Downstream Apoptotic Pathways Research Articles

Decoy peptides that inhibit TNF signaling by disrupting the TNF homotrimeric oligomer.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine and its functional homotrimeric form interacts with the TNF receptor (TNFR) to activate downstream apoptotic, necroptotic, and inflammatory signaling pathways. Excessive activation of these pathways leads to various inflammatory diseases, which makes TNF a promising therapeutic target. Here, 12-mer peptides were selected from the interface of TNF-TNFR based upon their relative binding energies and were named 'TNF-inhibiting decoys' (TIDs). These decoy peptides inhibited TNF-mediated secretion of cytokines and cell death, as well as activation of downstream signaling effectors. Effective TIDs inhibited TNF signaling by disrupting the formation of TNF's functional homotrimeric form. Among derivatives of TIDs, TID3c showed slightly better efficacy in cell-based assays by disrupting TNF trimer formation. Moreover, TID3c oligomerized TNF to a high molecular weight configuration. In silico modeling and simulations revealed that TID3c and its parent peptide, TID3, form a stable complex with TNF through hydrogen bonds and electrostatic interactions, which makes them the promising lead to develop peptide-based anti-TNF therapeutics.

Iodine-125 seed inhibits proliferation and promotes apoptosis of cholangiocarcinoma cells by inducing the ROS/p53 axis

With advances in radioactive particle implantation in clinical practice, Iodine-125 (125I) seed brachytherapy has emerged as a promising treatment for cholangiocarcinoma (CCA), showing good prognosis; however, the underlying molecular mechanism of the therapeutic effect of 125I seed is unclear. To study the effects of 125I seed on the proliferation and apoptosis of CCA cells. CCA cell lines, RBE and HCCC-9810, were treated with reactive oxygen species (ROS) scavenger acetylcysteine (NAC) or the p53 functional inhibitor, pifithrin-α hydrobromide (PFTα). Cell counting kit-8 (CCK-8) assay, 5-bromo-2-deoxy-uridine (BrdU) staining, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry assay were performed to test the radiation-sensitivity of 125I seed toward CCA cells at different radiation doses (0.4 mCi and 0.8 mCi). 2,7-dichlorofluorescein diacetate (DCF-DA) assay, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot analysis were performed to assess the effect of 125I seed on the ROS/p53 axis. A dose-dependent inhibitory effect of 125I seeds on the proliferation of CCA cells was observed. The 125I seed promoted apoptosis of CCA cells and induced the activation of the ROS/p53 pathway in a dose-dependent manner. NAC or PFTα treatment effectively reversed the stimulatory effect of 125I seed on the proliferation of CCA cells. NAC or PFTα suppressed apoptosis and p53 protein expression induced by the 125I seed. 125I seed can inhibit cell growth mainly through the apoptotic pathway. The mechanism may involve the activation of p53 and its downstream apoptotic pathway by up-regulating the level of ROS in cells.

MLK3 localizes mainly to the cytoplasm and promotes oxidative stress injury via a positive feedback loop.

Activation of mixed lineage kinase 3 (MLK3) by phosphorylation at Thr277/Ser281 stimulates downstream apoptotic pathways and ultimately leads to cell injury. MLK3 is reported to localize to both the cytoplasm and nucleus in human ovarian cancer cells and immortalized ovarian epithelial cells (T80 and T90 cells), and phosphorylation at Thr477 is required for the cytoplasmic retention of MLK3 in T80 cells. However, the subcellular distribution of MLK3 in other cell types has rarely been reported, and whether phosphorylation of MLK3 at Thr277/Ser281 affects its subcellular distribution is unknown. Here, our bioinformatics analysis predicted that MLK3 was mainly distributed in the cytoplasm and nucleus. In the human HEK293T embryonic kidney cell line and murine HT22 hippocampal neuronal cell line, endogenous MLK3 was more abundant in the cytoplasm and less abundant in the nucleus. In addition, overexpressed Myc-tagged MLK3 and EGFP-tagged MLK3 were also observed to localize mainly to the cytoplasm. MLK3 that was activated by phosphorylation at Thr277/Ser281 was mainly distributed in the cytoplasm, and phosphorylation deficient (T277A/S281A) and mimic (T277E/S281E) mutants both showed distributions similar to that of wild type (wt) MLK3, further proving that phosphorylation at Thr277/Ser281 was not involved in regulating MLK3 subcellular localization. In HEK293T cells, H2O2 stimulation accelerated MLK3 phosphorylation (activation), and this phosphorylation was reduced by the antioxidant N-acetylcysteine in a dose-dependent manner. Overexpressing wt MLK3 promoted the production of intracellular reactive oxygen species and increased cell apoptosis, both of which were enhanced by the phosphorylation-mimic (T277E/S281E) MLK3 variant but not by the phosphorylation-deficient (T277A/S281A) MLK3 variant. These findings provided additional evidence for the cytoplasmic and nuclear distribution of MLK3 in HEK293T cells or HT22 cells and revealed the pivotal role of MLK3 in the positive feedback loop of oxidative stress injury.

Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca2+-dependent gap junction intercellular communication.

Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca2+-related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

A novel NEDD4L-TXNIP-CHOP axis in the pathogenesis of nonalcoholic steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver diseases worldwide. There is a pressing clinical need to identify potential therapeutic targets for NASH treatment. Thioredoxin interacting protein (Txnip) is a stress responsive gene that has been implicated in the pathogenesis of NASH, but its exact role is not fully understood. Here, we investigated the liver- and gene-specific role of Txnip and its upstream/downstream signaling in the pathogenesis of NASH. Methods and Results: Using four independent NASH mouse models, we found that TXNIP protein abnormally accumulated in NASH mouse livers. A decrease in E3 ubiquitin ligase NEDD4L resulted in impaired TXNIP ubiquitination and its accumulation in the liver. TXNIP protein levels were positively correlated with that of CHOP, a major regulator of ER stress-mediated apoptosis, in NASH mouse liver. Moreover, gain- and loss-of-function studies showed that TXNIP increased protein not mRNA levels of Chop both in vitro and in vivo. Mechanistically, the C-terminus of TXNIP associated with the N-terminus of the α-helix domain of CHOP and decreased CHOP ubiquitination, thus increasing the stability of CHOP protein. Lastly, selective knockdown of Txnip by adenovirus-mediated shRNA (not targets Txnip antisense lncRNA) delivery in the livers of both young and aged NASH mice suppressed the expression of CHOP and its downstream apoptotic pathway, and ameliorated NASH by reducing hepatic apoptosis, inflammation, and fibrosis. Conclusions: Our study revealed a pathogenic role of hepatic TXNIP in NASH and identified a novel NEDD4L-TXNIP-CHOP axis in the pathogenesis of NASH.

APG115 Enhances the Sensitivity of Ibrutinib Via Promoting p53 to Target MCL-1 in Chronic Lymphocytic Leukemia

Introduction: APG115 is an orally active, selective, potent small molecule inhibitor of the MDM2-p53 interaction, which destabilizes MDM2-p53 complex and promotes p53 activation. Previous studies reported that APG115 promoted cell apoptosis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear. Methods: Peripheral blood samples from de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University. The effects of APG115 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE/7AAD staining and PI/RNase staining respectively. Related protein expression of p53 pathway in CLL cell lines and primary cells were detected by Western blot. Protein interactions were verified by COIP. Immunofluorescence was applied to detect protein localization. Results: To study the role of APG115, we added APG115 into CLL cell lines and primary cells to select appropriate concentration according to IC50. The cells viability of gradually decreased along with the extension of time or concentration augment, which suggested that CLL cells were time-dose-dependent on APG115. In addition, the protein levels of MDM2, p53, PUMA and p21 increased under the treatment of APG115, indicating that APG115 relieved the inhibition of MDM2 on p53 and activated downstream apoptotic pathways. Meanwhile, the APG115 induced cell apoptotic elevation and cell cycle arrest in G0/G1 phase. Subsequently, aimed to explore whether APG115 affects the ibrutinib sensitivity in CLL cells, the cell viability of combination group under both APG115 and ibrutinib was obviously suppressed than groups with single drug intervention. Immunofluorescence images revealed that p53 promoted MCL-1 into cytoplasm under the action of APG115. However, on the basis of ibrutinib intervention, APG115 facilitated the degradation of MCL-1 by p53. Furthermore, the invasion range of CLL cells in the combination group was smaller than that in the single agent group, and the invasion range of CLL cells in the single agent group was lower than that in the control group in vivo(Figure A). After injection of medication, both the monotherapy group and the combination group were lower than the control group, but there was no significant difference between the monotherapy group and the combination group. The survival rate of combination group was higher than that of single agent group (Figure B). Immunohistochemistry of the dissected spleens of mice showed that the malignancy of the cells in the single agent group was lower than that in the control group, while that in the combination group was lower than that in the monotherapy group. Finally, ibrutinib-resistant cell line was established to study the effect of APG115 on chemoresistance. After adding APG115, it was found that the survival rate of drug-resistant strain was lower than that of sensitive strain. It was proved that p53 induced the ubiquitination process when combined with MCL-1 to reduce the inhibitory effect of apoptosis via CO-IP. After APG115 was added, the apoptosis rate of drug-resistant strains was higher than that of sensitive strains. When APG115 was added on the basis of ibrutinib, the difference in apoptosis rate of drug-resistant strains was greater than that of sensitive strains. Conclusions: In summary, this study first revealed anticancer effects of the novel MDM2-p53 inhibitor APG115 in CLL. Promisingly, APG115 is expected to be a potential agent to improve the effects of BTK inhibitor treatment in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-proliferating and apoptosis-inducing activity of chemical compound FTI-6D in association with p53 in human cancer cell lines

