In silico identification and in vitro validation of alpha-hederin as a potent inhibitor of Wnt/ß-catenin signaling pathway in breast cancer stem cells.

Cancer stem cells (CSCs) play a vital role in metastasis, recurrence and chemoresistance in breast cancer. ß-catenin, which is a frequently over activated protein in CSCs, binds to T-cell factor/lymphoid enhancer factor (Tcf/Lef) family transcription factors leading to ectopic expression of Wnt pathway responsive genes necessary for the maintenance and action of CSCs. With the aim of identifying a small molecules that can effectively eliminate CSCs, molecular docking studies were performed against the Tcf/Lef binding hotspot on ß-catenin using a library of 100 natural or synthetic small molecules. Small molecule ligands giving docking energy better than - 7 kcal/mol were further investigated by binding interactions analysis and molecular dynamics (MD) simulations. These compounds were then investigated in vitro, for cytotoxicity against CSCs isolated from MDA-MB-231 triple negative breast cancer cells. Alpha-hederin (AH) was identified as the only compound in the selected library that has cytotoxicity against breast CSCs. AH was further investigated for it's ability to regulate Wnt pathway target genes (Cyclin D1 and CD44)and the tumor suppressor p53by real-time quantitative PCR. Absorption, distribution, metabolism, excretion and toxicity properties of the AH was predicted in silico. AH significantly down regulated the transcription of Cyclin D1 and CD44 while up-regulating the transcription of p53. AH was predicted to have acceptable drug likeness. Although AH is currently known to inhibit the growth of various cancer cells in vitro, present study demonstrated for the first time that it is a potent inhibitor of Wnt/ß-catenin signaling pathway and induce apoptosis in breast CSCs.

Autoantibodies in laryngeal cancer: detection and role as a biomarker.

OBJECTIVE: Diagnostic role of autoantibodies (AAb) as serological biomarkers has not been specifically investigated in laryngeal cancer (LC) previously. The study investigates the presence of anti-LC AAbs and their potential as a biomarker for early diagnosis of LC, to improve patient outcome. METHOD: Anti-LC AAb levels were investigated in LC patients (n = 30) and healthy individuals (n = 30) by indirect enzyme-linked immunosorbent assay (ELISA). Patient AAb levels were analyzed with various clinical factors, primarily tumor stage. RESULTS: AAb levels were significantly higher in LC patients than in the control group (P = .019). The diagnostic performance of AAb-level testing for LC detection presented a sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of 70% each. The positive likelihood (LR+) and negative likelihood (LR-) ratios were 2.33 and 0.43, respectively. AAb levels were independent of cancer stage (P = .708), duration since first appearance of symptoms (P = .228), duration of medical attention (P = .231), and degree of risk-factor exposure (P = .478). CONCLUSION: Significant level of AAbs could be detected among LC patients with good diagnostic performance, irrespective of stage. Thus, anti-LC AAbs reflect potential to be utilized as predictive biomarkers in early diagnostics of LC.

Factors affecting iron absorption and the role of fortification in enhancing iron levels.

Iron is an important micronutrient required for a number of biological processes including oxygen transport, cellular respiration, the synthesis of nucleic acids and the activity of key enzymes. The World Health Organization has recognised iron deficiency as the most common nutritional deficiency globally and as a major determinant of anaemia. Iron deficiency anaemia affects 40% of all children between the ages of 6 and 59 months, 37% of mothers who are pregnant and 30% of women between the ages of 15 and 49 years worldwide. Dietary iron exists in two main forms known as haem iron and non-haem iron. Haem iron is obtained from animal sources such as meat and shows higher bioavailability than non-haem iron, which can be obtained from both plant and animal sources. Different components in food can enhance or inhibit iron absorption from the diet. Components such as meat proteins and organic acids increase iron absorption, while phytate, calcium and polyphenols reduce iron absorption. Iron levels in the body are tightly regulated since both iron overload and iron deficiency can exert harmful effects on human health. Iron is stored mainly as haemoglobin and as iron bound to proteins such as ferritin and hemosiderin. Iron deficiency affects individuals at increased risk due to factors such as age, pregnancy, menstruation and various diseases. Different solutions for iron deficiency are applied at individual and community levels. Iron supplements and intravenous iron can be used to treat individuals with iron deficiency, while various types of iron-fortified foods and biofortified crops can be employed for larger communities. Foods such as rice, flour and biscuits have been used to prepare fortified iron products. However, it is important to ensure the fortification process does not exert significant negative effects on organoleptic properties and the shelf life of the food product.

