Screening of five Sri Lankan endemic plants for anti-cancer effects on breast cancer stem cells isolated from MCF-7 and MDA-MB-231 cell lines

Purpose: To screen selected endemic plants grown in Sri Lanka on breast cancer stem cells (bCSCs) for their anti-cancer propertiesMethods: Breast-CSCs expressing CD24-/CD44+ surface markers were isolated from MDA-MB-231 cells by magnetic-assisted cell sorting method and validated using flow cytometry. A panel of forty extracts from barks and leaves of Doona nervosa, Garcinia quaesita, Garcinia zeylanica, Memecylon rostratum and Schumacheria castaneifolia were obtained by sequential solvent extraction and tested on bCSC-mammospheres derived from MDA-MB-231 and MCF-7 cells and normal mammary epithelial (MCF-10A) cells. Proliferation and cell stemness were analyzed using WST-1 cell proliferation assay and alkaline phosphatase assay.Results: Hexane and chloroform extracts of G. zeylanica and G. quaesita barks showed dosedependent reduction in proliferation and stemness in both bCSCs tested with less effect on MCF-10A cells. Hexane, chloroform and ethyl acetate extracts of S. castaneifolia bark selectively inhibited mammospheres of triple negative bCSCs cells.Conclusion: This study demonstrates that the non-polar extracts of G. zeylanica and G. quaesita, S. castaneifolia barks inhibit the proliferation of bCSCs of triple negative and estrogen-progesterone positive breast cancers. Findings of the present study may useful for developing a future anti-cancer therapeutics which can target bCSCs.Keywords: Cancer stem cells, Garcinia zeylanica, Garcinia quaesita, Schumacheria castaneifolia, Mammosphere

In vitro Cytotoxic and Antioxidant Activity of Leaf Extracts of Mangrove Plant, Phoenix paludosa Roxb

Purpose: To investigate the anti-proliferative and antioxidant potentials of four different solvent extracts of Phoenix paludosa Roxb leaves.Methods: Four different solvent (hexane, chloroform, ethyl acetate and methanol) leaf extracts of the plant were tested for cytotoxicity against four cancer cells, viz, MCF-7 (oestrogen positive breast cancer cell line), MDA-MB-231 (triple negative breast cancer cell line), SK-BR-3 (breast adenocarcinoma) and ACHN (renal adenocarcinoma) as well as two normal cell lines, namely, HEK-293 (embryonic kidney cells) and MCF-10A (normal mammary epithelial cells)]. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and 2, 29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging assays were used to evaluate the antioxidant activity of the crude extracts.Results: The methanol extract showed the highest antioxidant activity (DPPH, half maximal inhibitory concentration (IC50) = 30.17 ± 6.21 μg/mL) and (ABTS, IC50 = 27.91 ± 3.21 μg/mL). Of the four extracts, methanol extract showed the strongest significant (p < 0.05) cytotoxicity to all four cancer cell lines at 24 and 48 h of incubation followed by the chloroform extract (IC50 of methanol extract (24 and 48 h): 36.71 ± 8.72 and 33.19 ± 5.53 μg/mL (MCF-7), 159.7 ± 32.09 and 141.9 ± 26.2 μg/mL (MDA-MB-231), 103.3 ± 18.9 and 75.39 ± 19.39 μg/mL (SKBR-3), 57.21 ± 3.72 and 43.16 ± 10.25 μg/mL (MCF-10A), 37.48 ± 5.75 and 26.99 ± 1.85 (ACHN) and 66.83 ± 14.26 and 60.34 ± 10.66 μg/mL (HEK-293)). Furthermore, the methanol extract was least cytotoxic to normal cell lines.Conclusion: The results obtained indicate that the methanol leaf extract of P. paludosa exhibit potent antioxidant and cytotoxic activities and has the potential of being developed into an anti-cancer agent.Keywords: Phoenix paludosa, antiproliferative, antioxidant, cytotoxicit

Isolation of cytotoxic triterpenes from the mangrove plant, Scyphiphora hydrophyllacea C.F.Gaertn (Rubiaceae)

