Oxidative stress induces the progression of cholangiocarcinoma by decreasing EBF1 and increasing ZNF423 expressions

Raynoo Thanan

Oxidative stress is a cause of inflammation–related diseases, including cholangiocarcinoma (CCA). Our previous studies in animal and human models indicated that oxidative stress is a major cause of CCA development. Oxidative stress damages biomolecules leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress–related carcinogenesis. In the present study, we established a hydrogen peroxide (H2O2)-resistant cell line (ox-MMNK1-L) from a cholangiocyte cell line (MMNK1) by chronic treatment with 25 M H2O2. The ox-MMNK1-L cell line had a significantly higher cell proliferation and migration rates than the parental cells. Moreover, the ox-MMNK1-L cells showed decreased expression of early B cell factor 1 (EBF1) whereas the expression of EBF1 inhibitor (ZNF423) was increased compared to the parental cells. These findings suggest that EBF1 and ZNF423 are the oxidative stress-responsive genes in CCA. Therefore, the expression patterns of EBF1 and ZNF423 were detected in CCA tissues. CCA patients who had low EBF1 expression and high ZNF423 in the tumor tissues was related with poor prognosis. Roles of EBF1 and ZNF423 in CCA progression were further investigated in MMNK1 and CCA cell lines using specific siRNAs. EBF1-knockdown-MMNK1 cells have shown to increase stem-like cell property, cell migration activity and estrogen response whereas ZNF423-knockdowned CCA cells showed reduced proliferation activity compared with the control cells. Thus, oxidative stress induces tumorigenic properties via suppression of EBF1 expression and activation of ZNF423 expression, resulting in CCA progression with poor prognosis.


Simultaneous targeting of oxidative stress and fibrosis as an effective strategy for treating cardiomyopathy-induced ventricular remodelling and dysfunction

Chrishan Samuel

This presentation outlines the background to why the therapeutic targeting of the interaction between transforming growth factor (TGF)-beta1 and oxidative stress would provide an effective means of reducing tissue remodelling-induced fibrosis progression and related dysfunction.
It also provides evidence from in vitro and in vivo studies conducted to demonstrate how the targeting of this interaction between TGF-beta1 and oxidative stress can effectively reduce left ventricular inflammation, remodelling and fibrosis, as well as cardiomyocyte hypertrophy and left ventricular dysfunction in a preclinical model of cardiomyopathy-induced fibrosis.
These findings have important implications for developing new drugs or repurposing currently-available drugs to target this TGF-beta1oxidative stress interaction.


Effects of acute physical exercise on oxidative stress and inflammatory status in young, sedentary obese subjects

Marta Greco

Obesity is typically associated with a chronic low-grade inflammation, characterized by increased levels of reactive oxygen species, contributing to an oxidative stress condition.
The health benefits of moderate and regular physical activity in obese patients, useful to prevent cardiovascular and metabolic complications, could be attributed, in part, to a stimulation of endogenous antioxidant defenses. Circulating oxidative stress and pro-inflammatory markers change after regular physical exercise; however, how a short session of acute physical activity affects the inflammatory status and redox balance in sedentary individuals is still unclear.
Aim of this study is to evaluate some indirect markers of oxidative stress and inflammatory parameters, both at rest and after acute exercise, in sedentary young men with or without obesity.
In these study, thirty sedentary male volunteers, aged 20–45 (mean age 32 ± 7 years), were recruited. The subject enrolled have been divided into 3 groups: normal weight (BMI < 25 kg/ m2); overweight to moderate obesity (25–35 kg/m2); severe obesity (35–40 kg/m2). The following analytes were determined in blood samples from the enrolled subjects: Glutathione Reductase, Glutathione Peroxidase, Superoxide Dismutase, Total Antioxidant Status, cytokines and growth factors (IL-2, IL-4, IL-6, IL-8, IL-10, IL-1α, IL-1β, TNFα, MCP-1, VEGF, IFNγ, EGF) before and after a 20-min run at ~ 70% of their VO2max. Inter-group comparisons demonstrated significantly higher Glutathione Reductase activity in severely obese subjects in the post-exercise period and higher EGF levels in normal weight individuals, either before and after exercise. Intra-group comparisons showed that the acute exercise stress induced a significant increase in Glutathione Reductase activity in severely obese subjects only, a significant decrease in MCP-1 in the normal weight group, and a decrease in EGF levels in all groups. Our results suggest that in sedentary individuals with different ranges of BMI, the activation of the endogenous antioxidant markers related to oxidative stress and distinct cytokines are differentially involved into the adaptive metabolic changes and redox responses induced by physical exercise. Therefore, these biomarkers may have the potential to identify individuals at higher risk for developing diseases pathophysiologically linked to oxidative stress.


