Trihydroxylated linear diterpenes from the brown alga Bifurcaria bifurcata

Two novel polar diterpenes were isolated from the brown alga Bifurcaria bifurcata collected off the Atlantic coast of Morocco, and their structures established by spectral methods. Both compounds are trihydroxylated acyclic diterpenes derived from 12-hydroxygeranylgeraniol. They were tested in vitro for their cytotoxicity and proved to be active against the NSCLC-N6 cell line. Their absolute configuration at the C-12 position has been determined with a modified Mosher's method [J. Am. Chem. Soc. 113 (1991) 4092] and that of the 12-hydroxygeranylgeraniol (bifurcadiol) has been revised.     

Preliminary study of the in vitro antiproliferative effect of a hydroethanolic extract from the subtropical seaweed Turbinaria ornata (Turner) J. Agardh on a human non-small-cell bronchopulmonary carcinoma line (NSCLC-N6)

An unusual sulfated fucan-like polysaccharidecontaining aminosugar constituent was isolated fromthe brown Fucales, Turbinaria ornata. Itsantiproliferative effect on asynchronous cells of ahuman non-small-cell bronchopulmonary carcinoma line(NSCLC-N6) was studied in vitro. The kinetics ofcell growth further to the continuous exposure ofcells to T. ornata extract was investigated andthe changes in the cell cycle were observed usingflow-cytometry. Cell growth appeared to be inhibitedin the G1 phase. Altogether our observations suggesta triggering of the terminal differentiation ofcancerous cells by this fucan-like polysaccharide.     

Bistramide A-induced irreversible arrest of cell proliferation in a non-small-cell bronchopulmonary carcinoma is similar to induction of terminal maturation

Summary— Bistramide A, a new toxin isolated from a New Caledonian Urochordata, shows an antiproliferative effect on a non-small-cell lung carcinoma line in vitro and G1-blockade. In this work, the growth arrest induced by bistramide A was shown to be irreversible as assessed by growth kinetics of pretreated cells. Furthermore, the drug caused an underexpression of the nuclear antigen Ki67. These events are similar to a G1-differentiation cell cycle step blockage and a terminal maturation induction.     

Seasonal variation in antifouling activity of crude extracts of the brown alga Bifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanus amphitrite and the marine bacteria Cobetia marina and Pseudoalteromonas haloplanktis

Previous studies have demonstrated that macroalgae from Brittany (France) contain products with antifouling activity against marine bacteria, fungi, diatoms, seaweeds and mussels. Little is known regarding the ecological function of these compounds and insufficient attention has been paid to evaluating the possible temporal variation in antifouling activity. Studies of chemical defenses in both terrestrial and marine organisms suggest that organisms vary widely in the production of chemical defenses associated with physical (temperature, light) and biological (e.g. grazing pressure) factors, season and geographical location. The present study aimed to investigate the antifouling activity of crude extracts of monthly collections of the brown alga, Bifurcaria bifurcata, against two marine bacteria, Cobetia marina and Pseudoalteromonas haloplanktis, and cypris larvae of the barnacle, Balanus amphitrite. The toxicity of the extracts was determined with a B. amphitrite nauplius assay.The antimicrobial activity of the extracts was found to be subject to seasonal variation, with the highest level of activity recorded from samples collected between April and September. Results of the anti-settlement experiments showed that the extracts of B. bifurcata (when tested from 0 to 100 μg/ml) can be divided into three groups on the basis of their minimum inhibitory concentrations (MICs): (1) extracts from plants collected from September to March reduced settlement at nontoxic concentrations (50-100 μg/ml); (2) extracts from plants collected from April to July (which were the most active extracts) reduced settlement significantly when tested at >5 μg/ml, but were toxic at 100 μg/ml; (3) the extract prepared from plants harvested in August was inhibitory at >25 μg/ml, but was toxic at 100 μg/ml. Toxicity tests on nauplii showed that LC₅₀ values of samples from the September to March collections were >100 μg/ml, demonstrating that they were nontoxic to nauplii. In contrast, samples obtained from the April to August collections were toxic to nauplii; the most toxic ones being from algae collected in May (LC₅₀=55.6 μg/ml) and in June (LC₅₀=38.3 μg/ml).The antifouling activity of extracts thus reached a peak in summer corresponding to maximal values for water temperature, light intensity and fouling pressure. It remains to be investigated whether this activity has an ecological role in the alga.     

