Ethoxyquin Inhibits the Progression of Murine Ehrlich Ascites Carcinoma through the Inhibition of Autophagy and LDH

Cancer cells exhibit an increased glycolysis rate for ATP generation (the Warburg effect) to sustain an increased proliferation rate. In tumor cells, the oxidation of pyruvate in the Krebs cycle is substituted by lactate production, catalyzed by LDH. In this study, we use ethoxyquin (EQ) as a novel inhibitor to target LDH in murine Ehrlich ascites carcinoma (EAC) and as a combination therapy to improve the therapeutic efficacy of the conventional chemotherapy drug, cisplatin (CIS). We investigated the anti-tumor effect of EQ on EAC-bearing mice and checked whether EQ can sustain the anti-tumor potential of CIS and whether it influences LDH activity. Treatment with EQ had evident anti-tumor effects on EAC as revealed by the remarkable decrease in the expression of the anti-apoptotic gene Bcl-2 and by a significant increase in the expression of apoptotic genes (BAX and caspase-3). EQ also caused a significant decrease in the autophagic activity of EAC cells, as shown by a reduction in the fluorescence intensity of the autophagosome marker. Additionally, EQ restored the altered hematological and biochemical parameters and improved the disrupted hepatic tissues of EAC-bearing mice. Co-administration of EQ and CIS showed the highest anti-tumor effect against EAC. Collectively, our findings propose EQ as a novel inhibitor of LDH in cancer cells and as a combinatory drug to increase the efficacy of cisplatin. Further studies are required to validate this therapeutic strategy in different cancer models and preclinical trials.

NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis

Background Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. Methods Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. Results We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. Conclusions This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.

189 GLUCOCORTICOID RECEPTOR AND SERUM- AND GLUCOCORTICOID-INDUCED KINASE-1 IN ESOPHAGEAL ADENOCARCINOMA AND ADJACENT BARRETT’S ESOPHAGUS

Barrett’s esophagus (BE) is a consequence of gastroesophageal reflux disease and is predisposed to esophageal adenocarcinoma (EAC). EAC is an exemplar model of inflammation-associated cancer. Glucocorticoids suppress inflammation through glucocorticoid receptor (GR) and serum- and glucocorticoid-induced kinase−1 (Sgk1) expressions. Methods We immunolocalized GR and Sgk1 in EAC and the adjacent BE tissues and studied their association with clinical disease course in 87 patients with EAC who underwent surgical resection (N = 58) or endoscopic submucosal dissection (N = 29). Results Low GR and Sgk1 expressions in adjacent BE tissues were associated with adverse clinical outcomes (P = 0.0008 and 0.034, respectively). Patients with low Sgk1 expression in EAC cells exhibited worse overall survival (P = 0.0018). In multivariate Cox regression analysis, low GR expression in the adjacent nonmalignant BE tissues was significantly associated with worse overall survival (P = 0.023). Conclusion The present study indicated that evaluation of GR and Sgk1 expressions in both the EAC cells and adjacent nonmalignant BE tissues could help to predict clinical outcomes following endoscopic and surgical treatments. In particular, the GR status in BE tissues adjacent to EAC was an independent prognostic factor.

Extract of Tagetes Erecta Could Be Used as a Potential Drug Candidate Against Cancer: A Study on the Anticancer Efficacy of Medicinal Plants Involving in Vitro and in Vivo Approach

