Cytotoxic effects of DON and T-2 toxin on plant cells

The effect of deoxynivalenol (DON) and T-2 toxin on mitotic index (MI) and relative division rate (RDR) in actively dividing onion (Allium cepa L.) root-tip cells was studied. Both these toxins resulted in decline of mitotic activity which was inversely proportional to the concentrations of these toxins. T-2 was more effective resulting in 59% RDR value at 2.5 ppm whereas DON treated root cells had 78% RDR at the same concentration as compared to respective sets of controls.     

Effects of kinetin riboside on proliferation and proapoptotic activities in human normal and cancer cell lines

Kinetin riboside (KR) is a N6‐substituted derivative of adenosine. It is a natural compound which occurs in the milk of coconuts on the nanomole level. KR was initially shown to selectively inhibit proliferation of cancer cells and induce their apoptosis. We observed that KR inhibited growth (20–80%) of not only human cancer, but also normal cells and that this effect strongly depended on the type of cells. The anti‐apoptotic Bcl‐2 protein was downregulated, while proapoptotic Bax was upregulated in normal as well as in cancer cell lines, upon exposure to KR. Cytochrome c level increased in the cytosol upon treatment of cells with KR. The activity of caspases (ApoFluor®Green Caspase Activity Assay), as well as caspase‐3 (caspase‐3 activation assay) were increased mainly in cancer cells. The expression of procaspase 9 and its active form in the nucleus as well as in cytosol of KR‐treated cells was elevated. In contrast, no effect of KR on caspase 8 expression was noted. The results indicated that non‐malignant cells were less sensitive to KR then their cancer analogs and that KR most likely stimulated apoptosis mechanism of cancer cells through the intrinsic pathway. J. Cell. Biochem. 112: 2115–2124, 2011. © 2011 Wiley‐Liss, Inc.     

Cytotoxic effect of human monocytes to mouse red cells

Human blood monocytes have been cultivated for various lengths of time. Within a few hours they are well spread on the glass and look very similar to peritoneal macrophages in vitro. After about 10 days of cultivation, they develop the capacity to exert cytotoxic effects against mouse red cells in the presence of foetal calf serum. Red cell ghosts as well as abnormal red cells are attached to the macrophages at this point. In the presence of human serum autologous to the monocytes, the rouse red cells are phagocytosed.     

Cytotoxic and mutagenic effects of emodin on cultured mouse carcinoma FM3A cells

Employing a suspension culture of a mouse mammary carcinoma cell line, FM3A cells, the cytotoxicity and induced mutagenicity of emodin (EM) were examined and compared with those of 2-hydroxy-emodin (2-OH-EM), which was identified as an active form of EM in the Ames/microsomes assay. EM was cytotoxic to FM3A cells in concentrations of 1-10 micrograms/ml, and induced 6-thioguanine-resistant (6TGr) mutation. 2-OH-EM was a little more toxic than EM, but induced little mutation.     

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture

Summary FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200μg/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40μg/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.     

Cytotoxic and genotoxic effects of Br-containing oxaphosphole on Allium cepa L. root tip cells and mouse bone marrow cells

The continuous production and release of chemicals into the environment has led to the need to assess their genotoxicity. Numerous organophosphorus compounds with different structures have been synthesized in recent years, and several oxaphosphole derivatives are known to possess biological activity. Such chemical compounds may influence proliferating cells and cause disturbances of the genetic material. In this study, we examined the cytotoxicity and genotoxicity of 4-bromo-N,N-diethyl-5,5-dimethyl-2,5-dihydro-1,2-oxaphosphol-2-amine 2-oxide (Br-oxph). In A. cepa cells, Br-oxph (10(-9) M, 10 (-6) M and 10 (-3) M) reduced the mitotic index 48 h after treatment with the two highest concentrations, with no significant effect at earlier intervals. Mitotic cells showed abnormalities 24 h and 48 h after treatment with the two lowest concentrations but there were no consistent changes in interphase cells. Bone marrow cells from mice treated with Br-oxph (2.82 x 10 (-3) μg/kg) also showed a reduced mitotic index after 48 h and a greater percentage of cells with aberrations (principally chromatid and isochromatid breaks). These findings indicate the cytotoxicity and genotoxicity of Br-oxph in the two systems studied.     

Cytotoxic and cytostatic effects induced by 4-hydroxynonenal in human osteosarcoma cells

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Furthermore, numerous results demonstrate that lipid peroxidation products affect central biochemical pathways and intracellular signalling at physiological concentrations. 4-Hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. This work has focused on the evaluation of HNE nuclear content, so far never directly measured, by electrospray-ionization-mass-spectrometry (ESI/MS) and on the correlation between its concentration and the induced effects after exogenous administration. In a human osteosarcoma cell line (SaOS2), HNE exhibited an early cytotoxic effect characterized by apoptosis, cytostatic and differentiating effects characterized by slow growth, increase in alkaline phosphatase (ALP), and alpha5 integrin subunit content with decrease in tumorigenicity.     