Cytotoxicity against Human Hepatocellular Carcinoma (HepG2) Cells and Anti-Oxidant Activity of Selected Endemic or Medicinal Plants in Sri Lanka.

Hepatocellular carcinoma (HCC) is the most fatal cancer globally with limited treatment options. Plants and herbs have been used to treat cancer and other diseases for a long time by traditional practitioners in Sri Lanka. In the present study, leaf and bark extracts of selected plants were investigated for cytotoxic properties on HepG2 cells. Anti-oxidant activity and total phenolic and flavonoid contents were also determined. Plant extracts that exerted cytotoxic effects on the HepG2 cell line with IC50 <100 µg/mL were tested on normal liver epithelial cells (THLE-3). Out of the 56 extracts, 21 exhibited potent cytotoxic effects (IC50 < 100 µg/mL) on HepG2 cells after 48 h exposure, and 12 were less toxic (IC50 > 100 µg/mL) to THLE-3 normal liver cells. Six extracts exhibited potent radical scavenging activity with EC50 < 100 µg/mL against 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, while 17 extracts showed potent anti-oxidant activity (Trolox equivalents > 100 mg/g) against ferric reducing anti-oxidant power (FRAP) assay. Out of the 56 extracts, 15 had total phenolic content above 100 mg/g of gallic acid equivalents, and 4 had flavonoid content above 100 mg/g of quercetin equivalents. Among the extracts screened, hexane, dichloromethane, ethyl acetate, and methanol extracts of Allophylus cobbe leaves (IC50 - 9.388, 6.8, 19.95, and 11.3 µg/mL, respectively), Madhuca longiflora bark (IC50 - 14.42 µg/mL), methanol extract of Munronia pinnata bark (IC50 - 52.06 µg/mL), and hexane, dichloromethane, ethyl acetate, and methanol extracts of Adenanthera bicolor (IC50 - 45.86, 27.35, 24.56, and 61.83 µg/mL, respectively) exerted potent cytotoxicity against HepG2 with less toxicity (IC50 > 100 µg/mL) to THLE-3 cells after 48 h of incubation. These findings provide a direction to isolate possible anti-cancer compounds for hepatocellular carcinoma.

A new liposomal nanocarrier for co-delivery of gedunin and p-glycoprotein siRNA to target breast cancer stem cells.

Gedunin is a secondary metabolite found in neem tree. Since the first discovery of this compound, its bio-active properties have been continuously evaluated. However, the low hydrophobicity of gedunin decreases its bioavailability and pharmacokinetic profile. In the present investigation, a new liposomal nanocarrier for co-delivery of gedunin and P-glycoprotein (P-gp) siRNA [siRNA coated liposomal gedunin (Lipo-Ged-siRNA)] was developed to improve the anti-proliferative activity of gedunin. Characteristics of prepared Lipo-Ged-siRNA demonstrated promising effects. Lipo-Ged-siRNA showed greater anti-proliferative effects (IC50-8.5 µg/mL) followed by pure gedunin (IC50- 40.2 µg/mL) in breast cancer stem cells (bCSCs). Immunofluorescence analysis demonstrated reduced expression of P-gp following exposure to Lipo-Ged-siRNA. Furthermore, Lipo-Ged-siRNA affected the expression of ABCB1, Cyclin D1, Bax, p53, and surviving genes in bCSCs.

Identification of 3-O-α-l-arabinosyl oleanolic acid, a triterpenoid saponin, as a new breast cancer stem cell growth inhibitor.

This study was conducted to determine the anti-cancer activity of 3-O-α-L-arabinosyl oleanolic acid (3-O-L-AO), a triterpenoid saponin, isolated from the leaves of Schumacheria castaneifolia Vahl in breast cancer stem cells (bCSCs) grown in hypoxia. Anti-proliferative effects of 3-O-L-AO in bCSCs were determined using WST-1 assay. Real-time PCR was employed to evaluate the effects of 3-O-L-AO on apoptosis. Compound 3-O-L-AO exerted greater anti-proliferative effect in bCSCs grown under hypoxic conditions. Treatment of bCSCs with 3-O-L-AO resulted in a significant up-regulation of Bax and p53 and a significant down-regulation of survivin, HIF-1α and HIF-2α. Activation of caspase 3/7 activity and apoptosis-related morphological changes in bCSCs exposed to 3-O-L-AO further confirmed that 3-O-L-AO can induce apoptosis. Collectively, the results obtained indicated that 3-O-L-AO can be considered as a new anti-cancer agent to target chemo- and radio-therapy-resistant bCSCs.

Hexane Extract of Garcinia quaesita Fruits Induces Apoptosis in Breast Cancer Stem Cells Isolated from Triple Negative Breast Cancer Cell Line MDA-MB-231.

