Fatty acid synthase (FASN) is overexpressed and hyperactivated in several human carcinomas, including lung cancer. We characterize and compare the anti-cancer effects of the FASN inhibitors C75 and ...(-)-epigallocatechin-3-gallate (EGCG) in a lung cancer model.
We evaluated in vitro the effects of C75 and EGCG on fatty acid metabolism (FASN and CPT enzymes), cellular proliferation, apoptosis and cell signaling (EGFR, ERK1/2, AKT and mTOR) in human A549 lung carcinoma cells. In vivo, we evaluated their anti-tumour activity and their effect on body weight in a mice model of human adenocarcinoma xenograft.
C75 and EGCG had comparable effects in blocking FASN activity (96,9% and 89,3% of inhibition, respectively). In contrast, EGCG had either no significant effect in CPT activity, the rate-limiting enzyme of fatty acid β-oxidation, while C75 stimulated CPT up to 130%. Treating lung cancer cells with EGCG or C75 induced apoptosis and affected EGFR-signaling. While EGCG abolished p-EGFR, p-AKT, p-ERK1/2 and p-mTOR, C75 was less active in decreasing the levels of EGFR and p-AKT. In vivo, EGCG and C75 blocked the growth of lung cancer xenografts but C75 treatment, not EGCG, caused a marked animal weight loss.
In lung cancer, inhibition of FASN using EGCG can be achieved without parallel stimulation of fatty acid oxidation and this effect is related mainly to EGFR signaling pathway. EGCG reduce the growth of adenocarcinoma human lung cancer xenografts without inducing body weight loss. Taken together, EGCG may be a candidate for future pre-clinical development.
An unexplored molecular scenario that might explain the inhibitory impact of the anti-diabetic drug metformin on the genesis of breast cancer relates to metformin's ability to modulate the expression ...status of micro (mi)RNAs. We here report the first miRNA expression profiling of human epithelial breast cancer cells cultured in the presence of metformin. We conducted real-time transcription polymerase chain reaction (qRT-PCR) Arrays to quantitatively compare the expression profile of 88 cancer-related miRNA sequences before and after treatment of MCF-7 cells, which were used as well-differentiated, epithelioid cell controls, with graded concentrations of metformin. Metformin-treated MCF-7 cells notably exhibited up to 18-fold increases in miRNA lethal-7a (let-7a) expression compared with untreated control cells. We confirmed that MCF-7 cells undergoing epithelial-to-mesenchymal (EMT) transition in response to the cytokine TGFβ notably up-regulated (~5-fold) miRNA-181a expression and exhibited better mammosphere-forming capabilities. We then explored the ability of metformin to impede TGFβ-enhanced propensity of breast cancer stem cells to form mammospheres in a miRNA-181a-related manner. Remarkably, TGFβ treatment failed to up-regulate miRNA-181a expression in the presence of metformin, which was able to fully abrogate TGFβ-enhanced mammosphere-forming ability. In addition, metformin co-treatment fully prevented TGFβ-induced down-regulation of the tumor suppressor miRNA-96 (~10-fold). Metformin's molecular functioning to prevent invasive breast cancer can be explained in terms of its previously unrecognized ability to efficiently up-regulate the tumor-suppressive miRNAs let-7a & miRNA-96 and inhibit the oncogenic miRNA-181a, thus epigenetically preserving the differentiated phenotype of mammary epithelium while preventing EMT-related cancer-initiating cell self-renewal.
The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming ...hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka's nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E
2
/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E
2
/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E
2
signaling that leads to nuclear phospho-Ser118-ERα, which ultimately exacerbates genomic ER signaling in response to E
2
. Because E
2
stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140, which targets SOX2, the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator, SOX2, and the local and systemic ability of E
2
to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy.
