PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations.

AIM: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. METHODS: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. RESULTS: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. CONCLUSION: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.

Kinetics and mechanistic study of competitive inhibition of thymidine phosphorylase by 5-fluoruracil derivatives.

In a previous investigation, cationic liposomes formulated with new 5-FU derivatives, differing for the length of the polyoxyethylenic spacer that links the N(3) position of 5-FU to an alkyl chain of 12 carbon atoms, showed a higher cytotoxicity compared to free 5-FU, the cytotoxic effect being directly related to the length of the spacer. To better understand the correlation of the spacer length with toxicity, we carried out initial rate studies to determine inhibition, equilibrium and kinetic constants (KI, KM, kcat), and get inside inhibition activity of the 5-FU derivatives and their mechanism of action, a crucial information to design structural variations for improving the anticancer activity. The experimental investigation was supported by docking simulations based on the X-ray structure of thymidine phosphorylase (TP) from Escherichia coli complexed with 3'-azido-2'-fluoro-dideoxyuridin. Theoretical and experimental results showed that all the derivatives exert the same inhibition activity of 5-FU either as monomer and when embedded in lipid bilayer.

Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Nanotherapy with hybrid liposomes for colorectal cancer along with apoptosis in vitro and in vivo.

AIM: We examined the therapeutic effects of hybrid liposomes (HL) composed of L-α-dimyristylphosphati-dylcholine (DMPC) and polyoxyethylene (25) dodecyl ether (C12(EO)25) on the growth of human colorectal cancer (WiDr) cells in vitro and in vivo. MATERIALS AND METHODS: HL composed of 95 mol% DMPC and 5 mol% C12(EO)25 were prepared by the sonication method and their therapeutic effects in xenograft mouse models of colorectal cancer liver metastases were examined in vivo. RESULTS: The inhibitory effects of HL-25 on the growth of WiDr cells along with apoptosis were assessed in vitro. Remarkable inhibitory effects of HL-25 for the liver metastasis of colorectal cancer cells along with apoptosis were revealed on the basis of histological analysis. Prolonged survival was attained for the xenograft mouse model of colorectal cancer after treatment with HL-25 in vivo. CONCLUSION: Therapeutic effects of HL-25 without any drugs on the liver metastasis of human colorectal cancer were obtained for the first time in vivo.

Hybrid liposomes inhibit tumor growth and lung metastasis of murine osteosarcoma cells.

Antitumor effects of hybrid liposomes (HL) composed of l-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) dodecyl ether (C12(EO)23) on the metastatic growth of murine osteosarcoma (LM8) cells were investigated in vitro and in vivo. Remarkable inhibitory effects of HL-23 on the growth of LM8 cells were obtained through the induction of apoptotic cell death in vitro. It was also indicated that HL-23 should dramatically suppress the invasion of LM8 cells and the formation of filopodia on the cell surface in vitro. Furthermore, significantly high therapeutic effects were observed in the homograft mouse models of LM8 cells with lung metastasis after the treatment with HL-23 in vivo. That is, the histological analysis demonstrated that the primary tumor growth of LM8 cells implanted subcutaneously into the mice was inhibited along with the induction of apoptosis. In addition, it was found that HL-23 significantly decreased the lung metastasis of LM8 cells in the mouse models through the inhibition of primary tumor invasion. These results suggest that HL-23 could be a novel agent for the chemotherapy of osteosarcoma.

WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs.

Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1(-/-) mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections.

Intravenous injection of hybrid liposomes suppresses the liver metastases in xenograft mouse models of colorectal cancer in vivo.

Therapeutic effects of hybrid liposomes (HL) composed of l-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(25) dodecyl ether (C(12)(EO)(25)) on the metastasis of human colon carcinoma (HCT116) cells were examined in vivo. Remarkably high therapeutic effects were obtained in the xenograft mouse models of colorectal cancer (CRC) liver metastases after treatment with HL-25 on the basis of relative liver weight and histological analysis of the liver tissue sections of mouse models with HE staining, and TUNEL staining for detection of apoptotic cells. The survival effects of HL-25 were obtained using xenograft mouse models of CRC liver metastases. Furthermore, with regard to pharmacokinetics, the accumulation of fluorescent labeled HL-25 was observed in the liver tissue of xenograft mouse models of CRC liver metastases for 24 h after the intravenous injection of fluorescent labeled HL-25. Therapeutic effects of HL without any drugs on the liver metastasis of human CRC were revealed for the first time in vivo.

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.

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.

[Specific accumulation and antitumor effects of hybrid liposomes on the growth of lung tumor cells].

