Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth,photosynthetic efficiency and toxin production

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed “starvation-feeding-dark”, was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.     

Caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis

Phosphorylation and dephosphorylation are important cellular events regulating major metabolic activities such as signal transduction, gene expression, cell cycle progression, and apoptosis. It is well documented that okadaic acid, a potent inhibitor of protein phosphatase-1 (PP-1) and -2A (PP-2A), can induce apoptosis in a variety of cell lines. Our recent studies have revealed that in the immortal rabbit lens epithelial cell line, N/N1003A, inhibition of PP-1, but not PP-2A, leads to rapid apoptosis of the lens epithelial cells. This induction of cell death is associated with up-regulated expression of a set of genes, including the tumor-suppressor gene, p53, and the proapoptotic gene, bax. In the present study, we demonstrate that inhibition of PP-1 by okadaic acid in the primary cultures of rat lens epithelial cells also leads to apoptotic death. Moreover, we show that the cysteine protease, caspase-3, is important in the execution of okadaic acid-induced apoptosis. Treatment of the primary cultures of rat lens epithelial cells with 100 nM okadaic acid up-regulates expression of caspase-3 at the mRNA, protein, and enzyme activity levels. Inhibition of the caspase-3 activity with a chemically synthesized inhibitor prevents okadaic acid-induced apoptosis in rat lens epithelial cells. Similar results are also observed in the immortal cell line N/N1003A. Furthermore, stable expression of the mouse gene encoding lens alphaB crystallin inhibits okadaic acid-induced apoptosis, and this inhibition is associated with repression of the okadaic acid-induced up-regulation of caspase-3 activity. Taken together, these results demonstrate that caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis.     

Evaluation of the use of two human cell lines for okadaic acid and DTX-1 determination by cytotoxicity assays and damage characterization.

Two human cell lines have been used, HEp-2 and (de)differentiated Caco-2, derived from a larynx and a colon carcinoma, respectively, with the aim of evaluating and characterizing the cytotoxicity of okadaic acid (OA) and related toxins. Effects of OA and dinophysistoxin-1 (DTX-1) on cell viability (neutral red uptake) and on cell morphology/cytoskeleton structure have been observed in both cell lines, though at different time exposures and with different concentrations. The morphological alteration was detected earlier than the viability inhibition in HEp-2 cells with both toxins and in Caco-2 cells with DTX-1. HEp-2 cells have shown to be more sensitive than the intestinal cell line and thus possibly suitable for screening of contaminated samples, while Caco-2 cells could be used for further investigating the possible mechanisms involved in diarrhoeic shellfish poisoning (DSP) toxins.     

The Formation of 2,6-Dihydroxy-phenylacetic Acid from Phenylacetic Acid by Various Fungi

Two principal toxins of diarrhetic shellfish poisoning, okadaic acid and dinophysistoxin-1, were esterified with 9-anthryldiazomethane in methanol. After cleaning with a Sep-pak silica cartridge column, the fluorescent esters were analyzed on a Develosil ODS column with MeCN- MeOH-H₂0 (8:1:1). The fluorescence intensities of both toxin derivatives measured at an excitation of 365 nm and an emission of 412 nm showed good linearity in the range 1 ~ 80ng.     

Design,synthesis, and screening of ortho-amino thiophene carboxamide derivatives on hepatocellular carcinomaas VEGFR-2Inhibitors

In this work, design, synthesis, and screening of thiophene carboxamides 4–13 and 16–23 as dual vascular endothelial growth factor receptors (VEGFRs) and mitotic inhibitors was reported. All compounds were screened against two gastrointestinal solid cancer cells, HepG-2 and HCT-116 cell lines. The most active cytotoxic derivatives 5 and 21 displayed 2.3- and 1.7-fold higher cytotoxicity than Sorafenib against HepG-2 cells. Cell cycle and apoptosis analyses for compounds 5 and 21 showed cells accumulation in the sub-G1 phase, and cell cycle arrest at G2/M phase. The apoptotic inducing activities of compounds 5 and 21were correlated to the elevation of p53, increase in Bax/Bcl-2 ratio, and increase in caspase-3/7.Compounds 5 and 21 showed potent inhibition againstVEGFR-2 (IC₅₀?=?0.59 and 1.29?μM) and β-tubulin polymerization (73% and 86% inhibition at their IC₅₀ values).Molecular docking was performed with VEGFR-2 and tubulin binding sites to explain the displayed inhibitory activities.     