The molecular mechanisms used by breast cancer stem cells (BCSCs) to survive and/or maintain their undifferentiated CD44
+
CD24
-/low
mesenchymal-like antigenic state remains largely unexplored. ...Autophagy, a key homeostatic process of cytoplasmic degradation and recycling evolved to respond to stress conditions, might be causally fundamental in the biology of BCSCs. Stable & specific knockdown of autophagy-regulatory genes by lentiviral-delivered small hairpin (sh) RNA drastically decreased the number of JIMT-1 epithelial BC cells bearing CD44
+
CD24
-/low
cell-surface antigens from ~75% in parental and control (-) shRNA-transduced cells to 26% and 7% in ATG8/LC3 shRNA- and ATG12 shRNA-transduced cells, respectively. Autophagy inhibition notably enhanced transcriptional activation of CD24 gene, potentiating the epithelial-like phenotype of CD44
+
CD24
+
cells versus the mesenchymal CD44
+
CD24
-/low
progeny. EMT-focused Real Time RT-PCR profiling revealed that genetic ablation of autophagy transcriptionally repressed the gene coding for the mesenchymal filament vimentin (VIM). shRNA-driven silencing of the ATG12 gene and disabling the final step in the autophagy pathway by the antimalarial drug chloroquine both prevented TGFb1-induced accumulation of vimentin in JIMT-1 cells. Knockdown of autophagy-specific genes was sufficient also to increase by up to 11-times the number of CD24
+
cells in MDA-MB-231 cells, a BC model of mesenchymal origin that is virtually composed of CD44
+
CD24
-/low
cells. Chloroquine treatment augmented the number of CD24
+
cells and concomitantly reduced constitutive overexpression of vimentin in MDA-MB-231 cells. This is the first report demonstrating that autophagy is mechanistically linked to the maintenance of tumor cells expressing high levels of CD44 and low levels of CD24, which are typical of BCSCs .
Display omitted .
•Poor water solubility limits clinical efficacy of silibinin in cancer.•Water-soluble silibinin meglumine prevents growth of lung cancer xenografts.•Silibinin meglumine impedes ...epithelial-to-mesenchymal transition in lung cancer.•Silibinin meglumine might be tested in clinical trials with lung cancer patients.
Silibinin is the primary active constituent of a crude extract (silymarin) from milk thistle plant (Silybum marianum) seeds. We explored the ability of an oral milk thistle extract formulation that was enriched with a water-soluble form of silibinin complexed with the amino-sugar meglumine to inhibit the growth of non-small-cell lung carcinoma (NSCLC) mouse xenografts. As a single agent, oral silibinin meglumine notably decreased the overall volumes of NSCLC tumors as efficiently as did the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Concurrent treatment with silibinin meglumine impeded the regrowth of gefitinib-unresponsive tumors, resulting in drastic tumor growth prevention. Because the epithelial-to-mesenchymal transition (EMT) is required by a multiplicity of mechanisms of resistance to EGFR TKIs, we evaluated the ability of silibinin meglumine to impede the EMT in vitro and in vivo. Silibinin-meglumine efficiently prevented the loss of markers associated with a polarized epithelial phenotype as well as the de novo synthesis of proteins associated with the mesenchymal morphology of transitioning cells. Our current findings with this non-toxic, orally active, and water-soluble silibinin formulation might facilitate the design of clinical trials to test the administration of silibinin meglumine-containing injections, granules, or beverages in combination with EGFR TKIs in patients with EGFR-mutated NSCLC.
The sole overexpression of pivotal regulators of the embryonic Epithelial-Mesenchymal Transition (EMT) genetic program ("EMT status") may be sufficient to efficiently drive the ontogeny of the breast ...cancer stem cell molecular signature independently of changes in EMT functioning ("EMT phenotype"). Using basal-like breast cancer models naturally enriched in either CD44
pos
CD24
low/neg
or CD44
pos
CD24
pos
tumor-initiating cell populations we herein illustrate that non-cytotoxic concentrations of the anti-diabetic drug metformin efficiently impedes the ontogeny of generating the stem cell phenotype by transcriptionally repressing the stem cell property EMT. Metformin treatment dynamically regulated the CD44
pos
CD24
neg/low
breast cancer stem cell immunophenotype, transcriptionally reprogrammed cells through decreased expression of key drivers of the EMT machinery including the transcription factors ZEB1, TWIST1 and SNAI2 (Slug) and the pleiotrophic cytokines TGFbs, and lastly impeded the propensity of breast cancer stem cells to form multicellular "microtumors" in non-adherent and non-differentiating conditions (i.e. "mammospheres"). These findings, altogether, provide strong motivation for the continued molecular understanding and clinical development of metformin as a non-toxic therapeutic aimed to interdict the breast cancer stem cell phenotype by targeting EMT, a molecular process that is central to the ontogenesis of the breast cancer stem cell molecular signature.
