Propolis Enhances 5-Fluorouracil Mediated Antitumor Efficacy and Reduces Side Effects in Colorectal Cancer: An in Vitro and in Vivo Study

In this study, we investigated the combined treatment of 5-fluorouracil (5-FU) and Anatolian propolis extract (PE) on colorectal cancer (CRC)using in vitro and in vivo studies. We exposed luciferase-transfected (Lovo-Luc CRC) cells and healthy colon cells (CCD-18Co) to varying concentrations of 5-FU and PE to assess their genotoxic, apoptotic, and cytotoxic effects, as well as their intracellular reactive oxygen species (iROS) levels. We also developed a xenograft model in nude mice and evaluated the anti-tumor effects of PE and 5-FU using various methods. Our findings showed that the combination of PE and 5-FU had selectivity against cancer cells, particularly at higher doses, and enhanced the anti-tumor effectiveness of 5-FU against colon CRC. The results suggest that PE can reduce side effects and increase the effectiveness of 5-FU through iROS generation in a dose-dependent manner.     

Synergistic effects of hormone therapy drugs and usnic acid on hormone receptor‐positive breast and prostate cancer cells

The aim of this study was to investigate the combined effects of usnic acid (UA) and Tamoxifen (Tam) or Enzalutamide (Enz) on hormone receptor‐positive breast and prostate cancer (BC and PC), respectively. The antiproliferative and apoptotic effects of Tam or Enz alone and in combination with UA on MCF7 and LNCaP cancer cells were detected. The results of the WST‐1 assay indicated that UA was a promising anticancer compound that significantly enhanced the effectiveness of hormone therapy drugs compared with each drug alone (combination index < 1). In addition, the combination of UA with Tam or Enz remarkably induced more cell cycle arrest at the G0/G1 phase and apoptosis than only drug‐treated cells (P < 0.01). Consequently, our findings suggest that the combination of UA with Tam or Enz may be a potential therapeutic approach for the treatment of BC and PC and further studies are required to exploit the potential mechanisms of synergistic effects.     

The two SAMP repeats and their phosphorylation state in Drosophila Adenomatous polyposis coli-2 play mechanistically distinct roles in negatively regulating Wnt signaling

The tumor suppressor Adenomatous polyposis coli (APC) plays a key role in regulating the canonical Wnt signaling pathway as an essential component of the β-catenin destruction complex. C-terminal truncations of APC are strongly implicated in both sporadic and familial forms of colorectal cancer. However, many questions remain as to how these mutations interfere with APC’s tumor suppressor activity. One set of motifs frequently lost in these cancer-associated truncations is the SAMP repeats that mediate interactions between APC and Axin. APC proteins in both vertebrates and Drosophila contain multiple SAMP repeats that lack high sequence conservation outside of the Axin-binding motif. In this study, we tested the functional redundancy between different SAMPs and how these domains are regulated, using Drosophila APC2 and its two SAMP repeats as our model. Consistent with sequence conservation–based predictions, we show that SAMP2 has stronger binding activity to Axin in vitro, but SAMP1 also plays an essential role in the Wnt destruction complex in vivo. In addition, we demonstrate that the phosphorylation of SAMP repeats is a potential mechanism to regulate their activity. Overall our findings support a model in which each SAMP repeat plays a mechanistically distinct role but they cooperate for maximal destruction complex function.     

Neuroprotection by mefenamic acid against D-serine: involvement of oxidative stress, inflammation and apoptosis

Mefenamic acid, a non-steroidal antiinflammatory drug (NSAID), directly and dose-dependently exhibits neuroprotective activity. In our study, we investigated the effects of mefenamic acid against d-serine on oxidative stress in the hippocampus, cortex and cerebellum of rats. Furthermore, the potential inflammatory and apoptotic effects of d-serine and potential protective effect of mefenamic acid were determined at mRNA and protein levels of TNF-α, IL-1β, Bcl-2 and Bax. We found that d-serine significantly increased oxidative stress, levels of inflammation- and apoptosis-related molecules in a region specific manner. Mefenamic acid treatment provided significant protection against the elevation of lipid peroxidation, protein oxidation, levels of TNF-α, IL-1β and Bax. As a conclusion, we suggest that d-serine, as a potential neurodegenerative agent, may have a pivotal role in the regulation of oxidative stress, inflammation and apoptosis; and NSAIDs, such as mefenamic acid, may assist other therapeutics in treating disorders where d-serine-induced neurotoxic mechanisms are involved in.     

To die or not to die: that is the autophagic question

Macroautophagy (commonly referred to as autophagy) is the process by which intact organelles and/or large portions of the cytoplasm are engulfed within double-membraned autophagic vacuoles for degradation. Whereas basal levels of autophagy ensure the physiological turnover of old and damaged organelles, the massive accumulation of autophagic vacuoles may represent either an alternative pathway of cell death or an ultimate attempt for cells to survive by adapting to stress. The activation of the autophagic pathway beyond a certain threshold may promote cell death directly, by causing the collapse of cellular functions as a result of cellular atrophy (autophagic, or type II, cell death). Alternatively, autophagy can lead to the execution of apoptotic (type I) or necrotic (type III) cell death programs, presumably via common regulators such as proteins from the Bcl-2 family. On the other hand, limited self-eating can provide cells with metabolic substrates to meet their energetic demands under stressful conditions, such as nutrient deprivation, or favor the selective elimination of damaged (and potentially dangerous) organelles. In these instances, autophagy operates as a pro-survival mechanism. The coordinate regulation of these opposite effects of autophagy relies upon a complex network of signal transducers, most of which also participate in non-autophagic signaling cascades. Thus, autophagy occupies a crucial position within the cell's metabolism, and its modulation may represent an alternative therapeutic strategy in several pathological settings including cancer and neurodegeneration. Here, we present a general outline of autophagy followed by a detailed analysis of organelle-specific autophagic pathways and of their intimate connections with cell death.     

DNA Methylation Mediates Persistent Epileptiform Activity In Vitro and In Vivo

Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5’ regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5’ region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5’ region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5’ region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5’ region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.     

Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16^INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.     

The effects of p53 on whole organism longevity are mediated by autophagy

The tumor suppressor protein p53 has a major impact on organismal aging. Recently it has become clear that p53 not only controls DNA damage responses, senescence and apoptosis but also plays a major role in the control of autophagy. Thus, deletion, depletion, or inhibition of p53 induces autophagy in human, mouse and nematode cells. We therefore tested the hypothesis that the mutation of the p53 orthologue CEP-1 might increase the life span of Caenorhabditis elegans through an increase in baseline autophagy. For this, we evaluated the survival of nematodes lacking cep-1, alone or in combination with RNA inference with the autophagy gene bec-1 (which encodes the orthologue of Atg6/Beclin 1). cep-1 mutants exhibited a prolonged life span. While BEC-1 depletion during adult life did not cause significant modification of the life expectancy of wild type controls, it did reduce the increased life span of cep-1 mutants down to approximately normal levels. These results indicate that the life span-extending effect of the cep-1 mutation is mediated by autophagy. These results lend support to the hypothesis that autophagy has a broad positive impact on organismal aging.