Enhanced radiosensitization by liposome-encapsulated pimonidazole for anticancer effects on human melanoma cells.

The nitroimidazole-related hypoxic radiosensitizer, pimonidazole (Pmz) was encapsulated in liposome composed of dipalmitoylphosphatidylcholine, cholesterol and dipalmitoylphosphatidylglycerol (molar ratio = 1:1:0.2; diameter = 112.9 nm), and the radiosensitization was evaluated in human melanoma cells HMV-II. Cell proliferation was examined by WST-8 assay after X-ray irradiation in the presence of liposomal Pmz or free-Pmz under hypoxic conditions. On 7th day after X-ray irradiation of 5 Gy, cell proliferation decreased more markedly in the administration of liposomal Pmz than free-Pmz at equivalent Pmz doses. Chromatin fragmentation or nuclear condensation was observed in liposomal Pmz-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with Pmz amounts of 250-2000 microM contained in liposomal Pmz. Intracellular uptake was more abundant for liposomal Pmz for 60-240 min than for free-Pmz. Thus liposomal Pmz has a potential to overcome radiation resistance in hypoxia, owing to enhanced intracellular uptake by melanoma cells.

Antitumor and anti-invasive effects of diverse new macrocyclic lactones, alkylolides and alkenylolides, and their enhancement by hyperthermia.

Alkylolides and alkenylolides of 198-254 Da such as hexadecan-16-olide and 9-hexadecen-16-olide were chemically synthesized in the present study as new macrocyclic lactones that are structurally different from widespread natural macrocyclic lactones including bryostatin (887 Da) and rhizoxin (613 Da), and were investigated for antitumor activity to Ehrlich ascites tumor cells by mitochondrial dehydroganase-based WST-1 assay and dye-exclusion assay. Of the alkylolides having 12, 15 or 16 carbon-atoms (D12:0, P15:0 or H16:0) and alkenylolides having 15 or 16 carbon-atoms with a double bond (P15:1 or H16:1), H16:0 was the most carcinostatic when administered at 37 degrees C for 20 h, with cell deformation and microvillus disappearance as detected by scanning electron microscopy. The carcinostatic activity was increased markedly for H16:0 and P15:0 when the administration period was prolonged to 72 h, but was not enhanced by intramolecular introduction of a double bond for P15:1 or H16:1. Hyperthermia at 42 degrees C for 30 min additively intensified the carcinostatic activity for H16:0 and P15:0, but scarcely for D12:0, and intensified the alkenyloides P15:1 and H16:1 only upon the subsequent 72-h treatment. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by alkyl- and alkenylolides even after the short-term exposure at 25 microM for 3 h without diminishing the cell viability. H16:0 also exhibited the most inhibitory activity to tumor invasion in addition to the highest carcinostatic activity. Both inhibitions were promoted by combination with hyperthermia. Thus diverse alkyl-/alkenylolides, may be potent multi-applicable anticancer agents in terms of either dual inhibitory activities against both tumor progression and invasion or hyperthermia-combined therapy.

Anti-tumor and anti-invasive effects of diverse delta-alkyllactones: dependence on molecular side-chain length, action period and intracellular uptake.

New delta-alkyllactones (DALs) with diverse side-chain lengths (184-254 Da), which are structurally different from the widespread, naturally occurring delta-lactones of higher molecular weight (348-439 Da), such as camptothecin and sultriecin, were chemically synthesized and analyzed for their carcinostatic activity. Of the DALs with 11, 12, 13, 14, or 16 carbon atoms, delta-hexadecalactone (DH16:0) was the most carcinostatic when administered to Ehrlich ascites tumor (EAT) cells at 37 degrees C for 20 h, and measured by the mitochondrial dehydrogenase-based WST-1 assay. Prolongation of the administration period to 72 h enhanced the carcinostatic activity more markedly for DH16:0 than for other DALs. The carcinostatic activity of DALs was unexpectedly augmented by increasing the number of carbon atoms, in contrast to the conventional view that carcinostatic activity is attenuated by the addition of carbon atoms to fatty acids. Intracellular accumulation of DH16:0, as analyzed by gas chromatography, was detected (1.5 Pg/cell), whereas other DALs studied were rarely found. The results indicate a close relationship between carcinostatic activity and intracellular accumulation. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by several DALs, even at doses as low as 5-10% of those necessary for carcinostatic activity, suggesting an invasive mechanism different from carcinostasis. The invasion-inhibitory activity was intensified by increasing the number of carbon atoms, in a manner similar to that for the carcinostatic activity. The lifespan of EAT-cell-transplanted mice was markedly prolonged with DH16:0, presumably due to excellent distribution throughout the body and tumor cells. Thus DH16:0 may be a potent anticancer agent, in term of its carcinostatic, anti-invasive, and lifespan-prolonging activities.