Ganoderic acid produced from submerged culture of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> induces cell cycle arrest and cytotoxicity in human hepatoma cell line BEL7402

Ganoderic acid (GA), produced by submerged culture of Ganoderma lucidum, at 500 μg/ml, caused nearly a 70% inhibition of the growth of human hepatoma cell line BEL7402 but not of a normal human liver cell line L02. Flow cytometry analyses showed that GA blocked the BEL7402 cell cycle at the transition from G₁ to S phase.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.     

Phototactic responses in the gametes of the brown alga,Ectocarpus siliculosus

The action spectrum of phototaxis was determined and the photoreceptive mechanism was studied in Ectocarpus gametes (Ectocarpales, Phaeophyceae) using a computerized cell-tracking system. The fine structures of the stigma and the flagellar swelling were analyzed, and the reflective function of the stigma was demonstrated for the first time. Under monochromatic light stimulation, Ectocarpus gametes show mainly positive phototaxis between 370 nm and 520 nm. The action spectrum has a minor peak near 380 nm, and two major peaks at 430 nm and 450 nm or 460 nm and a shoulder at 470 nm adjoining a remarkable depression near 440 nm. Under unilateral stroboscopic illumination with more than four pulses per second, the gametes show clear phototaxis. However, the response is disturbed at lower frequencies. Addition of methyl cellulose, which increases the viscosity of the medium and slows down gamete rotation, decreases the threshold frequency. These results indicate that rotation of the gamete plays an essential role in the photoreceptive mechanism. Under equal intensities of bilateral illumination at an angle of 90°, most of the gametes swim on the resultant between the two light beams. This response is disturbed when the angle of the two light beams is as large as 120°. Observations by transmission electron microscopy show that the flagellar swelling fits precisely into a concave depression of the chloroplast at the central region of the stigma. Electron-dense material is present in that sector of the flagellar swelling which faces away from the stigma. Epifluorescence microscopy without a barrier filter and epipolarization microscopy reveal that stigmata reflect blue light. A hypothesis is formulated which discusses the possibility that the reflected light is focused onto the flagellar swelling.We are grateful to Jochen Schäfer and Elke Reinecke for their technical assistance and Dr. G. Konerman for access to epipolarization microscopy. We are also grateful to the Alexander von Humboldt Foundation for a Research Fellowship to H.K. and the Deutsche Forschungsgemeinschaft (University of Freiburg, Freiburg, FRG) for financial aid to D.-P.H.To whom correspondence should be addressed.     

The antibacterial effect of a polyhydroxylated fucophlorethol from the marine brown alga,Fucus vesiculosus

An antibacterial compound was isolated from the brown alga Fucus vesiculosus. Purification consisted of extraction of plant material with 0.05% trifluoroacetic acid, concentration on a C₁₈ cartridge, and reverse-phase high performance liquid chromatography on a C₁₈ semi-preparative column. The isolated compound exhibited antibacterial activity against both the Gram-positive and the Gram-negative bacteria tested. Killing studies conducted indicated that the activity was bactericidal. The compound showed no haemolytic effect against human red blood cells. Results obtained by electrospray ionization mass spectrometry indicated that the antibacterial activity was caused by a polyhydroxylated fucophlorethol.     

Investigation of the antifouling constituents from the brown alga Sargassum muticum (Yendo) Fensholt

One of the most promising alternatives to toxic heavy metal-based paints is offered by the development of antifouling coatings in which the active ingredients are compounds naturally occurring in marine organisms and operating as natural antisettlement agents. Sessile marine macroalgae are remarkably free from settlement by fouling organisms. They produce a wide variety of chemically active metabolites in their surroundings, potentially as an aid to protect themselves against other settling organisms. In this study, a dichloromethane extract from the brown seaweed Sargassum muticum was tested in situ and, after 2 months of immersion, showed less fouling organisms on paints in which the extract was included, compared to paints containing only copper after 2 months of immersion. No barnacles or mussels have been observed on the test rack. Identification by NMR and GC/MS of the effective compound revealed the abundance of palmitic acid, a commonly found fatty acid. Pure palmitic acid showed antibacterial activity at 44 μg mL⁻¹, and also inhibited the growth of the diatom Cylindrotheca closterium at low concentration (EC₅₀ = 45.5 μg mL⁻¹), and the germination of Ulva lactuca spores at 3 μg mL⁻¹. No cytotoxicity was highlighted, which is promising in the aim of the development of an environmentally friendly antifouling paint.     

