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1.
Molecules ; 26(16)2021 Aug 19.
Article in English | MEDLINE | ID: mdl-34443605

ABSTRACT

Extracts derived from the Ceratonia siliqua L. (carob) tree have been widely studied for their ability to prevent many diseases mainly due to the presence of polyphenolic compounds. In this study, we explored, for the first time, the anti-cancer properties of Cypriot carobs. We produced extracts from ripe and unripe whole carobs, pulp and seeds using solvents with different polarities. We measured the ability of the extracts to inhibit proliferation and induce apoptosis in cancer and normal immortalized breast cells, using the MTT assay, cell cycle analysis and Western Blotting. The extracts' total polyphenol content and anti-oxidant action was evaluated using the Folin-Ciocalteu method and the DPPH assay. Finally, we used LC-MS analysis to identify and quantify polyphenols in the most effective extracts. Our results demonstrate that the anti-proliferative capacity of carob extracts varied with the stage of carob maturity and the extraction solvent. The Diethyl-ether and Ethyl acetate extracts derived from the ripe whole fruit had high Myricetin content and also displayed specific activity against cancer cells. Their mechanism of action involved caspase-dependent and independent apoptosis. Our results indicate that extracts from Cypriot carobs may have potential uses in the development of nutritional supplements and pharmaceuticals.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Fabaceae/chemistry , Phenols/chemistry , Phenols/pharmacology , Solvents/chemistry , Apoptosis/drug effects , Caspases/metabolism , Cell Line, Tumor , Fruit/chemistry , Humans , Seeds/chemistry
2.
Nanotheranostics ; 5(1): 113-124, 2021.
Article in English | MEDLINE | ID: mdl-33391978

ABSTRACT

Treatment of breast cancer underwent extensive progress in recent years with molecularly targeted therapies. However, non-specific pharmaceutical approaches (chemotherapy) persist, inducing severe side-effects. Phytochemicals provide a promising alternative for breast cancer prevention and treatment. Specifically, resveratrol (res) is a plant-derived polyphenolic phytoalexin with potent biological activity but displays poor water solubility, limiting its clinical use. Here we have developed a strategy for delivering res using a newly synthesized nano-carrier with the potential for both diagnosis and treatment. Methods: Res-loaded nanoparticles were synthesized by the emulsion method using Pluronic F127 block copolymer and Vitamin E-TPGS. Nanoparticle characterization was performed by SEM and tunable resistive pulse sensing. Encapsulation Efficiency (EE%) and Drug Loading (DL%) content were determined by analysis of the supernatant during synthesis. Nanoparticle uptake kinetics in breast cancer cell lines MCF-7 and MDA-MB-231 as well as in MCF-10A breast epithelial cells were evaluated by flow cytometry and the effects of res on cell viability via MTT assay. Results: Res-loaded nanoparticles with spherical shape and a dominant size of 179±22 nm were produced. Res was loaded with high EE of 73±0.9% and DL content of 6.2±0.1%. Flow cytometry revealed higher uptake efficiency in breast cancer cells compared to the control. An MTT assay showed that res-loaded nanoparticles reduced the viability of breast cancer cells with no effect on the control cells. Conclusions: These results demonstrate that the newly synthesized nanoparticle is a good model for the encapsulation of hydrophobic drugs. Additionally, the nanoparticle delivers a natural compound and is highly effective and selective against breast cancer cells rendering this type of nanoparticle an excellent candidate for diagnosis and therapy of difficult to treat mammary malignancies.


Subject(s)
Breast Neoplasms/drug therapy , Drug Carriers , Micelles , Resveratrol/therapeutic use , Antineoplastic Agents/therapeutic use , Female , Humans , MCF-7 Cells
3.
Nutr Cancer ; 73(8): 1302-1308, 2021.
Article in English | MEDLINE | ID: mdl-32698633

ABSTRACT

The natural isoforms of vitamin E γ-tocotrienol (γ-ΤΤ) and δ-tocotrienol (δ-ΤΤ) and the synthetic derivative α-tocopheryl polyethylene glycol 1000 succinate (TPGS) have promising anticancer potency in a variety of cancer cell lines and animal models of cancer. Ongoing clinical trials are investigating the anti-tumor effectiveness of TTs in combination with chemotherapeutic agents in patients suffering from breast, colon, non-small cell lung and ovarian cancers. Despite extensive research on different types of cancer, the anticancer potency of TTs and TPGS has not been thoroughly investigated in leukemias. Given the fact that certain types of leukemias have very low survival rates and that patients suffer significantly from the toxic side effects of chemotherapeutic drugs, there is a need to develop novel treatments with increased specificity against cancer cells and reduced toxicity to the patients. The aim of this review is to report current evidence on the anticancer potency of TTs and TPGS on leukemic cells lines and to discuss future studies that could be carried out to investigate the role of these agents in the management of leukemias.


