S. choloroleuca, S. mirzayanii and S. santolinifolia protect PC12 cells from H(2)O (2)-induced apoptosis by blocking the intrinsic pathway

Several studies have shown that neuronal cell death due to apoptosis is the major reason for cognitive decline in Alzheimer's disease. In this study, we report the anti-apoptotic effects of three Salvia species from Iran-S. choloroleuca, S. mirzayanii and S. santolinifolia-against H(2)O(2)-induced cytotoxicity in neuron-like PC12 cells. We showed that these antioxidant species could interfere with the intrinsic pathway of apoptosis by attenuating Bax/Bcl-2 ratio, decreasing outer mitochondrial membrane break and decreasing cytochrome c release to cytoplasm. Interestingly, we found that these species were able to replenish reduced glutathione level which affects cellular redox status and cytochrome c activity. Moreover, the decreased level of caspase-3, the executioner caspase, resulted in decrease of PARP-1 cleavage. Anti-apoptotic effects of these species along with their antioxidant effects, may represent a promising approach for treatment of neurodegenerative diseases.     

Promising anticancer activity of a lichen,Parmelia sulcata Taylor,against breast cancer cell lines and genotoxic effect on human lymphocytes

Plants are still to be explored for new anti-cancer compounds because overall success in cancer treatment is still not satisfactory. As a new possible source for such compounds, the lichens are recently taking a great attention. We, therefore, explored both the genotoxic and anti-growth properties of lichen species Parmelia sulcata Taylor. The chemical composition of P. sulcata was analyzed with comprehensive gas chromatography–time of flight mass spectrometry. Anti-growth effect was tested in human breast cancer cell lines (MCF-7 and MDA-MB-231) by the MTT and ATP viability assays, while the genotoxic activity was studied by assays for micronucleus, chromosomal aberration and DNA fragmentation in human lymphocytes culture. Cell death modes (apoptosis/necrosis) were morphologically assessed. P. sulcata inhibited the growth in a dose-dependent manner up to a dose of 100 μg/ml and induced caspase-independent apoptosis. It also showed genotoxic activity at doses (>125 μg/ml) higher than that required for apoptosis. These results suggest that P. sulcata may induce caspase-independent apoptotic cell death at lower doses, while it may be genotoxic at relatively higher doses.     

The mechanisms of lipoxygenase inhibitor-induced apoptosis in human breast cancer cells

Previous experimental studies have shown that high dietary fat intake is associated with mammary carcinogenesis. In the current study, the effect of 5-LOX or 12-LOX inhibitors on human breast cancer cell proliferation and apoptosis, as well as the possible mechanisms were investigated. The LOX inhibitors, NDGA, Rev-5901, and baicalein all inhibited proliferation and induced apoptosis in MCF-7 (ER+) and MDA-MB-231 (ER-) breast cancer cell in vitro. In contrast, the LOX products, 5-HETE and 12-HETE had mitogenic effects, stimulating the proliferation of both cell lines. These inhibitors also induced cytochrome c release, caspase-9 activation, as well as downstream caspase-3, caspase-7 activation, and PARP cleavage. LOX inhibitor treatment also reduced the levels of anti-apoptotic proteins Bcl-2 and Mcl-1 and increased the levels of the pro-apoptotic protein bax. In conclusion, blockade of both 5-LOX and 12-LOX pathways induces apoptosis in breast cancer cells through the cytochrome c release and caspase-9 activation, with changes in the levels of Bcl-2 family proteins.     

Anti-cancer effect of Cassia auriculata leaf extract in vitro through cell cycle arrest and induction of apoptosis in human breast and larynx cancer cell lines

The in vitro anti-cancer effect of Cassia auriculata leaf extract (CALE) was evaluated in human breast adenocarcinoma MCF-7 and human larynx carcinoma Hep-2 cell lines. CALE preferentially inhibited the growth of both the cell lines in a dose-dependent manner with IC₅₀ values of 400 and 500 μg for MCF-7 and Hep-2 cells, respectively. The results showed the anti-cancer action is due to nuclear fragmentation and condensation, associated with the appearance of A₀ peak in cell cycle analysis that is indicative of apoptosis. In addition, CALE treated MCF-7 and Hep-2 cells had decreased expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein, eventually leading a decrease in the Bcl-2/Bax ratio. These results demonstrated that CALE inhibits the proliferation of MCF-7 and Hep-2 cells through induction of apoptosis, making CALE a candidate as new anti-cancer drug.     