Pro-apoptotic Sorafenib signaling in murine hepatocytes depends on malignancy and is associated with PUMA expression in vitro and in vivo

The multi-kinase inhibitor Sorafenib increases the survival of patients with advanced hepatocellular carcinoma (HCC). Current data suggest that Sorafenib inhibits cellular proliferation and angiogenesis and promotes apoptosis. However, the underlying pro-apoptotic molecular mechanisms are incompletely understood. Here we compared the pro-apoptotic and anti-proliferative properties of Sorafenib in murine hepatoma cells and syngeneic healthy hepatocytes in vitro and in animal models of HCC and liver regeneration in vivo. In vitro, we demonstrate that cell cycle activity and expression of anti-apoptotic Bcl-2 like proteins are similarly downregulated by Sorafenib in Hepa1-6 hepatoma cells and in syngeneic primary hepatocytes. However, Sorafenib-mediated activation of caspase-3 and induction of apoptosis were exclusively found in hepatoma cells, but not in matching primary hepatocytes. We validated these findings in vivo by applying an isograft HCC transplantation model and partial hepatectomy (PH) in C57BL/6 mice. Sorafenib treatment activated caspase-3 and thus apoptosis selectively in small tumor foci that originated from implanted Hepa1-6 cells but not in surrounding healthy hepatocytes. Similarly, Sorafenib did not induce apoptosis after PH. However, Sorafenib treatment transiently inhibited cell cycle progression and resulted in mitotic catastrophe and enhanced non-apoptotic liver injury during regeneration. Importantly, Sorafenib-mediated apoptosis in hepatoma cells was associated with the expression of p53-upregulated-modulator-of-apoptosis (PUMA). In contrast, regenerating livers after PH revealed downregulation of PUMA and were completely protected from Sorafenib-mediated apoptosis. We conclude that Sorafenib induces apoptosis selectively in hepatoma cells but not in healthy hepatocytes and can additionally increase non-apoptotic hepatocyte injury in the regenerating liver.     

IRF-1 is an essential mediator in IFN-gamma-induced cell cycle arrest and apoptosis of primary cultured hepatocytes

IFN-gamma induces cell cycle arrest and p53-independent apoptosis in primary cultured hepatocytes. However, it is not yet understood what molecules regulate the mechanism. We report here that interferon regulatory factor 1 (IRF-1) is an essential molecule in these phenomena. Hepatocytes from IRF-1-deficient mice were completely resistant to IFN-gamma in apoptosis indicated by three different hallmarks such as LDH release, DNA fragmentation and the activation of caspase-3 family. Caspase-1 expression was little detected in hepatocytes, and constitutive and IFN-gamma-induced mRNA expression of Fas or caspase-3 did not change in between wild type and IRF-1-deficient hepatocytes. Expression of IFN-gamma-inducible caspase, caspase-11, did not change either. Thus, it is unlikely that these molecules directly regulate the mechanisms. Interestingly, IRF-1-deficient hepatocytes were also resistant to IFN-gamma-induced cell cycle arrest despite IFN-gamma-induced cell cycle arrest and apoptosis are regulated by independent pathways. Results by Northern blot analysis showed that IFN-gamma-induced but not constitutive p53 mRNA expression was regulated by IRF-1. In fact, IFN-gamma did not induce cell cycle arrest in p53-deficient hepatocytes. Taken together, IRF-1 mediates IFN-gamma signaling into primary hepatocytes for cell cycle arrest via p53 expression and for apoptosis.     

Determination of diarrhetic shellfish toxins in various dinoflagellate species

Sixteen species of unialgal samples of dinoflagellate, either wild or cultured, were tested for production of diarrhetic shellfish toxins such as okadaic acid (OA), dinophysistoxin-1 (DTX1), and pectenotoxins (PTXs). Determination of micro-quantities of the toxins was facilitated by fluorometry and UV HPLC. Seven Dinophysis species were confirmed to produce either OA or DTX1, or both. Toxin content and composition varied regionally and seasonally. Intraspecies variation was also observed among cultured strains of Prorocentrum lima. PTX2 was the only toxin detected among PTX family, and D. fortii was the only species to contain this toxin. author for correspondence     

Production of dinophysistoxin-1 and pectenotoxin-2 by a culture of Dinophysis acuminata (Dinophyceae)

The production of diarrhetic shellfish poisoning toxins (okadaic acid analogues and other lipophilic toxins) by a culture of Dinophysis acuminata, fed with the autotrophic ciliate Myrionecta rubra, was confirmed by LC–MS analysis, and the toxin profile compared with that in the field assemblage of the same species. The growth response of D. acuminata to the density of the food organism was also examined in laboratory experiments. In semi-continuous culture experiments, the growth rates of D. acuminata increased with increasing density of M. rubra and a maximum growth rate of 0.67 per day was calculated. In batch culture experiments; the cellular content of PTX2 and DTX1 were 14.7–14.8 and 2.5–4.8 pg cell^?1, respectively. Okadaic acid, dinophysistoxin-3, pectenotoxin-1, pectenotoxin-6, yessotoxin (YTX) and 45-OHYTX were not detected. PTX2 was detected (cellular toxin content: 22 pg cell^?1), but DTX1 was not detected, in an extract of D. acuminata collected from natural seawater at the same location where the cultured D. acuminata specimens were isolated. These results strongly suggest that D. acuminata produces these toxins during cell growth and that environmental factors influence variations in the toxin composition and specific cellular toxicity.     