Cytotoxic action of natural killer cells on human tumor cells

Natural killer cell cytotoxicity was studied in a 18-hour 51Cr-release assay in the cultures of human tumor target cells: K562 leukemia and lung adenocarcinoma (LAC) cells. The mean cytotoxic value was similar for K562 and LAC cells: 36.13 +/- 3.23% and 40.78 +/- 3.43%, respectively, although significant individual variability was recorded. The similar cytolytic action of blood mononuclear cells (MNC) on the two tumor lines was observed in 30% of normal donors. MNC from 30% donors produced more pronounced lytic action on K562 cells while MNC from other 30% donors lysed mainly LAC cells. In the competitive inhibition test cold K562 cells more effectively than cold LAC cells suppressed the MNC-induced lysis of both K562 and LAC cells.     

The binding of kinetin to plant ribosomes

The synthetic cytokinins kinetin and 6-benzylaminopurine exhibit equilibrium-type binding to purified chinese-cabbage leaf ribosomes. At 23mum and 4 degrees C one molecule of kinetin and 1.34 molecules of 6-benzylaminopurine are bound per ribosome. Adenine and adenine derivatives that are inactive as cytokinins showed much less affinity for ribosomes. Pretreatment of ribosomes with 0.5m-ammonium chloride or Triton X-100 did not decrease the extent of cytokinin binding. Binding appeared to be to the 83S ribosome species. A positive correlation between the extent of binding and the biological effect of various cytokinin analogues was demonstrated. These results are discussed in terms of cytokinin control of growth processes at the ribosomal level.     

Cytotoxic effects of a decoction of Nigella sativa, Hemidesmus indicus and Smilax glabra on human hepatoma HepG2 cells

A decoction of Nigella sativa seeds, Hemidesmus indicus root and Smilax glabra rhizome is used by traditional medical practitioners in Sri Lanka to treat cancer and has been shown to prevent chemically induced carcinogenesis in rats. The cytotoxicity of the decoction and the individual plant extracts were tested on the human hepatoma HepG2 cell line. The effects of 24 h incubation with different concentrations (0--50 mg/ml) of the extracts on HepG2 cells were determined. Results from MTT and SRB assays, and [(14)C]-leucine and [(3)H]-thymidine uptake demonstrated that the decoction had a strong dose-dependent cytotoxic activity. The greatest inhibitory effects were observed on DNA synthesis with both the decoction (91+/-S.E. 3.7% inhibition) and N. sativa plant extract (88+/-3.8%) even at low concentrations (5 mg/ml). The three individual plant extracts were cytotoxic in the order of potency N. sativa>H. indicus>S. glabra. Flow cytometric analysis using Annexin V and propidium iodide staining showed that after 24 h exposure to the decoction, cells were in the late stage of apoptosis and/or necrosis. Further experiments are worthwhile to determine the anticancer potential of this plant decoction and its components.     

Cytotoxic and apoptotic effects of menadione on rat hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common cancers, which may lead to death. Menadione shows cytotoxic activity thought affecting redox cycling in cancer cells. The aim of the present study was to investigate the effects of menadione on rat hepatocellular carcinoma (H4IIE) cell morphology, cytotoxicity, apoptosis and DNA damage or repair in vitro. Cell morphology evaluated by microscopy and cell viability was determined using the 3-[4,5-dimethylthiazol-2yl]-diphenyltetrazolium bromide test. Apoptotic cell death was assessed in H4IIE cells treated with menadione by 4′,6-diamidino-2-phenylindole staining. Quantitative real time polymerase chain reaction used to determine the expression level of poly (ADP-ribose) polymerase 1 (PARP1) gene. According to the results of this study menadione has got a cytotoxic activity (IC₅₀ 25 µM) and change the cell fate in H4IIE cells. Menadione treatments lead to PARP1 activation in a dose dependent manner and induce DNA damage and apoptosis, and this may suggest its use as a therapeutic agent in HCC treatment.     

