The chemosensitizing effect of aqueous extract of sweet fennel on cisplatin treated HeLa cells.

BACKGROUND: Cisplatin is an important chemotherapeutic agent that is widely used in treatment of several malignancies, but its side effects on normal tissues and organs limit its use. The aim of this study was to evaluate the effect of aqueous extract of sweet fennel alone and in combination with cisplatin on human cervical cancer adenocarcinoma cell line (HeLa cells) searching for an effective, inexpensive therapy with minimal side effects. MATERIALS AND METHODS: HeLa cell line was used to study the cytotoxic effect of different concentrations of the aqueous extract of sweet fennel alone and in combination with 50 µg/ml cisplatin. Quantitative measure of drug interaction was quantified by the combination index. Gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) were used to analyze the sweet fennel decoction. MTT assay was used to examine cell viability percentage. Electron microscopy was applied to study the ultrastructure of the cells. RESULTS: The phenyl propanoids (23%) and phenols (12%) constituted the highest percentage of the aqueous extract. Increasing the concentration of sweet fennel from 50 µg/ml to 80 µg/ml, decreased the percentage of the cell viability of HeLa cells from 86.74% to 78.28%, respectively. Further decrease to 11.31% was demonstrated when 50 µg/ml of fennel was combined with 50 µg/ml cisplatin (additive effect). In addition to the signs of apoptosis observed in HeLa cells at 50 µg/ml of fennel, disruption of both nuclear and cytoplasmic membranes and presence of autophagolysosomes were noticed at a dose of 80 µg/ml. Combination of 50 µg/ml of cisplatin with 60, 70, and 80 µg/ml of sweet fennel revealed no significant difference in comparison to cisplatin alone. The combination with 50 µg/ml of sweet fennel revealed marked vacuolization of the cytoplasm, fragmentation of the nucleus, and complete disruption of nuclear membrane. CONCLUSIOn: Combination of cisplatin and the 50 µg/ml of the fennel could enhance cervical cancer growth inhibition. This combination could be effective in lowering the dose of single or repeated cumulative courses of cisplatin and hence decreases its hazardous side effects. In vivo studies and the evaluation of different combination doses of cisplatin and sweet fennel are recommended.

Brazilin Isolated from Caesalpinia sappan suppresses nuclear envelope reassembly by inhibiting barrier-to-autointegration factor phosphorylation.

To date, many anticancer drugs have been developed by directly or indirectly targeting microtubules, which are involved in cell division. Although this approach has yielded many anticancer drugs, these drugs produce undesirable side effects. An alternative strategy is needed, and targeting mitotic exit may be one alternative approach. Localization of phosphorylated barrier-to-autointegration factor (BAF) to the chromosomal core region is essential for nuclear envelope compartment relocalization. In this study, we isolated brazilin from Caesalpinia sappan Leguminosae and demonstrated that it inhibited BAF phosphorylation in vitro and in vivo. Moreover, we demonstrated direct binding between brazilin and BAF. The inhibition of BAF phosphorylation induced abnormal nuclear envelope reassembly and cell death, indicating that perturbation of nuclear envelope reassembly could be a novel approach to anticancer therapy. We propose that brazilin isolated from C. sappan may be a new anticancer drug candidate that induces cell death by inhibiting vaccinia-related kinase 1-mediated BAF phosphorylation.

Obtusilactone B from Machilus Thunbergii targets barrier-to-autointegration factor to treat cancer.

Targeting specific molecules is a promising cancer treatment because certain types of cancer cells are dependent on specific oncogenes. This strategy led to the development of therapeutics that use monoclonal antibodies or small-molecule inhibitors. However, the continued development of novel molecular targeting inhibitors is required to target the various oncogenes associated with the diverse types and stages of cancer. Obtusilactone B is a butanolide derivative purified from Machilus thunbergii. In this study, we show that obtusilactone B functions as a small-molecule inhibitor that causes abnormal nuclear envelope dynamics and inhibits growth by suppressing vaccinia-related kinase 1 (VRK1)-mediated phosphorylation of barrier-to-autointegration factor (BAF). BAF is important in maintaining lamin integrity, which is closely associated with diseases that include cancer. Specific binding of obtusilactone B to BAF suppressed VRK1-mediated BAF phosphorylation and the subsequent dissociation of the nuclear envelope from DNA that allows cells to progress through the cell cycle. Obtusilactone B potently induced tumor cell death in vitro, indicating that specific targeting of BAF to block cell cycle progression can be an effective anticancer strategy. Our results demonstrate that targeting a major constituent of the nuclear envelope may be a novel and promising alternative approach to cancer treatment.

Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis.

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD50 of 0.9 µM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

Chronic morphine-induced neuronal morphological changes in the ventral tegmental area in rats are reversed by electroacupuncture treatment.

The aim of this study was to observe the effect of electroacupuncture (EA) on chronic morphine-induced neuronal morphological changes in the ventral tegmental area (VTA) in rats at electron-microscopic level. Fourteen days of administering escalating doses of morphine induced pathological morphological changes of neurons in the VTA: the rough endoplasmic reticulum swelled, membrane configuration of the nucleus and mitochondria blurred, and structure of myelin sheath changed. Both 2 and 100 Hz EA treatment reversed the morphological alterations induced by chronic morphine administration. The findings provide new evidence that EA may serve as a potential therapy in treating opiate addiction.

