Upregulation Of Bcl-2 Protein Research Articles

LncRNA LHFPL3-AS1 contributes to tumorigenesis of melanoma stem cells via the miR-181a-5p/BCL2 pathway

Long noncoding RNAs (lncRNAs) are recognized as a new area for cancer therapy. B-cell lymphoma-2 (Bcl-2)-mediated suppression of apoptosis is an important molecular hallmark of cancer. However, the influence of lncRNA on the regulation of oncogenic Bcl-2 in cancer stem cells has not been explored. In this study, our findings revealed that the lncRNA LHFPL3-AS1-long, generated from the polypyrimidine tract binding protein 1 (PTBP1)-mediated splicing of the LHFPL3-AS1 precursor, upregulated BCL2 protein to contribute to tumorigenesis of melanoma stem cells. The in vitro and in vivo results showed that LHFPL3-AS1-long directly interacted with miR-181a-5p to inhibit the mRNA degradation of Bcl-2 (the target of miR-181), thus suppressing apoptosis of melanoma stem cells. The splicing factor PTBP1 regulated the alternative splicing of LHFPL3-AS1 transcript by preferentially binding to the motifs located in exon3 of LHFPL3-AS1 precursor, leading to the biogenesis of LHFPL3-AS1-long in melanoma stem cells. In patients with melanoma, the expressions of PTBP1 and LHFPL3-AS1 were significantly upregulated compared with the healthy donors. Therefore, our study revealed a mechanistic crosstalk among an onco-splicing factor, lncRNA and tumorigenesis of melanoma stem cells, enabling PTBP1 and LHFPL3-AS1 to serve as the attractive therapeutic targets for melanoma.

Targeting NOX 4 by petunidin improves anoxia/reoxygenation-induced myocardium injury

Oxidative stress is the key factor of myocardial ischemia-reperfusion injury (MIRI). Anthocyanins are considered to be effective anti-oxidants. In this study, we observed the anti-MIRI effect of petunidin, one member of anthocyanins, and further explored its mechanism. In present study, anoxia/reoxygenation (A/R) models were replicated on Langendorff-perfused heart and neonatal rat primary cardiomyocytes by A/R treatment. The hemodynamic parameters of isolated hearts were monitored. The levels of oxidative stress and apoptosis in isolated heart and neonatal rat primary cardiomyocytes were evaluated. The expression levels of NADPH oxidase 2 (NOX 2), NOX 4, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax) and cytochrome c were detected by Western Blot. The results showed that petunidin could significantly improve isolated heart function, reduce oxidative stress, inhibit cardiomyocyte apoptosis, up-regulate Bcl-2 protein expression, down-regulate NOX4 and Bax expression, and reduce the level of cytoplasmic cytochrome c after A/R. However, it has no significant effect on NOX 2 protein expression, suggesting that NOX 4 may be the molecular target of petunidin. In vitro, petunidin had shown a consistent effect with that in isolated hearts. It also showed a significant inhibitory effect on reactive oxygen species (ROS) generation. However, the protective effects of petunidin on A/R injury were attenuated by over-expression of NOX 4 in neonatal rat primary cardiomyocytes. These data suggested that the protective effects of petunidin on MIRI may be achieved through targeting NOX 4, thus inhibiting the production of ROS, reducing oxidative stress, and regulating the Bcl-2 pathway to prevent cardiomyocytes apoptosis.

Hepatoprotective effects and mechanisms of Ixeris denticulate water extract on liver cirrhosis in experimental rat

BackgroundTo explore the protective effect and mechanisms of Ixeris denticulate water extract (IDWE) in the development of liver cirrhosis in experimental rat.MethodsSixty rats were randomly divided into five groups: control group, model group and IDWE (2, 4 and 8 g/kg) treatment groups. Alanine transferase (ALT), aspartate transaminase (AST), albumin (ALB), tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-8 in serum and superoxide dismutase (SOD), malondialdehyde (MDA) in liver tissue were evaluated, respectively. The liver index, liver morphology and liver histopathological analysis were detected as a supportive data. The liver protein expression of Bcl-2 and Bax were assessed by western blot, and NF-κB p65 protein expression was determined by immunohistochemistry analysis.ResultsThe result showed that a significantly decrease in the levels of serum AST, ALT and serum inflammatory factors TNF-α, IL-6 and IL-8 in IDWE-treated rats. The levels of serum ALB and SOD in liver tissue were markedly increased after IDWE treated, compared with model rats. Furthermore, IDWE-treated group also exhibited a down-regulated protein expression of NF-κB p65 and Bax, up-regulated Bcl-2 protein expression.ConclusionsIDWE could effectively alleviate the course of liver cirrhosis in rat model, which may be a potent hepatoprotective agent in clinical therapy in the future.