Protein Complex of Fibrinogen Gamma Chain and Complement Factor H in Ovarian Cancer Patient Plasma.

BACKGROUND: In the plasma of an advanced cancer patient, fibrinogen is sometimes increased with possible effects on red blood cells (RBCs). MATERIALS AND METHODS: The plasma fraction deteriorating osmotic resistance of RBCs was separated from a patient's plasma with advanced ovarian cancer by phenyl-sepharose column chromatography and analyzed with gel filtration chromatography. RESULTS: In the plasma fraction, we found a protein reactive against whole fibrinogen with a molecular weight higher than that of intact fibrinogen from a healthy volunteer. The-high molecular weight protein was immunoractive to an antibody against fibrinogen gamma chain but not to an antibody against alpha or beta chain. Complement factor H, identified by N-terminal sequencing of a 150-kDa protein separated from the protein, was also eluted from anti-fibrinogen gamma immunoaffinity column. CONCLUSION: Fibrinogen gamma chain and complement factor H were found to be bound as a protein complex in the plasma of a patient with advanced ovarian cancer.

Role of lactate in the brain energy metabolism: revealed by Bioradiography.

To elucidate the role of lactate in the brain, we used a novel method, 'Bioradiography', in which the dynamic process could be followed in living slices by use of positron-emitter-labeled compounds and imaging plates. We studied the incorporation of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) into rat brain slices incubated in oxygenated Krebs-Ringer solution. Under the glucose-free condition, [18F]FDG uptake rate in the cerebral cortex decreased with time and plateaued within 350 min but the addition of 5 mM lactate made the [18F]FDG uptake linear. When an inhibitor of the lactate transporter, 0.5 mM alpha-cyano-4-hydroxycinnamate (4-CIN) was applied to the glucose-free solution, the uptake rate decreased. Under the normal glucose condition, [18F]FDG uptake linearly increased for 6 h, but when 10 mM lactate was applied, the uptake rate decreased. In contrast, when 0.5 mM 4-CIN was applied to the normal glucose solution, [18F]FDG uptake rate increased. These results suggest that exogenous and endogenous lactate can substitute for glucose in the brain.

Enhancement of carcinostatic activity of omega-hydroxy fatty acids by their esterification through increased uptake into tumor cells.

Diverse omega-hydroxy fatty acids (omegaHFAs) and their derivatives were examined for their ability to diminish the cell viability of Ehrlich ascites tumor cells by the mitochondrial dehydrogenase-based WST-1 assay and trypan blue dye exclusion assay. Of the diverse omegaHFAs, hydroxyhexadecanoic acid (omegaH16:0) was appreciably carcinostatic, and hydroxypentadecanoic acid (omegaH15:0) or hydroxypentadecenoic acid (omegaH15:1) was weakly carcinostatic at a dose of 100 micro M, whereas hydroxydodecanoic acid (omegaH12:0) and hydroxyhexadecenoic acid (omegaH16:1) acid were scarcely carcinostatic at the same dose. In contrast their sodium salt derivatives except omegaH16:0 were not carcinostatic. Enhancement of the carcinostatic activity was markedly exerted by ethylesterization of omegaHFAs with the saturated fatty moiety such as omegaH16:0 and omegaH15:0, whereas ethylesters of the unsaturated omegaHFAs such as omegaH15:1 and omegaH16:1 were weakly carcinostatic. Thus intramolecular introduction of a double bond was shown to weaken the carcinostatic activity in case of either omegaHFAs or their ethylester, being in contrast to the conventional knowledge concerning the enhancement of carcinostatic activities of non-hydroxy fatty acids appendant with more double bonds. The intracellular uptake amount of each omegaHFA as quantified by gas chromatography was the following order: omegaH16:0 ethylester (10.1 pg/cell) > omegaH15:0 ethylester (6.4 pg/cell) > omegaH16:0 (3.4 pg/cell) > omegaH15:0 (2.8 pg/cell), which accords with the order of carcinostatic activities of four saturated omegaHFAs in contrast to discord between both the orders for unsaturated omegaHFAs which could be scarcely detected as the intact form within cells. The results indicate that enhancement of carcinostatic activity of omegaH16:0 by ethylesterization was attributed to an appreciable correlation between intracellular uptake amounts and carcinostatic activities for diverse omegaHFAs with saturated fatty moiety, being not true for unsaturated omegaHFAs.

Antitumor and anti-invasive effects of diverse musk-fragrant macrocyclic ketones and their enhancement by hyperthermia.

