ß-lapachone protects against doxorubicin-induced hepatotoxicity through modulation of NAD+ /SIRT-1/FXR/p-AMPK/NF-kB and Nrf2 signaling axis.

Doxorubicin (DOX) is a widely used antineoplastic drug, but its clinical use is limited by significant toxicities, such as hepatotoxicity. In this study, we evaluated the effects of ß-lapachone (ß-LAP), a natural quinone-containing compound, in a mouse model of DOX-induced hepatotoxicity. ß-LAP was orally administered at 1.25, 2.5, and 5 mg/kg for 4 days, and a single dose of DOX (20 mg/kg) was injected intraperitoneally on the second day. Histopathological changes, liver function markers, antioxidant and inflammatory markers were assessed. ß-LAP ameliorated liver injury and liver function markers evoked by DOX. ß-LAP also downregulated the mRNA expression of nuclear factor-kB-corresponding genes including interleukin-6, interleukin-1ß, and tumor necrosis factor-α. Moreover, ß-LAP increased the nuclear factor erythroid 2-related factor 2 target genes heme oxygenase-1 and NAD(P)H: quinone oxidoreductase 1, along with antioxidant enzymes including reduced glutathione, catalase, and superoxide dismutase with simultaneous reduction in the lipid peroxidation product malondialdehyde. Meanwhile, it recovered NAD+ /NADH ratios and subsequently elevated the protein levels of sirtuin-1 (SIRT-1), farnesoid X receptor (FXR), and phosphorylated AMP-activated protein kinase (p-AMPK). Collectively, these findings suggest a protective role of ß-LAP against DOX-induced hepatotoxicity by partly regulating the NAD+ /SIRT-1/FXR/p-AMPK axis.

Fatty acids of type 2 diabetic serum decrease the stemness properties of human adipose-derived mesenchymal stem cells.

In type 2 diabetes, dyslipidemia and increased serum free fatty acids (FFAs) exacerbate the development of the disease through a negative effect on insulin secretion. Adipose-derived mesenchymal stem cells (AdMSCs) play a key role in regenerative medicine, and these cells can potentially be applied as novel therapeutic resources in the treatment of diabetes. In this study, AdMSCs were treated with diabetic or nondiabetic serum FFAs isolated from women of menopausal age. Serum FFAs were analyzed using gas-liquid chromatography. The expression level of the stemness markers CD49e and CD90 and the Wnt signaling target genes Axin-2 and c-Myc were evaluated using real-time PCR. The proliferation rate and colony formation were also assessed using a BrdU assay and crystal violet staining, respectively. The level of glutathione was assessed using cell fluorescence staining. Compared to nondiabetic serum, diabetic serum contained a higher percentage of oleate (1.5-fold, p < 0.01). In comparison with nondiabetic FFAs, diabetic FFAs demonstrated decreasing effects on the expression of CD90 (-51%, p < 0.001) and c-Myc (-48%, p < 0.05), and proliferation rate (-35%, p < 0.001), colony formation capacity (-50%, p < 0.01), and GSH levels (-62%, p < 0.05). The negative effect of the FFAs of diabetic serum on the stemness characteristics may impair the regenerative capabilities of AdMSCs.

TGF-ß1 receptor blockade attenuates unilateral ureteral obstruction-induced renal fibrosis in C57BL/6 mice through attenuating Smad and MAPK pathways.

Renal fibrosis is characterized by accumulation of extracellular matrix components and collagen deposition. TGF-ß1 acts as a master switch promoting renal fibrosis through Smad dependent and/or Smad independent pathways. Thirty-five male C57BL/6 mice were divided into five groups of seven each; sham, unilateral ureteral obstruction (UUO), UUO+galunisertib (150 and 300 mg/kg/day), galunisertib (300 mg/kg/day). The UUO markedly induced renal fibrosis and injury as indicated by renal functional loss, increased levels of collagen Iα1, fibronectin and α-SMA; it also activated both the Smad 2/3 and MAPKs pathways as indicated by increased levels of TGF-ß1, p-Smad 2, p-Smad 3, p-p38, p-JNK and p-ERK. These UUO-induced changes were markedly attenuated by oral administration of galunisertib, the TGFßRI small molecule inhibitor. In conclusion, we demonstrated that TGF-ß1 receptor blockade can prevent UUO-induced renal fibrosis through indirect modulation of Smad and MAPKs signaling pathways and may be useful as a therapeutic agent in treatment and/or prevention of renal fibrosis.