Protective effects of autophagy inhibitor 3-methyladenine on ischemia-reperfusion-induced retinal injury.

To investigate the protective effects of autophagy inhibitor 3-methyladenine (3-MA) in a rat model of ischemic-reperfusion injury (IRI). Forty Sprague-Dawley male rats (weight 220-250g) were randomly divided into four groups: a control group (NC, n = 10), a Sham surgery group (n = 10), an IRI group (n = 10), and a 3-MA-treated IRI group [10 μL 3-MA (10mmol/L) was injected in vitreous after the injury, n = 10]. The retinal IRI was induced by elevating the intraocular pressure to 110mmHg for 60min. Hematoxylin and eosin (HE) staining was used to calculate the number of retinal ganglion cells (RGCs). The level of microtubule-associated protein 1A/1B light chain 3 (LC3), Beclin-1, and Caspase-3 in the retina was detected using the immunofluorescence staining method. The LC3, Beclin-1, B-cell lymphoma/leukemia-2 (Bcl-2), and Caspase-3 protein levels were examined by Western blotting. The number of RGCs in IRI group was significantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group was significantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA-treated IRI group was elevated and protein levels of LC3, Beclin-1 were down-regulated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA-treated IRI group presented down-regulated Caspase-3 and up-regulated Bcl-2 protein expression with comparison of IRI group (P < 0.05). Retina IRI-caused RGCs loss involved activated autophagy pathway and apoptosis, which could be prevented by autophagy inhibitor 3-MA. Autophagy inhibitor 3-MA may act as a potent therapeutic tool in attenuating retina IRI.

B7-H3 Induces Ovarian Cancer Drugs Resistance Through An PI3K/AKT/BCL-2 Signaling Pathway.

PurposeThis study was aimed to investigate the underlying mechanism of B7-H3 induced ovarian cancer proliferation and drugs resistance.Materials and methodsWe compared the expression of B7-H3 in ovarian tumor tissues from high-malignant or low-malignant patients by immunohistochemistry. We established B7-H3 overexpression and knockout ovarian cells by CRISPR-Cas9 technology and examined the expression of the PI3K/AKT/BCL-2 signals in tumor cells by Western blot or immunofluorescence. We detected the B7-H3 overexpression ovarian cancer cells drugs resistance by CCK8 cell proliferation analysis and Annexin V/PI staining. Tumor-bearing mice were used to investigate the anticancer effects of PI3K/AKT inhibitors in combination with B7-H3 neutralizing antibodies.ResultsEnhanced expression of B7-H3 was observed in ovarian tumor tissues from high-malignant patients compared to those from low-malignant patients. Notably, B7-H3 overexpression caused enhanced cells proliferation and chemo-resistance in vitro and in vivo through the activation of PI3K/AKT signaling pathways and up-regulation of BCL-2 protein. Combination of chemotherapeutic agents and B7-H3 neutralizing antibodies efficiently reverses the drugs resistance induced by B7-H3, resulting in improved anticancer effects in ovarian cancer.ConclusionB7-H3 expression induces the activation the PI3K/AKT signaling pathway and up-regulates BCL-2 in protein level, resulting in the sustained growth and chemo-resistance in ovarian cancer. Blockade of B7-H3 signals efficiently reverses the chemo-resistance, which provides an innovative target in ovarian cancer treatment.

Long non-coding RNA GAPLINC promotes angiogenesis by regulating miR-211 under hypoxia in human umbilical vein endothelial cells.

In this study, we investigated the role of a long non‐coding RNA GAPLINC in angiogenesis using human umbilical vein endothelial cells (HUVEC). We found that hypoxia and hypoxia‐inducible factor 1α (HIF‐1α) increased the expression of GAPLINC in HUVEC cells. Moreover, GAPLINC overexpression down‐regulated miR‐211 and up‐regulated Bcl2 protein expression. Further rescue experiments confirmed that hypoxia directly increased GAPLINC expression. GAPLINC overexpression also increased cell migration and vessel formation which promoted angiogenesis, and these changes were attributed to the increased expression of vascular endothelial growth factor receptors (VEGFR) and delta‐like canonical notch ligand 4 (DLL4) receptors. Finally, we demonstrated that GAPLINC promotes vessel formation and migration by regulating MAPK and NF‐kB signalling pathways. Taken together, these findings comprehensively demonstrate that overexpression of GAPLINC increases HUVEC cells angiogenesis under hypoxia condition suggesting that GAPLINC can be a potential target for critical limb ischaemia (CLI) treatment.

