Colibactin biosynthesis and biological activity depend on the rare aminomalonyl polyketide precursor.

The as-yet unidentified E. coli metabolite colibactin induces DNA damage in eukaryotic cells and promotes tumorigenesis. Its wide distribution in pathogenic and probiotic strains has raised great interest in its structure and biosynthesis. Here we show that colibactin formation involves a rare aminomalonyl unit used as a building block.

A monoclonal antibody against the glutaraldehyde-conjugated polyamine, putrescine: application to immunocytochemistry.

We developed a mouse monoclonal antibody (mAb; APUT-32, IgG1 subisotype mAb) against putrescine (Put) conjugated to bovine serum albumin using a glutaraldehyde (GA)-sodium borohydride procedure, for applications in immunocytochemistry (ICC). The antibody specificity was evaluated by an ELISA binding test, simulating the ICC of tissue sections. APUT-32 mAb was highly specific to Put, and distinguished alterations in the chemical structure of other polyamine (PA) analogs, showing 3.8% crossreaction with cadaverine, 3.3% with spermidine (Spd), and 2.3% with 1,3-diaminopropane. Comparable results in immunoreactivity of APUT-32 mAb were obtained with the ELISA inhibition test. By the indirect immunoperoxidase method using the APUT-32 mAb, Put-like immunoreactivities were observed in the cytoplasm of HeLa and MCF-7 cell lines fixed with GA in combination with NaBH4 reduction, but almost no immunoreaction was seen in the cytoplasm of the human melanoma BD cell line. On the other hand, the same method but using a previously prepared ASPM-29 mAb, specific for spermine (Spm) and Spd, produced intense immunostaining in the cytoplasm of all the three cell types. The Put-like immunoreaction was completely abolished by absorption of the APUT-32 mAb with 10 microg/ml Put-human serum albumin conjugate prepared using GA and NaBH4. HPLC analysis was also performed for the levels of each of the PAs in the three types of cell, showing that the levels of Put detected were much lower than those of Spm and Spd, and were strikingly different in the three cell lines among which the human melanoma BD cell line contained the lowest levels of Put. These results strongly suggest that APUT-32 mAb reacts specifically with Put in the tumor cells and therefore has the potential as a new tool for elucidating the biological roles of Put in cells and tissues.

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.

Marked therapeutic effect of dimyristoylphosphatidylcholine liposomes on carcinoma mice model in vivo.

Significantly prolonged survival rate was obtained for the first time using carcinoma mice models after the administration of single-component liposomes of dimyristoylphosphatidylcholine (DMPC) without any drug. An increase in lymphocytes under optical microscope was observed without any increase in the neutrophils count, suggesting that the DMPC liposomes might inhibit the tumor growth as well as increase in lymphocytes in vivo.

Chemotherapy with hybrid liposomes for melanomatosis.

The inhibitory effects of the hybrid liposomes on the growth of B-16 melanoma cells in vitro and in vivo were examined. The 50% inhibitory concentration of the hybrid liposomes composed of 90 mol% dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylenedodecyl ether (C12(EO)10) was one-twelfth of that of DMPC liposomes. It was noteworthy that for the first time significantly prolonged survival was obtained using a mouce model of carcinoma after the administration of the hybrid liposomes of 90 mol% DMPC/10 mol% C12(EO)n (n=10 or 23) without antitumor drugs.

Remarkably enhanced inhibitory effects of hybrid liposomes on the growth of specific tumor cells.

The highly specific inhibitory effects of the hybrid liposomes composed of 90 mol% L-alpha-dimyristoylphosphatidylglycerol and 10 mol% polyoxyethylene (10) dodecyl ether on the growth of lung adenocarcinoma and stomach tumor cells in vitro were obtained for the first time.

[Specific inhibitory effects of hybrid liposomes on the growth of various tumor cells].

The inhibitory effects of hybrid liposomes composed of lipids having a variety of head groups (zwitterionic L-alpha-dimyristoylphosphatidylcholine (DMPC), anionic L-alpha-dimyristoylphosphatidylglycerol (DMPG), and cationic 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP)) and polyoxyethylene (10) dodecyl ether (C12(EO)10) on the growth of tumor cells (human lung carcinoma (RERF-LC-OK), human hepatoma (Hep-G2), human stomach tumor (GT3TKB)) in vitro were examined. The hybrid liposomes of DMPC/10 mol% C12(EO)10 and DMPG/10 mol% C12(EO)10 were fairly more effective for inhibiting the growth of all tumor cells employed in this study as compared with liposomes (DMPC and DMPG) or micelles (C12(EO)10). Especially, it is attractive that the highly specific inhibitory effect of the hybrid liposomes of DMPG/10 mol% C12(EO)10 without any antitumor drugs on the growth of RERF-LC-OK and GT3TKB was obtained for the first time.

