Protein Bad Research Articles

Dimeric mimetic of BDNF loop 4 promotes survival of serum-deprived cell through TrkB-dependent apoptosis suppression

Brain-derived neurotrophic factor (BDNF) is involved in the regulation of neuronal cell growth, differentiation, neuroprotection and synaptic plasticity. Although aberrant BDNF/TrkB signaling is implicated in several neurological, neurodegenerative and psychiatric disorders, neurotrophin-based therapy is challenging and is limited by improper pharmacokinetic properties of BDNF. Dimeric dipeptide compound GSB-106 (bis-(N-monosuccinyl-l-seryl-l-lysine) hexamethylenediamide) has earlier been designed to mimic the TrkB-interaction 4 loop of BDNF. It displayed protective effect in various cell-damaging models in vitro. Animal studies uncovered antidepressive and neuroprotective properties upon GSB-106 per os administration. Current study shows that GSB-106 acts similarly to BDNF, promoting survival of serum-deprived neuronal-like SH-SY5Y cells. 100 nmol concentration of GSB-106 provided maximum neurotrophic effect, which corresponds to about 37% of the maximum effect provided by BDNF. Protective properties of GSB-106 arise from its ability to counteract cell apoptosis via activation of TrkB-dependent pro-survival mechanisms, including inactivation of pro-apoptotic BAD protein and suppression of caspases 9 and 3/7. Thus, our study has characterized neurotrophic activity of small dimeric compound GSB-106, which mimics certain biological functions of BDNF and neurotrophin-specific protective mechanisms. GSB-106 also displays similarities to some known low weight peptide and non-peptide TrkB ligands.

High ROS Production by Celecoxib and Enhanced Sensitivity for Death Ligand-Induced Apoptosis in Cutaneous SCC Cell Lines.

Incidence of cutaneous squamous cell carcinoma (cSCC) and actinic keratosis has increased worldwide, and non-steroidal anti-inflammatory drugs as celecoxib are considered for treatment. We show here strong anti-proliferative effects of celecoxib in four cSCC cell lines, while apoptosis and cell viability largely remained unaffected. Impeded apoptosis was overcome in combinations with agonistic CD95 antibody or TNF-related apoptosis-inducing ligand (TRAIL), resulting in up to 60% apoptosis and almost complete loss of cell viability. Proapoptotic caspase cascades were activated, and apoptosis was suppressed by caspase inhibition. TRAIL receptor (DR5) and proapoptotic Bcl-2 proteins (Puma and Bad) were upregulated, while anti-apoptotic factors (survivin, XIAP, cFLIP, Mcl-1, and Bcl-w) were downregulated. Strongly elevated levels of reactive oxygen species (ROS) turned out as particularly characteristic for celecoxib, appearing already after 2 h. ROS production alone was not sufficient for apoptosis induction but may play a critical role in sensitizing cancer cells for apoptosis and therapy. Thus, the full therapeutic potential of celecoxib may be better used in combinations with death ligands. Furthermore, the immune response against cSCC/AK may be improved by celecoxib, and combinations with checkpoint inhibitors, recently approved for the treatment of cSCC, may be considered.

BAD-Mediated Neuronal Apoptosis and Neuroinflammation Contribute to Alzheimer's Disease Pathology

Alzheimer's disease is the most common progressive neurodegenerative disease. However, the underlying molecular mechanism is incompletely understood. Here we report the identification of the pro-apoptotic BCL-2 family protein BAD as a key regulator for neuronal apoptosis, neuroinflammation and β-amyloid (Aβ) clearance in Alzheimer's disease. The pro-apoptotic activity of BAD is significantly increased in neurons of patients with Alzheimer's disease and 5XFAD mice. Conversely, genetic disruption of Bad alleles restores spatial learning and memory deficits in 5XFAD mice. Mechanistically, phosphorylation and inactivation of BAD by neurotropic factor-activated Akt is abrogated in cortical neurons in patients with Alzheimer's disease and 5XFAD mice, thereby triggering mitochondrial translocation of hypo-phosphorylated BAD to promote neuronal apoptosis and neurodegeneration. Through reactive oxygen species (ROS)-oxidized mitochondrial DNA (mtDNA) axis, BAD also promotes activation of microglial NLRP3 inflammasome, thereby skewing microglia toward neuroinflammatory microglia to inhibit microglial phagocytosis of Aβ plaques in 5XFAD mice. AAV-mediated Bad deletion in hippocampal neurons prevents neuronal apoptosis and rescues memory deficits in 5XFAD mice. In another Alzheimer's disease murine model, APP/PS1 mice, Bad loss also inhibits activation of microglial NLRP3 inflammasome and Aβ deposition, thereby restoring memory deficits. Our results support a model in which BAD contributes to memory deficits and neuropathology in Alzheimer's disease by driving neuronal apoptosis, neuroinflammation but suppressing microglial phagocytosis of Aβ plaques, suggesting that BAD is a potential therapeutic target for Alzheimer's disease.

