Agnieszka Sliwinska

Metformin in cancer prevention and therapy

The prevalence of diabetes is dramatically increasing worldwide. The results of numerous epidemiological studies indicate that diabetic population is not only at increased risk of cardiovascular complications, but also at substantially higher risk of many forms of malignancies. The use of metformin, the most commonly prescribed drug for type 2 diabetes, was repeatedly associated with the decreased risk of the occurrence of various types of cancers, especially of pancreas and colon and hepatocellular carcinoma. This observation was also confirmed by the results of numerous meta-analyses. There are however, several unanswered questions regarding the exact mechanism of the anticancer effect of metformin as well as its activity against various types of cancer both in diabetic and nondiabetic populations. In the present work we discuss the proposed mechanism(s) of anticancer effect of metformin and preclinical and clinical data suggesting its anticancer effect in different populations.

In vitro effect of gliclazide on DNA damage and repair in patients with type 2 diabetes mellitus (T2DM).

Type 2 diabetes mellitus is associated with elevated level of oxidative stress, which is one of the most important factors responsible for the development of chronic complications of this disease. Moreover, it was shown that diabetic patients had increased level of oxidative DNA damage and decreased effectiveness of DNA repair. These changes may be associated with increased risk of cancer in T2DM patients, since DNA damage and DNA repair play a pivotal role in malignant transformation. It was found that gliclazide, an oral hypoglycemic drug with antioxidant properties, diminished DNA damage induced by free radicals. Therefore, the aim of the present study was to evaluate the in vitro impact of gliclazide on: (i) endogenous basal and oxidative DNA damage, (ii) DNA damage induced by hydrogen peroxide and (iii) the efficacy of DNA repair of such damage. DNA damage and DNA repair in peripheral blood lymphocytes of 30 T2DM patients and 30 non-diabetic individuals were evaluated by alkaline single cell electrophoresis (comet) assay. The extent of oxidative DNA damage was assessed by DNA repair enzymes: endonuclease III and formamidopyrimidine-DNA glycosylase. The endogenous basal and oxidative DNA damages were higher in lymphocytes of T2DM patients compared to non-diabetic subjects and gliclazide decreased the level of such damage. The drug significantly decreased the level of DNA damage induced by hydrogen peroxide in both groups. Gliclazide increased the effectiveness of DNA repair in lymphocytes of T2DM patients (93.4% (with gliclazide) vs 79.9% (without gliclazide); P< or =0.001) and non-diabetic subjects (95.1% (with gliclazide) vs 90.5% (without gliclazide); P< or =0.001). These results suggest that gliclazide may protect against the oxidative stress-related chronic diabetes complications, including cancer, by decreasing the level of DNA damage induced by reactive oxygen species.

Gliclazide may have an antiapoptotic effect related to its antioxidant properties in human normal and cancer cells

Experimental and clinical studies suggest that gliclazide may protect pancreatic β-cells from apoptosis induced by an oxidative stress. However, the precise mechanism(s) of this action are not fully understood and requires further clarification. Therefore, using human normal and cancer cells we examined whether the anti-apoptotic effects of this sulfonylurea is due to its free radical scavenger properties. Hydrogen peroxide (H2O2) as a model trigger of oxidative stress was used to induce cell death. Our experiments were performed on human normal cell line (human umbilical vein endothelial cell line, HUVEC-c) and human cancer cell lines (human mammary gland cell line, Hs578T; human pancreatic duct epithelioid carcinoma cell line, PANC-1). To assess the effect of gliclazide the cells were pre-treated with the drug. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was employed to measure the impact of gliclazide on cell viability. Generation of reactive oxygen species, mitochondrial membrane potential (∆Ψm), and intracellular Ca2+ concentration [Ca2+] were monitored. Furthermore, the morphological changes associated with apoptosis were determined using double staining with Hoechst 33258-propidium iodide (PI). Gliclazide protects the tested cells from H2O2-induced cell death most likely throughout the inhibition of ROS production. Moreover, the drug restored loss of ΔΨm and diminished intracellular [Ca2+] evoked by H2O2. Double staining with Hoechst 33258-PI revealed that pre-treatment with gliclazide diminished the number of apoptotic cells. Our findings indicate that gliclazide may protect both normal and cancer human cells against apoptosis induced by H2O2. It appears that the anti-apoptotic effect of the drug is most likely associated with reduction of oxidative stress.

The levels of 7,8-dihydrodeoxyguanosine (8-oxoG) and 8-oxoguanine DNA glycosylase 1 (OGG1) - A potential diagnostic biomarkers of Alzheimer's disease.

