Implications of Translesion DNA Synthesis Polymerases on Genomic Stability and Human Health.

Replication fork arrest-induced DNA double strand breaks (DSBs) caused by lesions are effectively suppressed in cells due to the presence of a specialized mechanism, commonly referred to as DNA damage tolerance (DDT). In eukaryotic cells, DDT is facilitated through translesion DNA synthesis (TLS) carried out by a set of DNA polymerases known as TLS polymerases. Another parallel mechanism, referred to as homology-directed DDT, is error-free and involves either template switching or fork reversal. The significance of the DDT pathway is well established. Several diseases have been attributed to defects in the TLS pathway, caused either by mutations in the TLS polymerase genes or dysregulation. In the event of a replication fork encountering a DNA lesion, cells switch from high-fidelity replicative polymerases to low-fidelity TLS polymerases, which are associated with genomic instability linked with several human diseases including, cancer. The role of TLS polymerases in chemoresistance has been recognized in recent years. In addition to their roles in the DDT pathway, understanding noncanonical functions of TLS polymerases is also a key to unraveling their importance in maintaining genomic stability. Here we summarize the current understanding of TLS pathway in DDT and its implication for human health.

Nesfatin-1 peptide protects rat renal epithelial cells against high glucose and H2O2 induced injury via inhibition of oxidative stress, apoptosis, and fibrosis.

Nesfatin-1 is a potent polypeptide and plays a crucial role in many physiological functions. Nesfatin-1 levels are reported in both the central nervous system and peripheral organs. However, the expression of nesfatin-1 in the renal system under chronic oxidative stress-induced conditions and the direct effect of nesfatin-1 treatment on stress-induced pathological damage are not reported. Thus, the present study aimed to explore the role of nesfatin-1 in vitro in oxidative stress-induced renal epithelial cells. High glucose (HG) and H2O2 combination were used to induce oxidative stress (OS). MTT, crystal violet, and H and E staining were used to measure cell viability, cytotoxicity, and morphology. FACS analysis and confocal microscopy were used to measure OS and apoptosis. RT-PCR was done for gene expression analysis. Decreased nesfatin-1 expression was observed in renal epithelial cells induced with HG and H2O2 compared to an untreated control (0.16; p < 0.0001). Nesfatin-1 co-treatment with HG and H2O2 attenuated ROS, apoptosis, and fibrosis. SOD, Catalase, and Bcl-2 expression decreased (p < 0.0001) and Caspase-3 and TGF-ß1 expression increased in HG and H2O2-induced cells compared to control cells (p < 0.0001). Nesfatin-1 co-treatment attenuated these changes induced by HG and H2O2 (p < 0.0001). Nesfatin-1 expression was decreased in renal epithelial cells under stress-induced conditions. Moreover, nesfatin-1 co-treatment under stress-induced conditions protects the renal epithelial cells via inhibition of oxidative stress, apoptotic, and fibrotic signaling pathways.

Activation of Mas and pGCA receptor pathways protects renal epithelial cell damage against oxidative-stress-induced injury.

Over-activation of the renin-angiotensin-aldosterone system (RAAS) is a leading cause of cardio-renal complications. Oxidative stress is one of the major contributing factors in the over-activation of RAAS. Angiotensin-converting enzyme2/Angiotensin1-7/MasR and natriuretic peptide/particulate guanylyl cyclase receptor-A pathways play a key role in cardiorenal disease protection. Even though individual activation of these pathways possesses cardiorenal protective effects. However, the dual activation of these pathways under stress conditions and the underlying mechanism has not been explored. The study aimed to investigate whether activation of these pathways by dual-acting peptide (DAP) shows a protective effect in-vitro in oxidative stress-induced renal epithelial cells. Oxidative stress was induced in renal epithelial NRK-52E cells with H2O2. Co-treatment with Ang 1-7, BNP, and DAP was given for 30 min. AT1, MasR, and pGCA expression were measured by RT-PCR. The markers of oxidative stress and apoptosis were measured by confocal microscopy and FACS analysis. A significant increase in AT1, renin, α-SMA, and NFk-ß expression and a significant decrease in MasR and pGCA expression was observed in H2O2-induced cells. DAP improved H2O2-induced pathological changes in NRK-52E cells. The effect of DAP was superior to that of Ang1-7 and BNP alone. Interestingly, MasR and pGCA inhibitors could block the effect of DAP in H2O2-induced cells. DAP shows superior anti-RAAS activity, and it is effective against H2O2-induced oxidative stress, apoptosis, fibrosis, and inflammation compared to Ang1-7 and BNP alone. The protective effect is mediated by the dual activation of MasR and pGCA.