Effect of Melatonin on Expression of Apoptosis Regulator Proteins Bcl-2 and Bad in Ovarian Follicular Apparatus after High Temperature Exposure.

Expression of proapoptotic Bad and antiapoptotic Bcl-2 proteins in ovarian follicular apparatus of Wistar rats was evaluated on days 3, 7, and 14 after single experimental hyperthermia (EH) followed by therapeutic correction with subcutaneous melatonin (0.1 mg) dissolved in 0.2 ml physiological saline (PS) injected daily for 3 days. Duration of EH was less than 17 min; it was terminated when the rectal temperature attained 43.5°C. In acute posthyperthermia period (on experimental day 3), Bcl-2 and Bad expression area in folliculocytes of the experimental (EH+melatonin) and reference rats simultaneously increased in comparison with the corresponding values in control rats. At this, Bcl-2/Bad ratio of expression areas in experimental and reference rats did not differ from the control level. During the recovery period (on posthyperthermia day 7), Bad protein expression area significantly decreased in experimental rats resulting in elevation of Bcl-2/Bad ratio in comparison with control and reference groups on days 3 and 7. On day 14, Bcl-2 and Bad expression areas and Bcl-2/Bad ratio restored in experimental animals to the corresponding values assessed in control and reference groups. Thus, melatonin produced no effect on Bcl-2/Bad protein expression ratio during acute posthyperthermia period, but reduced the expression area of proapoptotic Bad protein and increased Bcl-2/Bad ratio on posthyperthermia day 7. Thus, melatonin can inhibit apoptosis via mitochondrial pathway in ovarian follicles on posthyperthermia day 7.

Cytotoxicity and Apoptosis Effects of Curcumin Analogue (2E,6E)-2,6-Bis(2,3-Dimethoxybenzylidine) Cyclohexanone (DMCH) on Human Colon Cancer Cells HT29 and SW620 In Vitro.

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. According to the Malaysian National Cancer Registry Report 2012–2016, colorectal cancer was the second most common cancer in Malaysia after breast cancer. Recent treatments for colon cancer cases have caused side effects and recurrence in patients. One of the alternative ways to fight cancer is by using natural products. Curcumin is a compound of the rhizomes of Curcuma longa that possesses a broad range of pharmacological activities. Curcumin has been studied for decades but due to its low bioavailability, its usage as a therapeutic agent has been compromised. This has led to the development of a chemically synthesized curcuminoid analogue, (2E,6E)-2,6-bis(2,3-dimethoxybenzylidine) cyclohexanone (DMCH), to overcome the drawbacks. This study aims to examine the potential of DMCH for cytotoxicity, apoptosis induction, and activation of apoptosis-related proteins on the colon cancer cell lines HT29 and SW620. The cytotoxic activity of DMCH was evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cell viability assay on both of the cell lines, HT29 and SW620. To determine the mode of cell death, an acridine orange/propidium iodide (AO/PI) assay was conducted, followed by Annexin V/FITC, cell cycle analysis, and JC-1 assay using a flow cytometer. A proteome profiler angiogenesis assay was conducted to determine the protein expression. The inhibitory concentration (IC50) of DMCH in SW620 and HT29 was 7.50 ± 1.19 and 9.80 ± 0.55 µg/mL, respectively. The treated cells displayed morphological features characteristic of apoptosis. The flow cytometry analysis confirmed that DMCH induced apoptosis as shown by an increase in the sub-G0/G1 population and an increase in the early apoptosis and late apoptosis populations compared with untreated cells. A higher number of apoptotic cells were observed on treated SW620 cells as compared to HT29 cells. Human apoptosis proteome profiler analysis revealed upregulation of Bax and Bad proteins and downregulation of Livin proteins in both the HT29 and SW620 cell lines. Collectively, DMCH induced cell death via apoptosis, and the effect was more pronounced on SW620 metastatic colon cancer cells, suggesting its potential effects as an antimetastatic agent targeting colon cancer cells.