Purpose: To isolate active cytotoxic compounds from the hexane and chloroform  extracts of the leaves of the mangrove plant, Scyphiphora hydrophyllacea C.F. Gaertn (Rubiaceae), grown in Sri Lanka.Methods: Dried pulverized leaves of S. hydrophyllacea were extracted with hexane and chloroform. Vacuum liquid chromatography (VLC), column chromatography (size exclusion chromatography, Sephadex LH-20) and reversed phase preparative recycling high performance liquid chromatography (HPLC) techniques were used to isolate three compounds (compounds 1, 2 and 3). The structures of the isolated compounds were established with the aid of 1H, 13C and two-dimensional nuclear magnetic resonance (2D-NMR) and electron ionization-mass spectrometry (EI-MS) techniques. 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effects of the compounds on oestrogen receptor positive breast (MCF-7) and non-small cell lung (NCIH- 292) cancer cells.Results: The isolated compounds were identified as oleanolic acid (1), ursolic acid (2) and eichlerianic acid (3). Ursolic acid and eichlerianic acid showed strong  cytotoxic effects {IC50- ursolic acid: 8.47 μg/mL (24 h, MCF-7), 7.78 μg/mL (24 h, NCI-H292) and eichlerianic acid: 8.86 μg/mL (24 h, MCF-7), 10.15 μg/mL (24 h, NCI-H292)} in MCF-7 and NCI-H292 cancer cells at 24, 48 and 72 h  post-incubation periods.Conclusion: Hexane and chloroform extracts of the leaves of S. hydrophyllacea yielded three compounds namely oleanolic acid, eichlerianic acid and ursolic acid. Ursolic acid and eichlerianic acid have been isolated for the first time from the leaves of S. hydrophyllacea grown in Sri Lanka and demonstrate in-vitro cytotoxic effects in oestrogen receptor positive (MCF-7) and non-small lung cancer (NCI-H-292) cells.Keywords: Scyphiphora hydrophyllacea, eichlerianic acid, ursolic acid, oleanolic acid, MCF-7, NCI-H-29

Anti-Inflammatory Activity Is a Possible Mechanism by Which the Polyherbal Formulation Comprised of Nigella sativa

The present study investigated the anti-inflammatory effects of a polyherbal decoction comprised of Nigella sativa, Hemidesmus indicus, and Smilax glabra in order to justify its claimed antihepatocarcinogenic activity. Activation of hepatic nuclear factor-kappa B (NF-κB), IκB kinase (IKK α/β) proteins, and TNFα and IL-6 expression was investigated in diethylnitrosamine- (DEN-) induced C3H mice-bearing early hepatocarcinogenic changes. Acute phase inflammatory response was evaluated by carrageenan-induced rat paw edema formation. Anti-inflammatory mechanisms were also assessed by determining effect on (a) membrane stabilization, (b) nitric oxide (NO) inhibitory activity, and (c) inhibition of leukocyte migration. A significant inhibition of the paw edema formation was observed in healthy rats as well as in rats bearing early hepatocarcinogenic changes with oral administration of the decoction. As with the positive control, indomethacin (10 mg/kg b.w.) the inhibitory effect was pronounced at 3rd and 4th h after carrageenan injection. A notable IKK α/β mediated hepatic NF-κB inactivation was associated with a significant hepatic TNFα downregulation among mice-bearing hepatocarcinogenic changes subjected to decoction treatment. Inhibition of NO production, leukocyte migration, and membrane stabilization are possible mechanisms by which anti-inflammatory effect is mediated by the decoction. Overall findings imply that anti-inflammatory activity could be one of the mechanisms by which the decoction mediates its antihepatocarcinogenic effects

Modulation of apoptosis in human hepatocellular carcinoma (HepG2 cells) by a standardized herbal decoction of Nigella sativa seeds, Hemidesmus indicus roots and Smilax glabra rhizomes with anti- hepatocarcinogenic effects

<p>Abstract</p> <p>Background</p> <p>A standardized poly-herbal decoction of <it>Nigella sativa </it>seeds, <it>Hemidesmus indicus </it>roots and <it>Smilax glabra </it>rhizomes used traditionally in Sri Lanka for cancer therapy has been demonstrated previously, to have anti-hepatocarcinogenic potential. Cytotoxicity, antioxidant activity, anti-inflammatory activity, and up regulation of p53 and p21 activities are considered to be some of the possible mechanisms through which the above decoction may mediate its anti-hepatocarcinogenic action. The main aim of the present study was to determine whether apoptosis is also a major mechanism by which the decoction mediates its anti-hepatocarcinogenic action.</p> <p>Methods</p> <p>Evaluation of apoptosis in HepG2 cells was carried out by (a) microscopic observations of cell morphology, (b) DNA fragmentation analysis, (c) activities of caspase 3 and 9, as well as by (d) analysis of the expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins associated with cell death.</p> <p>Results</p> <p>The results demonstrated that in HepG2 cells, the decoction can induce (a) DNA fragmentation and (b) characteristic morphological changes associated with apoptosis (nuclear condensation, membrane blebbing, nuclear fragmentation and apoptotic bodies). The decoction could also, in a time and dose dependent manner, up regulate the expression of the pro-apoptotic gene <it>Bax </it>and down regulate expression of anti-apoptotic <it>Bcl-2 </it>gene (as evident from RT-PCR analysis, immunohistochemistry and western blotting). Further, the decoction significantly (<it>p </it>< .001) enhanced the activities of caspase-3 and caspase-9 in a time and dose dependent manner.</p> <p>Conclusions</p> <p>Overall findings provide confirmatory evidence to demonstrate that the decoction may mediate its reported anti-hepatocarcinogenic effect, at least in part, through modulation of apoptosis.</p