Subject(s)
Antineoplastic Agents , Tocotrienols , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line , Cell Line, Tumor , Humans , Polyethylene Glycols , Succinates/pharmacology , Tocotrienols/pharmacology , Vitamin E/pharmacology
4.
Oncol Rep ; 44(1): 126-138, 2020 07.
Article in English | MEDLINE | ID: mdl-32377731

ABSTRACT

Retrotransposons copy their sequences via an RNA intermediate, followed by reverse transcription into cDNA and random insertion, into a new genomic locus. New retrotransposon copies may lead to cell transformation and/or tumorigenesis through insertional mutagenesis. Methylation is a major defense mechanism against retrotransposon RNA expression and retrotransposition in differentiated cells, whereas stem cells are relatively hypo­methylated. Epithelial­to­mesenchymal transition (EMT), which transforms normal epithelial cells into mesenchymal­like cells, also contributes to tumor progression and tumor metastasis. Cancer stem cells (CSCs), a fraction of undifferentiated tumor­initiating cancer cells, are reciprocally related to EMT. In the present study, the outcome of long terminal repeat (LTR)­Viral­Like 30 (VL30) retrotransposition was examined in mouse mammary stem­like/progenitor HC11 epithelial cells. The transfection of HC11 cells with a VL30 retrotransposon, engineered with an EGFP­based retrotransposition cassette, elicited a higher retrotransposition frequency in comparison to differentiated J3B1A and C127 mouse mammary cells. Fluorescence microscopy and PCR analysis confirmed the specificity of retrotransposition events. The differentiated retrotransposition­positive cells retained their epithelial morphology, while the respective HC11 cells acquired mesenchymal features associated with the loss of E­cadherin, the induction of N­cadherin, and fibronectin and vimentin protein expression, as well as an increased transforming growth factor (TGF)­ß1, Slug, Snail­1 and Twist mRNA expression. In addition, they were characterized by cell proliferation in low serum, and the acquisition of CSC­like properties indicated by mammosphere formation under anchorage­independent conditions. Mammospheres exhibited an increased Nanog and Oct4 mRNA expression and a CD44+/CD24­/low antigenic phenotype, as well as self­renewal and differentiation capacity, forming mammary acini­like structures. DNA sequencing analysis of retrotransposition­positive HC11 cells revealed retrotransposed VL30 copies integrated at the vicinity of EMT­, cancer type­ and breast cancer­related genes. The inoculation of these cells into Balb/c mice produced cytokeratin­positive tumors containing pancytokeratin­positive cells, indicative of cell invasion features. On the whole, the findings of the present study demonstrate, for the first time, to the best of our knowledge, that stem­like epithelial HC11 cells are amenable to VL30 retrotransposition associated with the induction of EMT and CSC generation, leading to tumorigenesis.


Subject(s)
Cell Transformation, Neoplastic/pathology , Mammary Neoplasms, Experimental/pathology , Neoplastic Stem Cells/metabolism , Retroelements , Animals , Biomarkers, Tumor/metabolism , Cell Line , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Epithelial-Mesenchymal Transition , Female , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/metabolism , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Transfection
5.
J Biochem Mol Toxicol ; 34(3): e22443, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31909879

ABSTRACT

The aim of this study was to evaluate the impact that 6-O-(3″, 4″-di-O-trans-cinnamoyl)-α- l-rhamnopyranosylcatalpol (Dicinn) and verbascoside (Verb), two compounds simultaneously reported in Verbascum ovalifolium, have on tumor cell viability, apoptosis, cell cycle kinetics, and intracellular reactive oxygen species (ROS) level. At 100 µg/mL and 48 hours incubation time, Dicinn and Verb produced good cytotoxic effects in A549, HT-29, and MCF-7 cells. Dicinn induced cell-cycle arrest at the G0 /G1 phase and apoptosis, whereas Verb increased the population of subG1 cells and cell apoptosis rates. Furthermore, the two compounds exhibited time-dependent ROS generating effects in tumor cells (1-24 hours). Importantly, no cytotoxic effects were induced in nontumor MCF-10A cells by the two compounds up to 100 µg/mL. Overall, the effects exhibited by Verb in tumor cells were more potent, which can be correlated with its structural features, such as the presence of phenolic hydroxyl groups.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cytotoxins/pharmacology , Neoplasms/drug therapy , Reactive Oxygen Species/metabolism , Verbascum/chemistry , A549 Cells , Antineoplastic Agents, Phytogenic/chemistry , Cytotoxins/chemistry , Drug Screening Assays, Antitumor , HT29 Cells , Humans , MCF-7 Cells , Neoplasms/metabolism , Neoplasms/pathology
6.
Sci Rep ; 9(1): 14375, 2019 10 07.
Article in English | MEDLINE | ID: mdl-31591437