Transfersome Hydrogel Containing 5-Fluorouracil and Etodolac Combination for Synergistic Oral Cancer Treatment.

Oral cancer is one of the most common malignancies with an increased rate of incidence. 5-Fluorouracil (5FU) is an effective chemotherapeutic indicated for oral cancer treatment. Etodolac (Et), a cyclooxygenase-2 inhibitor, can be used as an adjuvant agent to sensitize cancer cells to chemotherapy. The aim of this work was to prepare and characterize 5FU and Et dual drug-loaded transfersomes to treat oral cancer. Transfersomes were prepared by thin-film hydration method and characterized for the average particle size and zeta-potential using dynamic light scattering and scanning electron microscopy techniques. The prepared transfersomes were further characterized for their drug loading, entrapment efficiencies using amicon centrifuge tubes and drug release behavior using cellulose membrane. The synergistic activity of dual drug-loaded transfersomes was studied in FaDu oral cancer cells. Results showed that the average particle size, polydispersity index, and zeta potential were 91±6.4 nm, 0.28±0.03, and (-)46.9±9.5 mV, respectively, for 5FU- and Et (1:1)-loaded transfersomes. The highest encapsulation efficiency achieved was 36.9±3.8% and 79.8±6.4% for 5FU and Et (1:1), respectively. Growth inhibition studies in FaDu cells using different concentrations of 5FU and Et showed a combination index of 0.36, indicating a synergistic effect. The FaDu cell uptake of drug-loaded transfersomes was significantly (p<0.05) greater than that of free drugs. The transfersome hydrogel made of HPMC (2% w/w) showed similar flux, lag time, and permeation coefficient as that of drug-loaded transfersomes across excised porcine buccal tissue. In conclusion, 5FU and Et transfersome hydrogel can be developed for localized delivery to treat oral cancer.

Syzygium cumini (jamun) fruit-extracted phytochemicals exert anti-proliferative effect on ovarian cancer cells.

BACKGROUND: The medicinal properties of Syzygium sp., especially the antidiabetic property, date back to the ancient times. However, in the recent past, extracts from different parts of the Syzygium sp. have demonstrated promising anticancer activities in diverse cancer types, and now, attempts are being made to identify the active phytochemicals. AIMS AND OBJECTIVES: In this study, we intended to test the anticancer properties of phytochemicals extracted from the fruit of Syzygium cumini plant in ovarian cancer cells. MATERIALS AND METHODS: A total of nine phytochemicals extracted from the S. cumini fruits using chloroform were tested for their anticancer activity in the ovarian cancer cell line PA-1. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium assay was performed to calculate the 50% inhibition (IC50) concentration and cell cytotoxicity values. Cell scratch assay was performed to assess the proliferation inhibition activity of the phytochemicals. Cisplatin was used as positive control. RESULTS: Out of the nine phytochemicals tested, quercetin (QC), gallic acid (GA), and oleanolic acid (OA) were found active. QC and GA were most effective with more than 90% cell cytotoxicity at 2.5 µ g/ml and above concentrations and OA moderately effective up to 5 µg/ml serial concentrations. Cell proliferation was significantly inhibited by QC and GA and moderately but significantly by OA. CONCLUSION: Our data demonstrate the anticancer activity of QC, GA, and OA phytochemicals, which is consistent with the previous reports. However, this is the first report showing the anticancer activity of these phytochemicals derived from S. cumini in the ovarian cancer cells. These data suggest that there is a potential to develop these phytochemicals as anticancer therapeutic agents either as monotherapeutic agents or in combination with commonly used chemotherapeutic agents, which needs to be explored.

Inhibition of double stranded RNA dependent protein kinase (PKR) abrogates isoproterenol induced myocardial ischemia in vitro in cultured cardiomyocytes and in vivo in wistar rats.