Globally, the burden of cancer is increasing consistently. Modern cancer therapies include lots of toxicity in the non-targeted organs reducing the life expectancy of the patients. Therefore, the development of safer alternative medicines with less toxicity and high efficacy is of immense importance. The present study was designed to evaluate the anticancer activity of a medicinal plant, “Tagetes erecta” (TE), in established cancer cell lines in vitro and in animal models in vivo. GC-MS analysis was performed that revealed hexadecanoic acid, Linolenic acid, Quinic acid, 2,3- dihydrobenzofuran (Coumaran), and β-stigmasterol as major bioactive compounds in TE leaves. Aqueous extract of Tagetes erecta (AETE) treatment potentially reduced the tumor weight (TW) and tumor volume (TV) and increased the life span in EAC-induced tumor-bearing Swiss albino mice. Side effect analysis confirmed the lack of toxicity of AETE to non-targeted organs in normal Swiss albino mice. Studies in cancer cell lines indicated dose and time-dependent cytotoxicity in Human laryngeal carcinoma (HEp-2) and Ehrlich ascites carcinoma (EAC) cells. Flow cytometric analysis established significant induction of apoptosis in EAC cells without arresting the cell cycle. In addition, AETE treatment led to a significant increase in cells with depolarised mitochondrial membrane potential. The present study indicated that AETE potentially inhibits tumor progression without disturbing normal body physiology. Thus, we conclude that AETE can be used as a potential therapeutic agent against cancer.

Nrf2/Keap1-Pathway Activation and Reduced Susceptibility to Chemotherapy Treatment by Acidification in Esophageal Adenocarcinoma Cells

Chronic acid reflux causes cellular damage and inflammation in the lower esophagus. Due to these irritating insults, the squamous epithelium is replaced by metaplastic epithelium, which is a risk factor for the development of esophageal adenocarcinoma (EAC). In this study, we investigated the acid susceptibility in a Barrett’s cell culture in vitro model, using six cell lines, derived from squamous epithelium (EPC1 and EPC2), metaplasia (CP-A), dysplasia (CP-B), and EAC (OE33 and OE19) cells. Cells exposed to acidic pH showed a decreased viability dependent on time, pH, and progression status in the Barrett’s sequence, with the highest acid susceptibility in the squamous epithelium (EPC1 and EPC2), and the lowest in EAC cells. Acid pulsing was accompanied with an activation of the Nrf2/Keap1- and the NFκB-pathway, resulting in an increased expression of HO1—independent of the cellular context. OE33 showed a decreased responsiveness towards 5-FU, when the cells were grown in acidic conditions (pH 6 and pH 5.5). Our findings suggest a strong damage of squamous epithelium by gastroesophageal reflux, while Barrett’s dysplasia and EAC cells apparently exert acid-protective features, which lead to a cellular resistance against acid reflux.

In vivo toxicity and antitumor activity of newly green synthesized reduced graphene oxide/silver nanocomposites

A novel biosynthesis of dual reduced graphene oxide/silver nanocomposites (rGO/AgNC) using the crude metabolite of Escherichia coli D8 (MF06257) strain and sunlight is introduced in this work. Physicochemical analysis of these rGO/AgNC revealed that they are sheet-like structures having spherically shaped silver nanoparticles (AgNPs) with an average particle size of 8 to 17 nm, and their absorption peak ranged from 350 to 450 nm. The biosynthesized rGO/AgNC were characterized by UV–vis and FT-IR spectra, X-ray diffraction, Zeta potential and transmission electron microscopy. After the injection of these nanocomposites to mice, their uptake by the kidney and liver has been proven by the ultrastructural observation and estimation of the hepatic and renal silver content. These nanocomposites caused a moderate toxicity for both organs. Changes in the liver and kidney functions and histopathological effects had been observed. The rGO/AgNC revealed a remarkable antitumor effect. They showed a dose-dependent cytotoxic effect on Ehrlich ascites carcinoma (EAC) cells in vitro. Treatment of mice bearing EAC tumors intraperitoneally with 10 mg/kg rGO/AgNC showed an antiproliferative effect on EAC cells, reduced ascites volume, and maintained mice survival. The results indicate that this green synergy of silver nanoparticles with reduced graphene oxide may have a promising potential in cancer therapy.