ABSTRACT

Breast cancer is the second in mortality rate malignancy among women. Despite the many advances in breast cancer treatment, there is still a need to improve drug efficacy and reduce non-specific effects. D-alpha-tocopheryl polyethylene glycol succinate (TPGS) is frequently used in the development of drug delivery systems to improve the pharmacokinetics of anti-cancer drugs and reduce multi-drug resistance. We have previously shown that TPGS not only acts as a carrier molecule but also exerts anti-cancer effects. As part of this study, we investigated the effect of TPGS with YM155, a small molecule suppressant of Survivin, in various breast cancer cell lines representing different subtypes of the disease. We aimed to evaluate the presumed synergistic effect of the TPGS-YM155 combination and reveal its mechanism of action. Our results show that the TPGS-YM155 combination acts synergistically to reduce specifically the viability of SKBR3 cells. The combination of these agents reduced activation of the AKT pathway, decreased Survivin and Bcl-2 levels, and induced caspase-dependent and independent apoptosis via the mitochondrial pathway. Importantly, the TPGS-YM155 combination did not significantly affect the viability of MCF-10A normal immortalized cells. In conclusion, the combination of YM155 and TPGS could be a promising approach against SKBR3-type breast cancer.


Subject(s)
Breast Neoplasms/drug therapy , Imidazoles/pharmacology , Naphthoquinones/pharmacology , Survivin/genetics , Vitamin E/pharmacology , Apoptosis/drug effects , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Drug Delivery Systems/methods , Drug Synergism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Survivin/antagonists & inhibitors
7.
Food Chem Toxicol ; 134: 110820, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31539616

ABSTRACT

The Romanian coastlines of the Black Sea have abundant seaweed resources, but little effort has been done to investigate their biological potential. The aim of the present study was to assess the in vitro antioxidant and anti-proliferative effects of Cystoseira barbata (Stackhouse) C. Agardh (Sargassaceae), a brown alga inhabiting the Black Sea coast of Romania. The 70% acetone, methanol and water extracts of C. barbata were evaluated for their total phenolic content, antioxidant activity and anti-proliferative potential against human tumor cell lines (pulmonary A549, colon HT-29, mammary MCF-7) and the non-tumor mammary epithelial MCF-10A cell line. C. barbata 70% acetone extract (CBAE) displayed the highest antioxidant and cytotoxic activities. The mechanism of CBAE anti-proliferative activity involved initially increased intracellular ROS accumulation, followed by increased DNA content in the subG1 phase and DNA fragmentation leading to excessive apoptosis. Thus, our study provides a theoretical basis for the use of CBAE as a tumor preventive agent. Furthermore, UHPLC-DAD-QTOF-MS analysis of CBAE tentatively identified 18 phlorotannins as fucophlorethol and eckol derivatives, containing three up to seven phloroglucinol units. In conclusion, C. barbata represents a valuable source for the development of macroalgal-based products with putative use as nutraceuticals and pharmaceuticals.


Subject(s)
Biological Products/pharmacology , Seaweed/chemistry , A549 Cells , Antioxidants/pharmacology , Apoptosis/drug effects , Biological Products/isolation & purification , Cell Proliferation/drug effects , Female , HT29 Cells , Humans , Inhibitory Concentration 50 , MCF-7 Cells , Reactive Oxygen Species/metabolism , Romania , Tannins/metabolism
8.
BMC Cancer ; 16: 279, 2016 Apr 20.
Article in English | MEDLINE | ID: mdl-27098354