Detection of diarrhetic shellfish poisoning toxins from tropical shellfish using liquid chromatography-selected reaction monitoring mass spectrometry

A negative mode liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) method was developed to detect low concentrations of the diarrhetic shellfish poisoning (DSP) toxins okadaic acid and dinophysistoxin-1 (DTX-1). Detection relies upon monitoring the transition of negative precursor ions [M - H]- to a common fragment ion of m/z 255. Our limit of detection for okadaic acid with this method is 0.5 pg on column. LC-SRM MS has allowed us to detect persistent, low concentrations of DSP toxins from Singapore shellfish.     

Inhibition of protein phosphatases-1 and -2A with acanthifolicin: Comparison with diarrhetic shellfish toxins and identification of a region on okadaic acid important for phosphatase inhibition

Acanthifolicin (9,10-epithio-okadaic acid from Pandoras acanthifolium) inhibited protein phosphatase-1 (PP1) similarly to okadaic acid (IC₅₀ = 20 nM and 19 nM, respectively) but was slightly less active against protein phosphatase-2A (PP2A) (IC₅₀ 1 nM and 0.2 nM, respectively). Methyl esterification of acanthifolicin sharply reduced its activity. PP2A was inhibited with an IC₅₀ = 5.0 μM, whilst PP1 was inhibited < 10% at 250 μM toxin. Okadaic acid methyl ester was similarly inactive whereas dinophysistoxin-1 (35-methyl okadaic acid) inhibited PP1/2A almost as potently as okadaic acid. Pure acanthifolicin/okadaic acid methyl ester may be useful as specific inhibitors of PP2A at 1–10 μM concentrations in vitro and perhaps in vivo. The data also indicate that a region on these toxins important for PP1/2A inhibition comprises the single carboxyl group.     

Prevention of toxin-induced cytoskeletal disruption and apoptotic liver cell death by the grapefruit flavonoid, naringin

The protein phosphatase-inhibitory algal toxins, okadaic acid and microcystin-LR, induced overphosphorylation of keratin and disruption of the keratin cytoskeleton in freshly isolated rat hepatocytes. In hepatocyte cultures, the toxins elicited DNA fragmentation and apoptotic cell death within 24 h. All these toxin effects could be prevented by the grapefruit flavonoid, naringin. The cytoprotective effect of naringin was apparently limited to normal hepatocytes, since the toxin-induced apoptosis of hepatoma cells, rat or human, was not prevented by the flavonoid.     

Okadaic acid co-induces vimentin expression and cell cycle arrest in MPC-11 mouse plasmacytoma cells

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC-11 mouse plasmacytoma cells was compared with that of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell cycle progression of asynchronously grown MPC-11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA-caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid-treated relative to TPA-treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 × 10^?9M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC-11 cells by okadaic acid. © 1995 Wiley-Liss, Inc.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse)(159.3 ± 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse)(906 ± 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Antiproliferative activity of rosamultic acid is associated with induction of apoptosis,cell cycle arrest,inhibition of cell migration and caspase activation in human gastric cancer (SGC-7901) cells

BackgroundGastric cancer is the second leading cause of cancer related deaths after lung cancer globally. Among natural products, natural triterpenes represent a structurally diverse group of organic compounds with potent antitumor activity.PurposeThe objective of the present research work demonstrated the antiproliferative and apoptotic activity of rosamultic acid, a natural triterpenoid, in human gastric cancer (SGC-7901) cells. Its effect on cellular morphology, cell cycle arrest, DNA fragmentation and expression levels of caspase-3, caspase-8 and caspase-9 were also determined.MethodsAntiproliferative activity of rosamultic acid was evaluated by MTT assay. Phase contrast, fluorescence microscopy as well as flow cytometry using Hoechst 33342, acridine orange/ethidium bromide and Annexin V-FITC as cellular probes were used to evaluate the induction of apoptosis by rosamultic acid. Protein level expressions were analyzed by western blot analysis.ResultsThe results revealed that rosamultic acid induced dose-dependent as well as time dependent cytotoxic effects in SGC-7901 gastric cancer cells. It also led to a reduction in clonogenic activity along with inhibiting the cell migration. Characteristic features of apoptosis induced by rosamultic acid were observed and quantified. Cell cycle arrest at sub-G1 phase was induced by rosamultic acid along with downregulation of expression levels of CDK4, CDK6 and cyclin D1. Rosamultic acid also significantly led to the activation of caspase-3, -8 and -9 during the 48 h treatment along with cleaving PARP in a dose-dependent manner. DNA fragmentation following rosamultic acid treatment was also observed in these cells.ConclusionThe current study strongly reveals that rosamultic acid inhibits gastric cancer proliferation by inducing apoptosis mediated through cell cycle arrest, downregulation of cell cycle related protein expressions, inhibition of cell migration, DNA damage, and activation of caspases.     