In general, chemotherapeutic effects were low for non-small cell lung cancer (NSCLC) in the lung tumor. We examined the accumulation and antitumor effects of hybrid liposomes (HL-23) composed of phospholipid (L-α-dimyristoylphosphatidylcholine: DMPC) and PEG surfactant [polyoxyethylene(23)dodecyl ether: C12(EO)23] on NSCLC cells in vitro. Accumulation of HL-23 including a fluorescence probe [1-Palmitoyl-2-[12(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phosphocholine: NBDPC] was observed for NSCLC cells using a confocal laser microscope, but no accumulation of HL-23 in normal lung cells was observed. Furthermore, inhibitory effects of HL-23 on the growth of NSCLC cells were obtained on the basis of a WST-1 assay. It was also clarified that HL-23 induced apoptosis for NSCLC cells on the basis of Annexin-V binding and TUNEL assay. These results suggest that HL-23 could be applied in effective chemotherapies for NSCLC.

Histological bioanalysis for therapeutic effects of hybrid liposomes on the hepatic metastasis of colon carcinoma in vivo.

Therapeutic effects of hybrid liposomes (HL) composed of l-alpha-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (23) dodecylether (C(12)(EO)(23)) on the metastasis of colon carcinoma (Colon26) cells were examined in vivo. Fluorescent labeled Colon26 cells were observed in the liver tissue of hepatic metastasis mouse models after the intrasplenic inoculation of the cells. Remarkably high therapeutic effects were obtained in the hepatic metastasis mouse models after the treatment with HL on the basis of relative liver weight and histological analysis of the liver tissue sections of mouse models with hematoxylin-eosin staining, Masson trichrome staining, and CEA immunostaining as a histochemical marker of metastatic colon carcinoma. Furthermore, no toxicity was observed in the hepatic metastasis mouse models after the intravenous injection of HL. Therapeutic effects of HL without any drugs on the hepatic metastasis were revealed on the basis of histological analysis for the first time in vivo.

Inhibitory effects of cationic hybrid liposomes on the growth of human renal cell carcinoma.

Cationic hybrid liposomes (HL) can be prepared by simply ultrasonicating a mixture of L-alpha-dimyristoylphosphatidylcholine (DMPC), polyoxyethylene (21)dodecyl ether (C(12)(EO)(21)) and O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl) diethanolamine chloride (2C(14)ECl) in a buffer solution. The inhibitory effects of cationic HL containing 2C(14)ECl on the growth of OS-RC-2 human renal carcinoma cells in vitro and in vivo were examined. The 50% inhibitory concentration of cationic HL was remarkably reduced compared with that of DMPC liposomes. Induction of apoptosis by cationic HL in OS-RC-2 cells was verified on the basis of flow cytometric analysis and fluorescence microscopy in vitro. In addition, the effects on survival of cationic HL along with apoptosis in vivo were obtained using a mouse model of renal carcinoma. Chemotherapy with drug-free cationic HL for renal cell carcinoma was developed for the first time through the interaction between cationic HL and anionic surface region of tumor cell membranes, without any side-effects. The results obtained might be applied in chemotherapies used at present for patients with renal carcinoma.

[Inhibitory effects of hybrid liposomes on the growth of gastric tumor cells established from cotton rats].

We established cell-line (CoRa 622 G6) of gastric carcinoma using cotton rats with spontaneous malignant gastric carcinoma with hypergastrinaemia. Inhibitory effects of hybrid liposomes (HL) composed of dimyristoylphosphatidylcoline (DMPC) and polyoxyethylene (n) dodecyl ether (C(12)(EO)(n): n=21, 23, 25) on the growth of CoRa 622 G6 cells were clarified on the basis of WST-1 assay. Fusion and accumulation of HL including fluorescence probe into CoRa 622 G6 cell membrane were clarified using confocal laser microscopy and total internal reflection fluorescence microscopy. Induction of apoptosis of CoRa 622 G6 cells after the treatment with HL was observed in fluorescence micrographs on the basis of Annexin-V binding assay and TUNEL method using confocal laser microscopy. The results in this study could contribute to the chemotherapy for patients with gastric carcinoma.

Induction of apoptosis of human tumor cells by hybrid liposomes including docosahexaenoic acid.

Inhibitory effects of hybrid liposomes (HL) composed of L-alpha-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(20) sorbitan monooleate (Tween 80) including polyunsaturated fatty acids on the growth of human tumor cells were examined in vitro. Remarkably high inhibitory effects of HL including docosahexaenoic acid (HL-DHA) on the growth of lung carcinoma (RERF-LC-OK), colon tumor (WiDr), and stomach tumor (MKN45) cells were obtained. The induction of apoptosis by HL-DHA was revealed on the basis of fluorescence microscopic and flow cytometric analyses.