ABSTRACT

BACKGROUND: Acquired resistance towards apoptosis is a hallmark of cancer. Elimination of cells bearing activated oncogenes or stimulation of tumor suppressor mediators may provide a selection pressure to overcome resistance. KC-53 is a novel biyouyanagin analogue known to elicit strong anti-inflammatory and anti-viral activity. The current study was designed to evaluate the anticancer efficacy and molecular mechanisms of KC-53 against human cancer cells. METHODS: Using the MTT assay we examined initially how KC-53 affects the proliferation rates of thirteen representative human cancer cell lines in comparison to normal peripheral blood mononuclear cells (PBMCs) and immortalized cell lines. To decipher the key molecular events underlying its mode of action we selected the human promyelocytic leukemia HL-60 and the acute lymphocytic leukemia CCRF/CEM cell lines that were found to be the most sensitive to the antiproliferative effects of KC-53. RESULTS: KC-53 promoted rapidly and irreversibly apoptosis in both leukemia cell lines at relatively low concentrations. Apoptosis was characterized by an increase in membrane-associated TNFR1, activation of Caspase-8 and proteolytic inactivation of the death domain kinase RIP1 indicating that KC-53 induced mainly the extrinsic/death receptor apoptotic pathway. Regardless, induction of the intrinsic/mitochondrial pathway was also achieved by Caspase-8 processing of Bid, activation of Caspase-9 and increased translocation of AIF to the nucleus. FADD protein knockdown restored HL-60 and CCRF/CEM cell viability and completely blocked KC-53-induced apoptosis. Furthermore, KC-53 administration dramatically inhibited TNFα-induced serine phosphorylation on TRAF2 and on IκBα hindering therefore p65/NF-κΒ translocation to nucleus. Reduced transcriptional expression of pro-inflammatory and pro-survival p65 target genes, confirmed that the agent functionally inhibited the transcriptional activity of p65. CONCLUSIONS: Our findings demonstrate, for the first time, the selective anticancer properties of KC-53 towards leukemic cell lines and provide a detailed understanding of the molecular events underlying its dual anti-proliferative and pro-apoptotic properties. These results provide new insights into the development of innovative and targeted therapies for the treatment of some forms of leukemia.


Subject(s)
Cell Proliferation/drug effects , Leukemia/drug therapy , Neoplasm Proteins/biosynthesis , Sesquiterpenes/administration & dosage , Spiro Compounds/administration & dosage , Apoptosis/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HL-60 Cells , Humans , Leukemia/genetics , Leukemia/pathology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/pathology , Mitochondria/drug effects , Mitochondria/genetics , NF-kappa B/biosynthesis , NF-kappa B/genetics , Neoplasm Proteins/genetics , Phosphorylation , Receptors, Tumor Necrosis Factor, Type I/biosynthesis , Receptors, Tumor Necrosis Factor, Type I/genetics , Sesquiterpenes/chemistry , Signal Transduction/drug effects , Spiro Compounds/chemistry
9.
Breast Cancer Res ; 17: 98, 2015 Jul 25.
Article in English | MEDLINE | ID: mdl-26208975

ABSTRACT

INTRODUCTION: Basal-like breast cancer (BLBC) is an aggressive subtype often characterized by distant metastasis, poor patient prognosis, and limited treatment options. Therefore, the discovery of alternative targets to restrain its metastatic potential is urgently needed. In this study, we aimed to identify novel genes that drive metastasis of BLBC and to elucidate the underlying mechanisms of action. METHODS: An unbiased approach using gene expression profiling of a BLBC progression model and in silico leveraging of pre-existing tumor transcriptomes were used to uncover metastasis-promoting genes. Lentiviral-mediated knockdown of interleukin-13 receptor alpha 2 (IL13Ralpha2) coupled with whole-body in vivo bioluminescence imaging was performed to assess its role in regulating breast cancer tumor growth and lung metastasis. Gene expression microarray analysis was followed by in vitro validation and cell migration assays to elucidate the downstream molecular pathways involved in this process. RESULTS: We found that overexpression of the decoy receptor IL13Ralpha2 is significantly enriched in basal compared with luminal primary breast tumors as well as in a subset of metastatic basal-B breast cancer cells. Importantly, breast cancer patients with high-grade tumors and increased IL13Ralpha2 levels had significantly worse prognosis for metastasis-free survival compared with patients with low expression. Depletion of IL13Ralpha2 in metastatic breast cancer cells modestly delayed primary tumor growth but dramatically suppressed lung metastasis in vivo. Furthermore, IL13Ralpha2 silencing was associated with enhanced IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and impaired migratory ability of metastatic breast cancer cells. Interestingly, genome-wide transcriptional analysis revealed that IL13Ralpha2 knockdown and IL-13 treatment cooperatively upregulated the metastasis suppressor tumor protein 63 (TP63) in a STAT6-dependent manner. These observations are consistent with increased metastasis-free survival of breast cancer patients with high levels of TP63 and STAT6 expression and suggest that the STAT6-TP63 pathway could be involved in impairing metastatic dissemination of breast cancer cells to the lungs. CONCLUSION: Our findings indicate that IL13Ralpha2 could be used as a promising biomarker to predict patient outcome and provide a rationale for assessing the efficacy of anti-IL13Ralpha2 therapies in a subset of highly aggressive basal-like breast tumors as a strategy to prevent metastatic disease.