Chemical hypoxia induces pro-inflammatory signals in fat-laden hepatocytes and contributes to cellular crosstalk with Kupffer cells through extracellular vesicles

BackgroundObstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD.MethodsPrimary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo.ResultsHypoxia induced HIF-1α in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1β, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1.ConclusionHypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD. [Abstract word count: 257].     

Occurrence of Prorocentrum lima,a DSP toxin-producing species from the Atlantic coast of Canada

A species of Prorocentrum (Dinophyta, Prorocentrales), isolated from a phytoplankton net sample from the Atlantic coast of Nova Scotia, has been brought into unialgal culture. The sample was collected at an aquaculture site immediately following an incident of diarrhetic shellfish poisoning (DSP) due to the consumption of contaminated mussels. This clonal isolate has been identified as P. lima, based on its morphological characteristics. Analysis of the culture extract, using high performance liquid chromatography (HPLC) with fluorescence detection, indicated the presence of the DSP toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1).     

Differential regulation of the JNK/AP-1 pathway by S-adenosylmethionine and methylthioadenosine in primary rat hepatocytes versus HuH7 hepatoma cells

S-adenosylmethionine (AdoMet) and 5'-methylthioadenosine (MTA) exert a protective action on apoptosis induced by okadaic acid in primary rat hepatocytes but not in human transformed HuH7 cells. In the present work, we analyzed the role played by the JNK/activator protein (AP)-1 pathway in this differential effect. Okadaic acid induced the phosphorylation of JNK and c-Jun and the binding activity of AP-1 in primary hepatocytes, and pretreatment with either AdoMet or MTA prevented those effects. In HuH7 cells, pretreatment with either AdoMet or MTA did not affect JNK and c-Jun activation or AP-1 binding induced by okadaic acid. In both cell types, p38 was activated by okadaic acid, but neither AdoMet nor MTA presented a significant effect on its activity. Therefore, the differential effect of both AdoMet and MTA on the JNK/AP-1 pathway could explain their antiapoptotic effect on primary hepatocytes and the lack of protection they show against okadaic acid-induced apoptosis in hepatoma cells.     

Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells

Combretastatin A-4 (CA-4), a natural stilbenoid isolated from Combretum caffrum, is a new vascular targeting agent (VTA) known for its antitumor activity due to its anti-tubulin properties. We investigated the molecular mechanisms leading to cell death in non-small cell lung cancer H460 cells induced by natural (CA-4) and synthetic stilbenoids (ST2151) structurally related to CA-4. We found that both compounds induced depolymerization and rearrangement of spindle microtubules, as well as an increasingly aberrant organization of metaphase chromosomes in a dose- and time-dependent manner. Prolonged exposition to ST2151 led cells to organize multiple sites of tubulin repolymerization, whereas tubulin repolymerization was observed only after CA-4 washout. H460 cells were arrested at a pro-metaphase stage, with condensed chromosomes and a triggered spindle assembly checkpoint, as evaluated by kinetochore localization of Bub1 and Mad1 antibodies. Persistent checkpoint activation led to mitochondrial membrane permeabilization (MMP) alterations, cytochrome c release, activation of caspase-9 and -3, PARP cleavage and DNA fragmentation. On the other hand, caspase-2, and -8 were not activated by the drug treatment. The ability of cells to reassemble tubulin in the presence of an activated checkpoint may be responsible for ST2151-induced multinucleation, a recognized sign of mitotic catastrophe. In conclusion, we believe that discovery of new agents able to trigger mitotic catastrophe cell death as a result of mitotic block and prolonged spindle checkpoint activation is particularly worthwhile, considering that tumor cells have a high proliferative rate and mitotic failure occurs irrespective of p53 status. Electronic Supplementary Material Supplementary material is available in the online version of this article at . Ilio Vitale and Antonio Antoccia contribuited equally to this work.