Ryuichi Ueoka

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.

Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells.

Human hepatic cancer cells, HepG2, formed spheroids on a poly-L-glutamic acid-coated dish. Doxorubicin (DOX) efflux activity of the cells in spheroid culture was higher than that in monolayer culture due to the higher expression of MDR1 protein of the cells in spheroids compared with those in monolayer. The amount of MDR1 per cell in spheroids was similar to that of hepatic tumor tissue in vivo. Consequently, it was suggested that the drug efflux activity of cells in spheroid culture reflected the activity of hepatic cancer cells. Furthermore, the IC(50) of DOX and epirubicin (EPI) in HepG2 cells, both of which are known to be exported by MDR1, were higher in spheroid compared with monolayer cells, while IC(50) of 5-fluorouracil (5-FU), which is not exported by MDR1 protein, was almost the same in both types of culture. The higher IC(50) of DOX and EPI in HepG2 cells in spheroid culture was associated with a higher efflux activity of the drugs in the spheroid-cultured cells, which appeared to reflect the IC(50) of DOX and EPI in cancer cells in vivo. Therefore, a spheroid culture of hepatic cancer cells seems to provide a promising cell-based in vitro assay system for examining the proper IC(50) values of anticancer agents that would reflect the drug resistance of cancer cells in vivo. In addition, the system would be useful in screening for inhibitors of MDR1 activity, which will help to overcome the multidrug resistance of cancer cells.

Specific accumulation and growth inhibitory effects of hybrid liposomes to hepatoma cells In vitro

Specific accumulation and growth inhibitory effects of hybrid liposomes composed of 90 mol% dimyristoylphosphatidylcholine and 10 mol% polyoxyethylene(23)dodecyl ether were obtained in human hepatoma cells without affecting normal liver cells at all.

Highly specific inhibitory effect of three-component hybrid liposomes including sugar surfactants on the growth of glioma cells.

Three-component hybrid liposomes composed of L-alpha-dimyristoylphosphatidylcholine, micellar surfactant (Tween 20), and beta-D-fructofuranosyl-alpha-D-glucopyranoside monododecanoate were found to be highly effective for inhibiting the growth of glioma cells without any drug.

Chemotherapy with hybrid liposomes for human breast tumors along with apoptosis in vivo

Hybrid liposomes (HL-n) can be prepared by dissolving both vesicular and micellar molecules in buffer solution with ultrasonication. A clear solution of HL-n composed of 95 mol% dimyristoylphosphatidylcholine (DMPC) and 5 mol% polyoxyethylenedodecyl ether (C(12)(EO)(n), n=21 and 25) having hydrodynamic diameter of 100 nm could be kept over 1 month on the basis of dynamic light scattering measurements was obtained. (1) Increases of fusion and accumulation of HL-n including NBDPC as a fluorescence probe into human breast tumor (MDA-MB-453) cell membranes were observed. (2) Reduction of mitochondrial membrane potential and activation of caspase-8, caspase-9, and caspase-3 were observed, indicating that apoptotic signal by HL-n should pass through mitochondria and these caspases. (3) Remarkable reduction of tumor volume in a xenograft mice model intravenously treated with HL-n without drugs after the subcutaneously inoculation of MDA-MB-453 cells was verified in vivo. Induction of apoptosis in tumor of xenograft mice treated with HL-n was observed in micrographs on the basis of TUNEL method. It was noteworthy that the therapeutic effects of HL-n along with apoptosis were obtained for xenograft mice model of human breast tumor in vivo.

Remarkably high inhibitory effects of docosahexaenoic acid incorporated into hybrid liposomes on the growth of tumor cells along with apoptosis.

Inhibitory effects of hybrid liposomes composed of l-alpha-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (20) sorbitan monooleate (Tween 80) including polyunsaturated fatty acids or their ethyl esters (HL-PUFA) on the growth of human tumor cells were examined in vitro. Remarkably high inhibitory effects of HL including docosahexaenoic acid (HL-DHA) and alpha-linolenic acid ethyl ester (HL-ALAE) on the growth of lung carcinoma (RERF-LC-OK and A549) cells, colon tumor (WiDr) cells and stomach tumor (MKN45) cells were obtained. The addition of vitamin E (alpha-tocopherol) to HL-DHA and -ALAE prevented almost completely the growth inhibition of A549 cells distinct from the other tumor cells used in this study. On the other hand, fluorescence microscopic and flow cytometric analyses indicated that the inhibitory effects of HL-DHA on the growth of RERF-LC-OK, WiDr and MKN45 cells could be attained through the induction of apoptosis.

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.

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.

Chemotherapy with hybrid liposomes for acute lymphatic leukemia leading to apoptosis in vivo

Hybrid liposomes (HL) composed of 95 mol% l-α-dimyristoylphosphatidylcholine (DMPC) and 5 mol% polyoxyethylene (25) dodecyl ether (C(12)(EO)(25)) were prepared by sonication. A clear solutions of HL-25 having hydrodynamic diameter of about 50 nm could be maintained over 3 weeks. Remarkable reduction of tumor volume in model mice of acute lymphatic leukemia (ALL) intravenously treated with HL-25 without drugs after the subcutaneously inoculation of human ALL (MOLT-4) cells was verified in vivo. Induction of apoptosis in tumor of model mice of ALL treated with HL-25 was observed in micrographs on the basis of TUNEL method. Remarkable decrease of the ascites in ALL model mice treated with HL-25 was observed. It is noteworthy that prolonged survival (>400%) was obtained in model mice of ALL after the treatment with HL-25 without drugs.

Remarkable inhibitory effects of hybrid liposomes on growth of human colon cancer cells through induction of cell cycle arrest along with apoptosis

Background Hybrid liposomes can be prepared by simply sonicating a mixture of vesicular and micellar molecules in buffer solutions. In this study, we investigated the effects of hybrid liposomes on the growth of human colon cancer cells in vitro. Methods Hybrid liposomes (HL-n, n = 21, 23, 25) composed of L-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(n) dodecyl ethers (C12(EO)n, n = 21, 23, 25) were prepared by the sonication method and their inhibitory effects on growth of human colon cancer HCT116 cells were examined in vitro. Results Significant growth inhibition of HCT116 cells was observed in the presence of HL-n. The fifty percent inhibitory concentration (IC50) of HL-n was less than half that of DMPC liposomes. Furthermore, fluorescence microscopic and flow cytometric analyses indicated that the markedly inhibitory effects of HL-n on the growth of HCT116 cells could be attained through the induction of cell cycle arrest at the G0/G1 phase along with apoptotic cell death. Conclusion It was found for the first time that HL-n can induce both cell cycle arrest and apoptosis in colon cancer cells. The findings in this study should contribute to novel chemotherapy for colon cancer.