Aldo-Keto Reductase 1C3 Mediates Chemotherapy Resistance in Esophageal Adenocarcinoma via ROS Detoxification

Esophageal adenocarcinoma (EAC) is one of the most lethal malignancies, and limits promising treatments. AKR1C3 represents a therapeutic target to combat the resistance in many cancers. However, the molecular mechanism of AKR1C3 in the chemotherapy resistance of EAC is still unclear. We found that the mRNA level of AKR1C3 was higher in EAC tumor tissues, and that high AKR1C3 expression might be associated with poor overall survival of EAC patients. AKR1C3 overexpression decreased cell death induced by chemotherapeutics, while knockdown of AKR1C3 attenuated the effect. Furthermore, we found AKR1C3 was inversely correlated with ROS production. Antioxidant NAC rescued chemotherapy-induced apoptosis in AKR1C3 knockdown cells, while the GSH biosynthesis inhibitor BSO reversed a protective effect of AKR1C3 against chemotherapy. AKT phosphorylation was regulated by AKR1C3 and might be responsible for eliminating over-produced ROS in EAC cells. Intracellular GSH levels were modulated by AKR1C3 and the inhibition of AKT could reduce GSH level in EAC cells. Here, we reported for the first time that AKR1C3 renders chemotherapy resistance through controlling ROS levels via AKT signaling in EAC cells. Targeting AKR1C3 may represent a novel strategy to sensitize EAC cells to conventional chemotherapy.

Identification of TLR2 Signalling Mechanisms Which Contribute to Barrett’s and Oesophageal Adenocarcinoma Disease Progression

Chronic inflammation plays an important role in the pathogenesis of oesophageal adenocarcinoma (EAC) and its only known precursor, Barrett’s oesophagus (BE). Recent studies have shown that oesophageal TLR2 levels increase from normal epithelium towards EAC. TLR2 signalling is therefore likely to be important during EAC development and progression, which requires an inflammatory microenvironment. Here, we show that, in response to TLR2 stimulation, BE organoids and early-stage EAC cells secrete pro-inflammatory cytokines and chemokines which recruit macrophages to the tumour site. Factors secreted from TLR2-stimulated EAC cells are shown to subsequently activate TLR2 on naïve macrophages, priming them for inflammasome activation and inducing their differentiation to an M2/TAM-like phenotype. We identify the endogenous TLR2 ligand, HMGB1, as the factor secreted from EAC cells responsible for the observed TLR2-mediated effects on macrophages. Our results indicate that HMGB1 signalling between EAC cells and macrophages creates an inflammatory tumour microenvironment to facilitate EAC progression. In addition to identifying HMGB1 as a potential target for early-stage EAC treatment, our data suggest that blocking TLR2 signalling represents a mechanism to limit HMGB1 release, inflammatory cell infiltration and inflammation during EAC progression.

In vivo Toxicity and Antitumor Activity of Newly Green Synthesized Reduced Graphene Oxide / Silver Nanocomposites

A novel biosynthesis of dual reduced graphene oxide/silver nanocomposites (rGO/AgNC) using the crude metabolite of Escherichia coli D8 (MF06257) strain and sunlight is introduced in this work. Physicochemical analysis of these rGO/AgNC revealed that they are sheet-like structures having spherically shaped AgNPs with an average particle size of 8 to 17 nm, and their absorption peak ranged from 350 to 450 nm. The biosynthesized rGO/AgNC were characterized by UV-Vis and FT-IR spectra, X-ray diffraction and transmission electron microscopy. After the injection of these nanocomposites to mice, their uptake by the kidney and liver has been proven by the ultrastructural observation and estimation of the hepatic and renal silver content. These nanocomposites caused a moderate toxicity for both organs. Changes in the liver and kidney functions and histopathological effects had been observed. The rGO/AgNC revealed a remarkable antitumor effect. They showed a dose-dependent cytotoxic effect on Ehrlich ascites carcinoma (EAC) cells in vitro. Treatment of mice bearing EAC tumors intraperitoneally with 10 mg/kg rGO/AgNC showed an antiproliferative effect on EAC cells, reduced ascites volume, and maintained mice survival. The results indicate that this green synergy of silver nanoparticles with reduced-graphene oxide may have a promising potential in cancer therapy.