Protective Effect of Resveratrol Against Corticosterone-induced Neurotoxicity in PC12 Cells.

ObjectiveResveratrol(RES) is a natural polyphenol which possesses an anti-depressant effect. However, the mechanisms of its anti-depressant effect remain unclear. The aim of the study is to investigate the potential mechanisms in the neuro-protective efficiency in the corticosterone-induced pheochromacytoma 12 (PC12) cells.MethodsPC12 cells were treated with 200 μM of corticosterone in the absence or presence of different concentrations of RES for 24 h. Then, cell viability was measured by Cell Counting Kit-8 assay. Apoptosis of PC12 cells was measured by Annexin V-FITC and Propidium iodide (PI) labelling. The expression of apoptosis-related proteins including Bax, Bcl-2, caspase-3 was determined by western blotting.ResultsThe results showed that treatment with 200 μM of corticosterone induced cytotoxicity in PC12 cells. However, different concentrations of RES (2.5μmol/L, 5μmol/L and 10 μmol/L) significantly increased the cell viability, suppressed the apoptosis of PC12 cells, down-regulated Bax and caspase-3 protein expression, and up-regulated Bcl-2 protein expression, compared to the model group (p<0.05).ConclusionResveratrol has a protective effect on corticosterone-induced neurotoxicity in PC12 cells, which may be related to the apoptosis via inhibition of apoptosis-related proteins and displays the antidepressant-like effect.

Effects and Mechanisms of Vitamin C Post-Conditioning on Platelet Activation after Hypoxia/Reoxygenation

Background: Platelet activation occurs upon ischemia/reperfusion and is related to the generation of reactive oxygen species (ROS) during this process. Vitamin C (VC) is a powerful antioxidant. VC scavenges ROS, reduces platelet activation, and attenuates reperfusion injury. However, the effects of VC on platelets undergoing hypoxia/reoxygenation (H/R) remain unclear. Objectives: Herein, we evaluated the effects of VC on platelets in vitro following H/R and the related mechanisms. Method: Fresh platelets were collected from 67 volunteers at the Blood Center of Hebei Province. Platelets were diluted with saline to a concentration of 2.00 × 10¹¹/L. Aggregation and the curve slope were evaluated within 4 h with a whole-blood impedance analyzer. To determine the optimal experimental time, platelets were treated with hypoxia or reoxygenation for different times, and impedance aggregometry was carried out by measuring changes in electrical impedance induced by arachidonic acid (0.5 mM) and adenosine diphosphate (10 µM), thereby establishing the H/R model. Three antioxidants (VC, melatonin, and probucol) were used to treat platelets after H/R, and impedance aggregometry was used to determine their effects on platelet aggregation. The influence of VC on apoptosis-related indicators was detected. ROS and the mitochondrial membrane potential were observed by inverted fluorescence microscopy and flow cytometry, respectively. Related protein levels were detected by Western blotting. Results: ROS scavengers inhibited platelet activation and aggregation in a concentration-dependent manner. VC post-conditioning scavenged ROS, downregulated cytochrome C, Bax, and caspase-9 proteins, and upregulated Bcl-2 protein. These effects collectively blocked platelet apoptosis and inhibited platelet aggregation. Conclusions: VC inhibited platelet aggregation by blocking apoptosis. Thus, VC may have applications in the treatment of platelet-related diseases.

Sericin alleviates restraint stress induced depressive- and anxiety-like behaviors via modulation of oxidative stress, neuroinflammation and apoptosis in the prefrontal cortex and hippocampus

This study was aimed to examine the effects of sericin administration on restraint stress induced anxiety- and depressive-like behaviors, oxidative stress, inflammation and apoptosis in the prefrontal cortex (PFC) and hippocampus (HIP) of mice. Animals were subjected to chronic restraint stress (3 h/day for 21 days) to induce a depressive-like model. Sericin was administered at different doses (100, 150, and 200 mg/kg/day, gavage for 21 days) along with immobilization. Elevated plus maze (EPM) and open field test (OFT) were performed to assess anxiety; while, the forced swim test (FST) and tail suspension test (TST) were implemented to evaluate depressive-like behaviors. Mitochondrial membrane potential (MMP), and markers of oxidative stress, neuroinflammation, and apoptosis were evaluated in the PFC and HIP regions. Moreover, serum levels of corticosterone were measured. Results showed that sericin increased number of central entries in OFT and prolonged time spent in open arms of EPM apparatus, while it reduced immobility time in TST and FST. Moreover, sericin treatments decreased oxygen species (ROS) and lipid peroxidation levels, restored MMP, and enhanced total antioxidant capacity (TAC) and enzyme activity of GPx and SOD in both brain regions. Furthermore, sericin reduced serum corticosterone concentration and suppressed neuroinflammatory response in the HIP and PFC, shown by decreased NF-κB, TNF-α, and IL-1β protein levels. Finally, sericin inhibited mitochondrial-dependent apoptosis pathway through down-regulation of Bax, cytochrome c, caspase-9 and -3, and up-regulation of Bcl-2 protein. These findings provide evidence for the protective effect of sericin therapy against psychopathological and behavioral changes induced by restraint stress.