Effect of Standardized Decoction of Nigella sativa Seed, Hemidesmus indicus Root and Smilax glabra Rhizome on the Expression of p53 and p21 Genes in Human Hepatoma Cells (HepG2) and Mouse Liver with Chemically-Induced Hepatocarcinogenesis

Purpose: To evaluate in vitro (using human hepatoma HepG2 cells) and in vivo (using mouse liver with diethlynitrosamine (DEN)-induced hepatocarcinogenesis) effect of a standardized decoction on the expression of p53 (tumour suppressor) and p21 (cyclin kinase inhibitor) genes with the long-term goal of developing the formulation into a globally acceptable therapy for hepatocellular carcinoma (HCC). Methods: The effect of the decoction on (a) mRNA and (b) protein expression of p53 and p21 genes in HepG2 cells and mouse livers with DEN-induced early hepatocarcinogenesis were evaluated by (a) reverse transcription PCR (RT-PCR) and (b) immunohistochemical and Western blot analysis, respectively. Results: The results demonstrated that the decoction significantly (p <0.001) enhanced the expression of p53 and p21 genes in a time- and dose-dependent manner in HepG2 cells. A dose of 75 μg/ml significantly increased p53 mRNA at 24 and 48 h and p21 mRNA at 12, 24, 48 h of incubation with the decoction (p <0.01). Induction of hepatocarcinogenesis in mice significantly increased hepatic expression of both p53 and p21 compared to distilled water control (p <0.001), while treatment with the decoction further enhanced expression of both genes in DEN-induced hepatocarcinogenesis (p <0.01). Conclusion: Overall, the findings demonstrate that the decoction may mediate its reported antihepatocarcinogenic effect, at least in part, through the modulating activities of genes involved in tumour suppression and cell cycle arrest

The Genome of Setaria digitata: A Cattle Nematode Closely Related to Human Filarial Parasites

Here we present the draft genome sequence of Setaria digitata, a parasitic nematode affecting cattle. Due to its similarity to Wuchereria bancrofti, the parasitic nematode that causes lymphatic filariasis in humans, S. digitata has been used as a model organism at the genomic level to find drug targets which can be used for the development of novel drugs and/or vaccines for human filariasis. Setaria digitata causes cerebrospinal nematodiasis in goats, sheep, and horses posing a serious threat to livestock in developing countries. The genome sequence of S. digitata will assist in finding candidate genes to use as drug targets in both S. digitata and W. bancrofti. The assembled draft genome is similar to 90 Mb long and contains 8,974 genomicscaffolds with a G+C content of 31.73%

Novel sequence variants and a high frequency of recurrent polymorphisms in <it>BRCA1 </it>gene in Sri Lankan breast cancer patients and at risk individuals

Abstract Background Breast Cancer is the most commonly diagnosed cancer among Sri Lankan women. Germline mutations in the susceptibility genes BRCA1 and BRCA2 in hereditary breast/ovarian cancer, though low in prevalence, are highly penetrant and show geographical variations. There have been only a few reports from Asia on mutations in BRCA1/2 genes and none from Sri Lanka. Methods A total of 130 patients with (N = 66) and without (N = 64) a family history of breast cancer, 70 unaffected individuals with a family history of breast cancer and 40 control subjects were analysed for BRCA1 mutations. All but exon 11 were screened by single strand conformation analysis (SSCP) and heteroduplex analysis. PCR products which showed abnormal patterns in SSCP were sequenced. Exon 11 was directly sequenced. Results Nineteen sequence variants were found in BRCA1 gene. Two novel deleterious frame-shift mutations; c.3086delT/exon11 (in one patient) and c.5404delG/exon21 (in one patient and two of her family members) were identified. A possibly pathogenic novel missense mutation (c.856T>G/exon 11) and three novel intronic variants (IVS7+36C>T, IVS7+41C>T, IVS7+49del15) were characterised. Ten previously reported common polymorphisms and three previously reported intronic variants were also observed. Conclusion After screening of 66 patients with family history and 64 sporadic breast cancer patients, 2 deleterious mutations (c.3086delT and c.5404delG) in two families were identified and two more possibly pathogenic mutations (c.856T>G and IVS17-2A>T) in two families were identified. Data base BRCA1 - Gene Bank: Accession # U14680 Version # 14680.1</p