Transforming Growth Factor-b (TGFb) is a major driving force of the Epithelial-to-Mesenchymal (EMT) genetic program, which becomes overactive in the pathophysiology of many age-related human ...diseases. TGFb-driven EMT is sufficient to generate migrating cancer stem cells by directly linking the acquisition of cellular motility with the maintenance of tumor-initiating (stemness) capacity. Chronic diseases exhibiting excessive fibrosis can be caused by repeated and sustained infliction of TGFb-driven EMT, which increases collagen and extracellular matrix synthesis. Pharmacological prevention and/or reversal of TGFb-induced EMT may therefore have important clinical applications in the management of cancer metastasis as well as in the prevention and/or treatment of end-state organ failures. Earlier studies from our group have revealed that clinically-relevant concentrations of the biguanide derivative metformin, the most widely used oral agent to lower blood glucose concentration in patients with type 2 diabetes and metabolic syndrome, notably decreased both the self-renewal and the proliferation of trastuzumab-refractory breast cancer stem cell populations. Given that: a.) tumor-initiating cancer stem cells display a significant enrichment in the expression of basal/mesenchymal or myoepithelial markers, including an increased secretion of TGFb; b.) metformin treatment impedes the ontogeny of generating the stem cell phenotype by transcriptionally repressing key drivers of the EMT genetic program (e.g. ZEB1, TWIST1, SNAIL2 Slug, TGFbs), we recently hypothesized that prevention of TGFb-induced EMT might represent a common molecular mechanism underlying the anti-cancer stem cells and anti-fibrotic actions of metformin. Remarkably, metformin exposure not only impedes TGFb-promoted loss of the epithelial marker E-cadherin in MCF-7 breast cancer cells but it prevents further TGF-induced cell scattering and accumulation of the mesenchymal marker vimentin in Madin-Darby canine kidney (MDCK) cells. We now propose that metformin, by weakening the ability of TGFb signaling to fully induce mesenchymal cell states in a variety of pathological processes including fibrosis (e.g. chronic renal disease, non-alcoholic steatohepatitis, heart failure or sclerosis) and malignant progression (and likely by reducing TGFb-regulated inflammation and immune responses -inflamm-aging-), molecularly behaves as a bona fide anti-aging modality.
Pressurized media filters are the most effective means of preventing emitter clogging when reclaimed effluents are used in drip irrigation systems. In these filters, water pollutants are retained in ...a granular bed, which needs to be replaced once its life span has been reached. Silica sand is the most common material used as a filtration bed, but the use of alternative materials which may improve filtration efficiency and reduce environmental impact, should be explored. Thus, the aim of this study was to compare the effect of two different granular media (silica sand and recycled glass) used in three filters with different underdrain designs (collector arms, inserted domes, and porous media) on emitter clogging. Experiments were carried out by filtering a reclaimed effluent for the duration of 1000 h for each filter and material. Four irrigation laterals 90 m in length with a 2.3 l/h pressure-compensating emitter placed every 0.40 m along the dripline were placed after each filter. Filter performances were continuously assessed while emitter discharges at 8 selected locations across the laterals were measured at the beginning, after 500 h, and at the end of the experiment. Filtration cycles lasted longer with recycled glass, but turbidity removal was affected by the combination of bed material and underdrain. Only after 1000 h of irrigation, was the discharge significantly lower for those emitters protected with porous underdrain using glass compared with sand. Emitter discharge was considerably reduced at the end of the lateral due to a higher number of completely clogged emitters at this location, but there were not significant differences between granular materials and filter designs. Overall, the results show that using recycled glass does not significantly increase emitter clogging compared with silica sand.
•Recycled glass is an alternative material to silica sand as a filter bed.•Three different filter underdrain designs using a reclaimed effluent were tested.•Filtration cycles were longer using recycled glass.•More differences in emitter discharge between filters appeared using recycled glass.•Emitter discharge depended on the interaction between media, filter, and time.
PDF