Protein kinase R (PKR) plays a main role in inflammation, insulin resistance, and glucose balance. It is activated by various stress signals and is key mediators of diabetes and associated complications. In the present study, we investigated the effect of PKR inhibition on myocardial dysfunction, inflammatory, cell death and interrelated signalling pathways in isoproterenol induced myocardial ischemia in vivo in wistar rats and in vitro in cultured cardiomyocytes. H9C2 rat cardiomyocytes were treated with 10 µM Isoproterenol (ISO). For in vivo studies, rats were divided into 4 groups: control, ischemic group (ISO), preventive group, curative group and each group consist of 8 rats. Myocardial Ischemia (MI) was induced with two subsequent doses of ISO (100 mg/kg, s.c.). The rats were treated with PKR inhibitor, C16 (166.5 µg/kg, i.p.) for 14 days. Heart rate, systolic, diastolic and mean arterial pressures were measured by non-invasive BP apparatus. Cardiac biomarkers were measured by commercial kits. Ischemic Zone, Morphological abnormalities and fibrosis of heart was detected by TTC, haematoxylin & eosin staining, Masson's and Sirius red staining respectively. Protein expression was done by western blotting and immune histochemistry. mRNA expression was done by RT-PCR. MI was characterized by declined myocardial performance along with elevation of cardiac biomarkers and associated with increased expression of PKR, oxidative-nitrosative stress, activated various inflammatory pathways (nuclear factor kappa light chain enhancer of activated B cells -NF-κB); Mitogen-activated protein kinases-MAPK; c-Jun N-terminal kinase-JNK), increased expression of inflammatory markers (Tumour necrosis factor alpha-TNF-α), markers of fibrosis (Alpha smooth muscle actin -α-SMA; Transforming growth factor beta-TGF-ß), enhanced cell death (Ischemic zone) and increased expression of extracellular regulated-kinases (ERK-1/2) and advanced glycation end products (AGE's). Interestingly, inhibition of PKR attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrosative stress, inflammation, cell death, and inter-related signalling pathways. Our findings report that inhibition of PKR improves the ischemic mediated inflammation, apoptosis, cardiac hypertrophy and fibrosis in MI induced rats. Hence, inhibition of PKR might be one of intervention therapy for the treatment of myocardial ischemia.

Inhibition of protein kinase R protects against palmitic acid-induced inflammation, oxidative stress, and apoptosis through the JNK/NF-kB/NLRP3 pathway in cultured H9C2 cardiomyocytes.

BACKGROUND AND PURPOSE: Double-stranded RNA-dependent protein kinase (PKR) is a critical regulator of apoptosis, oxidative stress, and inflammation under hyperlipidemic and insulin resistance conditions. Saturated free fatty acids, such as palmitic acid (PA), are known inducers of apoptosis in numerous cell types. However, the underlying molecular mechanism is not fully understood. The aim of the present study was to examine the effect of PA on cultured rat H9C2 cardiac myocytes cells and to investigate the PKR mediated harmful effects of PA in vitro in cultured cardiomyocytes. EXPERIMENTAL APPROACH: PKR expression was determined by immunofluorescence and immunoblotting. Oxidative stress and apoptosis were determined by flow cytometry and assay kits. The expression of different gene markers of apoptosis, oxidative stress, and inflammation were measured by Western blot analysis and reverse transcription polymerase chain reaction. KEY RESULTS: PKR expression, reactive oxygen species levels as well as apoptosis were increased in PA-treated cultured H9C2 cardiomyocytes. The harmful effects of PA were attenuated by a selective PKR inhibitor, C16. Moreover, we observed that upregulation of c-Jun N-terminal kinase (JNK), nuclear factor-kB (NF-kB) and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) pathways is associated with increased expression of interleukin 6 and tumor necrosis factor-α in PA-treated cardiomyocytes and attenuation by a selective PKR inhibitor. CONCLUSION AND IMPLICATIONS: Our study reports, for the first time, that PKR-mediated harmful effects of PA in cultured cardiomyocytes via activation of JNK, NF-kB, and NLRP3 pathways. Inhibition of PKR is one of the possible mechanistic approaches to inhibit inflammation, oxidative stress, and apoptosis in lipotoxicity-induced cardiomyocyte damage.

Pharmacological evaluation of novel PKR inhibitor indirubin-3-hydrazone in-vitro in cardiac myocytes and in-vivo in wistar rats.

AIMS: Double stranded protein kinase R cellular response is associated with various stress signals such as nutrients, endoplasmic stress, cytokines and mechanical stress. Increased PKR activity has been observed under diabetic and cardiovascular disease conditions. Most of the currently available PKR inhibitors are non-specific and have other effects as well. Thus, the aim of the present study was to examine the effect of novel PKR inhibitor indirubin-3-hydrazone (IHZ) in cultured rat H9C2 cardiomyocytes and wistar rats. MATERIALS AND METHODS: PKR expression was determined by Q-PCR, immunofluorescence and immunoblotting. The expression of different gene markers for apoptosis was measured by RT-PCR. Apoptosis and oxidative stress were determined by flow cytometry. KEY FINDINGS: High glucose (HG) treated H9C2 cardiomyocytes and high fructose (HF) treated wistar rats developed a significant increase in PKR expression. A significant increase in apoptosis and generation of reactive oxygen species was also observed in HG treated H9C2 cells and HF treated rats. Reduced vacuole formation and prominent nuclei were also observed in high glucose treated cells. Cardiac hypertrophy and increased fibrosis were observed in HF treated rats. All these effects of HG and HF were attenuated by novel PKR inhibitor, indirubin-3-hydrazone. SIGNIFICANCE: Our results indicate IHZ as an effective inhibitor of PKR in vitro and in-vivo, thus it may prove very useful in blocking the multiple harmful effects of PKR.