Effects of lentivirus-mediated astrocyte elevated gene-1 overexpression on proliferation and apoptosis of human retinoblastoma cells.

To investigate the effect of astrocyte elevated gene-1 (AEG-1) overexpression on the biological behaviour of human retinoblastoma (RB) cells and its possible mechanism. Three human RB cell lines (SO-RB50, Y79 and WERI-RB1) were infected with AEG-1-GFP recombinant lentiviral vectors to induce AEG-1 overexpression, while the cells infected with negative lentiviral vectors and cells without any intervention formed control groups. All three RB cell lines showed an overexpression of AEG-1 after lentivirus infection (p<0.001 for all three cell lines). The survival rate of RB cells increased (all p<0.001) in the AEG-1 overexpressed groups when compared with the control groups. There was a decrease in G0/G1 cell cycle phase arrest and an accumulation in G2/M cell cycle phase in all three RB cell lines (p<0.001), with an induction in the S phase in WERI-RB1 cells. It was paralleled by a downregulation of p21 and p27 proteins and an upregulation of the Cdc2 protein. The apoptosis rate of RB cells declined (p<0.001) when AEG-1 was overexpressed, in association with an upregulation of Bcl-2 protein and a downregulation of Bax protein and cleaved caspase-3 proteins. A lentivirus-mediated AEG-1 overexpression in RB cells led invitro to a growth promotion and an apoptosis inhibition of human RB cells, associated with an upregulation of the Bcl-2 protein, a downregulation of the Bax protein and of cleaved caspase-3 proteins, and with alterations of the cell cycle. AEG-1 may be involved in the development and progression of RB.

Adenosine protects pancreatic beta cells against apoptosis induced by endoplasmic reticulum stress.

Chronic exposure to high glucose induces endoplasmic reticulum (ER) stress in pancreatic beta cells (PBCs). The previous evidence showed that adenosine modulate PBCs viability and insulin secretion. The aim of this study was to evaluate possible involvement of adenosine in protection of MIN6 β-cells from Tunicamycin (Tu)-induced ER stress. MIN6 cells were cotreated with Tu and different concentrations of adenosine. Cell viability, proliferation, and apoptosis were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), 5-bromo-2'-deoxyuridine (Brdu), and colony formation assays. Caspase-12 activity was assayed using the fluorometric method. Thioflavin T (ThT) staining was used for the evaluation of protein aggregation. Insulin secretion was evaluated using specific an ELISA kit. Ca2+ mobilization assayed using Fura2/AM probe. BIP, CHOP, XBP-1, and XBP-1s expression in both messenger RNA (mRNA) and protein levels were evaluated using the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Bcl-2, p-eIF2α/eIF2α, and GADD34 levels also determined with Western blot analysis. Adenosine protected MIN6 cells against Tu-induced ER stress in a dose-dependent manner and increased their proliferation. Decreased caspase-12 activity and upregulated Bcl-2 protein may explain antiapoptotic effects of adenosine. ThT staining indicated an attenuated aggregation of misfolded proteins. Adenosine effectively increased insulin secretion in Tu-treated cells. BIP, CHOP, XBP1, and sXBP1 expression were decreased significantly in cotreated cells, indicating alleviation of ER stress. However, adenosine potentiated the expression of GADD34 and decreased p-eIF2α/eIF2α ratio. Adenosine increased cytosolic Ca 2+ levels, which may promote adenosine triphosphate (ATP) synthesis in mitochondria, helping ER to preserve protein hemostasis. Taken together, adenosine upregulated Bcl-2 and GADD34 to protect PBCs against Tu-induced apoptosis and increase Insulin secretion.

Acteoside Attenuates Oxidative Stress and Neuronal Apoptosis in Rats with Focal Cerebral Ischemia-Reperfusion Injury.