Cytotoxic effect of Erythroxylum daphnites extract is associated with G1 cell cycle arrest and apoptosis in oral squamous cell carcinoma

Plant-derived molecules showing antineoplastic effects have recently gained increased attention as potential adjuvants to traditional therapies for various cancers. Cerrado biome in Brazil contains high floral biodiversity, but knowledge about the potential therapeutic effects of compounds derived from that flora is still limited. The present study investigated the antineoplastic activity of Erythroxylum daphnites Mart., a Brazilian native plant from Cerrado biome, in the SCC-9 oral squamous cell carcinoma cell line. Cells were treated with various concentrations of hexane extract of Erythroxylum daphnites leaves (EDH) and assessed for cytotoxicity, proliferation, and apoptosis. Thin layer chromatography was conducted to characterize the substances present in EDH. Our results showed that EDH exerted anti-proliferative effects in SCC-9 cells by stabilizing the cell cycle at G₁ phase in association with reduced intracellular levels of cyclins D and E and increased level of p21. EDH also demonstrated pro-apoptotic properties, as shown by an increased expression of caspase-3. Triterpenes were the major constituents of EDH. Our findings demonstrated a cytotoxic effect of EDH against SCC-9 cells in vitro mediated by the restraint of cellular proliferation and induction of apoptosis. Taken together, these findings support EDH constituents as potential therapeutic adjuvants for oral cancer.     

Fish cell culture: Characteristics of a cell line from the silver perch,Bairdiella chrysura

Summary A cell line designated SP-1 was established from tissue of the silver perch,Bairdiella chrysura. Cells were fibroblast-like and grew best at 26°C in Leibovitz medium (L-15) containing 15% fetal bovine serum and 0.150m sodium chloride. Passage 1 to passage 9 SP-1 cells contained a chromosome number of 48; at passages 27 and 50 the modal numbers were 51 and 54, respectively. Confirmation of the origin of SP-1 cells was made by the cytotoxic antibody dye-exclusion test. This cell line supported the growth of lymphocystis virus from the silver perch but was not found to replicate various other fish and mammalian viruses. This study was supported in part by fund supplied by the Faculty Research Council of the University of Southern Mississippi and by the National Oceanographic and Atmospheric Administration, Office of Sea Grant No. 04-3-158-58. A portion of these results were presented at the 26th Annual Meeting of the Tissue Culture Association, Motreal, Canada, 1975.     

Nonhomologous end-joining in a cell-free extract from the cultured silkworm cell line BmN4

Nonhomologous end-joining (NHEJ) is one of the repair pathways for double-strand breaks (DSBs) in eukaryotic cells. By using linearized plasmid substrates, we have detected intramolecular NHEJ activity in a cell-free extract from the cultured silkworm cell line BmN4. The efficiency of NHEJ differed according to the structure of DNA ends; approximately 1% of input DNA was repaired when the substrate had cohesive ends. The reaction required the hydrolysis of nucleotide triphosphate; interestingly, all of four rNTPs or four dNTPs could support the reaction. A substrate with non-complementary DNA ends was mainly repaired by the DNA polymerase-mediated pathway. These results indicate that the present cell-free system will be useful to analyze the molecular mechanisms of DSB repair and NHEJ in insect cells.     