Subject(s)
Breast Neoplasms/genetics , Interleukin-13 Receptor alpha2 Subunit/genetics , Lung Neoplasms/genetics , Neoplasm Metastasis/genetics , STAT6 Transcription Factor/genetics , Signal Transduction/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Disease-Free Survival , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/genetics , Humans , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Neoplasm Metastasis/pathology , Phosphorylation/genetics , Prognosis
10.
Biomed Res Int ; 2015: 584862, 2015.
Article in English | MEDLINE | ID: mdl-26137487

ABSTRACT

Vitamin E isoforms have been extensively studied for their anticancer properties. Novel drug delivery systems (DDS) that include liposomes, nanoparticles, and micelles are actively being developed to improve Vitamin E delivery. Furthermore, several drug delivery systems that incorporate Vitamin E isoforms have been synthesized in order to increase the bioavailability of chemotherapeutic agents or to provide a synergistic effect. D-alpha-tocopheryl polyethylene glycol succinate (Vitamin E TPGS or TPGS) is a synthetic derivative of natural alpha-tocopherol which is gaining increasing interest in the development of drug delivery systems and has also shown promising anticancer effect as a single agent. This review provides a summary of the properties and anticancer effects of the most potent Vitamin E isoforms and an overview of the various formulations developed to improve their efficacy, with an emphasis on the use of TPGS in drug delivery approaches.


Subject(s)
Antineoplastic Agents/administration & dosage , Neoplasms/drug therapy , Vitamin E/administration & dosage , alpha-Tocopherol/administration & dosage , Antineoplastic Agents/chemistry , Cell Line, Tumor , Drug Delivery Systems , Humans , Liposomes/administration & dosage , Liposomes/chemistry , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Neoplasms/pathology , Vitamin E/analogs & derivatives , Vitamin E/chemistry , alpha-Tocopherol/analogs & derivatives , alpha-Tocopherol/chemistry
11.
Comb Chem High Throughput Screen ; 18(3): 281-95, 2015.
Article in English | MEDLINE | ID: mdl-25747448

ABSTRACT

Modern methods of drug discovery and development in recent years make a wide use of computational algorithms. These methods utilise Virtual Screening (VS), which is the computational counterpart of experimental screening. In this manner the in silico models and tools initial replace the wet lab methods saving time and resources. This paper presents the overall design and implementation of a web based scientific workflow system for virtual screening called, the Life Sciences Informatics (LiSIs) platform. The LiSIs platform consists of the following layers: the input layer covering the data file input; the pre-processing layer covering the descriptors calculation, and the docking preparation components; the processing layer covering the attribute filtering, compound similarity, substructure matching, docking prediction, predictive modelling and molecular clustering; post-processing layer covering the output reformatting and binary file merging components; output layer covering the storage component. The potential of LiSIs platform has been demonstrated through two case studies designed to illustrate the preparation of tools for the identification of promising chemical structures. The first case study involved the development of a Quantitative Structure Activity Relationship (QSAR) model on a literature dataset while the second case study implemented a docking-based virtual screening experiment. Our results show that VS workflows utilizing docking, predictive models and other in silico tools as implemented in the LiSIs platform can identify compounds in line with expert expectations. We anticipate that the deployment of LiSIs, as currently implemented and available for use, can enable drug discovery researchers to more easily use state of the art computational techniques in their search for promising chemical compounds. The LiSIs platform is freely accessible (i) under the GRANATUM platform at: http://www.granatum.org and (ii) directly at: http://lisis.cs.ucy.ac.cy.


Subject(s)
High-Throughput Screening Assays , Internet , Medical Informatics , Algorithms , Biological Science Disciplines , Quantitative Structure-Activity Relationship
12.
Molecules ; 19(11): 19114-36, 2014 Nov 19.
Article in English | MEDLINE | ID: mdl-25415475

ABSTRACT

The roots of Vernonia kotschyana Sch. Bip. ex Walp. (Asteraceae) are used in Malian traditional medicine in the treatment of gastroduodenal ulcers and gastritis. Since oxidative stress is involved in gastric ulceration, the aim of this study was to screen the root extracts for their in vitro antioxidant activity and phenolic content. The roots were extracted successively with chloroform, ethyl acetate, ethanol and water. The antioxidant activity of root extracts was evaluated in both cell-free and cell-based assays. Their chemical characterization was performed by Fourier transform infrared spectroscopy (FT-IR) whereas the total phenolic content was determined by the Folin-Ciocalteu method. The ethyl acetate extract displayed the highest phenolic content and was found to be the most active in the free radical scavenging and lipid peroxidation inhibition assays; it also showed a high antioxidant activity in MCF-12F cells. This study suggests a potential use of the ethyl acetate extract of Vernonia kotschyana not only as an antioxidant agent in gastroduodenal ulcers and gastritis, but also in other disorders characterized by high levels of oxidative stress.