Compounds with 3,4-fused tricyclic indole (FTI) frameworks are attractive scaffolds for drug discovery. We synthesized FTI-6D, a compound with this framework, which was cytotoxic in several human cancer cell lines. FTI-6D induced apoptosis via activation of the p53 downstream mitochondria-related apoptotic pathway, characterized by an increased ratio of pro-apoptotic Bcl-2 family members to anti-apoptotic members. This change was followed by caspase-9 and caspase-3 cleavage and activation in two cancer cell lines, RKO and AGS. The anti-proliferating effect of FTI-6D was remarkably detected in eight cancer cells with wild-type TP53 (TP53_wt), including RKO and AGS, but not in seven cancer cells with mutated TP53 (TP53_mut). Additionally, p53 protein levels increased after FTI-6D treatment in TP53_wt cancer cells, and the cytotoxic effect of FTI-6D was decreased by TP53 knockdown. Accordingly, the expression of p53 downstream genes involved in apoptotic signaling pathways, such as BBC3 and TP53INP1, and those involved in cell growth inhibition, such as CDKN1A, was upregulated in TP53_wt cancer cells. These results suggest that the anti-proliferative and apoptosis-inducing activities of FTI-6D rely on p53 and the corresponding signaling processes. This study demonstrated that FTI-6D shows anti-cancer activity against TP53_wt cancer cells. FTI-6D may have potential as a prototype compound for a new drug to utilize a functional p53 pathway in TP53_wt cancer cells.

Eftozanermin alfa (ABBV-621) monotherapy in patients with previously treated solid tumors: findings of a phase 1, first-in-human study

Eftozanermin alfa (eftoza), a second-generation tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL-R) agonist, induces apoptosis in tumor cells by activation of death receptors 4/5. This phase 1 dose-escalation/dose-optimization study evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary activity of eftoza in patients with advanced solid tumors. Patients received eftoza 2.5–15 mg/kg intravenously on day 1 or day 1/day 8 every 21 days in the dose-escalation phase, and 1.25–7.5 mg/kg once-weekly (QW) in the dose-optimization phase. Dose-limiting toxicities (DLTs) were evaluated during the first treatment cycle to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Pharmacodynamic effects were evaluated in circulation and tumor tissue. A total of 105 patients were enrolled in the study (dose-escalation cohort, n = 57; dose-optimization cohort, n = 48 patients [n = 24, colorectal cancer (CRC); n = 24, pancreatic cancer (PaCA)]). In the dose-escalation cohort, seven patients experienced DLTs. MTD and RP2D were not determined. Most common treatment-related adverse events were increased alanine aminotransferase and aspartate aminotransferase levels, nausea, and fatigue. The one treatment-related death occurred due to respiratory failure. In the dose-optimization cohort, three patients (CRC, n = 2; PaCA, n = 1) had a partial response. Target engagement with regard to receptor saturation, and downstream apoptotic pathway activation in circulation and tumor were observed. Eftoza had acceptable safety, evidence of pharmacodynamic effects, and preliminary anticancer activity. The 7.5-mg/kg QW regimen was selected for future studies on the basis of safety findings, pharmacodynamic effects, and biomarker modulations. (Trial registration number: NCT03082209 (registered: March 17, 2017)).