Cimicifuga racemosa extract BNO 1055 inhibits proliferation of the human prostate cancer cell line LNCaP

Extracts from black cohosh (Cimicifuga racemosa, CR) exert an anti-proliferative action in human breast cancer cell cultures, which has been attributed to an anti-estrogenic effect. However, CR constituents do not bind to either of the known estrogen receptors. Thus, the anti-tumor effect of CR me be mediated by mechanisms not involving these receptors. Polycyclic aromatic hydrocarbons are toxic environmental pollutants, which indirectly act as anti-estrogens by activating the aryl hydrocarbon receptor (AhR). The AhR is widely expressed in mammalian tissues and tumors. A recent screening study demonstrated activation of the AhR by a variety of herbal extracts, among others, CR. Since activation of the AhR causes inhibition of growth of prostate cancer cells, we addressed the question, whether CR may not only inhibit growth of breast cancer--but also of prostate cancer cells. In the AhR ligand assay, the CR extract BNO 1055 reduced tracer binding to 71% of the control demonstrating interaction of constituents of this extract with the receptor. Under basal as well as under estradiol- and dihydrotestosterone stimulated conditions, the CR extract dose dependently inhibited proliferation of LNCaP cells. A significant reduction of cell growth was observed at a concentration as low as 50 ng/ml. Thus, it is demonstrated for the first time that CR compounds potently inhibit the growth of human prostate cancer cells in vitro. This anti-proliferative effect may be mediated via the AhR.     

A role of activated Sonic hedgehog signaling for the cellular proliferation of oral squamous cell carcinoma cell line

Sonic hedgehog (Shh) is a secreted morphogen crucial for appropriate cellular proliferation during mammalian development. The activated Shh signaling is known to predispose to human tumors such as medulloblastoma and basal cell carcinoma, while a role of Shh signaling in the other common tumors is still controversial. Here we showed the overexpression of Shh in five cell lines among 14 human oral squamous cell carcinoma (OSCC) cell lines. One of the Shh-expressing OSCC cell lines HSQ-89 showed the inhibition of G1/S transition and apoptotic cell death by treatment with Cyclopamine, a steroidal alkaloid that blocks the intracellular Shh signaling. Furthermore, we found that treatment with Y-27632, a specific inhibitor of Rho-associated kinase, mimicked the effect of Cyclopamine on the cell cycle progression of HSQ-89. Our study revealed the involvement of activated Shh signaling in the cellular proliferation of OSCC cells, indicating Shh signaling might be a good therapeutic target for OSCC.     

PSPH induces cell autophagy and promotes cell proliferation and invasion in the hepatocellular carcinoma cell line Huh7 via the AMPK/mTOR/ULK1 signaling pathway

Phosphoserine phosphatase (PSPH), a key enzyme of the l -serine synthesis pathway, has been involved in cancer progression and survival. However, limited evidence revealed the PSPH influence on hepatocellular carcinoma (HCC). Herein, we observed that PSPH expression was upregulated in both HCC tissues and cell lines, which was determined by western blotting. TCGA database showed that the PSPH protein levels were significantly upregulated and affected patient survival rates in HCC. Then gain- and loss-of-function manipulations were performed by transfection with a pcDNA-PSPH expression vector or a specific short interfering RNA against PSPH in Huh7 cells. Huh7 cell proliferation, stemness, invasion, and apoptosis were assessed by using CCK-8 test, colony formation assay, Transwell assay, and Flow cytometry analysis, respectively, and levels of autophagy-related proteins were detected by using western blotting. The results showed that PSPH could induce Huh7 cell autophagy, promote cell proliferation and invasion, and inhibit apoptosis. The knockdown of PSPH could inhibit Huh7 cell proliferation, invasion, and autophagy. Furthermore, PSPH activated Liver kinase B1 (LKB1) and TGF beta-activated kinase 1 (TAK1), affected the adenosine 5′-monophosphate-activated protein kinase (AMPK)/mTOR/ULK1 signaling pathway, but could not activate calcium/calmodulin-dependent protein kinase kinase (CaMKK) in Huh7 cells. Inhibition of either LKB1, TAK1, or AMPK could eliminate the effect of PSPH overexpression on Huh7 cell behaviors. However, inhibition of CaMKK could not influence the effect of PSPH overexpression on Huh7 cell behaviors. In conclusion, PSPH could induce autophagy, promote proliferation and invasion, and inhibit apoptosis in HCC cells via the AMPK/mTOR/ULK1 signaling pathway.     