Subject(s)
Antioxidants/chemistry , Antioxidants/pharmacology , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plant Roots/chemistry , Vernonia/chemistry , Cell Line , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Gastritis/drug therapy , Humans , Lipid Peroxidation/drug effects , Oxidative Stress/drug effects , Phenols/chemistry , Phenols/pharmacology , Stomach Ulcer/drug therapy
13.
Nat Prod Commun ; 9(4): 481-2, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24868861

ABSTRACT

A raw extract and four extractive fractions were obtained from Cedrus brevifolia (Cyprus cedar) bark. They were all studied regarding the phenolic content and profile using spectrophotometry and HPLC-DAD-ESI-MS. The antioxidant activity was investigated using in vitro assays: DPPH and ABTS radicals scavenging and reducing power assays. The ethyl acetate fraction had the highest total phenolic and proanthocyanidin contents; a taxifolin-O-hexoside, catechin, epicatechin and procyanidin oligomers (three dimers, two trimers) were identified in this fraction. The ethyl acetate fraction was found to possess the highest DPPH and ABTS radicals scavenging effects (EC50 = 13.9 +/- 0.3 and 2.3 +/- 0.0 microg/mL, respectively) and reducing capacity (EC50 = 9.1 +/- 0.1 microg/mL). Antioxidant effects were highly correlated with total phenolic and proanthocyanidin contents (r = 0.89-0.99). These results suggest that Cedrus brevifolia bark is a new source of antioxidants.


Subject(s)
Antioxidants/chemistry , Cedrus/chemistry , Phenols/chemistry , Plant Bark/chemistry , Benzothiazoles/chemistry , Biphenyl Compounds/chemistry , Chromatography, High Pressure Liquid/methods , Free Radical Scavengers , Picrates/chemistry , Plant Extracts/chemistry , Proanthocyanidins/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Sulfonic Acids/chemistry
14.
Biochem Pharmacol ; 89(1): 31-42, 2014 May 01.
Article in English | MEDLINE | ID: mdl-24560876

ABSTRACT

D-alpha-tocopheryl polyethylene glycol succinate (TPGS) is a vitamin E derivative that has been intensively applied as a vehicle for drug delivery systems to enhance drug solubility and increase the oral bioavailability of anti-cancer drugs. Recently, it has been reported that TPGS acts as an anti-cancer agent alone or synergistically with chemotherapeutic drugs and increases the efficacy of nanoparticle formulations. In this study, we investigated the antitumor efficacy and the molecular mechanism of action of TPGS in breast cancer cell lines. Our results show that TPGS can induce G1/S cell cycle arrest and apoptosis in breast cancer cell lines (MCF-7 and MDA-MB-231) but not in "normal" (non-tumorigenic) immortalized cells (MCF-10A and MCF-12F). An investigation of the molecular mechanism of action of TPGS reveals that induction of G1/S phase cell cycle arrest is associated with upregulation of P21 and P27Kip1 proteins. Induction of apoptosis by TPGS involves the inhibition of phospho-AKT and the downregulation of the anti-apoptotic proteins Survivin and Bcl-2. Interestingly, our results also suggest that TPGS induces both caspase -dependent and -independent apoptotic signaling pathways and that this vitamin E derivative is selectively cytotoxic in breast cancer cell lines. When compared to the Survivin inhibitor YM155, TPGS was shown to be more selective for cancer cell growth inhibition. Overall our results suggest that TPGS may not only be useful as a carrier molecule for drug delivery, but may also exert intrinsic therapeutic effects suggesting that it may promote a synergistic interaction with formulated chemotherapeutic drugs.


Subject(s)
Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Cycle/drug effects , Inhibitor of Apoptosis Proteins/metabolism , Vitamin E/analogs & derivatives , Base Sequence , Blotting, Western , Breast Neoplasms/metabolism , Cell Line, Tumor , DNA Primers , Enzyme-Linked Immunosorbent Assay , Female , Humans , Polyethylene Glycols/pharmacology , Real-Time Polymerase Chain Reaction , Survivin , Vitamin E/pharmacology
15.
J Med Food ; 16(11): 984-91, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24195749