Modulation of PI-specific phospholipase C by membrane curvature and molecular order.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus has been assayed on large and small unilamellar vesicles consisting of PI, either pure or in mixtures with other lipids. Vesicle diameter (in the 50-300 nm range) influences PI-PLC activity, enzyme rates increasing with decreasing curvature radii. With sonicated unilamellar vesicles of pure PI, two apparent K(s) values are observed, one in the 0-2 mM concentration range and the other in the 2-12 mM concentration range. The latter ( approximately 4.2 mM) corresponds to previously published values, while the low-concentration K(s) is on the same order of magnitude as the single apparent K(m) value found with large unilamellar liposomes ( approximately 0.30 mM). PI-PLC appears to be very sensitive to bilayer composition. Certain nonsubstrate lipids, e.g., galactosylceramide or cholesterol, inhibit PI-PLC in a dose-dependent way, at least up to 33 mol % in the bilayers, under conditions with a constant PI concentration. Simultaneous measurements of enzyme activity, interfacial enzyme binding, and fluorescence of different probes, on a variety of bilayer compositions, reveal that both the level of enzyme binding and activity decrease with increasing lipid order, as measured by the fluorescence polarization of the hydrophobic probe diphenylhexatriene. In contrast, no correlation is found for enzyme activity with fluorescence changes of probes, e.g., laurdan, that report on phenomena occurring mainly at the lipid-water interface. Sphingomyelin has a dual effect. Up to 40 mol %, it increases PI-PLC activity, with little effect on bilayer molecular order. At higher proportions, the increased lipid chain order causes a decrease in enzyme activity. The same effects are observed for distearoylphosphatidylcholine when added to PI bilayers. These results support the "two-stage model" for binding of PI-PLC to lipid bilayers, and underline the significance of the enzyme partial penetration into the membrane hydrophobic matrix for its catalytic activity.

Novel mechanism of hybrid liposomes-induced apoptosis in human tumor cells.

Hybrid liposomes can be prepared by simply ultrasonicating a mixture of vesicular and micellar molecules in a buffer solution. The physical properties of these liposomes, such as size, membrane fluidity, phase transition temperature and hydrophobicity can be controlled by changing the composition. Hybrid liposomes composed of dimyristoylphosphatidylcholine and polyoxyethylene (10) dodecyl ether were found to inhibit the growth of human promyelocytic leukemia (HL-60) cells without using any drugs. Induction of apoptosis by hybrid liposomes in HL-60 cells was verified on the basis of fluorescence microscopy and flow cytometry analysis, after fusion and accumulation of hybrid liposomes, which was revealed on the basis of microphysiometer. We elucidated the pathways of apoptosis induced by the hybrid liposomes. That is, hybrid liposomes fused and accumulated in tumor cell membranes, and the apoptosis signal first passed through mitochondria, caspase-9 and caspase-3, second through Fas, caspase-8, caspase-3 and then reached the nucleus. Hybrid liposomes themselves can induce apoptosis in human tumor cells along with high inhibitory effects on the growth of tumor cells.

A novel role for an insect apolipoprotein (apolipophorin III) in beta-1,3-glucan pattern recognition and cellular encapsulation reactions.

Lipoproteins and molecules for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study we describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide production in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study we show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and beta-1,3-glucan and therefore may act as a pattern recognition molecule for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiological levels, and, with the notable exception of beta-glucan binding, are only observed with the lipid-associated form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition molecule and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunology, particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.

Remarkably enhanced inhibitory effects of three-component hybrid liposomes including sugar surfactants on the growth of lung carcinoma cells.

Sugar parts play important roles in recognizing molecules on the cell membranes. We successfully produced sugar-type micellar surfactants, lactonoalkylamide (LacCn), for the first time. Spherical vesicles, three-component hybrid liposomes, were obtained after the sonication of the mixture of L-alpha-dimyristoyl-phosphatidylcholine (DMPC), Tween 20 and LacCn (DMPC:Tween 20:LacCn=65:7:28). It is noteworthy that high inhibitory effects of the three-component hybrid liposomes composed of DMPC, Tween 20, and LacCn (DMPC:Tween 20:LacCn=65:7:28) on the growth of glioma (U251) and lung adenocarcinoma (RERF-LC-OK) cells were attained in vitro without any drug, although no significant inhibitory effects of any individual component (DMPC, Tween 20, LacCn) or the two-component hybrid liposomes of DMPC and Tween 20 on the growth of tumor cells examined were obtained.