Silibinin Therapy Improves Cholangiocarcinoma Outcomes by Regulating ERK/Mitochondrial Pathway.

Background: Silibinin is widely utilized drug in various cancer treatments, though its application in cholangiocarcinoma has not yet been explored. For the first time, we evaluated the anticancer potential and underlying molecular mechanism of silibinin in treatment of cholangiocarcinoma treatment. Methods: HuCCT-1 and CCLP-1 cells were chosen to be an in vitro study model and were exposed to various concentrations of silibinin for indicated times. Cell viability was evaluated by the cell counting kit-8 (CCK-8) assay and half maximal inhibitory (IC50) concentrations were calculated. Cell proliferation capacity was determined through the use of colony formation and 5-Ethynyl-2′- deoxyuridine (EdU) assays. Cell apoptosis and cycle arrest were assessed by Live/Dead staining assay and flow cytometry (FCM). The protein levels of extracellular regulated protein kinases (ERK)/mitochondrial apoptotic pathway were evaluated through western blotting (WB). Mitochondrial membrane potential changes were determined via 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1). A cholangiocarcinoma cell line xenograft model was used to assess the anti-tumor activity of silibinin in vivo. Results: Inhibition of the ERK protein by silibinin led to a significant decrease in mitochondrial membrane potential, which, in turn, caused Cytochrome C to be released from the mitochondria. The activation of downstream apoptotic pathways led to apoptosis of cholangiocarcinoma cells. In general, silibinin inhibited the growth of cholangiocarcinoma cell line xenograft tumors. Conclusions: Silibinin is able to inhibit cholangiocarcinoma through the ERK/mitochondrial apoptotic pathway, which makes silibinin a potential anti-tumor drug candidate for cholangiocarcinoma treatment.

Study on the protective effect of Lycopene on ischemia-reperfusion myocardium through Inhibiting the opening of mitochondrial MPTP and the activation of apoptotic pathway

Through in vitro experiments, the paper investigated whether Lycopene can mitigate cardiac ischemia/reperfusion injury, and further explored whether the underlying mechanism works by inhibiting the opening of MPTP.A hypoxia-reoxygenation model was established. CCK8 was employed to determine the viability of H9c2 cardiomyocytes. LDH was used to determine the degree of damage of H9c2 cardiomyocytes. Trypan blue staining was applied to determine the survival rate of H9c2 cardiomyocytes. Flow cytometry was used to determine the apoptosis rate of H9c2 cardiomyocytes. Rhodamine 123 staining method was applied to evaluate the changes of mitochondrial membrane potential of H9c2 cardiomyocytes. Western blot was employed to determine the protein expressions of related factors of MPTP downstream apoptosis pathway in H9c2 cardiomyocytes. Calcium induction method was used to evaluate the opening degree of MPTP of H9c2 cardiomyocytes. Western blot was applied to determine the protein expressions of the related factors regulating MPTP opening in H9c2 cardiomyocytes.The vitality of H9c2 cardiomyocytes in the Lycopene group was significantly improved, the LDH activity was significantly reduced, and the cell survival rate was significantly improved. The apoptosis rate was significantly reduced, and the membrane potential decreased significantly. The expression levels of Bax, cytochrome C, APAF-1, caspase 9 and caspase-3 proteins were significantly reduced. The sensitivity of MPTP opening was significantly decreased. Lycopene can effectively alleviate the hypoxia/reoxygenation injury of H9c2 cardiomyocytes. By up-regulating Bcl-2 and down-regulating Bax, Lycopene can inhibit the opening of mitochondrial MPTP and the activation of downstream apoptotic pathways, thereby reducing apoptosis.