ABSTRACT

A crude hydromethanolic extract from Pinus brutia bark and its fractions (diethyl ether, ethyl acetate, n-butanol, and aqueous fractions) were studied with regard to their phenolic content and antioxidant activities. The total phenolics and proanthocyanidins in each extract were quantified by spectrophotometric methods; the polyphenolic profile was analyzed by RP-HPLC-DAD-ESI-MS. All extracts were tested with regard to their ability to scavenge free radicals (ABTS radical cation, superoxide and hydroxyl radicals), reduce ferric ions, and inhibit 15-lipoxygenase. P. brutia bark extracts had high phenolic contents (303.79±7.34-448.90±1.39 mg/g). Except diethyl ether extract, all other extracts contained proanthocyanidins ranging from 225.79±3.94 to 250.40±1.44 mg/g. Several polyphenols were identified by RP-HPLC-DAD-ESI-MS: taxifolin in diethyl ether extract, a taxifolin-O-hexoside, catechin, procyanidin dimers, and trimers in ethyl acetate extract. Except diethyl ether extract, all other extracts were effective scavengers of superoxide and hydroxyl radicals (EC50=33.5±1.1-54.93±2.85 µg/mL and 0.47±0.06-0.6±0.0 mg/mL, respectively). All extracts had noticeable 15-lipoxygenase inhibitory effects (EC50=22.47±0.75-34.43±2.25 µg/mL). We conclude that P. brutia bark is very promising for the dietary supplements industry due to its high free radical scavenging and 15-lipoxygenase inhibitory effects.


Subject(s)
Antioxidants/pharmacology , Arachidonate 15-Lipoxygenase/metabolism , Enzyme Inhibitors/pharmacology , Free Radicals/metabolism , Pinus/chemistry , Plant Extracts/pharmacology , Polyphenols/pharmacology , Antioxidants/analysis , Enzyme Inhibitors/analysis , Plant Bark/chemistry , Polyphenols/analysis
16.
Cancer Lett ; 337(2): 167-76, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23752064

ABSTRACT

The purpose of this work is to determine the molecular mechanisms underlying tamoxifen resistance. We show here that ER-ß is epigenetically silenced in a cell line with acquired tamoxifen resistance (MCF-7/TAM-R) and this could be reversed by 5-AZA-deoxycytidine (5-AZA) and trichostatin-A (TSA) pre-treatment. Subsequent treatment with 4-hydroxy-tamoxifen (4-OHT) induced ER-ß nuclear translocation, upregulated pS2 and p21 levels and reduced cell viability. Transfection with an ER-ß expression vector sensitized MCF-7/TAM-R cells to the growth inhibitory and pro-apoptotic effects of 4-OHT, indicating that ER-ß re-expression alone is sufficient to restore sensitivity to tamoxifen. This novel finding reveals that ER-ß is fundamental in overcoming acquired tamoxifen resistance and provides insights for new therapeutic protocols against breast cancer.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/genetics , Chromatin Assembly and Disassembly/drug effects , Drug Resistance, Neoplasm , Estrogen Receptor beta/genetics , Gene Silencing , Histone Deacetylase Inhibitors/pharmacology , Tamoxifen/analogs & derivatives , Active Transport, Cell Nucleus , Apoptosis/drug effects , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Survival/drug effects , Decitabine , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Estrogen Receptor beta/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hydroxamic Acids/pharmacology , MCF-7 Cells , Tamoxifen/pharmacology , Time Factors , Transfection
17.
BMC Cancer ; 13: 238, 2013 May 15.
Article in English | MEDLINE | ID: mdl-23675643

ABSTRACT

BACKGROUND: Soy phytoestrogens, such as daidzein and its metabolite equol, have been proposed to be responsible for the low breast cancer rate in Asian women. Since the majority of estrogen receptor positive breast cancer patients are treated with tamoxifen, the basic objective of this study is to determine whether equol enhances tamoxifen's anti-tumor effect, and to identify the molecular mechanisms involved. METHODS: For this purpose, we examined the individual and combined effects of equol and tamoxifen on the estrogen-dependent MCF-7 breast cancer cells using viability assays, annexin-V/PI staining, cell cycle and western blot analysis. RESULTS: We found that equol (>50 µM) and 4-hydroxy-tamoxifen (4-OHT; >100 nM) significantly reduced the MCF-7 cell viability. Furthermore, the combination of equol (100 µM) and 4-OHT (10 µM) induced apoptosis more effectively than each compound alone. Subsequent treatment of MCF-7 cells with the pan-caspase inhibitor Z-VAD-FMK inhibited equol- and 4-OHT-mediated apoptosis, which was accompanied by PARP and α-fodrin cleavage, indicating that apoptosis is mainly caspase-mediated. These compounds also induced a marked reduction in the bcl-2:bax ratio, which was accompanied by caspase-9 and caspase-7 activation and cytochrome-c release to the cytosol. Taken together, these data support the notion that the combination of equol and tamoxifen activates the intrinsic apoptotic pathway more efficiently than each compound alone. CONCLUSIONS: Consequently, equol may be used therapeutically in combination treatments and clinical studies to enhance tamoxifen's effect by providing additional protection against estrogen-responsive breast cancers.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Apoptosis/drug effects , Caspases/metabolism , Equol/pharmacology , Phytoestrogens/pharmacology , Tamoxifen/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Caspase 9/metabolism , Caspase Inhibitors/pharmacology , Cell Survival/drug effects , Cytochromes c/metabolism , Drug Synergism , Humans , MCF-7 Cells , Proto-Oncogene Proteins c-bcl-2/metabolism , Serpins/metabolism , Viral Proteins/metabolism , bcl-2-Associated X Protein/metabolism
18.
Cancer Lett ; 330(1): 113-21, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23201137