The porcupine inhibitor WNT974 provokes ectodermal lineage differentiation of human embryonic stem cells.

Porcupine (Porcn) enzyme plays an essential role in Wnt signaling activation. Stearoyl-CoA desaturase-1 (SCD1) is required to provide Porcn substrates. The aim of this study was to determine the effect of a novel Porcn inhibitor on the fate of human embryonic stem cells (hESCs) and the reliance of Porcn on SCD1 activity. hESCs were cultured on a feeder layer or Matrigel-coated plates. Small molecules WNT974 (LGK-974) and CAY10566 were used to inhibit Porcn and SCD1 activity, respectively. We assessed the effect of Porcn inhibition on viability, expression of Wnt signaling targets, pluripotency markers, proliferation, differentiation, and protein fatty acylation. hESCs' conditioned medium (CM) containing secreted Wnt proteins were applied in rescue experiments. To examine the catalytic dependency of Porcn on SCD1, the results of combined inhibitor treatment were compared with the SCD1 inhibitor alone. LGK-974 at the selected concentrations showed mild effects on hESCs viability, but significantly reduced messenger RNA and protein expression of Wnt signaling targets (Axin-2 and c-Myc) and pluripotency markers (OCT-4 and SOX-2) (p < .05). Adding 1 µM of Porcn inhibitor reduced proliferation (p = .03) and enhanced differentiation capacity into ectodermal progenitors (p = .02), which were reverted by CM. Click chemistry reaction did not show significant alteration in protein fatty acylation upon LGK-974 treatment. Moreover, combined inhibitor treatment caused no further substantial reduction in Wnt signaling targets, pluripotency markers, and protein fatty acylation relative to CAY10566-treated cultures. The substrate availability for Porcn activity is regulated by SCD1 and targeting Porcn by LGK-974 prompts the transition of hESCs from self-renewal state to ectodermal lineage.

A small molecule modulating monounsaturated fatty acids and Wnt signaling confers maintenance to induced pluripotent stem cells against endodermal differentiation.

BACKGROUND: Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs). METHODS: The hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction. RESULTS: When SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased ß-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells. CONCLUSION: The small molecule SCD1 inhibitor attenuates the Wnt/ß-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism. The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. ß-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, ß-catenin increases the expression of the endoderm markers Sox17 and CXCR4.

Amygdalin Decreases Adhesion and Migration of MDA-MB-231 and MCF-7 Breast Cancer Cell Lines.

BACKGROUND: Cell adhesion, as dynamic interactions between cell-cell and cell-matrix, has an essential role in cancer cell migration. Integrins as cell membrane receptors are involved in cell adhesion and signal transduction. Aberrant expression of integrins is associated with the cancer cell adhesion. OBJECTIVE: Targeting the process of cell adhesion and migration could be helpful to prevent cancer cell metastasis. Amygdalin is a cyanoglycoside compound with anti-cancer properties, while its effect on cancer cell adhesion is not completely clear. METHODS: The cytotoxic effect of amygdalin on breast cancer cell lines (MCF-7 and MDA-MB- 231) and human skin fibroblast cell line as a normal cell, was evaluated through MTT assay. The cell adhesion assay and wound healing assay were performed to determine amygdalin effects on adhesion and migration of cancer cells. Further analysis was carried out to evaluate integrin α and ß levels through real-time PCR. RESULTS: We demonstrated that amygdalin diminished the cell viability of both cell lines in a time and dose-dependent manner, while amygdalin did not have any toxicity on the human skin fibroblast cell line in the same dosages. Following amygdalin treatment, the adhesion of both studied cell lines to fibronectin and collagen I decrease, and this reduction is significantly greater in the case of binding to fibronectin compared to binding to collagen. The MDA-MB-231 cell migration was decreased greater than MCF-7 cells. The levels of α and ß integrin were differentially regulated by amygdalin in both cancer cell lines. CONCLUSION: These results suggest that depending on cancer cell lines, amygdalin affects cancer cell adhesion and migration.