Ethyl pyruvate attenuates isoproterenol-induced myocardial infarction in rats: Insight to TNF-α-mediated apoptotic and necroptotic signaling interplay

The current study investigated the prophylactic effect of ethyl pyruvate (EP) in Isoproterenol (ISO) - induced myocardial infarction (MI). Ethyl pyruvate (EP) was given at a dose of 100 mg/kg i.p for 7 days, while isoproterenol (ISO) was administered at a dose of 10 mg/kg s.c. on the 6th and 7th days to induce MI. All parameters were assessed 24 and 48 h following treatment. Interestingly, EP pre-treatment significantly improved ISO-induced hemodynamic alterations and remarkably ameliorated serum levels of cardiac injury markers, Cardiac Troponin I (cTnI) and Cardiac Creatine Kinase (CK-MB). Also, EP notably suppressed levels of oxidative stress markers, total antioxidants (TAO) and malondialdehyde (MDA) as compared to ISO-treated group. Cardioprotective effects of EP were confirmed by histopathological examination. Moreover, EP remarkably attenuated ISO-induced elevation in Tumor Necrosis Factor Alpha (TNF-α) and Nuclear factor kappa-B p65 (NF-κB) expression, along with Interleukin-6 (IL-6), Monocyte chemoattractant protein 1 (MCP-1) and Inducible nitric oxide synthase (i-NOS) levels. Also, EP significantly diminished expression of apoptotic markers; caspase 8, cleaved caspase 3 and apoptotic regulator; cellular FLICE-like inhibitory protein (cFLIP). Finally, EP notably mitigated necroptotic mediators, phosphorylated receptor-interacting serine/threonine protein kinase 1 and 3 (p-RIPK1 and p-RIPK3), phosphorylated mixed lineage kinase domain-like protein (p-MLKL) and heat shock protein 70 (HSP 70) expression as compared to the ISO-treated group. Our study was the first to investigate the effect of EP on the necroptotic signaling. Taken together, EP conferred its cardioprotective effect against ISO-induced MI partially through mitigation of TNF-α and its downstream inflammatory, apoptotic and necroptotic signaling pathways.

Anti-Tumor Effects of Ginsenoside 20(S)-Protopanaxadiol and 1,25-Dihydroxyvitamin D3 Combination in Castration Resistant Prostate Cancer.

In spite of possessing desirable anticancer properties, currently, limited clinical success has been achieved with 20(S)-protopanaxadiol (aPPD) and 1,25-dihydroxyvitamin D3 (calcitriol). This study is designed to evaluate if the combination of aPPD with calcitriol can inhibit human prostate cancer xenograft growth by using nuclear receptor signaling. Athymic male nude mice were utilized to establish an androgen-independent human prostate cancer C4-2 cell castration-resistant prostate cancer (CRPC) xenograft model. Mice were treated orally for six weeks with 70 mg/kg aPPD administered once daily or three times per week with 4 µg/kg calcitriol or in combination or only with vehicle control. Contrary to our expectations, calcitriol treatment alone increased C4-2 tumor growth. However, the addition of calcitriol substantially increased aPPD-mediated tumor growth suppression (76% vs. 53%, combination vs. aPPD alone). The combination treatment significantly increased levels of cleaved caspase-3 apoptotic marker compared to vehicle-treated or aPPD-treated C4-2 tumors. The mechanistic elucidations indicate that tumor inhibition by the aPPD and calcitriol combination was accompanied by elevated vitamin D receptor (VDR) protein expression. In silico data suggest that aPPD weakly binds to the native LBD pocket of VDR. Interestingly, the combination of aPPD and calcitriol activated VDR at a significantly higher level than calcitriol alone and this indicates that aPPD may be an allosteric activator of VDR. Overall, aPPD and calcitriol combination significantly inhibited tumor growth in vivo with no acute or chronic toxic effects in the C4-2 xenograft CRPC nude mice. The involvement of VDR and downstream apoptotic pathways are potential mechanistic routes of antitumor effects of this combination.

4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, a Major Active Metabolite of Bisphenol A, Triggers Pancreatic β-Cell Death via a JNK/AMPKα Activation-Regulated Endoplasmic Reticulum Stress-Mediated Apoptotic Pathway.