Induction of vesicle formation in a cell line derived from imaginal discs

Summary The ability of insect hemolymph to induce vesicles in a high passage insect cell line, IAL-TND1, is described. The factor responsible, designated VPA for ‘vesicle-promoting activity’, was determined to be heat sensitive, nondialyzable, and protease Type XIV sensitive but insensitive to trypsin digestion. In efforts to determine the source of VPA, hemolymph was collected from different developmental stages ofTrichoplusia ni, and certain tissues fromT. ni were cocultured with IAL-TND1 cells. Hemolymph from every developmental stage tested exhibited VPA although the effect was somewhat reduced in spinning-stage larvae. Additionally, several tissue, including fat body, tess, and imaginal discs, released VPA into the culture medium. Neural tissues and endocrine glands did not induce vesicle formation.     

An established cell line from the beetle, Xylotrechus pyrrhoderus (coleoptera: Cerambycidae)

Summary A continuous cell line has been established from larval fat body tissues of the cerambycid beetle Xylotrechus pyrrhoderus Bates. These cells were cultured in MGM-450 medium. The cell line, designated as XP-1, showed a heterogeneous population consisting of spherical and spindle-shaped cells with some capacity to adhere and a doubling time of 5 d. The chromosome number of the cell line ranged from 18 to 42 with a mode of 20. Isozyme analysis showed that the cells had patterns distinctive from those of other insect cell lines. The cells were sensitive to insect hormones, and when continuously treated with 20-hydroxyecdysone and juvenile hormone, they assumed a floating elongated-spindle shape and became strongly adherent, respectively.     

Establishment and characterization of a cell line from the american opossum (Didelphys virginiana)

Summary A permanent tissue-cultured cell line (designated OK) has been established from kidney tissue of an adult American opossum. The OK line has been characterized with respect to morphology, chromosome constitution, tissue-culture requirements, and attainable mitotic arrest. The cells are epithelial-like with a stable nondiploid chromosomal modal number of 23. Cells grown in Eagle's minimal essential medium with 10% fetal bovine serum have a mean doubling time of 18 hr. The cell line OK is potentially useful for the isolation and purification of the mammalian X chromosome because of the size differential between the smaller X's and the larger autosomes. This work was supported by NIH grant HD-04420.     

Protocatechuic acid induces cell death in HepG2 hepatocellular carcinoma cells through a c-Jun N-terminal kinase-dependent mechanism

Protocatechuic acid (PCA), chlorogenic acid (CA) and luteolin (LT) are plant phenols found in Chinese medicinal herbs such as Lonicera japonica. Cytotoxicity assays showed that PCA, CA and LT (at 100 μmol/L) effectively killed the HepG2 hepatocellular carcinoma cells. Among these three naturally occurring compounds, only PCA was capable of stimulating the c-Jun N-terminal kinase (JNK) and p38 subgroups of the mitogen-activated protein kinase (MAPK) family. Coincidently, PCA-induced cell death was rescued by specific inhibitors for JNK and p38, while the cytotoxicities of CA and LT were partially eliminated by the antioxidant effect of N-acetyl-L-cysteine (NAC). Further investigation demonstrated that the aqueous extract of Lonicera japonica also triggered HepG2 cell death in a JNK-dependent manner, but the amount of PCA alone in this herbal extract was insufficient to contribute the subsequent cytotoxic effect. Collectively, our results suggest that PCA is a naturally occurring compound capable of inducing JNK-dependent hepatocellular carcinoma cell death. An erratum to this article is available at.     

Establishment and characterization of a cell line derived from a human serous surface papillary carcinoma of the ovary

A novel serous surface papillary carcinoma of the ovary (SSPC) cell line, HYKSSPC, was established successfully. Carcinoma cells were obtained from ascitic fluid of a 60-year-old Japanese woman. The population doubling time was 51.4 h. A phase contrast micrograph showed a pavement stone-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy, and the mode was in 46-47. An immunocytochemical study showed that CA125, BerER4 and cytokeratin were positive and that CEA, calretinin and thrombomodulin were negative. This cell line preserved some characters of the adenocarcinoma while growing in vitro. A chemosensitivity test revealed that HYKSSPC cells were sensitive to CDDP (cis-platinum), 5-fluorouracil, mitomycin C, paclitaxel and irinotecan. To our knowledge, HYKSSPC is the first established cell line derived from SSPC, and it may offer some useful information for investigating this disease.