In vitro cytotoxic potential of Polyalthia longifolia on human cancer cell lines and induction of apoptosis through mitochondrial-dependent pathway in HL-60 cells

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. We are reporting first time the anticancer potential of P. longifolia leaves extract (A001) and its chloroform fraction (F002). Both inhibited cell proliferation of various human cancer cell lines in which colon cancer cells SW-620 showed maximum inhibition with IC(50) value 6.1 microg/ml. Furthermore, F002 induce apoptosis in human leukemia HL-60 cells as measured by several biological end points. F002 induce apoptotic bodies formation, DNA ladder, enhanced annexin-V-FITC binding of the cells, increased sub-G(0) DNA fraction, loss of mitochondrial membrane potential (DeltaPsi(mt)), release of cytochrome c, activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. All the above parameters revealed that F002-induced apoptosis through the mitochondrial-dependent pathway in HL-60 cells.     

Cytotoxic and pro-apoptotic effects of Abrus precatorius L. on human metastatic breast cancer cell line, MDA-MB-231

Abrus precatorius is highly regarded as a universal panacea in the herbal medicine with diverse pharmacological activity spectra. This experimental study on the mechanism of the anticancer activity of A. precatorius leaf extracts, may offer new evidence for A. precatorius in the treatment of breast cancer in clinical practice. Cell death was determined by using MTT assay. Further analyses were carried out by doing DNA laddering, PARP cleavage, FACS, semi-quantitative RT-PCR and detection of cellular reactive oxygen species (ROS) by DCFDA assay. A. precatorius showed very striking inhibition on MDA-MB-231 cells. MTT assay showed more than 75 % inhibition of the cells and treated cells indicated visible laddering pattern with thick compact band. PARP cleavage produced 89 kDa cleavage product which was associated with apoptosis. Flow cytometer exhibited a sub-G0/G1 peak as an indicative of apoptosis. mRNA expression level of apoptosis-related genes p21 and p53 was markedly increased in cells treated with the extract as compared to control. The up-regulation of p21 and p53 may be the molecular mechanisms by which A. precatorius extract which induces apoptosis. An increase in the concentration of A. precatorius extract does not generate ROS, instead it reduces ROS formation in MDA-MB-231 cells, as evident from the shift in fluorescence below untreated control. This is the first report showing that A. precatorius leaf extract exhibits a growth inhibitory effect by induction of apoptosis in MDA-MB-231 cells. Our results contribute towards validation of the A. precatorius extract as a potentially effective chemopreventive or therapeutic agent against breast cancer.     

Description of Tylenchorhynchus thermophilus n. sp. (Nematoda: Tylenchina) from Saltgrass in Death Valley,California

A stunt nematode, Tylenchorhynchus thermophilus n. sp., is described and illustrated from soil collected around roots of saltgrass (Distichlis spicata) in Death Valley, California. It is distinguished from the similar species, T. ewingi, T. mexicanus, and T. mashoodi, in having a longer female body, longer tail with more annules, and larger phasmids. Physical and chemical analysis of soil from saltgrass roots showed it to consist of 71% sand and possess high salinity (salt content of 0.51%) and a pH of 9.3.     