Imoxin attenuates high fructose-induced oxidative stress and apoptosis in renal epithelial cells via downregulation of protein kinase R pathway.

Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a ubiquitously expressed serine/threonine kinase, is a key inducer of inflammation, insulin resistance, and glucose homeostasis in obesity. Recent studies have demonstrated that PKR can respond to metabolic stress in mice as well as in humans. However, the underlying molecular mechanism is not fully understood. The aim of this study was to examine the effect of high fructose (HF) in cultured renal tubular epithelial cells (NRK-52E) derived from rat kidney and to investigate whether inhibition of PKR could prevent any deleterious effects of HF in these cells. PKR expression was determined by immunofluorescence staining and Western blotting. Oxidative damage and apoptosis were measured by flow cytometry. HF-treated renal cells developed a significant increase in PKR expression. A significant increase in reactive oxygen species generation and apoptosis was also observed in HF-treated cultured renal epithelial cells. All these effects of HF were attenuated by a selective PKR inhibitor, imoxin (C16). In conclusion, our study demonstrates PKR induces oxidative stress and apoptosis, is a significant contributor involved in vascular complications and is a possible mediator of HF-induced hypertension. Inhibition of PKR pathway can be used as a therapeutic strategy for the treatment of cardiovascular and metabolic disorders.

Down regulation of brain cellular prion protein in an animal model of insulin resistance: possible implication in increased prevalence of stroke in pre-diabetics/diabetics.

The risk of stroke is drastically increased in diabetic and pre-diabetic patients. The worldwide spread of obesity and insulin resistance increases the incidence of stroke. The direct effect of insulin resistance, as it pertains to stroke, on the central nervous system is not well understood. Since one of the physiological functions of the cellular prion protein (PrP(C)) is neuroprotection, we studied effects of brain insulin resistance on the expression of PrP(C) in fructose-fed rats as an animal model of prediabetes. Compared with control chow-fed animals, rats fed a high-fructose diet (FF), exhibited compromised tyrosine phosphorylation of insulin receptor ß subunit (IRß) and reduced serine phosphorylation of Akt, PI3K activity, and decreased PIP3 levels in cortices indicating the induction of brain insulin resistance. We also observed that both mRNA and protein expression of the PrP(C) was significantly decreased whereas protein level of NR2B subunit of NMDA glutamate receptors profoundly increased in the brain of fructose-fed rats compared to control chow-fed rats. Considering a neuroprotective role for PrP(C) and the involvement of NR2B subunit in the excitotoxicity-induced neuronal apoptosis, these phenomena may contribute to the severity and poor prognosis of ischemic stroke in diabetes/prediabetes.

Increased methylglyoxal formation with upregulation of renin angiotensin system in fructose fed Sprague Dawley rats.

The current epidemic of obesity and type 2 diabetes is attributed to a high carbohydrate diet, containing mainly high fructose corn syrup and sucrose. More than two thirds of diabetic patients have hypertension. Methylglyoxal is a highly reactive dicarbonyl generated during glucose and fructose metabolism, and a major precursor of advanced glycation end products (AGEs). Plasma methylglyoxal levels are increased in hypertensive rats and diabetic patients. Our aim was to examine the levels of methylglyoxal, mediators of the renin angiotensin system and blood pressure in male Sprague-Dawley rats treated with a high fructose diet (60% of total calories) for 4 months. The thoracic aorta and kidney were used for molecular studies, along with cultured vascular smooth muscle cells (VSMCs). HPLC, Western blotting and Q-PCR were used to measure methylglyoxal and reduced glutathione (GSH), proteins and mRNA, respectively. Fructose treated rats developed a significant increase in blood pressure. Methylglyoxal level and protein and mRNA for angiotensin II, AT1 receptor, adrenergic α1D receptor and renin were significantly increased, whereas GSH levels were decreased, in the aorta and/or kidney of fructose fed rats. The protein expression of the receptor for AGEs (RAGE) and NF-κB were also significantly increased in the aorta of fructose fed rats. MG treated VSMCs showed increased protein for angiotensin II, AT1 receptor, and α1D receptor. The effects of methylglyoxal were attenuated by metformin, a methylglyoxal scavenger and AGEs inhibitor. In conclusion, we report a strong association between elevated levels of methylglyoxal, RAGE, NF-κB, mediators of the renin angiotensin system and blood pressure in high fructose diet fed rats.

