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.

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.

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 without drugs for rheumatoid arthritis.

Hybrid liposomes (HLs) can be prepared by simply sonicating a mixture of vesicular and micellar molecules in buffer solutions. This study aims to demonstrate inhibitory effects of HLs on the growth of fibroblast-like synoviocytes along with apoptosis and therapeutic effects of HLs in a mouse model with rheumatoid arthritis (RA). HLs composed of 95 mol% L-α-dimyristoylphosphatidylcholine (DMPC) and 5 mol% polyoxyethylene(23)dodecyl ether (C12(EO)23) were prepared by the sonication method. The inhibitory effects of HLs on the growth of human fibroblast-like synoviocytes-RA (HFLS-RA) cells in vitro and their inhibitory mechanism were examined. High inhibitory effects of HLs on the growth of HFLS-RA cells were observed. The induction of apoptosis by HLs was revealed on the basis of flow cytometric analysis. Furthermore, therapeutic effects of HLs in the mouse model with RA were examined in vivo. Our results demonstrate that HLs showed inhibitory effects on the growth of HFLS-RA cells in vitro along with apoptosis and therapeutic effects in mouse models of RA in vivo.

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.

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.

Growth inhibition by novel liposomes including trehalose surfactant against hepatocarcinoma cells along with apoptosis.

Novel liposomes composed of L-α-dimyristoylphosphatidylcholine (DMPC) and trehalose surfactant (DMTreCn) were produced by the method of sonication in buffer solution. The thickness of fixed aqueous layer of DMTreCn was larger than that of DMPC liposomes and increased in a dose-dependent manner. The remarkable inhibitory effects of DMTreCn on the growth of human hepatocellular carcinoma (HCC) (Hep-G2 and HuH-7) cells were obtained along with apoptosis, without affecting the growth of normal cells. DMTreCn induced apoptosis of Hep-G2 and HuH-7 cells through the activation of caspase-3, 8 and 9. Release of cytochrome c from mitochondria and activation of Bcl-2 family protein (BAX) were recorded, indicating that DMTreCn induced apoptosis of Hep-G2 and HuH-7 cells through mitochondrial pathway via BAX. It is noteworthy that the remarkable inhibitory effects of DMTreCn on the growth of human HCC cells were obtained along with apoptosis for the first time.

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.

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.

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.

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.

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.

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.

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.

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.

Residual powders from Shochu distillation remnants induce apoptosis in human hepatoma cells via the caspase-independent pathway.

Shochu distillation remnants (SDR) are by-products in the manufacturing process of the Japanese liquor Shochu and include various useful organic compounds derived from the fermentation of grains. We have obtained valuable powder (PSDR) from freeze-dried SDR by the treatment with ethanol. In this study, we examined the anticancer effects of barley-, rice-, and sweet potato-PSDR against HepG2 and HuH-7 cells of human hepatocellular carcinoma (HCC) in vitro. All PSDR inhibited the growth of both these HCC cells through the induction of apoptosis. Especially, barley-PSDR was the most effective for the growth inhibition and apoptosis induction of HCC cells of all PSDR. We next examined the apoptotic mechanisms induced by barley-PSDR. Decrease in mitochondrial membrane potential and release of cytochrome c from mitochondria were observed in HCC cells after the treatment with barley-PSDR. Furthermore, barley-PSDR induced the nuclear translocation of apoptosis-inducing factor (AIF) from mitochondria, while it did not significantly affect the activities of caspase-3, -8, and -9. The results suggested that barley-PSDR induced apoptosis against HCC cells via the caspase-independent mitochondrial pathway. The findings in this study suggest that PSDR has the possibility of therapeutic and/or preventive agents of HCC.

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.