Identification of kinetin and kinetin riboside in coconut (Cocos nucifera L.) water using a combined approach of liquid chromatography-tandem mass spectrometry, high performance liquid chromatography and capillary electrophoresis

Kinetin (free base and riboside), which was assumed by many scientists to be a synthetic cytokinin plant growth hormone, has been detected for the first time in the endosperm liquid of fresh young coconut fruits ("coconut water"). To facilitate the study, we developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of kinetin and kinetin riboside in purified coconut water extract sample. Following a solid-phase extraction of cytokinins in coconut water using C18 columns, the samples were further purified by Oasis MCX columns and analyzed by LC-MS/MS for kinetin and kinetin riboside. Detection by mass spectrometry was carried out using selected reaction monitoring (SRM) mode, by identifying the putative kinetin and kinetin riboside based on their characteristic fragments. Based on a signal-to-noise ratio of 3, the limits of detection in SRM mode were 0.02 microM and 0.005 microM for kinetin and kinetin riboside, respectively. Furthermore, optimal conditions for a baseline chromatographic separation of 18 cytokinin standards by high performance liquid chromatography (HPLC) were developed. The HPLC method had been employed for the confirmation and further fractionation of kinetin in coconut water extracts. The confirmation and fractionation of kinetin riboside was carried out using a further modified HPLC program due to the presence of other interfering material(s) in the sample matrix. Finally, fractions of putative kinetin and kinetin riboside collected from HPLC eluate of coconut water sample were further authenticated by independent capillary zone electrophoresis (CZE) experiment.     

Cytotoxic effects of 2-arylbenzofuran phytoestrogens on human cancer cells: modulation by adrenal and gonadal steroids

Although 2-arylbenzofuran phytoalexins are known for decades, their anticancer activity has not been studied systematically. We have previously reported on the isolation and the estrogen receptor (ER) modulation properties of three new 2-arylbenzofurans from Onobrychis ebenoides, ebenfuran I [2-(2,4-dihydroxyphenyl)-5-hydroxy-6-methoxy-benzofuran], ebenfuran II [2-(2,4-dihydroxyphenyl)-3-formyl-4-hydroxy-6-methoxy-benzofuran] and ebenfuran III [2-(2,4-dihydroxyphenyl)-3-formyl-4-hydroxy-6-methoxy-5-(3-methyl-buten-2-yl)-benzofuran]. We now show that, while I and II could stimulate the proliferation of MCF-7 cells, III was inhibitory in a proliferation-dependent manner. III inhibited the growth of all human cancer cells examined, regardless of ER or multidrug resistance status. Estradiol rendered MCF-7 cells more sensitive to III, and this coincided with the ability of the hormone at concentrations ≥0.1 nM to bind to the ER of the cells and stimulate their proliferation in the presence of III. Cell proliferation stimulating concentrations of I and II also enhanced the effect of III on MCF-7 cells. However, dehydroepiandrosterone and dihydrotestosterone were ineffective in this respect. III-treated MCF-7 cells exhibited G1 phase arrest followed by detachment-induced cell death and/or apoptosis in the adherent fraction, pronounced induction of Bax and suppression of estradiol induction of Bcl-2. Our data indicate that the largely unexplored pool of benzofuran phytoalexins includes entities potentially suitable for chemoprevention and treatment of human cancer.     

Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus, in tumour cells

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca²⁺ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca²⁺ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.     

Cytotoxic effects of phallolysin on a human heteroploid cell line

The effect of Phallolysin on cellular growth, macromolecular biosyntheses and cellular membrane structure was analysed using cells from the EUE line. Concentrations of the toxin that do not affect cellular growth, as determined by plating efficiency, have no effect on RNA or protein synthesis, but stimulate DNA synthesis. The doses of Phallolysin that inhibit cell survival do not affect macromolecular biosyntheses, but greatly increase the percentage of cells stainable with Trypan blue after 1 hour of incubation. At the same dose of the toxin the cells, analysed by electron microscopy, show increased vacuolization indicating an alteration of the membrane apparatus.     

The effects of lead and kinetin on greening barley leaves

The content of lead in greening etiolated barley leaves remained the same, regardless the time of incubation of excised leaves in the presence of lead ions (8–24 h). The lead deposits have not been detected within mesophyll cells, but were found in intercellular spaces of mesophyll, in guard cells and in cuticle covering stomata. This suggests that lead may be transported in the leavesvia transpiration stream. Lead reduced the content of chlorophyll, especially chlorophyllb content and the average number of grana, whereas in the presence of kinetin the content of chlorophyll increased. In the combined treatment (lead + kinetin) kinetin diminished the inhibitory effect of lead on the chlorophyll content. The number of chloroplasts in mesophyll cells remained unchanged after lead treatment, whereas kinetin alone or applied together with lead increased the average chloroplasts number. The thylakoids system in chloroplasts of kinetin and kinetin + lead treated plants was similar to that observed in control, although the grana number was smaller. Both lead and kinetin increased the content of condensed chromatin in nuclei.