Development of therapy resistance is a major clinical issue in breast cancer treatments. Breast cancer stem cells (bCSCs) have a clearly defined role in the development of breast cancer therapy resistance and tumor recurrence. Therefore, discovery of new treatment strategies to circumvent cancer therapy resistance and tumor recurrence by targeting bCSCs is desperately needed. Fruits of many Garcinia species are edible and, possess a range of health benefits. Garcinia quaesita, a species in the genus Garcinia, is endemic to Sri Lanka. Dried fruits of G. quaesita are commonly used to flavor dishes in Sri Lanka. The present study assessed the potential anticancer and apoptotic properties of G. quaesita fruit extracts in bCSCs using WST-1 cell proliferation assay, sphere formation assay, caspase 3/7 assay, real-time PCR and fluorescent and phase-contrast microscopy. DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and FRAP (Ferric Reducing Anti-oxidant Power) assays were used as anti-oxidant assays. The hexane extract of G. quaesita fruits was found to mediate cytotoxicity in bCSCs through induction of apoptosis. Furthermore, the hexane extract showed free radical scavenging ability. This pilot investigation provides a rationale to consume G. quaesita fruits as an anticancer dietary supplement for breast cancer patients.

Evaluation of anticancer effects of a pharmaceutically viable extract of a traditional polyherbal mixture against non-small-cell lung cancer cells.

OBJECTIVE: The present work tested organic solvents to prepare an extract with anticancer properties from a polyherbal mixture containing Nigella sativa (seeds), Hemidesmus indicus (roots) and Smilax glabra (rhizomes). We evaluate anticancer effects in non-small-cell lung cancer cells (NCI-H292), and discuss optimization for pharmaceutical use in the context of efficacy, yield and toxicity. METHODS: Using different organic solvents, six extracts were prepared from the polyherbal mixture. Based on the cytotoxic effects of these extracts on NCI-H292 cells and normal lung cells (MRC-5), as evaluated by the sulphorhodamine B assay, the total ethyl acetate (T-EA) extract was selected for further analysis. The possible anticancer mechanisms were assessed by evaluating the extract's effects on apoptosis (through fluorescent microscopic analysis, DNA fragmentation analysis, caspase 3/7 assay and analysis of expression levels of apoptosis-related genes p53, Bax, survivin, Hsp70 and Hsp90), colony formation and antioxidant activity. RESULTS: The extract had cytotoxic effects against NCI-H292 cells in a time- and dose-dependent manner. Significant antioxidant activity and inhibition of colony formation were also observed. The expression level of caspase 3/7 significantly (P < 0.001) increased in NCI-H292 cells treated with 50 µg/mL of the extract. The same dosage led to a significant increase in expression levels of Bax and p53 (P < 0.05 and P < 0.01 respectively), accompanied by a significant decrease (P < 0.0001) in survivin, Hsp70 and Hsp90. CONCLUSION: T-EA extract of the above polyherbal mixture has cytotoxicity against NCI-H292 cells via induction of apoptosis, antioxidant effects and inhibition of colony formation.

In Vitro Anticancer Effect of Gedunin on Human Teratocarcinomal (NTERA-2) Cancer Stem-Like Cells.

Gedunin is one of the major compounds found in the neem tree (Azadirachta indica). In the present study, antiproliferative potential of gedunin was evaluated in human embryonal carcinoma cells (NTERA-2, a cancer stem cell model) and peripheral blood mononuclear cells (PBMCs), using Sulforhodamine (SRB) and WST-1 assays, respectively. The effects of gedunin on expression of heat shock protein 90 (HSP90), its cochaperone Cdc37, and HSP client proteins (AKT, ErbB2, and HSF1) were evaluated by real-time PCR. Effects of gedunin on apoptosis were evaluated by (a) apoptosis associated morphological changes, (b) caspase 3/7 expression, (c) DNA fragmentation, (d) TUNEL assay, and (e) real-time PCR of apoptosis related genes (Bax, p53, and survivin). Gedunin showed a promising antiproliferative effect in NTERA-2 cells with IC50 values of 14.59, 8.49, and 6.55 µg/mL at 24, 48, and 72 h after incubations, respectively, while exerting a minimal effect on PBMCs. Expression of HSP90, its client proteins, and survivin was inhibited and Bax and p53 were upregulated by gedunin. Apoptosis related morphological changes, DNA fragmentation, and increased caspase 3/7 activities confirmed the proapoptotic effects of gedunin. Collectively, results indicate that gedunin may be a good drug lead for treatment of chemo and radiotherapy resistant cancer stem cells.