4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), a major active metabolite of bisphenol A (BPA), is generated in the mammalian liver. Some studies have suggested that MBP exerts greater toxicity than BPA. However, the mechanism underlying MBP-induced pancreatic β-cell cytotoxicity remains largely unclear. This study demonstrated the cytotoxicity of MBP in pancreatic β-cells and elucidated the cellular mechanism involved in MBP-induced β-cell death. Our results showed that MBP exposure significantly reduced cell viability, caused insulin secretion dysfunction, and induced apoptotic events including increased caspase-3 activity and the expression of active forms of caspase-3/-7/-9 and PARP protein. In addition, MBP triggered endoplasmic reticulum (ER) stress, as indicated by the upregulation of GRP 78, CHOP, and cleaved caspase-12 proteins. Pretreatment with 4-phenylbutyric acid (4-PBA; a pharmacological inhibitor of ER stress) markedly reversed MBP-induced ER stress and apoptosis-related signals. Furthermore, exposure to MBP significantly induced the protein phosphorylation of JNK and AMP-activated protein kinase (AMPK)α. Pretreatment of β-cells with pharmacological inhibitors for JNK (SP600125) and AMPK (compound C), respectively, effectively abrogated the MBP-induced apoptosis-related signals. Both JNK and AMPK inhibitors also suppressed the MBP-induced activation of JNK and AMPKα and of each other. In conclusion, these findings suggest that MBP exposure exerts cytotoxicity on β-cells via the interdependent activation of JNK and AMPKα, which regulates the downstream apoptotic signaling pathway.

Dexmedetomidine Attenuates Cellular Injury and Apoptosis in H9c2 Cardiomyocytes by Regulating p-38MAPK and Endoplasmic Reticulum Stress.

BackgroundMyocardial ischaemia-reperfusion injury (IRI) has been confirmed to induce endoplasmic reticulum stress (ERS) when myocardial cell function continues to deteriorate to a certain degree. The clinical applications of effective tested strategies are sometimes inconsistent with the applications evaluated in experiments, although reasonable mechanisms and diverse signalling pathways have been broadly explored. Dexmedetomidine (DEX) has been shown to attenuate IRI of the heart in animal studies. This study aimed to determine whether DEX can protect injured cardiomyocytes under hypoxia/reoxygenation (H/R) at the cellular level and whether the mechanism is related to ERS and the p38 MAPK pathway.MethodsH9c2 cells were subjected to H/R or thapsigargin (TG) to build a model. DEX or 4-PBA was added to the medium either 1 h or 24 h before modelling, respectively. Model parameters were determined by assessing cell viability and injury, which were measured by assessing cell counting kit-8 (CCK8), lactate dehydrogenase (LDH) release and flow cytometry results, and the expression of GRP78, CHOP and caspase-12. In addition, the protein expression of p38MAPK and p-p38MAPK was examined, and SB202190, a negative regulator, was also preincubated in medium.ResultsCompared to that of cells in the control group, the activity of cells in the H/R and TG groups was decreased dramatically, and the LDH concentration and proportion of apoptotic cells were increased. DEX could correspondingly reverse the changes induced by H/R or TG. Additionally, DEX effectively attenuated ERS defined as increased expression of GRP78, CHOP and caspase-12. Additionally, DEX could obviously depress the P38 MAPK phosphorylation and high p-p38 MAPK expression in the TG group, indicating DEX has a function similar to that of SB202190.ConclusionH/R injury in H9c2 cells can lead to abnormal ERS and apoptosis, as well as activation of the p38MAPK signalling pathway. DEX can protect cardiomyocytes by intervening in ERS, regulating p38MAPK and the downstream apoptotic signalling pathway.

Adipose-Derived Stem Cells Protect Ischemia-Reperfusion and Partial Hepatectomy by Attenuating Endoplasmic Reticulum Stress.

Ischemia-reperfusion (IR) is an inevitable complication of liver surgery. Recent studies indicate a critical role of endoplasmic reticulum stress (ERS) in hepatic IR. Mesenchymal stem cells (MSCs) have proven to be an effective tool for tissue regeneration and treatment of various diseases, including that of the liver. However, the mechanisms underlying the therapeutic effects of stem cells on hepatic IR injury (IRI) are still poorly understood, especially in the context of ERS. In this study, we established a porcine model of hepatic IRI and partial hepatectomy, and transplanted the animals with adipose-derived mesenchymal stem cells (ADSCs) isolated from miniature pigs. ADSCs not only alleviated the pathological changes in the liver parenchyma following IRI, but also protected the resident hepatocytes from damage. Mechanistically, the ADSCs significantly downregulated ERS-related proteins, including GRP78, p-eIF2α, ATF6 and XBP1s, as well as the proteins involved in ERS-induced apoptosis like p-JNK, ATF4 and CHOP. Taken together, ADSCs can alleviate hepatic IRI by inhibiting ERS and its downstream apoptotic pathways in the hepatocytes, indicating its therapeutic potential in liver diseases.