The antitumor and anti-invasive activities of the low-molecular-weight macrocyclic ketones (MCKs), such as musk secreted from the mammalian genital glands and musk released from relatively unkown plants, were investigated comparatively together with the enhancement of the effects in combination with hyperthermia. Ehrlich ascites tumor cells were treated with each MCK and cultured, followed by evaluation of the cell viability using the mitochondrial dehydrogenase-based WST-8 assay. The number of HT-1080 human fibrosarcoma cells cultured with the MCKs or invading through a reconstituted basement membrane was measured using microscopy. The order of the efficiency was as follows: (Z)-g-cycloheptadecen-1-one (Hp) (17:1, musk rats), 8-cyclohexadecen-1-one (16:1, musk ferns), cyclopentadecanone (15:0, musk rats) and 3-methylcyclopentadecanone (16:0, musk deer), having 15-17 carbon atoms with and without a double bond, which exhibited a carcinostatic effect either at 100 µM for 20-h culture or at 50 µM for 72-h culture. The effects were markedly enhanced by heat treatment at 42˚C. MCKs were not found in the cells by gas-liquid chromatographic determination, indicating that the carcinostatic effects were attributed to their surface activity on the cell membrane. Invasion of HT-1080 cells was inhibited by MCKs at doses scarcely diminishing the cell viability, indicating that the suppression of invasiveness did not ensue from the secondary action due to carcinostasis. The order of invasion-inhibitory efficacy of the MCKs was, however, similar to that of their carcinostatic effects. Hp17:1 also exhibited the highest anti-invasive activity in addition to the highest carcinostatic activity. The two inhibitory effects were promoted by combination with hyperthermia. MCKs with a double bond, particularly Hp17:1 rather than 8-Hx16:1, but not saturated-aliphatic MCKs, may be potent multi-applicable antitumor agents due to their dual inhibitory activities against tumor progression and invasion and in hyperthermia-combined therapy.

Carcinostatic effects of diverse ascorbate derivatives in comparison with aliphatic chain moiety structures: Promotion by combined hyperthermia and reduced cytotoxicity to normal cells.

In this study, using human tongue squamous carcinoma cells (HSC-4) carcinostatic activity was compared for diverse L-ascorbic acid (Asc) derivatives, including the 'straight-C(16)-chain types', 6-O-palmitoyl-Asc (A6-P) and Asc-2-phosphate-6-O-palmitate sodium salt (APPS), as well as the 'branched-C(16)-chain types', Asc-2-phosphate-6-O-(2'-hexyl)decanoate (APHD), an isomer of APPS, and Asc-2,3,5,6-O-tetra-(2'-hexyl)decanoate (VCIP). The order of magnitude of the carcinostatic effects at 37°C was: APPS>A6-P = APHD>VCIP and at 42°C was APPS = A6-P>APHD>VCIP. Therefore, the two straight-C(16)-chain derivatives, APPS and A6-P, had a greater effect compared to the two branched-C(16)-chain Asc derivatives, which are considered to have more difficulty with 'orientation along cell-membrane-glycerolipid direction'. APPS-treated HCS-4 cells were observed for a decrease in cell number, cell shrinkage, pycnosis indicative of apoptosis and cell deformation. The order of cytotoxicity for the normal human dermal fibroblasts (OUMS-36) at 37°C was: A6-P (50% inhibitory concentration: 150-300 µM)>APHD (450-600 µM)>>Asc = APPS (800-1000 µM). Accordingly, APHD was more cytotoxic than APPS, since the straight-C(16)-chain type, which was eliminated after the enzymatic esterolysis of APPS, is metabolized via the 'fatty acid ß-oxidation cycle' more efficiently in normal cells. Thus, APPS had a greater advantage over APHD, A6-P and VCIP in terms of carcinostatic effects at 37°C, carcinostasis promotion at 42°C and a decrease of cytotoxicity to normal cells. This observation suggests a marked potential for aliphatic chain-moiety structures as anticancer agents, due to their cancer-selective carcinostasis and combined efficacy with hyperthermia, without causing side effects.

Anticancer effects of 6-o-palmitoyl-ascorbate combined with a capacitive-resistive electric transfer hyperthermic apparatus as compared with ascorbate in relation to ascorbyl radical generation.