A mechanical non-enzymatic method for isolation of mouse embryonic fibroblasts.

Mouse embryonic fibroblasts (MEFs) accessibility coupled with their simple generation make them as a typical embryonic cell model and feeder layer for in vitro expansion of pluripotent stem cells (PSCs). In this study, a mechanical isolation technique was adopted to isolate MEFs and the efficiency of this technique was compared with enzymatic digestion method. The suspended MEFs were prepared either by mechanical method or 0.25% trypsin enzymatic digestion. The effect of tissue processing on cell apoptosis/necrosis, morphology, viable cell yield, population doubling time, surface marker expression, and the capacity to support PSCs were determined. The mechanical method yielded a significantly higher number of viable cells. However, it showed similar morphology and proliferation characteristics as compared to enzymatic digestion. The mechanical method induced slight apoptosis in MEFs; however, it did not exert the necrotic effect of trypsinization. Treatment of tissue slurry with trypsin solution caused cell lysis and subsequently cell clump formation. Mechanically isolated cells exhibited a higher expression of the MEF surface antigens Sca1, CD106, and CD105. The PSCs on mechanically isolated MEFs displayed a higher expression of pluripotency genes, and formed more compact colonies with a stronger tendency to crowding compared with those cultured on cells isolated by enzymatic digestion. The mechanical method based on tissue inter-syringe processing is relatively a rapid and simple method for MEF isolation. Compared to the enzymatic digestion, the cells obtained from this method show higher expression of embryonic fibroblasts markers and a more functional capacity in supporting PSCs culture.

Inhibition of USP14 induces ER stress-mediated autophagy without apoptosis in lung cancer cell line A549.

Non-small cell lung cancer is the most common type of lung cancer, accounting for more than 80% of this tumor. Ubiquitin-specific protease (USP) 14 is one of the 100 deubiquitinating enzymes that is overexpressed in lung cancer and has been validated as a therapeutic target. The aim of this study is to determine whether the accumulation of ubiquitinated proteins results in endoplasmic reticulum (ER) stress-mediated autophagy. To inhibit USP-14, A549 lung cancer cells were treated with USP-14 siRNA and IU1-47 (20 µM). The protein level, mRNA expression, and cell cycle analysis were evaluated using Western blot, real-time PCR, and flow cytometry, respectively. We found that treating A549 cells with USP14 inhibitors significantly reduced the proliferation rate and induced cell cycle arrest at G2/M phase. We also found that USP14 inhibitors did not induce apoptosis but actually induced autophagy through accumulation of ubiquitinated proteins/ER stress/unfolded protein response (UPR) axis. Moreover, we have for the first time demonstrated that the USP14 inhibition induces ER stress-mediated autophagy in A549 cells by activation of c-Jun N-terminal kinase 1 (JNK1). In conclusion, the current investigation represents a new mechanism by which inhibition of USP14 triggers autophagy via ER stress-mediated UPR in A549 cells.

Application of porcupine inhibitors in stem cell fate determination.

Porcupine (Porcn), a membrane-bound O-acyltransferase, is an endoplasmic reticulum-located protein that has catalytic activity. Porcn is involved in post-translational lipid modification of wingless-Int (Wnt) proteins and serves as an indispensable step in the Wnt proper secretion and signaling. Small-molecule inhibitors targeting Porcn catalytic function in vitro and in vivo are of great interest not only for treating cancer and fibrotic disorders but also in the field of regenerative medicine. Although a number of studies have been conducted, the exact role of Porcn in stem cell fate is not entirely clear. In some cases, Porcn inhibition declined differentiation rate, and in others, it induced stem cell differentiation toward specific lineages. In this review, we first elaborated the Porcn catalytic activity and its inhibitors. Then, we discussed about the recently reported results of Porcn inhibitors in stem cells self-renewal and differentiation.

ß-LAPachone ameliorates doxorubicin-induced cardiotoxicity via regulating autophagy and Nrf2 signalling pathways in mice.