Nerve Growth Factor Protects Retinal Ganglion Cells Related to Inhibiting Endoplasmic Reticulum Stress by Inhibiting IRE1-JNK-CHOP Signaling Pathway

ABSTRACT Background Under various physiological conditions, endoplasmic reticulum stress can induce apoptotic cell death, leading to brain and retinal neuronal cell death, but the relations of ER stress-induced apoptosis and the nerve growth factor’s therapeutic effect in Glaucoma optic neuropathy still unclear. Methods An endoplasmic reticulum stress model was established in ganglion cells using TG, the endoplasmic reticulum stress inducer. MTT assay and flow cytometry were used to detect the protective effect of NGF on retinal ganglion cells. Western blot was used to detect apoptosis-related proteins Bcl-2, Bad and endoplasmic reticulum stress-related proteins GRP78, IRE1, JNK and CHOP. Results MTT assay and flow cytometry showed NGF can protect the apoptosis of ganglion cells. Western blot analysis showed the level of pro-apoptotic protein Bad was decreased and anti-apoptotic protein Bcl-2 was increased after NGF treatment. Endoplasmic reticulum stress-induced proteins GRP78, IRE1, JNK and CHOP are counter- acted by NGF. Conclusion NGF protects retinal ganglion cells related to inhibiting endoplasmic reticulum stress by inhibiting IRE1-JNK-CHOP signaling pathway.

Sulphonamide‐Containing Oxazoline Hybrids as New Class of Neuroprotective Agents and Lead Molecule as Autophagy Inducer

AbstractSulphonamide derived oxazoline hybrid compounds showed neuroprotective effects by inducing autophagy. In the present study, a series of sulphonamide containing oxazoline hybrids were synthesized and their neuroprotective efficacy in neuronal cell lines was explored. All the compounds showed moderate to good neuroprotection against adrenal medulla cell lines (PC 12). Compound 6 j had good neuroprotective activity against PC 12 cell lines showing increased expression of autophagy regulatory proteins (ATG7, LC 3B, p62, Beclin‐1) and anti‐apoptotic proteins (Bcl‐2) and also decreased expression of pro‐apoptotic proteins (Bax and Bad). This study demonstrated clearly that compound 6 j showed neuroprotective effect through the induction of autophagy and opens up new avenues for a new class of compounds as neuroprotective therapeutic agents.

Preclinical Evaluation of Gilteritinib on NPM1-ALK-Driven Anaplastic Large Cell Lymphoma Cells.

Anaplastic large cell lymphoma (ALCL) is an aggressive type of non-Hodgkin lymphoma. More than three-fourths of anaplastic lymphoma kinase (ALK)-positive ALCL cases express the nucleophosmin 1 (NPM1)-ALK fusion gene as a result of t(2;5) chromosomal translocation. The homodimerization of NPM1-ALK fusion protein mediates constitutive activation of the chimeric tyrosine kinase activity and downstream signaling pathways responsible for lymphoma cell proliferation and survival. Gilteritinib is a tyrosine kinase inhibitor recently approved by the FDA for the treatment of FMS-like tyrosine kinase mutation-positive acute myeloid leukemia. In this study, we demonstrate for the first time gilteritinib-mediated growth inhibitory effects on NPM1-ALK-driven ALCL cells. We utilized a total of five ALCL model cell lines, including both human and murine. Gilteritinib treatment inhibits NPM1-ALK fusion kinase phosphorylation and downstream signaling, resulting in induced apoptosis. Gilteritinib-mediated apoptosis was associated with caspase 3/9, PARP cleavage, the increased expression of proapoptotic protein BAD, and decreased expression of antiapoptotic proteins, survivin and MCL-1. We also found downregulation of fusion kinase activity resulted in decreased c-Myc protein levels. Furthermore, cell-cycle analysis indicated gilteritinib induced G0-G1-phase cell-cycle arrest and reduced CD30 expression. In summary, our preclinical studies explored the novel therapeutic potential of gilteritinib in the treatment of ALCL cells expressing NPM1-ALK and potentially in other ALK or ALK fusion-driven hematologic or solid malignancies. IMPLICATIONS: Our preclinical results explore the use of gilteritinib for the treatment of NPM1-ALK-driven ALCL cells and pave a path for developing future clinical trials. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/913/F1.large.jpg.