Distinct patterns of mitochondrial changes precede induction of apoptosis by all-trans-retinoic acid and N-(4-hydroxyphenyl)retinamide in MCF7 breast cancer cells

The biochemical mechanisms of apoptosis-induction by all-trans-retinoic acid (atRA) and N-(4-hydroxyphenyl)retinamide (4HPR) in cultured MCF7 cancer cells were studied by multiparameter flow cytometry. Retinoid treatment induced formation of two biochemically distinct cell subpopulations, which preceded the appearance of cells with fragmented nuclei. Exposure to atRA led to a transient increase in NADH level and mitochondrial oxidative turnover and a slow decline in reduced thiol level and mitochondrial membrane potential, suggesting that atRA treatment induces a transient defense mechanism. The synthetic retinoid 4HPR, in contrast, caused a gradual decrease in mitochondrial oxidative turnover and cardiolipin level together with a small decline in mitochondrial membrane potential, suggesting that 4HPR induces oxidation of cardiolipin and subsequent leakage of the mitochondria. Co-incubation with cyclosporin A, an inhibitor of the mitochondrial permeability transition, did not prevent formation of fragmented nuclei or induction of changes in mitochondrial parameters by retinoids. Thus, the mitochondrial permeability transition does not appear to be involved in retinoid induction of apoptosis in MCF7 cells. Retinoid exposure of diploid human mammary epithelial cells induced mild oxidative stress but did not lead to formation of two cell subpopulations. We conclude that atRA and 4HPR induce apoptosis in MCF7 cells by two distinct and novel biochemical mechanisms.     

Short-term effect on intestinal epithelial Na(+)/H(+) exchanger by Gi(alpha1,2)-coupled 5-HT(1A) and G(q/11)-coupled 5-HT(2) receptors

The present study evaluated the effect of 5-hydroxytryptamine (5-HT) on intestinal Na(+)/H(+) exchanger (NHE) activity and the cellular signaling pathways involved in T84 cells. T84 cells express endogenous NHE1 and NHE2 proteins, detected by immunoblotting, but not NHE3. The rank order for inhibition of NHE activity in acid-loaded T84 cells was 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; IC(50)=519 [465, 579] nM)>cariporide (IC(50)=630 [484, 819] nM)>amiloride (IC(50)=19 [16, 24] microM); the NHE3 inhibitor S3226 was found to be devoid of effect. This different inhibitory sensitivity indicates that both NHE1 and NHE2 isoforms may play an active role in Na(+)-dependent intracellular pH (pH(i)) recovery in T84 cells. Short-term exposure (0.5 h) of T84 cells to 5-HT increased NHE activity in a concentration-dependent manner. The stimulation induced by 5-HT (30 microM) was partially inhibited by both WAY 100135 (300 nM) and ketanserin (300 nM), antagonists of 5-HT(1A) and 5-HT(2) receptors, respectively. NHE activity was significantly increased by 8-OH-DPAT and alpha-methyl-5-HT, agonists of, respectively, 5-HT(1A) and 5-HT(2) receptors. An incubation of T84 cells with anti-G(s) and anti-G(beta) antibodies complexed with lipofectin did not prevent the 5-HT-induced stimulation of NHE activity. Overnight treatment with anti-G(ialpha1,2) and anti-G(q/11) antibodies complexed with lipofectin blocked the stimulatory effect induced by 8-OH-DPAT and alpha-methyl-5-HT, respectively. It is concluded that in T84 cells 5-HT enhances intestinal NHE activity through stimulation of G(ialpha1,2)-coupled 5-HT(1A) and G(q/11)-coupled 5-HT(2) receptors.     

Suppression of growth of highly-metastatic human breast cancer cells by norcantharidin and its mechanisms of action

The effects of norcantharidin (NCTD) on the growth of highly-metastatic human breast cancer cells were investigated by in vitro and ex vivo assays. Our results indicated that norcantharidin inhibited the in vitro growth of human breast cancer MDA-MB-231 cell line in dose- and time-dependent manners after the cancer cells were treated with norcantharidin at the concentrations of 6, 30 and 60 μmol/L for 24, 48 and 72 h. Moreover, the sera from the NCTD-treated rabbits after intravenous injection of NCTD at 15 and 30 min significantly suppressed the growth of the cancer cells ex vivo. The analyses by Hoechst 33258 staining and flow cytometry showed that the typical apoptotic morphological changes appeared and cell cycles arrested at G2/M phase in MDA-MB-231 cells after the cells were treated for 48 h with NCTD. In addition, NCTD down-regulated the expressions of anti-apoptotic protein Bcl-2 and up-regulated the expressions of pro-apoptotic protein Bax, eventually leading to the reduction of Bcl-2/Bax ratio in MDA-MB-231 cells. Furthermore, NCTD at concentrations of 6, 30 and 60 μmol/L dose-dependently reduced the phosphorylation of Akt and NF-κB expression in the breast cancer cell line. Induction of apoptosis and cell cycle arrest as well as reduction of Bcl-2/Bax ratio by NCTD may be the important mechanisms of action of NCTD suppressing the growth of MDA-MB-231 cells, which are associated with inhibition of the Akt and NF-κB signaling. Our findings suggest that norcantharidin may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of human breast cancer.     