Cytotoxic properties of Diospyros seychellarum extract.

During a recent botanical expedition in the Seychelles archipelago we identified healers using Diospyros seychellarum as a tonic. Since this plant lacks any medicinal record in the current literature, we assessed the cytotoxic potential of D. seychellarum. Using Jurkat cells as a model system we show, by flow cytometry, that treatment with the leaf extract results in mitochondrial depolarization and subsequent loss of cellular membrane integrity. Additionally, by transmission electron microscopy, we show that treatment with the extract results in chromatin condensation, mitochondrial swelling, and loss of nuclear membrane integrity. Through these morphological and biochemical observations we concluded that the extract of Diospyros seychellarum is able to induce apoptosis. While it is difficult to extrapolate a potential pharmacologic function based on the ethnomedical use as a tonic, the ability of this extract to induce apoptosis warrants further investigation of the medicinal properties of this plant.

Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism.

PGs produced from arachidonic acid by the action of cyclooxygenase enzymes play a pivotal role in the regulation of both inflammatory and immune responses. Because leukotriene B4 (LTB4), a product of 5-lipoxygenase (5-LO) pathway, can exert numerous immunoregulatory and proinflammatory activities, we examined the effects of PGs on LTB4 release from dendritic cells (DC) and from peritoneal macrophages. In concentration-dependent manner, PGE1 and PGE2 inhibited the production of LTB4 from DC, but not from peritoneal macrophage, with an IC50 of 0.04 microM. The same effect was observed with MK-886, a 5-LO-activating protein (FLAP)-specific inhibitor. The decreased release of LTB4 was associated with an enhanced level of IL-10. Furthermore, the inhibition of LTB4 synthesis by PGs was significantly reversed by anti-IL-10, suggesting the involvement of an IL-10-dependent mechanism. Hence, we examined the effects of exogenous IL-10 on the 5-LO pathway. We demonstrate that IL-10 suppresses the production of LTB4 from DC by inhibiting FLAP protein expression without any effect on 5-LO and cytosolic phospholipase A2. Taken together, our results suggest links between DC cyclooxygenase and 5-LO pathways during the inflammatory response, and FLAP is a key target for the PG-induced IL-10-suppressive effects.

Sex bodies and synaptonemal complexes of Balb/C mice: antioxidant intervention of oxyradical insult.

Spermatogenesis is inhibited in Balb/C mice as a result of oxyradical insult. However, mammalian spermatocytes and synaptonemal complexes retain their structure and function after oxyradical insult due to protection afforded by the antioxidant vitamin E. Control groups were compared with experimental groups which were fed various vitamin E-deficient diets and subjected to varying times in an humidified 100% oxygen (hyperoxia) chamber. Measurements were made of sex body volume (SBV), nuclear envelope aberrations (NEA), and synaptonemal complex structure in spermatocytes during pachytene of meiosis prophase I. Changes in the volume of the sex body were positively correlated with increased oxyradical insult. The structure of the synaptonemal complex was not altered in any of the experimental groups which is a significant observation. It is suggested that vitamin E affords antioxidant protection and inhibits the alteration of membranes and sex chromosomes in mice during meiosis.

The selectivity and specificity of the actions of the lipido-sterolic extract of Serenoa repens (Permixon) on the prostate.

PURPOSE: To investigate the effects of the phytotherapeutic agent, Permixon(R), on primary cultures of fibroblast and epithelial cells from the prostate, epididymis, testes, kidney, skin and breast and to determine the selectivity and specificity of the action of the drug. MATERIALS AND METHODS: All primary cultures were examined by electron microscopy before and following treatment with Permixon(R) (10 microg./ml.). In addition the apoptotic index was assessed by flow cytometry employing propidium iodide as a fluorophore. The impact of the drug on 5alpha-reductase (5alphaR) isoenzymes was also tested utilizing a pH specific assay. RESULTS: There were changes in the morphology of prostate cells after treatment including accumulation of lipid in the cytoplasm and damage to the nuclear and mitochondrial membranes; no similar changes were observed in other cells. Permixon(R) increased the apoptotic index for prostate epithelial cells by 35% and 12% in the prostate stromal/fibroblast. A lesser apoptotic effect was demonstrated in skin fibroblast (3%) whereas none of the other primary cultures showed any increase in apoptosis when compared with the controls. Permixon(R) was also an effective inhibitor of both 5alphaR type I and II isoenzymes in prostate cells, but other cells showed no inhibition of 5alphaR activity following treatment with the plant extract. CONCLUSIONS: This investigation demonstrated the selectivity of the action of Permixon(R) for prostate cells. The morphological changes in the prostate are accompanied by an increase in the apoptotic index along with an inhibition in the activity of the nuclear membrane bound 5alphaR isoenzymes. No similar changes were observed in any of the other cells under investigation.