Acteoside (ACT) has been shown to exert antioxidant and neuroprotective effects in neurodegenerative diseases. However, the effect of ACT on cerebral ischemia-reperfusion (I/R) injury is not yet clear. In this study, we found that ACT administration reduced infarct volume and brain edema, and improved neurological deficits, as indicated by the decreased modified neurological severity score. Administration of ACT strikingly reduced oxidative stress, accompanied by decreased levels of reactive oxygen species and malondialdehyde and increased levels of superoxide dismutase and catalase in a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). Furthermore, ACT administration reduced the number of terminal deoxynucleotidyl transferase uridine 5'-triphosphate (UTP) nick-end labeling-positive cells in the cerebral cortex of ischemic side of MCAO/R rats, accompanied by downregulation of B cell lymphoma 2 (Bcl-2) associated X protein and cleaved caspase-3 proteins and upregulation of Bcl-2 protein. Additionally, ACT treatment inhibited the protein kinase R/eukaryotic initiation factor-2α stress pathway in the brains of MCAO/R rats. Our results demonstrated that ACT attenuates oxidative stress and neuronal apoptosis in MCAO/R rats, suggesting that ACT may serve as a novel therapeutic candidate for the treatment of I/R brain injury.

EBV-BZLF1 Promotes Gastric Cancer Growth Through Activating the PI3K/AKT Pathway

Background: EBVaGC (EBV associated gastric cancer) had been paid much attentions once classified by TCGA and were considered as the suitable group for immunotherapy, which hold distinguishing characteristics with EBVnGC (EBV nonassociated gastric cancer). This study aims to explore the biologic behavior and its possible mechanisms to provide treatment options for EBVaGC. Methods: Lentiviral infection system was used to construct the control and BZLF1 stably expressing gastric cancer cell lines (AGS and HGC27). The cell-derived xenografts (CDX) were constructed to detect the tumor growth and functional experiment including CCK8 assay, cell cycle assay, apoptosis assay, cell migration and invasion assay were implemented to detect the proliferation and transfer ability in vitro. Western blot and immunohistochemistry were used to detect the expression of related molecules. Results: The BZLF1 overexpression cells and the corresponding xenografts grew faster than control groups with enhanced pAKT and pS6 expression. BZLF1 induced “G0/1 phase” arrest in AGS and “S phase” arrest in HGC27 cell, inhibited cell apoptosis through upregulating bcl-2 protein, meanwhile, slightly increased P53 and p-P53 expression in both BZLF1 overexpression cells. However, no significant effects were observed on the malignant biologic behaviors in terms of migration and invasion ability. Conclusion: BZLF1 may promote the growth of gastric cancer through activating the PI3K/AKT pathway, regulating the cell cycle especially regarding P53 signaling network. PI3K/AKT pathway inhibitor probably become promising treatment options for EBVaGC.

Isoquercetin attenuates oxidative stress and neuronal apoptosis after ischemia/reperfusion injury via Nrf2-mediated inhibition of the NOX4/ROS/NF-κB pathway

Isoquercetin (Iso) has been found to have neuroprotective effects against cerebral ischemic stroke. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. In this study, we aimed to evaluate the neuroprotective effects of Iso in primary culture of rat hippocampal neurons exposed to oxygen and glucose deprivation and reperfusion (OGD/R) injury and in rats subjected to middle cerebral artery occlusion and reperfusion (MCAO/R) injury. We found that rats treated with Iso exhibited a lower degree of infarct volume, and brain water content than the vehicle-treated rats. Treatment with Iso also improved the neurological deficits in MCAO/R rats as shown by the decreased modified neurological severity score. Iso treatment decreased the reactive oxygen species (ROS) and malondialdehyde (MDA) production, and increased the activity of superoxide dismutase (SOD) and catalase (CAT) in brains of MCAO/R rats and primary culture of rat hippocampal neurons exposed to OGD/R. Iso treatment prevents I/R-induced neuronal apoptosis in vivo and in vitro as indicated by increased cell viability and decreased number of TUNEL-positive cells, accompanying with downregulation of cleaved caspase-3 protein and upregulation of Bcl-2 protein. Moreover, Nrf2 knockdown weakened the anti-apoptotic and anti-oxidant activities of Iso in primary culture of rat hippocampal neurons exposed to OGD/R. Interestingly, we found that Iso could induce Nrf2 translocation from cytoplasm to nucleus in primary culture of rat hippocampal neurons exposed to OGD/R. Iso activated the NOX4/ROS/NF-κB signaling pathway in in vivo and in vitro cerebral I/R injury models. Nrf2 knockdown blocked the inhibitory effect of Iso on protein expression of NOX4, p-IκBα and p-p65 in primary culture of rat hippocampal neurons exposed to OGD/R. All the data suggested that Iso protected against oxidative stress and neuronal apoptosis in in vivo and in vitro cerebral I/R injury models via Nrf2-mediated inhibition of the NOX4/ROS/NF-κB signaling pathway. Our findings suggested that Iso could be a potential agent for I/R brain injury.