Comparative cellular and molecular analysis of cytotoxicity and apoptosis induction by doxorubicin and <Emphasis Type="Italic">Baneh</Emphasis> in human breast cancer T47D cells

It is now widely accepted that dietary phytochemicals inhibit cancer progression and enhance the effects of conventional chemotherapy. In this report, we comparatively studied the cellular and molecular aspects of apoptosis induction by the methanolic extract of Baneh fruit skin in comparison to Doxorubicin (Dox), a well-known anticancer drug, in human breast cancer T47D cells. The MTT assay was used to determine the antiproliferative effects. The flow cytometric and microscopic analyses were done to evaluate the apoptosis induction. Furthermore, western blot analyses have been done to study the role of key molecular players of apoptosis including caspase 3 and PARP. The Baneh extract showed strong antiproliferative activity against T47D cells in a dose- and time-dependent manner that was comparable to and even stronger than Dox in certain concentrations. Analysis of Baneh-treated cells by flow cytometry and fluorescence microscopy indicated strong apoptosis induction and nuclear morphological alterations similar to or greater than Dox. Finally, molecular analysis of apoptosis by western blotting proved activation of caspase 3 followed by poly ADP ribose polymerase (PARP) cleavage more efficiently in Baneh than in Dox treated cancer cells. These findings indicate that Baneh extract contains phytochemicals which act as inhibitor of cell proliferation and inducer of apoptosis in human breast cancer T47D cells that makes it a potentially good candidate for new anticancer drug development.     

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.     

Selective fraction of Atractylodes lancea (Thunb.) DC. and its growth inhibitory effect on human gastric cancer cells

The purpose of this study was to investigate the pharmacological effect of fraction of Atractylodes lancea (Thunb.) DC. (A. lancea) extract. In this study, we isolated different polarity fractions, including petroleum ether (PE), ethyl acetate, n-butanol, and the remaining H₂O fractions from the water extract of A. lancea. The antigastric cancer properties of the different fractions in BGC-823 and SGC-7901 cells were evaluated. Apoptotic cells were treated with PE fraction and stained with Hoechst 33342 and 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide. The cell cycle was analyzed via flow cytometry. The main compounds of PE fraction were determined by HPLC–ESI–MS. Results of this study showed that the PE fraction of A. lancea inhibited the growth of BGC-823 and SGC-7901 cells in a dose- and time-dependent manner. The morphological and mitochondrial transmembrane potential changes suggested that the cells showed preliminary apoptosis characteristics after treatment with the three different polarities. The main compounds of PE fraction include two sesquiterpene compounds: eudesm-4(15),7-diene-9α,11-diol and eudesm-4(15)-ene-7a,11-diol; three sesquiterpene lactone compounds: atractylenolid I, atractylenolid III and 3-β-acetyl-atractylenolid III and one polyacetylenic compound: 4,6,12-tetradecatriene-8,10-diyne-1,3,14-triol.     

Evaluation of the effects of nicorandil and its molecular precursor (without radical NO) on proliferation and apoptosis of 786-cell