Cofactors in in vitro induction of apoptosis in HL60 cells by all-trans retinoic acid (ATRA).

The aim of this study was to determine the culture conditions that could modulate the induction of apoptosis by all-trans retinoic acid (ATRA). Cell viability was evaluated by trypan blue test, differentiation by nitro blue tetrazolium test, and apoptosis by morphological analysis. ATRA induced apoptosis in HL60 cells only when more than 100,000 cells/mL were seeded, while differentiation was induced regardless of the seeded cell concentration. Reduction in the concentration of foetal calf serum or glutamine in the medium led to a weak increase in apoptosis. In contrast, a dramatic enhancement of apoptosis occurred when the culture medium was supplemented with glucose or when the culture pH was decreased. These characteristics were independent of the mechanism of action of ATRA, but the action of glucose could be of significance in diabetic patients. An exchange of supernatants after 3 days of culture showed that supernatants from control cultures seeded at high cell density were better apoptosis inducers than supernatants from cultures treated with ATRA, but seeded at low cell density. Factor(s) in this supernatant which induced apoptosis was (were) removed by ultrafiltration. In conclusion, our results showed that ATRA alone cannot induce apoptosis, but can do so in conjunction with cofactors. The depletion of some components of the medium and the appearance of secreted macromolecule(s) could be cofactor(s) in the induction of apoptosis.

Relationship between fatty acid accretion, membrane composition, and biologic functions.

Dietary fat affects metabolic pathways for phospholipid biosynthesis in tissues in a coordinated fashion. This may be important to aspects of development that concern phosphatidylcholine metabolism or regulatory processes that depend on signals from a changing milieu in the microenvironment of the membrane. Dietary fat influences the phosphatidylethanolamine (PE) composition in many membranes of the brain and retina and may by altered by small changes in the content of 20:4(6) and 22:6(3). Membrane PE fatty acids that contain one, four, or six double bonds and the ratio of 22:5(6) to 22:6(3) in PE that contains four to six double bonds are also affected. An increase in the omega 6 fatty acid content of membranes is associated with increased PE methyltransferase activity and decreased phosphocholine transferase activity, thus indicating a mechanism by which change in an exogenous factor (e.g., dietary fat intake) may alter neural phospholipid biosynthesis. Small changes in the composition of dietary fat intake change the composition of brain membranes during development. It is provocative to ponder whether diet could be used to induce formation of membrane structures that are more resistant to specific insults that cause degeneration of brain structural material, to ensure optimal functional compositions, or to reverse degenerative changes that occur in neural membrane structure and function.

Morphologic effects of artemisinin in Plasmodium falciparum.

Ultrastructural changes induced in Plasmodium falciparum by artemisinin were studied in vitro. Electron microscopic autoradiography was performed on infected erythrocytes that were exposed in vitro to 3H-dihydroartemisinin and 14C-artemisinin. These drugs consistently were located in food vacuoles and mitochondria. Two hours after administration, changes were observed in parasite mitochondria, rough endoplasmic reticulum, and nuclear envelope. At four hours, in addition to the earlier changes, nuclear membranes and, to a lesser extent, some plasma membranes formed myelin figures. In addition, there was a disappearance of ribosomes, and a destruction of food vacuole membranes. These changes may lead to the total disorganization of the parasites. Approximately 30% of the parasites manifested these alterations.

Dietary fat and 3-MC induction of hepatic nuclear and microsomal cytochrome P-450.

Hepatic microsomes from male Holtzman albino rats fed a synthetic fat-free diet for 21 days had significantly less cytochrome P-450 and exhibited less binding capacity (delta Amax/mg protein) for aniline and octylamine than microsomes from similar rats fed a diet containing 10% corn oil. Treatment with 3-methylcholanthrene (3-MC) increased the concentrations of cytochrome P-450 (as measured by CO binding spectra) to nearly equal levels in both dietary groups, but the binding of aniline and octylamine to microsomes of rats fed the fat diet exceeded the increase in cytochrome P-450 concentration. Nuclear envelope concentrations of cytochrome P-450 were unaffected by diet. The administration of 3-MC to rats fed a fat-free diet failed to induce nuclear envelope P-450; however, in rats fed the corn oil diet, 3-MC increased this CO binding pigment over twofold. The affinity of nuclear envelope P-450 towards type II substrates was at least equal to that of microsomes, except in control rats fed the fat-free diet. In general, 3-MC pretreatment increased the binding affinity of nuclear envelop and microsomes toward aniline, while increasing affinity for SKF 525-A binding only to nuclear envelope. Molecular weight species in the region known to contain the cytochrome P-450 were quantified by fluorescence gel electrophoresis. Molecular weight species of 48,000 and 53,000 in the nuclear envelope had their counterparts in the microsomal preparation, but a 50,000 dalton component of nuclear envelope was not detected in microsomes. 3-Methylcholanthrene increased only a species with molecular weight 45,500 in the microsomal and nuclear envelope preparations. Rats fed the diet containing corn oil had microsomes with increased capacity for binding CO, but this was not accompanied by increased cytochrome P-450 protein concentration, as measured by quantitative fluorescence gel electrophoresis.