Evidence indicates that oxidative stress contributes to neuronal cell death in Alzheimers disease (AD). Increased oxidative DNA damage l, as measured with 8-oxoguanine (8-oxoG), and reduced capacity of proteins responsible for removing of DNA damage, including 8-oxoguanine DNA glycosylase 1 (OGG1), were detected in brains of AD patients. In the present study we assessed peripheral blood biomarkers of oxidative DNA damage, i.e. 8- oxoG and OGG1, in AD diagnosis, by comparing their levels between the patients and the controls. Our study was performed on DNA and serum isolated from peripheral blood taken from 100 AD patients and 110 controls. For 8-oxoG ELISA was employed. The OGG1 level was determined using ELISA and Western blot technique. Levels of 8-oxoG were significantly higher in DNA of AD patients. Both ELISA and Western blot showed decreased levels of OGG1 in serum of AD patients. Our results show that oxidative DNA damage biomarkers detected in peripheral tissue could reflect the changes occurring in the brain of patients with AD. These results also suggest that peripheral blood samples may be useful to measure oxidative stress biomarkers in AD.

Analysis of oxidative DNA damage and its repair in Polish patients with diabetes mellitus type 2: Role in pathogenesis of diabetic neuropathy

PURPOSE Distal symmetric polyneuropathy (DSPN) is common complication of type 2 diabetes (T2DM). In this work we investigated the role of oxidative damage in connection with particular polymorphisms of DNA repair genes and their repair capacity. MATERIAL/METHODS Materials constitute the peripheral blood of patients with T2DM with and without DSPN and control subjects without disturbance of the carbohydrate fraction. The study of gene polymorphisms which products take part in base excision repair (BER) pathway: 726 Val/Ala adenosine diphosphate ribosyl transferase (ADPRT), 324 His/Glu MutYhomolog (MUTYH) and 148 Asp/Glu human apurinic/apyrimidinic endonuclease (APE) was carried out using restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) method. The study of DNA damage induced by hydrogen peroxide and the efficiency of their repair was carried out using comet assay. RESULTS None of the 3 polymorphisms were associated with the risk of DSPN. However, in group of patients together with T2DM and T2DM/DSPN 726 Ala ADPRT allele was significantly susceptible to increased risk of T2DM (OR=1.59; 95% CI: 1.08-2.36). Investigation of DNA damage and repair revealed that T2DM patients have decreased ability to DNA repair. This capacity even drops down in the group of T2DM/DSPN patients compared to subjects with diabetes alone. ADPRT and APE polymorphisms were significantly associated with higher DNA damages (P<0.05) in heterozygous and mutant homozygous in correlation to homozygous wild type, but for MUTYH polymorphism relation was not confirmed. CONCLUSIONS Pathogenesis of T2DM and development of DSPN may be related to oxidative stress connected with BER gene polymorphisms.

Genotoxicity and cytotoxicity of ZnO and Al2O3 nanoparticles

Abstract Objectives: Metal oxide nanoparticles (ZnO-NPs and Al2O3-NPs) are used in many fields, including consumer products and biomedical applications. As a result, exposure to these NPs is highly frequent, however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms are available. For this reason, we studied cytotoxic and genotoxic effects of ZnO-NPs and Al2O3-NPs on human peripheral blood lymphocytes. Materials and methods: We obtained our goals by using MTT assay, Annexin V-FITC flow cytometry, and alkaline, neural and pH 12.1 versions of comet assay. Results: Exposure of lymphocytes to both NPs for 24 h slightly decreased viability of lymphocytes at ≥0.5 mM. For the first time, we revealed using the comet assays that both ZnO-NPs and Al2O3-NPs caused a concentration-dependent increase of DNA single-strand breaks, but not alkali-labile sites. Treatment with DNA glycosylases showed that the NPs induced oxidative DNA damage. DNA damage caused by both nanoparticles at 0.05 mM was removed within 120 min, however lymphocytes did not repair DNA damage induced by 0.5 mM NPs. Studied nanoparticles did not induce apoptosis in lymphocytes. Conclusion: Our results suggest that ZnO-NPs and Al2O3-NPs at concentration up to 0.5 mM did not exhibit cytotoxic effect but may exert genotoxic effect on lymphocytes, at least partially by the generation of oxidative DNA damage and strand breaks.

Impact of Single Nucleotide Polymorphisms of Base Excision Repair Genes on DNA Damage and Efficiency of DNA Repair in Recurrent Depression Disorder

Elevated level of DNA damage was observed in patients with depression. Furthermore, single nucleotide polymorphisms (SNPs) of base excision repair (BER) genes may modulate the risk of this disease. Therefore, the aim of this study was to delineate the association between DNA damage, DNA repair, the presence of polymorphic variants of BER genes, and occurrence of depression. The study was conducted on peripheral blood mononuclear cells of 43 patients diagnosed with depression and 59 controls without mental disorders. Comet assay was used to assess endogenous (oxidative) DNA damage and efficiency of DNA damage repair (DRE). TaqMan probes were employed to genotype 12 SNPs of BER genes. Endogenous DNA damage was higher in the patients than in the controls, but none of the SNPs affected its levels. DRE was significantly higher in the controls and was modulated by BER SNPs, particularly by c.977C>G–hOGG1, c.972G>C–MUTYH, c.2285T>C–PARP1, c.580C>T–XRCC1, c.1196A>G–XRCC1, c.444T>G–APEX1, c.-468T>G–APEX1, or c.*50C>T–LIG3. Our study suggests that both oxidative stress and disorders in DNA damage repair mechanisms contribute to elevated levels of DNA lesions observed in depression. Lower DRE can be partly attributed to the presence of specific SNP variants.