Remarkable inhibitory effects of hybrid liposomes on the growth of HL-60 cells coupled to induction of apoptosis.

The hybrid liposomes (90 mol% DMPC/10 mol% C12(EO)10 or C12(EO)12) have a highly inhibitory effect on the growth of tumor cells (HL-60). The induction of apoptosis by the hybrid liposomes in HL-60 cells was revealed on the basis of flow cytometry and DNA electrophoresis.

[Specific inhibitory effect of hybrid liposomes on the growth of hybridoma cells in vitro].

Remarkably high inhibitory effects of the hybrid liposomes composed of L-alpha-dimyristoyl-phosphatidylcholine (DMPC) and polyoxyethylenealkyl ether (C14(EO)n, n = 6-8 and C12(EO)n, n = 8-12)) on the growth of human lymphoma-human B-lymphocyte hybridoma (HF) cells in vitro were obtained. The hybrid liposomes composed of 90 mol% DMPC/10 mol% C14(EO)n (n = 6-8) or C12(EO)n (n = 8-12) were more fluid as compared with 90 mol% DMPC/10 mol% C14(EO)4 or C12(EO)n (n = 4, 23) hybrid liposomes on the basis of fluorescence polarization measurements. These results suggest that the inhibitory effects of the hybrid liposomes on the growth of HF cells should be related to the membrane fluidity. No toxicity to normal rats in vivo was observed in the experiment using 90 mol% DMPC/10 mol% C14(EO)7 or 90 mol% DMPC/10 mol% C12(EO)12 hybrid liposomes.

Intrathecal chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea encapsulated into hybrid liposomes for meningeal gliomatosis: an experimental study.

1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), one of the chloroethyl nitrosoureas, is effective against malignant glioma. To develop its use in intrathecal chemotherapy, we encapsulated BCNU in hybrid liposomes composed of dimyristoylphosphatidylcholine and micellar surfactants (Tween 20) and dissolved it in artificial cerebrospinal fluid (lipo-BCNU). We then studied the toxicity of hybrid liposomes and cellular proliferation inhibition of lipo-BCNU in vitro. We found that 3 mM hybrid liposomes did not affect the viability of human endothelial cells and that lipo-BCNU inhibited the proliferation of human glioma cell lines U-105MG, U-251MG, and U-373MG, and rat glioma cell lines C6 and 9L in a concentration-dependent fashion. Wistar rats that were administered lipo-BCNU intracisternally showed no weight loss, neurological symptoms, or histological changes of the brain and spinal cord. A Wistar rat model of meningeal gliomatosis was established by intracisternal inoculation of 0.1 ml cell suspension containing 1 x 10(6) or 5 x 10(6) viable C6 glioma cells. Two days after inoculation, lipo-BCNU (BCNU, 2.5 mg/kg) was administered intracisternally. When 1 x 10(6) glioma cells were inoculated (experiments 1 and 2), the median survival times were 24.5 and 26 days in the control groups and 32 and 45 days in the lipo-BCNU-treated groups. respectively. When 5 x 10(6) glioma cells were inoculated (experiments 3-6), the median survival times were 17-29.5 days in the control groups and 23-44 days in the treated groups, respectively. Significantly prolonged survival was obtained in three of six experimental groups. After the administration of 1 ml lipo-BCNU (BCNU, 4.67 mM) or 1 ml BCNU solubilized with 5% dextrose/water (BCNU, 4.67 mM) into the cisterna magna of dogs, the cisterna magna cerebrospinal fluid was sampled, and the BCNU concentrations were assayed by high-performance liquid chromatography. The half-life of the lipo-BCNU was longer than that of BCNU solubilized with 5% dextrose/water. These results suggest that the intrathecal administration of lipo-BCNU may be possible for the treatment of meningeal gliomatosis.

Specific hybrid liposomes composed of phosphatidylcholine and polyoxyethylenealkyl ether with markedly enhanced inhibitory effects on the growth of tumor cells in vitro.

The hybrid liposomes (90mol% DMPC/10mol% C12(EO)8 and 90mol% DMPC/10mol% C12(EO)12) have a highly inhibitory action against the growth of tumor cells. The uniform and stable structure of the hybrid liposomes was revealed on the basis of electron microscopy and dynamic light scattering measurements.

High affinity of hybrid liposomes for normal human epidermal keratinocytes in vitro.

A high affinity of hybrid liposomes towards normal human epidermal keratinocytes was observed. The fluorescence micrograph showed that hybrid liposomes may be incorporated into the keratinocytes by fusion.