ß-LAPachone (B-LAP) is a naphthoquinone that possesses antioxidant properties. In the present investigation, the protective effect of B-LAP against doxorubicin (DOX)-induced cardiotoxicity was examined in mice. Thirty-five mice were divided into 5 groups: control group, B-LAP (5 mg/kg) group, DOX (15 mg/kg) group, DOX+B-LAP (2.5 mg/kg) group and DOX+B-LAP (5 mg/kg) group. B-LAP was administered orally for 14 days of experimental period. A single dose of DOX (15 mg/kg) was injected intraperitoneally on day 3. Cardiac function, histoarchitecture, indices of oxidative stress and circulating markers of cardiac injury were examined. B-LAP (5 mg/kg) decreased serum levels of lactate dehydrogenase (LDH), creatine kinase MB (CK-MB) and cardiac troponin I (cTnI), and ameliorated cardiac histopathological alterations. In addition to increasing cellular NAD+ /NADH ratio, B-LAP up-regulated the cardiac levels of SIRT1, beclin-1, p-LKB1 and p-AMPK, and reduced the cardiac levels of p-mTOR, interleukin (IL)-1ß, TNF (tumour necrosis factor)-α and caspase-3. B-LAP also elevated the nuclear accumulation of Nrf2 and simultaneously up-regulated the protein levels of haem oxygenase (HO-1) and glutathione S-transferase (GST) in the hearts of DOX mice. While B-LAP reduced malondialdehyde concentrations in heart of DOX-treated mice, it further promoted the activities of cardiac superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT).In accordance with increased cell survival, B-LAP significantly improved the cardiac function of DOX mice. Collectively, these findings underline the protective potential of B-LAP against DOX-induced cardiotoxicity by regulating autophagy and AMPK/Nrf2 signalling pathway in mice.

Oxidative stress and Parkinson's disease: conflict of oxidant-antioxidant systems.

Parkinson's disease (PD) is defined as a chronic neurodegenerative disorder which is diagnosed mostly by its clinical manifestations. Reactive oxygen species (ROS) are considered as key modulators in the development of PD. Despite the intensive investigations, antioxidant-dependent molecular mechanisms of initiation and development of PD are controversial. Free radicals cause serious damage and death of dopamine producing cells when antioxidant capacity of the cells is reduced against oxidative stress (OxS). Many intracellular reactions create ROS, including activation of NADPH oxidase (NOX), mitochondrial dysfunction, and hydrogen peroxide (H2O2) decomposition. On the contrary, natural antioxidants, vitamins, proteins, and antioxidant signaling pathways are major factors to neutralize ROS and its destructive effects. The functional role of nuclear factor E2-related factor 2, Heme oxygenase-1, and selenium against ROS-dependent initiation and progression of PD is elucidated. In this review, we collected multiple factors that play the main role in the initiation, development, and pathogenesis of PD and we discussed their function in the PD.

Tangeretin protects renal tubular epithelial cells against experimental cisplatin toxicity.

OBJECTIVES: Cisplatin is an effective antineoplastic agent; its clinical utility, however, is limited by a few salient toxic side effects like nephrotoxicity. This study aimed to determine the potential protective effects of tangeretin, a citrus-derived flavonoid, against renal tubular cell injury in cisplatin-induced renal toxicity of rats. MATERIALS AND METHODS: Tangeretin was injected intraperitoneally at 2.5 and 5 mg/kg doses for 10 days, and a single dose of cisplatin (8 mg/kg) was injected on the 7th day. Tests of kidney function and tubular injury in renal tissues and urine together with oxidative stress and inflammation markers were examined. RESULTS: Tangeretin ameliorated cisplatin-induced elevations in serum creatinine, BUN, and histopathologic changes. It also attenuated kidney oxidative stress elicited by cisplatin as demonstrated by reduced MDA and increased GSH, CAT, and SOD activities, elevated Nrf2 expression and protein levels of its downstream effectors, HO-1 and NQO-1. Tangeretin further alleviated inflammation evoked by cisplatin as indicated by reduced NF-κB p65 subunit phosphorylation with a simultaneous decrement in its downstream effectors IL-1ß and TNF-α expression and protein levels. Moreover, it declined caspase-3 protein levels and TUNEL positive cells in the kidneys, the markers of apoptosis and DNA fragmentation, thus improving renal endurance. Additionally, tangeretin mitigated renal levels of KIM-1 and NGAL, as well as urinary cystatin C and ß2-microglobulin concentrations, the markers of renal tubular injury. CONCLUSION: Collectively, these data signify the binary profit of tangeretin: enhancement of renal protective mechanisms against cisplatin and attenuation of renal tubular cell injuries induced by the agent.