SIRT1 as a Therapeutic Target in Diabetic Complications

BACKGROUND Sirtuin1 is an epigenetic enzyme involved in histone and nonhistone protein deacetylation. It acts primarily as a metabolic sensor, which responses to changing energy status by deacetylating crucial transcription factors and cofactors. In this way, Sirtuin1 regulates mitochondrial function and biogenesis, oxidative stress, inflammation, apoptosis and cellular senescence. Disturbance of all of these phenomena promotes the pathogenesis of diabetic complications. These disorders are inseparably connected with chronic hyperglycemia, which possesses a strong epigenetic determinant. OBJECTIVE To summarize the contemporary knowledge regarding the role of Sirtuin1 in the development, progression and therapy of diabetic complications. METHODS We extensively searched literature describing the importance of Sirtuin1 in pathophysiology and treatment of all kinds of diabetic complications till September 2017. We focused on the examples of synthetic and natural compounds-mediated Sirtuin1 upregulation along with Sirtuin1-associated epigenetics. RESULTS Reduction of Sirtuin1 is implicated in endothelial dysfunction and metabolic memory, underlying the development of micro- and macrovascular complications. Declined Sirtuin1 also participates in diabetic testicular and erectile dysfunction. Sirtuin1 is elevated by naturally occurring anti-oxidant and anti-inflammatory compounds such as resveratrol, trans-δ-viniferin, vitamin D and more. Similarly, Sirtuin1 level increases after treatment with standard antihyperglycemic (metformin, exenatide, liraglutide), antihypertensive (sartans), lipid-lowering (fibrates, statins) and anticoagulant (fidarestat) drugs. Regarding epigenetics, a number of miRNAs trigger Sirtuin1 decrease, which further contributes to histone acetylation of Sirtuin1-regulated and relevant for diabetes genes. CONCLUSION Evidence strongly suggest that Sirtuin1 upregulation may serve as a potent therapeutic approach against development and progression of diabetic complications.

Is p53 Involved in Tissue-Specific Insulin Resistance Formation?

p53 constitutes an extremely versatile molecule, primarily involved in sensing the variety of cellular stresses. Functional p53 utilizes a plethora of mechanisms to protect cell from deleterious repercussions of genotoxic insults, where senescence deserves special attention. While the impressive amount of p53 roles has been perceived solely by the prism of antioncogenic effect, its presence seems to be vastly connected with metabolic abnormalities underlain by cellular aging, obesity, and inflammation. p53 has been found to regulate multiple biochemical processes such as glycolysis, oxidative phosphorylation, lipolysis, lipogenesis, β-oxidation, gluconeogenesis, and glycogen synthesis. Notably, p53-mediated metabolic effects are totally up to results of insulin action. Accumulating amount of data identifies p53 to be a factor activated upon hyperglycemia or excessive calorie intake, thus contributing to low-grade chronic inflammation and systemic insulin resistance. Prominent signs of its actions have been observed in muscles, liver, pancreas, and adipose tissue being associated with attenuation of insulin signalling. p53 is of crucial importance for the regulation of white and brown adipogenesis simultaneously being a repressor for preadipocyte differentiation. This review provides a profound insight into p53-dependent metabolic actions directed towards promotion of insulin resistance as well as presenting experimental data regarding obesity-induced p53-mediated metabolic abnormalities.

Associations between DNA Damage, DNA Base Excision Repair Gene Variability and Alzheimer's Disease Risk.

Background: Increased oxidative damage to DNA is one of the pathways involved in Alzheimers disease (AD). Insufficient base excision repair (BER) is in part responsible for increased oxidative DNA damage. The aim of the present study was to assess the effect of polymorphic variants of BER-involved genes and the peripheral markers of DNA damage and repair in patients with AD. Material and Methods: Comet assays and TaqMan probes were used to assess DNA damage, BER efficiency and polymorphic variants of 12 BER genes in blood samples from 105 AD patients and 130 controls. The DNA repair efficacy (DRE) was calculated according to a specific equation. Results: The levels of endogenous and oxidative DNA damages were higher in AD patients than controls. The polymorphic variants of XRCC1 c.580C>T XRCC1 c.1196A>G and OGG1 c.977C>G are associated with increased DNA damage in AD. Conclusion: Our results show that oxidative stress and disturbances in DRE are particularly responsible for the elevated DNA lesions in AD. The results suggest that oxidative stress and disruption in DNA repair may contribute to increased DNA damage in AD patients and risk of this disease. In addition, disturbances in DRE may be associated with polymorphisms of OGG1 and XRCC1.