Increased renal methylglyoxal formation with down-regulation of PGC-1α-FBPase pathway in cystathionine γ-lyase knockout mice.

We have previously reported that hydrogen sulfide (H(2)S), a gasotransmitter and vasodilator has cytoprotective properties against methylglyoxal (MG), a reactive glucose metabolite associated with diabetes and hypertension. Recently, H(2)S was shown to up-regulate peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a key gluconeogenic regulator that enhances the gene expression of the rate-limiting gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase). Thus, we sought to determine whether MG levels and gluconeogenic enzymes are altered in kidneys of 6-22 week-old cystathionine γ-lyase knockout (CSE(-/-); H(2)S-producing enzyme) male mice. MG levels were determined by HPLC. Plasma glucose levels were measured by an assay kit. Q-PCR was used to measure mRNA levels of PGC-1α and FBPase-1 and -2. Coupled-enzymatic assays were used to determine FBPase activity, or triosephosphate levels. Experimental controls were either age-matched wild type mice or untreated rat A-10 cells. Interestingly, we observed a significant decrease in plasma glucose levels along with a significant increase in plasma MG levels in all three age groups (6-8, 14-16, and 20-22 week-old) of the CSE(-/-) mice. Indeed, renal MG and triosephosphates were increased, whereas renal FBPase activity, along with its mRNA levels, were decreased in the CSE(-/-) mice. The decreased FBPase activity was accompanied by lower levels of its product, fructose-6-phosphate, and higher levels of its substrate, fructose-1,6-bisphosphate in renal extracts from the CSE(-/-) mice. In agreement, PGC-1α mRNA levels were also significantly down-regulated in 6-22 week-old CSE(-/-) mice. Furthermore, FBPase-1 and -2 mRNA levels were reduced in aorta tissues from CSE(-/-) mice. Administration of NaHS, a H(2)S donor, increased the gene expression of PGC-1α and FBPase-1 and -2 in cultured rat A-10 cells. In conclusion, overproduction of MG in CSE(-/-) mice is due to a H(2)S-mediated down-regulation of the PGC-1α-FBPase pathway, further suggesting the important role of H(2)S in the regulation of glucose metabolism and MG generation.

Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose.

BACKGROUND AND PURPOSE: Endothelial dysfunction is a feature of hypertension and diabetes. Methylglyoxal (MG) is a reactive dicarbonyl metabolite of glucose and its levels are elevated in spontaneously hypertensive rats and in diabetic patients. We investigated if MG induces endothelial dysfunction and whether MG scavengers can prevent endothelial dysfunction induced by MG and high glucose concentrations. EXPERIMENTAL APPROACH: Endothelium-dependent relaxation was studied in aortic rings from Sprague-Dawley rats. We also used cultured rat aortic and human umbilical vein endothelial cells. The MG was measured by HPLC and Western blotting and assay kits were used. KEY RESULTS: Incubation of aortic rings with MG (30 µM) or high glucose (25 mM) attenuated endothelium-dependent, acetylcholine-induced relaxation, which was restored by two different MG scavengers, aminoguanidine (100 µM) and N-acetyl cysteine (NAC) (600 µM). Treatment of cultured endothelial cells with MG or high glucose increased cellular MG levels, effects prevented by aminoguanidine and NAC. In cultured endothelial cells, MG and high glucose reduced basal and bradykinin-stimulated nitric oxide (NO) production, cGMP levels, and serine-1177 phosphorylation and activity of endothelial NO synthase (eNOS), without affecting threonine-495 and Akt phosphorylation or total eNOS protein. These effects of MG and high glucose were attenuated by aminoguanidine or NAC. CONCLUSIONS AND IMPLICATIONS: Our results show for the first time that MG reduced serine-1177 phosphorylation, activity of eNOS and NO production. MG caused endothelial dysfunction similar to that induced by high glucose. Specific and safe MG scavengers have potential to prevent endothelial dysfunction induced by MG and high glucose concentrations.