Rosmarinic acid elicits neuroprotection in ischemic stroke via Nrf2 and heme oxygenase 1 signaling.

Rosmarinic acid (RA) can elicit a neuroprotective effect against ischemic stroke, but the precise molecular mechanism remains poorly understood. In this study, an experimental ischemic stroke model was established in CD-1 mice (Beijing Vital River Laboratory Animal Technology, Beijing, China) by occluding the right middle cerebral artery for 1 hour and allowing reperfusion for 24 hours. After intraperitoneally injecting model mice with 10, 20, or 40 mg/kg RA, functional neurological deficits were evaluated using modified Longa scores. Subsequently, cerebral infarct volume was measured using TTC staining and ischemic brain tissue was examined for cell apoptosis with TUNEL staining. Superoxide dismutase activity and malondialdehyde levels were measured by spectrophometry. Expression of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), Bcl-2, Bax, Akt, and phospho-Ser473 Akt proteins in ischemic brain tissue was detected by western blot, while mRNA levels of Nrf2, HO-1, Bcl-2, and Bax were analyzed using real time quantitative PCR. In addition, HO-1 enzyme activity was measured spectrophotometrically. RA (20 and 40 mg/kg) greatly improved neurological function, reduced infarct volume, decreased cell apoptosis, upregulated Bcl-2 protein and mRNA expression, downregulated Bax protein and mRNA expression, increased HO-1 and Nrf2 protein and mRNA expression, increased superoxide dismutase activity, and decreased malondialdehyde levels in ischemic brain tissue of model mice. However, intraperitoneal injection of a HO-1 inhibitor (10 mg/kg zinc protoporphyrin IX) reversed the neuroprotective effects of RA on HO-1 enzyme activity and Bcl-2 and Bax protein expression. The PI3K/Akt signaling pathway inhibitor LY294002 (10 mM) inhibited Akt phosphorylation, as well as Nrf2 and HO-1 expression. Our findings suggest that RA has anti-oxidative and anti-apoptotic properties that protect against ischemic stroke by a mechanism involving upregulation of Nrf2 and HO-1 expression via the PI3K/Akt signaling pathway.

Effects of simvastatin on aortic vascular endothelial cell apoptosis and Bcl-2 protein expression in a rat model of atherosclerosis

To explore the effects of simvastatin on vascular endothelial cell apoptosis and Bcl-2 protein expression in the aorta in a rat model of atherosclerosis. Thirty-six rats were randomized into control group (n=10), atherosclerosis model group (n=13) and simvastatin intervention group (n=13). In the latter two groups, rat models of atherosclerosis were established by intraperitoneal injection of vitamin D3 combined with high-fat feeding for 6 weeks, and the control rats were fed with regular diet. In the intervention group, the rats were further fed with high-fat diet with daily simvastatin treatment for 4 weeks. After the treatments, the pathological changes and plaque in the thoracic aorta were observed, and the expression of Bcl-2 protein was detected with immunohistochemistry. TUNEL assay was used to determine the apoptosis index (AI) of the vascular endothelial cells. Compared with that in the control group, Bcl-2 protein expression in the aorta of atherosclerotic rats was significantly decreased (P<0.05); simvastatin treatment obviously increased the expression of Bcl-2 protein in atherosclerotic rats (P<0.05) to a level similar to that in the control group. The AI was the highest in the model group (P<0.05) and comparable between the control and simvastatin treatment group. The therapeutic effect of simvastatin against atherosclerosis is probably mediated by up-regulation of Bcl-2 protein, which inhibits vascular endothelial cell apoptosis in rats with aortic atherosclerosis.

Role of p-MKK7 in myricetin-induced protection against intestinal ischemia/reperfusion injury