Lipoprotein Preparations: A Potential Routes for Cell-Targeted Delivery of Polyphenols and other drugs

Hanna Lewandowska

The potential of lipoproteins as drug delivery systems is presently underestimated. Lipoproteins may be of great use in drug delivery due to their unique characteristics: Cells have lipoprotein receptors, more or less specific for each type of lipoprotein, which (especially LDL receptors) are particularly strongly expressed in rapidly proliferating cells. Another advantage connected to the application of lipoproteins as a drug carriers is their large (several to several dozen nm diameter) size. While normal tissue is not permeable to big particles, tumor tissue tends to accumulate macromolecules and especially lipids. This phenomenon has been characterized and termed the tumor-selective enhanced permeability and retention (EPR) effect [1]. The next attractive feature of lipoproteins as a bio carrier is their biosafety. While synthetic materials can cause toxicology problems, the lipoproteins are fully biodegradable in the body by way of natural mechanisms. As solid particles, they have greater structural stability than, for example, the liposomes that are currently a very popular medium in medical preparations [2]. It is also worth to mention, lipoproteins are easily isolated from blood plasma. Taking into account the above-described findings and conclusions, it seems a reasonable scientific goal to look into the potential of lipoproteins as carriers of polyphenolics, metals, and other drugs, and into the effects that modified lipoproteins have in cells.

Polyphenols (PPs) were shown to be able to exhibit numerous regulatory functions in mammalian cells. Nevertheless, the potential regulatory effects of native PPs that are shown in vitro in numerous papers, are not likely to occur in the tissue, due to the poor ADMET (absorption, distribution, metabolism, excretion, and toxicology) qualities of PPs [3]. Especially, the issue are their poor water solubility and a high potential for modification by both first and second phase metabolism. Therefore many attempts are made to make polyphenols more available.  Apart from chemical modifications of the PPs many attempts have focused on looking for the appropriate drug carriers. Among the successful solutions are the liposomes, polymeric micelles, phospholipids, and other nanoparticle-based drug delivery systems [4]. For instance, a highly absorptive curcumin dispersed with colloidal nano-particles, was demonstrated to yield more than 30-fold higher bioavailability via oral administration compared with conventional curcumin in rat models [5].

As an alternative to the above-mentioned synthetic and semi-synthetic vehicles for PPs delivery, we proposed [6] a fully natural carrier capable of carrying the lipophilic payload, the LDL particle. Highly enhanced uptake of LDL in cancer, potentially makes it an ideal carrier for PP, which has been shown in the literature to have beneficial antioxidant effects at low concentrations, while pro-oxidative and cytostatic at higher doses [7].

In our recent work work [6], new polyphenol-containing LDL nano-preparations were prepared. Modulation of lipophilicity through the use of carriers allowed for excellent improvement of the therapeutic properties of such drugs as paclitaxel and doxorubicin (Abraxane [8], Doxil [9]). The proposed series of popular PPs, with increasing lipophilic properties, applied with a fully natural lipophilic carrier, are an attempt to find optimal conditions for the administration of drugs with different lipophilicity on the specific example of popular dietary supplements. The procedure for the synthesis of PP-saturated LDL nanoparticles, and their anti- and pro-oxidative activity and toxicity to human cancer cells will be presented. Along with the short summary of the obtained results, the possibility to use lipoproteins in drug delivery (including e.g. metal complexes, potential nanozymes) will be further discussed.

The work was supported by the National Science Centre, Poland (grant no. 2018/31/B/NZ7/03083).