ABSTRACT

The olive polyphenols oleuropein and hydroxytyrosol were reported recently to produce extracellular hydrogen peroxide (H2O2) under standard culture conditions. The precise factors responsible for this production and the conditions promoting or retarding it are critical for the interpretation of the in vitro results. In this study, a systematic evaluation of the components of the most commonly used culture media revealed that sodium bicarbonate is the defining cause for the production of H2O2 by these polyphenols. The produced H2O2 caused extensive oxidative DNA damage and significant decrease in cell viability of cancer (MDA-MB-231) and normal (MCF-10A, STO) cells alike. Sodium pyruvate and the antioxidant N-acetyl cysteine (NAC) totally reversed these effects. Therefore, we conclusively identified the culture conditions that promote H2O2 production by these polyphenols, producing artifacts that may be misinterpreted as a specific anticancer activity. Our findings raise considerable questions regarding the use of culture media with sodium bicarbonate or sodium pyruvate as components, for the in vitro study of these and possibly other plant polyphenols.


Subject(s)
Olea/chemistry , Oxidants/pharmacology , Phenylethyl Alcohol/analogs & derivatives , Pyrans/pharmacology , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Culture Media , Female , Humans , Hydrogen Peroxide/metabolism , Iridoid Glucosides , Iridoids , Mice , Oxidative Stress/drug effects , Phenylethyl Alcohol/pharmacology , Plant Extracts/pharmacology , Plant Oils/pharmacology , Reactive Oxygen Species/metabolism
19.
Biochem Biophys Res Commun ; 425(1): 76-82, 2012 Aug 17.
Article in English | MEDLINE | ID: mdl-22820195

ABSTRACT

Indirubin derivatives gained interest in recent years for their anticancer and antimetastatic properties. The objective of the present study was to evaluate and compare the anticancer properties of the two novel bromo-substituted derivatives 6-bromoindirubin-3'-oxime (6BIO) and 7-bromoindirubin-3'-oxime (7BIO) in five different breast cancer cell lines. Cell viability assays identified that 6BIO and 7BIO are most effective in preventing the proliferation of the MDA-MB-231-TXSA breast cancer cell line from a total of five breast cancer cell lined examined. In addition it was found that the two compounds induce apoptosis via different mechanisms. 6BIO induces caspase-dependent programmed cell death through the intrinsic (mitochondrial) caspase-9 pathway. 7BIO up-regulates p21 and promotes G(2)/M cell cycle arrest which is subsequently followed by the activation of two different apoptotic pathways: (a) a pathway that involves the upregulation of DR4/DR5 and activation of caspase-8 and (b) a caspase independent pathway. In conclusion, this study provides important insights regarding the molecular pathways leading to cell cycle arrest and apoptosis by two indirubin derivatives that can find clinical applications in targeted cancer therapeutics.


Subject(s)
Apoptosis/drug effects , Breast Neoplasms/pathology , Indoles/pharmacology , Oximes/pharmacology , Breast Neoplasms/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Female , Humans , Neoplasm Invasiveness
20.
Biochem Pharmacol ; 78(1): 33-44, 2009 Jul 01.
Article in English | MEDLINE | ID: mdl-19447221

ABSTRACT

2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21(Cip1/Waf1) and up-regulating p16(INK4A). Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27(Kip1). Bcl-2 and p27(Kip1) were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-kappaB activity and p50 levels were increased by 2-ME2 and suppression of NF-kappaB signaling reduced p27(Kip1) expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27(Kip1) in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27(Kip1)-dependent G1/S phase arrest in conjunction with activating NF-kappaB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.


Subject(s)
Apoptosis/drug effects , Estradiol/analogs & derivatives , G1 Phase/physiology , Jurkat Cells/drug effects , NF-kappa B/physiology , Proto-Oncogene Proteins c-bcl-2/physiology , S Phase/physiology , 2-Methoxyestradiol , Cell Cycle/drug effects , Cell Cycle/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinases/antagonists & inhibitors , Estradiol/pharmacology , G1 Phase/drug effects , Humans , Jurkat Cells/cytology , S Phase/drug effects
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