Nicorandil is a nitric oxide (NO) donor used in the treatment of angina symptoms. It has also been reported to protect cells and affect the proliferation and death of cells in some tissues. The molecules that interfere with these processes can cause dysfunction in healthy tissues but can also assist in the therapy of some disorders. In this study we examined the effect of nicorandil and of the molecular precursor that does not have the NO radical (N-(beta-hydroxyethyl) nicotinamide) on the cell proliferation and death of human renal carcinoma cells (786-O) under normal oxygenation conditions. The molecular precursor was used in order to analyze the effects independents of NO. In the cytotoxicity test, nicorandil was shown to be cytotoxic at very high concentrations and it was more cytotoxic than its precursor (cytotoxic at concentrations of 2,000 and 3,000 μg/mL, respectively). We propose that the lower cytotoxicity of the precursor is due to the absence of the NO radical. In this study, the cells exposed to nicorandil showed neither statistically significant changes in cell proliferation nor increases in apoptosis or genotoxicity. The precursor generated similar results to those of nicorandil. We conclude that nicorandil causes no changes in the proliferation or apoptosis of the cell 786-O in normal oxygenation conditions. Moreover, the lack of NO radical in the precursor molecule did not show a different result, except in the cell cytotoxicity.     

Descriptions of Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina) from Haryana,India

Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and spermathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b’ = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c’ = 2.0 to 2.9, stylet = 11 to 12 µm, excretory pore in the region of median pharyngeal bulb, 43 to 47 µm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b’ = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c’ = 2.7 to 3.5, stylet = 6 to 7 µm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.     

Histological Comparisons of Parasitism by Schistonchus spp. (Nemata: Aphelenchoididae) in Neotropical Ficus spp.

Syconia (enclosed infructescences) infested with host-specific species of Schistonchus (Aphelenchoididae) were collected from six species of Ficus (Moraceae) native to Florida or Panama. They were sectioned and histologically examined to assess the effects of parasitism. Parasitism by Schistonchus spp. was associated with hypertrophied cells, tissue necrosis, and the presence of an exudate in all species. Occasional hypertrophy of the outer epidermal cells occurred on seed florets, wasp florets, and on the endothecial cells of male florets in F. aurea (subgenus Urostigma) from Florida. Aberrations of the inner mesocarp occurred under the hypertrophied cells on seed florets. In F. laevigata (subgenus Urostigma) from Florida, Schistonchus sp. infested immature male florets and was associated with hypertrophy of endothecial cells, epidermal cells of the anther filaments, and anthers. Schistonchus sp. also caused aberrations of the anther filament, anthers, and pollen. Ficus poponoei (subgenus Urostigma) and F. glabrata (subgenus Pharmacosycea), both from Panama, had hypertrophied outer epidermal cells on seed florets. Ficus poponoei also had Schistonchus sp. within the pedicel of an aborted floret, with hypertrophy of the cortical parenchyma. Ficus trigonata (subgenus Urostigma) from Panama had hypertrophy of the outer epidermis of seed florets. When the outer epidermis on these florets was missing, the inner mesocarp was hypertrophied. Ficus maxima (subgenus Pharmacosycea) from Panama had hypertrophy on the outer epidermis of seed and aborted florets. Schistonchus spp. were not found in wasp larvae or pupae in any of the Ficus spp. examined. Hypertrophy was never observed in the absence of Schistonchus spp.     

The effect of folate-related SNPs on clinicopathological features,response to neoadjuvant treatment and survival in pre- and postmenopausal breast cancer patients

This study aimed to investigate the relationship of ten single nucleotide polymorphisms (SNPs) in the MTHFR, MTR, MTRR, DHFR, MTHFD1, TS, RFC1 and DNMT3b genes with cancer survival, therapeutic response to neoadjuvant chemotherapy and clinicopathological characteristics in 300 pre- and postmenopausal breast cancer patients of a Russian Western Siberian population. We found that the MTHFR 677CT genotype as well as combination of MTHFR 677CT and 677TT genotype was related to tumor size and estrogen-positive status in postmenopausal group. The RFC1 80А allele was associated with an increased risk of lymph node metastases among postmenopausal women. The MTHFR 677TT genotype was significantly correlated with a better progression-free survival in premenopausal patients. In contrast, a worse outcome was observed in this group patient with MTHFD1 1958AA genotype. In the multivariate analysis, the MTHFD1 1958AA genotype was identified as an independent prognostic factor for premenopausal breast cancer survival. Our findings provide evidence for associations of breast cancer survival with folate-related SNPs in a population of Western Siberian region of Russia and the MTHFD1 (1958G>A) may have additional prognostic value especially among premenopausal patients.