  1. Maeda, H. The Enhanced Permeability and Retention (EPR) Effect in Tumor Vasculature: The Key Role of Tumor-Selective Macromolecular Drug Targeting. Advances in Enzyme Regulation 2001, 41, 189–207, doi:10.1016/S0065-2571(00)00013-3.
  2. Feng, T.; Wei, Y.; Lee, R.J.; Zhao, L. Liposomal Curcumin and Its Application in Cancer. International journal of nanomedicine 2017, 12, 6027–6044.
  3. Nelson, K.M.; Dahlin, J.L.; Bisson, J.; Graham, J.; Pauli, G.F.; Walters, M.A. The Essential Medicinal Chemistry of Curcumin: Miniperspective. Journal of Medicinal Chemistry 2017, 1620–1637.
  4. Gera, M.; Sharma, N.; Ghosh, M.; Huynh, D.L.; Lee, S.J.; Min, T.; Kwon, T.; Jeong, D.K. Nanoformulations of Curcumin: An Emerging Paradigm for Improved Remedial Application. Oncotarget 2017, 8, 66680–66698.
  5. Sasaki, H.; Sunagawa, Y.; Takahashi, K.; Imaizumi, A.; Fukuda, H.; Hashimoto, T.; Wada, H.; Katanasaka, Y.; Kakeya, H.; Fujita, M. Innovative Preparation of Curcumin for Improved Oral Bioavailability. Biological and Pharmaceutical Bulletin 2011, 34, 660–665.
  6. Lewandowska, H.; Kalinowska, M. New Polyphenol-Containing LDL Nano-Preparations in Oxidative Stress and DNA Damage: A Potential Route for Cell-Targeted PP Delivery. Materials 2020, 13, 5106.
  7. Martin, K.R.; Appel, C.L. Polyphenols as Dietary Supplements: A Double-Edged Sword. Nutrition and Dietary Supplements 2009, 2, 1–12.
  8. Abraxane (Paclitaxel Protein-Bound Particles for Injectable Suspension) Available online: https://www.centerwatch.com/directories/1067-fda-approved-drugs/listing/3061-abraxane-paclitaxel-protein-bound-particles-for-injectable-suspension (accessed on 24 January 2020).
  9. Doxil (Doxorubicin HCl Liposome Injection) Available online: https://www.centerwatch.com/directories/1067-fda-approved-drugs/listing/3407-doxil-doxorubicin-hcl-liposome-injection (accessed on 24 January 2020).

Oxidative potential, cytotoxicity, and intracellular oxidative stress generating capacity of PM10 at an urban background site in Italy

Maria Giulia Lionetto

It is widely recognized that long and short-term exposure to atmospheric particulate matter (PM) has detrimental effects on human health, in particular on pulmonary and cardiovascular systems. Recent studies suggest that several effects of atmospheric PM on human health may be mediated by the induction of oxidative stress, which has been considered as an important underlying mechanism of action for the outcome of adverse health effects. The aim of the study was to investigate the toxicological properties of PM10 sampled at an urban background site (the Environmental-Climate Observatory of ISAC-CNR in Lecce, Southern Italy) focusing on the intrinsic oxidative potential (OP), measured with the acellular dithiothreitol (DTT) assay test, and on health-related cellular outcomes such as the induction of intracellular oxidative stress, assessed by the ROS sensitive fluorescent probe CM-H2DCFDA, cytotoxicity (measured as reduced cell viability by the MTT assay), and genotoxicity (measured by comet test). In particular, the study wanted to assess whether the PM10 intrinsic OP is correlated with cellular endpoints, and whether cellular and acellular endpoints are correlated with chemical properties of atmospheric PM10 such as concentration and carbon content. The cellular endpoints were investigated on A549 cell line, representative of the Alveolar Type II pneumocytes of the human lung, and widely used as a cellular model.
The obtained results showed a detectable intrinsic OP, cytotoxicity, and intracellular oxidative stress generating capacity (OSGC) in aqueous extracts of PM10 samples. A statistically significant correlation was observed between OP, cytotoxicity, and OSGC with the carbon content of PM10. This suggests that combustion sources at this site play an important role in determining cellular oxidative stress and cytotoxicity of PM10. The OP was correlated to OSCG, suggesting that the ability of PM10 to generate intracellular oxidative stress conditions is related to its intrinsic oxidative potential depending on the physico-chemical properties of the particles. The OSGC results are well correlated with cell mortality and, a lower, but still statistically significant correlation is observed between intrinsic OP and reduced cellular viability, suggesting that the prooxidant properties of PM10 play a key role in the multiple mechanisms underlying PM10 cytotoxicity. Genotoxicity results, although limited to a subset of samples, are well correlated with OSGC, cytotoxicity and OP and this also suggest the relationship between the prooxidant properties of PM and genotoxic effects. In conclusions, obtained results demonstrate the correlation between the intrinsic oxidative potential of particulate matter and health related cellular outcomes.


Limited Oxidative Stress Favors Resistance to Skeletal Muscle Atrophy in Hibernating Brown Bears (Ursus Arctos)