Systems biology approaches based discovery of a small molecule inhibitor targeting both c-Met/PARP-1 and inducing cell death in breast cancer.

Breast cancer is the second most common types of cancer worldwide. Molecular strategies have developed rapidly; however, novel treatments strategies with high efficacy and lower toxicity are still urgently demanded. Notably, biological networks estimated from microarray data and functional activity network analysis could be utilized to identify and validate potential targets. In this study, two microarray data (GSE13477, GSE31192) were firstly selected, and analyzed by multi-functional activity network analysis to generate the core protein-protein-interaction (PPI) network. Several potential targets were subsequently identified and c-Met and poly (ADP-ribose) polymerase-1 (PARP-1) were manually chosen as the key targets in breast cancer. Furthermore, virtual screening and molecular dynamics (MD) simulations were utilized to recognize novel c-Met/PARP-1 inhibitors in Specs products database. Three small molecules, namely, ZINC19909930, ZINC20032678 and ZINC13562414 were selected. Additionally, these compounds were synthesized, and two breast cancer cell lines, MDA-MB-231 and MCF-7 cells were used to validate our bioinformatic findings in vitro. MTT assay and Hoechst staining showed that ZINC20032678 significantly induced breast cancer cell death, which was mediated through apoptosis by flow cytometry. Furthermore, ZINC20032678 was shown to target the active sites of the both targets and recruitment of downstream apoptotic signaling pathways, eventually inducing breast cancer cell apoptosis. Collectively, our findings not only offer systems biology approaches based drug target identification, but also provide the new clues for developing novel inhibitors for future breast cancer research.

Inflammation-dependent downregulation of miR-532-3p mediates apoptotic signaling in human sarcopenia through targeting BAK1.

Inflammation and apoptosis are considered as two major pathological causes of human sarcopenia. The current understanding based on different models recognizes that apoptosis does not trigger inflammation, while emerging evidence indicates that inflammation can induce apoptosis. Here, we provide solid evidence to suggest that the inflammation-dependent downregulation of miR-532 causes apoptosis through targeting a proapoptotic gene BAK1 (BCL2 antagonist/killer 1). To identify miRNAs and genes that are aberrantly expressed in the muscle tissues of sarcopenia patients, we conducted two independent microarray analyses. In total, we identified 53 miRNAs and 69 genes with differential expression levels. Of these aberrantly expressed miRNAs, miR-532-3p showed the most obvious changes in sarcopenia tissues, and more importantly, it can be repressed by the well-known inflammatory inducer lipopolysaccharide (LPS) in vitro. According to gene-based microarray results and the predicted targets of miR-532-3p, we presumed that BAK1 was a putative target of miR-532-3p. Further in vitro and in vivo analyses verified that miR-532-3p could directly bind to the three prime untranslated region (3'-UTR) of BAK1 through the seed sequence CUCCCAC. In addition, we found that NFKB1 (also known as p50), a subunit of the transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), could specifically bind to the promoter region of miR-532-3p and repress its expression. Further analysis revealed that the activation of TLR4 (Toll-like receptor 4) signaling led to the translocation of p50 from the cytoplasm to the nucleus, where it repressed miR-532-3p expression and thus led to an increase of BAK1. The accumulated BAK1 activated its downstream apoptotic signaling pathways and resulted in apoptosis, eventually causing the pathogenesis underlying sarcopenia. Overall, our results uncovered a new mechanism by which the inflammation-dependent downregulation of miR-532-3p contributed to the pathogenesis of sarcopenia through mediating BAK1 expression.

Ursolic Acid Protected Lung of Rats From Damage Induced by Cigarette Smoke Extract.