Micheliolide Protects Against Doxorubicin-Induced Cardiotoxicity in Mice by Regulating PI3K/Akt/NF-kB Signaling Pathway.

Micheliolide (MCL) is a naturally derived anti-inflammatory agent. In the present investigation, we examined the protective potential of MCL against doxorubicin (DOX)-induced cardiotoxicity in mice. Mice were injected with a single 15-mg/kg intraperitoneal dose of DOX at day 1 and the study groups received daily 12.5, 25, and 50 mg/kg doses of MCL for 7 days. Cardiac histopathology, cardiac function, serum markers of cardiac injury, and tissue markers of inflammation, and oxidative stress were examined. MCL decreased serum levels of creatinine kinase MB (CK-MB) and cardiac troponin I (cTnI) levels, ameliorated cardiac tissue architecture, and improved cardiac stroke volume. Apart from reducing the activities of NF-kB p65 subunit, MCL attenuated the cardiac levels of PI3K, phosphorylated (p)-Akt, p-Bad, and caspase-3 levels and simultaneously elevated p-PTEN levels. While the gene expressions of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß) were decreased, the tissue activities of superoxide dismutase (SOD) as well as gene expressions of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase-1 (NQO1) were increased after treatment with MCL. Furthermore, tissue levels of malondialdehyde (MDA) were also decreased. Collectively, these findings point to the protective effects of MCL against DOX-induced cardiotoxicity by regulating PI3K/Akt/NF-kB signaling pathway in mice.

ß-Lapachone protects against doxorubicin-induced nephrotoxicity via NAD+/AMPK/NF-kB in mice.

ß-Lapachone (B-LAP) is a natural naphtaquinone with established anti-oxidative stress and anti-cancer activities. We aimed to investigate B-LAP protective potential against doxorubicin (DOX)-induced nephrotoxicity in mice. The mice received an oral dose of B-LAP followed by a single intraperitoneal injection of 20 mg/kg DOX a day later. They were then treated for 4 days with 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg doses of B-LAP. Renal levels of NAD+/NADH ratios, p-AMPKα, p-NF-κB p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) along with renal expressions of TNF-α, IL-1ß, and IL-6 were examined. Serum levels of kidney function markers as well as renal histopathology were also investigated. In addition to increasing the activities of p-AMPKα, B-LAP elevated NAD+/NADH ratios in the kidneys and decreased the renal levels of nuclear p-NF-κB and its correspondent downstream effectors TNF-α, IL-1ß, IL-6, and iNOS in the kidneys. Also, B-LAP effectively ameliorated renal architectural changes and attenuated serum levels of urea, creatinine, and cystatin C. Collectively, these findings suggest the protective actions of B-LAP against DOX-induced nephrotoxicity in mice.

Candidate biomarkers for Parkinson's disease.

Parkinson's disease (PD) is one of the most common diseases associated with neurodegenerative disorders. It affects 3% to 4% of the population over the age of 65 years. The neuropathological dominant symptoms of PD include the destruction of neurons in the substantia nigra, thus causing striatal dopamine deficiency and the presence of intracellular inclusions that contain aggregates of α­synuclein. The premature form of PD is familial and is known as early onset PD (EOPD). It involves a small portion of patients with PD, displaying symptoms before the age of 60 years. Although individuals who are suffering from the EOPD may have genetic changes, the molecular mechanisms that differentiate between EOPD and late onset PD (LOPD) remain unclear. Owing to the complexity of discriminating between the different forms, treatment, and management of PD, the identification of biomarkers for early diagnosis seems necessary. For this purpose, many studies have been undertaken for the introduction of several biological molecules through various techniques as potential biomarkers. The main focus of these studies was on α-synuclein. However, there are other molecules that are potential biomarkers, such as microRNAs and peptoids. In this article, we tried to review some of these studies.