The aim of the present study is to determine the anti-proliferative activity of 6-o-palmitoyl-L: -ascorbic acid (Asc6Palm) that is a lipophilic derivative of L: -ascorbic acid (Asc), on human tongue squamous carcinoma HSC-4 cells by combined use of hyperthermia in comparison to Asc. Asc6Palm or Asc were administered to HSC-4 cells for 1 h, to which hyperthermia at 42 °C was applied for initial 15 min. After further 1-72 h incubation at 37 °C, cell proliferation was determined with Crystal Violet staining. Ascorbyl radical (AscR) in HSC-4 cell suspension was measured by electron spin resonance (ESR), and cell morphology was observed with scanning electron microscopy (SEM). At 37 °C, 4 mM Asc or 0.35 mM Asc6Palm were enough to suppress proliferation of HSC-4 cells. By combined use of hyperthermia at 42 °C, cell proliferation was decreased when compared to 37 °C. After Asc of 4 mM was incubated with HSC-4 cell suspensions at 37 °C or 42 °C for 0-180 min, the signal intensity of ascorbyl radical (AscR) by ESR was not different regardless of the presence or absence of cells at 37 °C, whereas AscR signal was enlarged in the presence of HSC-4 cells at 42 °C. It was suggested that oxidation of Asc occurred rapidly in HSC-4 cells by hyperthermia, and thereby enhanced the anti-proliferative activity. By SEM observation, the surface of HSC-4 cells treated with Asc6Palm revealed distinct morphological changes. Thus, the combined regimen of Asc6Palm and hyperthermia is expected to exert a marked antitumor activity.

Antitumor effects of nano-bubble hydrogen-dissolved water are enhanced by coexistent platinum colloid and the combined hyperthermia with apoptosis-like cell death.

In order to erase reactive oxygen species (ROS) related with the proliferation of tumor cells by reducing activity of hydrogen, we developed functional water containing nano-bubbles (diameters: <900 nm for 71%/population) hydrogen of 1.1-1.5 ppm (the theoretical maximum: 1.6 ppm) with a reducing ability (an oxidation-reduction potential -650 mV, normal water: +100-200 mV) using a microporous-filter hydrogen-jetting device. We showed that hydrogen water erased ROS indispensable for tumor cell growth by ESR/spin trap, the redox indicator CDCFH-DA assay, and was cytotoxic to Ehrlich ascites tumor cells as assessed by WST-8 assay, crystal violet dye stain and scanning electron microscopy, after 24-h or 48-h incubation sequent to warming at 37°C or 42°C. Hydrogen water supplemented with platinum colloid (0.3 ppm Pt in 4% polyvinylpyrrolidone) had more antitumor activity than hydrogen water alone, mineral water alone (15.6%), hydrogen water plus mineral water, or platinum colloid alone as observed by decreased cell numbers, cell shrinkage and pycnosis (nuclear condensation)/karyorrhexis (nuclear fragmentation) indicative of apoptosis, together with cell deformation and disappearance of microvilli on the membrane surface. These antitumor effects were promoted by combination with hyperthermia at 42°C. Thus, the nano-bubble hydrogen water with platinum colloid is potent as an anti-tumor agent.

The antitumor activities of the structurally-similar two-species aromatics Tonalide and Pearlide and the enhancement of their effects by hyperthermia.

Antitumor activities have been reported for the aromatics Tonalide (6-acetyl-1,1,2,4,4,7-hexamethyl-tetrahydronaphthalene, AHTN) and Pearlide (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-γ-2-benzopyran, HHCB), which are contained in detergents. In this study, their carcinostatic activities in Ehrlich ascites tumor (EAT) cells were evaluated by mitochondrial dehydroganase-based WST-1 assay and dye-exclusion assay. The viability of EAT cells treated at 37 or 42°C for 30 min and sequentially cultured at 37°C was assayed at graded times. Immediately after treatment at 37°C, neither Tonalide nor Pearlide had an effect on EAT cells, even at a concentration as high as 200 µM. However, cell viability was reduced to 40% versus the control after 20 h of culture with Tonalide at 50 µM, and to below 20% at 25 µM after 72 h. In contrast, Pearlide was nearly inactive, even at a dose of 100 µM after 20 h of culture, and only reduced cell viability to 41.2% after 72 h. After treatment at 42°C without culture, neither of the aromatics was effective, even at a dose of 200 µM. The viability of cells cultured with Tonalide for 20 h after treatment at 42°C was reduced to nearly half of that at 37°C, and to 10% of the control after culture for 72 h. These values for the reduction of cell viability were also acheived by the Trypan blue dye-exclusion assay. The lifespan-prolonging effects of Tonalide on mice implanted with EAT cells were also examined. The lifespan of mice administrated 1.8 mg/day of Tonalide was prolonged by up to 28 days, while mice that did not receive Tonalide died within a mean of 15 days. Thus, Tonalide exhibited marked carcinostatic effects in vitro and in vivo and may prove to be a potent multipurpose antitumor agent in combination with hyperthermia.