Intestinal ischemia reperfusion (I/R) may cause inflammation-, oxidative stress-, and apoptosis-related tissue injuries and facilitate bacterial infection, leading to multiple organ failure. Myricetin, a flavonoid, is found to have diverse biological effects including anti-inflammatory, anti-oxidative, and anti-bacterial effects. Based on our pre-experiment, we proposed that myricetin pretreatment (25, 50mg/kg) could ameliorate intestinal I/R injury and myricetin-induced modulation on MKK7/JNK signal pathway might play a key role in the amelioration. The present study was designed to verify the proposal by using both rat intestinal I/R model in vivo and hypoxia/reoxygenation (H/R)-injured intestinal epithelial cell line (IEC-6 cells) model in vitro. The results confirmed our proposal. Myricetin selectively ameliorated I/R- and H/R-induced injuries in vivo and in vitro respectively without significantly affecting the corresponding normal controls. Myricetin significantly alleviated I/R-induced rat intestinal injury by reducing the generation of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 and by reducing MPO activity. Myricetin significantly reduced oxidative stress through decreasing MDA level and increasing the levels of SOD and GSH in the intestinal tissues compared with I/R control rats. Myricetin significantly decreased apoptosis by selectively down-regulating the expression of p-MKK7 and p-JNK without affecting MKK7 and JNK, inhibiting Bax, caspase-3 protein expression, and up-regulating Bcl-2 protein expression in I/R-injured jejunum of rats. In vitro study indicated that MKK7 siRNA transfection significantly decreased both MKK7 and p-MKK7 and other apoptosis-related proteins, partially simulating myricetin-induced anti-apoptotic effects. MKK7 siRNA transfection+myricetin could not further decrease MKK7, p-MKK7, and other apoptosis-related proteins, suggesting that inhibition of MKK7/JNK pathway plays a key role in myricetin-induced protection against intestinal I/R. MKK7 overexpression by cDNA transfection abrogated myricetin-reduced apoptosis-related protein expression, confirming that the MKK7/JNK signal pathway is the key target for myricetin-induced amelioration. The present study indicated that pretreatment of myricetin induced selective protection against intestinal I/R injury without significantly affecting corresponding normal controls and p-MKK7 was the key target, suggesting that myricetin is worth further translational studies.

Abstract A34: The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies

Abstract Introduction: It was shown that BCR inhibitors such as ibrutinib interrupt microenvironmental interactions and mobilize malignant B cells of chronic lymphocytic leukemia (CLL) from immune niches into the blood stream. It has been suggested that this mobilization will sensitize malignant B cells to other drugs, providing a possibility to introduce synthetically lethal combinations of drugs with an acceptable toxicity profile. This might be limited to a subgroup of CLL patients with specific underlying molecular mechanisms of disease biology or reaction to BCR inhibitors. The aim of this study was to test for the effect of ibrutinib on the expression of genes and activity of pathways that could be potentially targeted by clinically available drugs to achieve highly potent combinations. Results: We performed gene expression profiling in samples obtained from CLL patients treated with ibrutinib as a single agent (pre-ibrutinib vs. day 15 and/or week 5/12/15; N=12 pairs) to identify potential therapeutic targets. We have primarily focused on analyzes of cell-surface proteins and anti-apoptotic Bcl2 family members, since therapeutic antibodies and small molecular inhibitors for these molecules are available. We have analyzed changes in &amp;gt;30 cell-surface molecules, 8 members of the Bcl2 family, and members of other potentially relevant pathways (chemokines, adhesion, Akt, Erk, Ras, NFkB). Notably, we have observed that the mRNA and B cell-surface levels of CD20 are strongly down-modulated with ibrutinib, which indicates that the previously suggested combination of ibrutinib and anti-CD20 antibodies (such as rituximab or ofatumumab) is likely not ideal. Indeed, we showed that CLL cells with lower levels of CD20 are more resistant to rituximab in vitro, but also in vivo (P&amp;lt;0.01). We also showed that the effect of ibrutinib on CD20 levels is mediated by inhibition of CXCR4/SDF-1 axis, which impairs the SDF-1α (ligand for CXCR4) mediated regulation of CD20 expression (P&amp;lt;0.01). Notably, it has been shown that addition of rituximab to ibrutinib in vivo is able to partially eliminate CLL cells despite lower levels of CD20 suggesting that there must be other mechanism of ibrutinib action that allows for the effect of rituximab. The screening of anti-apoptotic molecules revealed that Mcl1 levels were significantly down-modulated after ibrutinib treatment in vivo (P=0.002), and ibrutinib also inhibited Mcl1 induction in CLL cells co-cultured with stromal cells (HS-5). As Mcl1 was described to directly protect CLL cells from rituximab, we suggest that ibrutinib-mediated Mcl1 reduction is a mechanism that facilitates rituximab efficacy. On the other hand we have shown that several members of Bcl2 family are up-regulated in CLL samples obtained after &amp;gt;3 weeks on ibrutinib therapy. We have also shown that interactions of stromal cells (HS-5) with CLL cells lead to up-regulation of these molecules while ibrutinib inhibits this in vitro (P&amp;lt;0.01). This suggest that the observed in vivo up-regulation of specific Bcl2 family members is due to an adaptation of B cells or selection of malignant B cells with high-level expression of anti-apoptotic molecules sufficient to survive the BCR signalling inhibition. We observed that the survival of CLL cells treated with ibrutinib is dependent on Bcl2, and subsequent Bcl2 inhibition can act synergistically with ibrutinib in B cell lines and CLL samples. These anti-apoptotic molecules therefore represent primary candidates with synthetically lethal potential in combination with ibrutinib. Additionally, we have identified dependence of a subtype of CLL cases on integrin signalling. The inhibition of integrin VLA-4 with anti-VLA4 antibody or integrin-associated kinase overcame the resistance of CLL cells to chemotherapy drugs and also to anti-CD20 antibodies, and synergized with the effects of ibrutinib. Conclusion: We have performed gene expression analysis of primary samples obtained from patients on ibrutinib, and identified gene expression changes that are associated with adaptation of B cells and/or selection of B cell resistant to BCR inhibition. We have shown that malignant B cells are dependent on integrin-signalling and the up-regulation of Bcl2 protein, which makes them synergistically lethal targets. Supported by: The results of this research have been acquired within CEITEC 2020 (LQ1601) project with financial contribution made by the Ministry of Education, Youths and Sports of the Czech Republic within special support paid from the National Programme for Sustainability II funds. Citation Format: Gabriela Pavlasova, Katerina Musilova, Vaclav Seda, Katerina Cerna, Eva Vojackova, Veronika Svobodova, Marek Mraz. The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A34.