Fabrice Bertile

Muscle atrophy is an inevitable part of ageing, disuse, starvation, or microgravity. Its molecular bases are well deciphered thanks to studies in mice under weightlessness or using the bed rest model in humans. However, a fully effective therapy is still lacking. Screening biodiversity holds potential to find new ways of potential medical interest. Indeed, muscles are fairly well preserved in hibernating animals despite long inactive and fasting periods. To unravel the underlying mechanisms, we compared brown bear (Ursus arctos) tissues collected during the summer-active season versus hibernation using a combination of omics techniques and biochemistry methods. We identified several mechanisms likely involved in brown bear resistance to muscle atrophy during hibernation, and possibly transferable to humans. Examination of the general and regional oxidant/antioxidant balance and oxidative damages during summer versus winter, notably highlited greater plasma-antioxidant capacity, and both white adipose tissue and skeletal muscles appeared protected from oxidative stress during hibernation. Because, oxidative stress promotes proteolysis and inhibits protein synthesis, its limitation during hibernation likely contributes to muscle protein sparing. Additionally, we highlighted inhibition of TGF signalling, maintenance of BMP signalling, and elicitation of a myogenic microRNA response via MEF2A. Despite a preference for lipid substrates during hibernation, muscle glycolysis was maintained and elevated levels of muscle glycogen stores were measured. Carbohydrate metabolism and protein sparing in hibernating bears could be controlled by the higher concentrations of plasma docosahexaenoic acid during winter. We finally observed that the serum from hibernating bears strongly inhibits proteasomal and lysosomal proteolysis in human muscle cells in vitro. The natural resistance of hibernating bears to muscle atrophy appears controlled through a coordinated regulatory program. Transfer of bear protein sparing strategies to humans, notably after identification of serum antiproteolytic factors, is expected to help better fight muscle wasting in aged or sedentary people, and in astronauts.


[email protected] Nanoparticles Promoted Oxidative Stress-Induced Cell Death in Caco-2 Human Colon Cancer Cells

Chenxu Yu

Natural antioxidants such as fucoxanthine (FX) and phlorotannin (PhT) are known for their bioactivities and potential health benefits. However, both suffer from poor water solubility which limits their bioavailability. In this study, nanoencapsulation with polyvinylpyrrolidone (PVP) was utilized as a nano-enabling technique to improve the bioaccessibility of both FX and PhT, which also bring the benefit of protecting the antioxidants against unwanted oxidation. Different PVP Loading ratios were investigated to evaluate the impact on bioavailability, and it was found that for both FX and PhT, 1:8 loading ratio (payload:PVP) was optimal in bring about the best performance in terms of encapsulation efficiency (EE) and loading capacity (LV). Dynamics release profile of PhT from PhT-PVP nanocomplexes in simulated gastrointestinal fluids was characterized, and it was shown that these nanocomplexes were non-toxic against HaCaT keratinocytes in vitro, while effectively reducing endogenous oxygen species (ROS) to reduce oxidative stress-induced cell death. Meanwhile, FX-PVP nanocomplexes were shown to effectively transport into Caco-2 colon cancer cells and deliver high dosage of FX, which, at high dosage, became pro-oxidant, and indeed accelerated H2O2-induced cell death in caco-2 cells. Apparently, by carefully designing and utilizing nano-enabled encapsulation techniques, natural antioxidants can be manipulated effectively to behave as either anti- or pro-oxidant to achieve desirable health-improving results (either reducing oxidative-stress induced cell death, or promote it for cancer cells). The technique has the potential to become an effective means for producing functional foods and therapeutic out of natural ingredients.


Systemic Oxidative Stress Is Increased in Postmenopausal Women and Independently Associates with Homocysteine Levels

Jumana Saleh

Circulating Markers of Oxidative Stress in Metabolic Diseases: An update of clinical relevance. Abdel-Hadi DH1, Al-Lawati M 2, Al Farhan H2 National University of Science & Technology1, Sultan Qaboos University Hospital1, Muscat, Oman.
Metabolic disease is often marked with dyslipidemia and increased cardiovascular risk markers. These markers include conventional risk factors, such as increased LDL cholesterol, decreased HDL levels, increased liver enzymes, and elevated HBA1c, inflammatory and oxidative stress markers. Oxidative stress is the hallmark of atherosclerosis. Oxidation markers in metabolic disease are not routinely utilized as diagnostic tools or therapeutic targets due to their instability, oxidizability, intracellular abundance and low serum levels. Also, findings regarding these oxidation marker measures are inconsistent and subject to large variations.
Importantly, considerable evidence shows significant associations between conventional serum metabolic markers and oxidative stress. However, knowledge regarding the role of these metabolic risk factors in enhancing oxidative stress is limited, and often overlooked.
Formation of protein carbonyls and lipid oxidation products are fairly stable and may overcome the shortcomings of oxidative modifications. Notably, proteins and lipids constitute the major components of circulating proteins and lipoproteins that significantly increase, or are altered, in the dyslipidemic state of metabolic disease. Therefore, measuring oxidatively modified serum markers of the dyslipidemic state and metabolic disease may be useful for the early detection of predisposing atherosclerosis risk factors, and provides new perspectives regarding their clinical relevance to cardiovascular disease progression.
Here we present updates and new perspectives considering circulating metabolic risk markers as potential triggers of oxidative stress, their possible mechanisms of action and prospective diagnostic and clinical applications.
Key words: Dyslipidemia, Oxidative Stress, Systemic markers, Cardiovascular Disease, Diagnostic markers.