Background: We found previously that ursolic acid (UA) administration could alleviate cigarette smoke-induced emphysema in rats partly through the unfolded protein response (UPR) PERK-CHOP and Nrf2 pathways, thus alleviating endoplasmic reticulum stress (ERS)-associated oxidative stress and cell apoptosis. We hypothesized that other UPR pathways may play similar roles in cigarette smoke extract (CSE)-induced emphysema. So, we sought to investigate the dynamic changes and effects of UPR and the downstream apoptotic pathways. Further, we investigated whether UA could alleviate CSE-induced emphysema and airway remodelling in rats, whether and when it exerts its effects through UPR pathways as well as Smads pathways. Methods: One hundred eight Sprague Dawley (SD) rats were randomly divided into three groups: Sham group, CSE group, and UA group, and each group was further divided into three subgroups, administered CSE (vehicle) for 2, 3, or 4 weeks; each subgroup had 12 rats. We examined pathological changes, analyzed the three UPR signaling pathways and subsequent ERS, intrinsic and extrinsic apoptotic pathway indicators, as well as activation of Smad2,3 molecules in rat lungs. Results: Exposure to CSE for 3 or 4 weeks could apparently induce emphysema and airway remodeling in rats, including gross and microscopic changes, alteration of mean alveolar number (MAN), mean linear intercept (MLI), and mean airway thickness in lung tissue sections. UA intervention could significantly alleviate CSE-induced emphysema and airway remodeling in rats. UA exerted its effects through ameliorating apoptosis by down regulating UPR signalling pathways and subsequent apoptosis pathways, as well as, downregulating p-Smad2 and p-Smad3 molecules. Conclusions: UA attenuated CSE-induced emphysema and airway remodeling, exerting its effects partly through regulation of three UPR pathways, amelioration downstream apoptotic pathways, and alleviating activation of Smad2 and Smad3.

C‑Jun N‑terminal kinase inhibition attenuates early brain injury induced neuronal apoptosis via decreasing p53 phosphorylation and mitochondrial apoptotic pathway activation in subarachnoid hemorrhage rats.

Early brain injury (EBI)-induced neuronal apoptosis is primarily responsible for the subsequent complications of aneurysmal subarachnoid hemorrhage (aSAH), which may increase the risk of mortality in patients with aSAH. c-Jun N-terminal kinase (JNK) has been demonstrated to be a promoter of EBI-induced cell apoptosis, although the mechanism has yet to be fully elucidated. The present study aimed to explore whether the role of JNK1 is associated with tumor protein p53 (p53), which is one of the most important factor that triggers cell apoptosis. JNK1 expression was downregulated via in vivo small interfering RNA transfection in an aSAH rat model in order to assess differences in the behavior, survival times, morphology and genetics of the experimental animals. The results revealed that JNK1 inhibition improved the neurological scores and survival times of SAH rats by interrupting cascaded neuronal apoptosis. The interruption of EBI-induced neuronal apoptosis may originate from a decrease in the level of p53 phosphorylation and deactivation of the downstream mitochondrial apoptotic pathway. Taken together, these results suggest that JNK1 may be a promising target for improving the prognosis of patients with aSAH.

Mutations in the P10 region of procaspase-8 lead to chemotherapy resistance in acute myeloid leukemia by impairing procaspase-8 dimerization

Caspase-8 activation initiates apoptotic signaling cascades, and certain mutations in procasepase-8 have been reported to be associated with the progression and prognosis of different types of tumors. In this study, we have identified four novel mutations, which are highly correlated with chemotherapy resistance and poor prognosis of acute myeloid leukemia (AML) patients, within the P10 subunit of procaspase-8. These newly discovered mutations cause premature termination of translation, resulting in truncated procaspase-8 protein, which is incapable of forming dimer to initiate apoptosis signaling pathway. Further biochemical analysis reveals that the segment of P10 subunit of procaspase-8 consisting of three amino acid residues from L491 to F493 is crucial for the formation of procaspase-8 interdimer, and the aberration of this segment disrupts the dimerization and consequently precludes the activation of caspase-8 and downstream apoptotic signaling pathway. Therefore, the patients with AML who bear these types of P10 mutations were more likely to develop chemotherapy resistance due to impaired apoptotic signaling in cellular system, leading to significantly reduced overall survival (OS) as compared with patients carrying no such types of P10 mutations. Taken together, these newly identified P10 mutations in procaspase-8 could be used as novel biomarkers for predicting response and survival of chemotherapy-treated AML patients, as well as potential therapeutic targets for medical intervention in the future.