Sinapic acid ameliorate cadmium-induced nephrotoxicity: In vivo possible involvement of oxidative stress, apoptosis, and inflammation via NF-κB downregulation.

Cadmium (CD), an environmental and industrial pollutant, generates reactive oxygen species (ROS) and NOS responsible for oxidative and nitrosative stress that can lead to nephrotoxic injury, including proximal tubule and glomerulus dysfunction. Sinapic acid (SA) has been found to possess potent antioxidant and anti-inflammatory effects in vitro and in vivo. We aimed to examine the nephroprotective, anti-oxidant, anti-inflammatory, and anti-apoptotic effects of SA against CD-induced nephrotoxicity and its underlying mechanism. Kidney functional markers (serum urea, uric acid, creatinine, LDH, and calcium) and histopathological examinations of the kidney were used to evaluate CD-induced nephrotoxicity. Oxidative stress markers (lipid peroxidation and total protein), renal nitrosative stress (nitric oxide), antioxidant enzymes (catalase and NP-SH), inflammation markers (NF-κB [p65], TNF-α, IL-6, and myeloperoxidase [MPO]), and apoptotic markers (caspase 3, Bax, and Bcl-2) were also assessed. SA (10 and 20mg/kg) pretreatment restored kidney function, upregulated antioxidant levels, and prevented the elevation of lipid peroxidation and nitric oxide levels, significantly reducing oxidative and nitrosative stress. CD upregulated renal cytokine levels (TNF-α, IL-6), nuclear NF-κB (p65) expression, NF-κB-DNA-binding activity, and MPO activity, which were significantly downregulated upon SA pretreatment. Furthermore, SA treatment prevented the upregulation of caspase 3 and Bax protein expression and upregulated Bcl-2 protein expression. SA pretreatment also alleviated the magnitude of histological injuries and reduced neutrophil infiltration in renal tubules. We conclude that the nephroprotective potential of SA in CD-induced nephrotoxicity might be due to its antioxidant, anti-inflammatory, and anti-apoptotic potential via downregulation of oxidative/nitrosative stress, inflammation, and apoptosis in the kidney.

Fish Scale Collagen Peptides Protect against CoCl2/TNF-α-Induced Cytotoxicity and Inflammation via Inhibition of ROS, MAPK, and NF-κB Pathways in HaCaT Cells.

Skin diseases associated with inflammation or oxidative stress represent the most common problem in dermatology. The present study demonstrates that fish scale collagen peptides (FSCP) protect against CoCl2-induced cytotoxicity and TNF-α-induced inflammatory responses in human HaCaT keratinocyte cells. Our study is the first to report that FSCP increase cell viability and ameliorate oxidative injury in HaCaT cells through mechanisms mediated by the downregulation of key proinflammatory cytokines, namely, TNF-α, IL-1β, IL-8, and iNOS. FSCP also prevent cell apoptosis by repressing Bax expression, caspase-3 activity, and cytochrome c release and by upregulating Bcl-2 protein levels in CoCl2- or TNF-α-stimulated HaCaT cells. In addition, the inhibitory effects of FSCP on cytotoxicity and the induction of proinflammatory cytokine expression were found to be associated with suppression of the ROS, MAPK (p38/MAPK, ERK, and JNK), and NF-κB signaling pathways. Taken together, our data suggest that FSCP are useful as immunomodulatory agents in inflammatory or immune-mediated skin diseases. Furthermore, our results provide new insights into the potential therapeutic use of FSCP in the prevention and treatment of various oxidative- or inflammatory stress-related inflammation and injuries.