To study the mechanism of Cuscuta chinensis Lam. And Lycium barbarum L. in the treatment of asthenospermia based on network pharmacology

Ethnopharmacological relevanceCuscuta chinensis Lam. and Lycium barbarum L. (SC-FL) is a commonly used kidney tonic Chinese medicine combination that is widely used in the clinical treatment of oligoasthenospermia.However, its specific mechanism remains unclear and requires in-depth study. Aim of the studyTo explore the potential targets of SC-FL in the treatment of oligoasthenospermia using network pharmacology, and to verify the results with in vivo and in vitro experiments. Materials and methodsA herb-compound-target-disease network and PPI network were constructed with Cytoscape software. The targets of SC-FL for the treatment of male sterility were introduced into a bioinformatics annotation database, and the GO and KEGG databases were used for pathway enrichment analysis. Subsequently, Tripterygium wilfordii Hook. f. (GTW) polyglycoside was used to induce a spermatogenic dysfunction model in GC-1 spg cells and SD male rats in in vitro and in vivo experiments, respectively. The SC-FL and PI3K pathway inhibitor LY294002 was used to intervene in the spermatogenic dysfunction model to detect the expression of proteins and mRNA related to the PI3K pathway and to detect the indicators related to proliferation and apoptosis. ResultsIn in vitro experiments, the percentage of spermatogenic cells and the proportion of GC-1 spg cells at G0/G1 and G2/M stages in the model group (GTW group) and the inhibitor group (LY group) were significantly decreased (P < 0.01) compared with the blank control group (NC group). The apoptosis rate of the GTW group was significantly increased (P < 0.01). The ultrastructures of GC-1 spg cells in the GTW group and LY group were obviously destroyed. Compared with the GTW group, the SC-FL group had a significantly reduced apoptosis rate of GC-1 spg cells, reduced percentage of cells in S phase, and a significantly improved mitochondrial membrane potential. SC-FL can repair the ultrastructure of GC-1 spg cells damaged by GTW. The above effects of SC-FL are closely related to up-regulation of GFRa1, RET, PI3K, p-AKT, and Bcl-2 and down-regulation of BAD and BAX proteins and mRNA expression. In vivo, compared with the GTW group, the body mass, testicular mass, and epididymal weight of the GTW + SC-FL group were significantly increased (P < 0.01). Sperm concentrations and the PR + NP of GTW + SC-FL were significantly higher than in the GTW group (P < 0.01 or P < 0.05). FSH, LH, and T levels in the GTW + SC-FL and LY + SC-FL groups were significantly higher than those in the GTW and LY group (P < 0.01 or P < 0.05). HE staining results showed that the morphology of testicular tissue in the GTW + SC-FL and LY + SC-FL groups was superior to that in the GTW and LY group. The above effects of SC-FL are closely related to the up-regulation of proteins and mRNA expression of PI3K, p-AKT, and Bcl-2. ConclusionThrough the PI3K/Akt signaling pathway, SC-FL up-regulates GFRa1, RET, PI3K, p-AKT, and Bcl-2, and down-regulates the expression of BAD and BAX proteins and mRNA, thus reducing the percentage of GC-1 spg cells in S-phase, significantly increasing the mitochondrial membrane potential, significantly reducing cell apoptosis, and improving sperm counts and viability.

Survivin Regulates Bad Gene Expression by Binding to Its Promoter and Modulates Cell Cycle and Apoptosis in Esophageal Carcinoma Cell.

Esophageal cancer (EC) is the eighth most prevalent cancer and the sixth leading cause of cancer-related mortality worldwide. As an antiapoptotic and a proapoptotic protein, respectively, survivin and Bad play an important role in carcinogenesis of the most human cancers including EC. However, the regulatory relationships between them remain unclear. We sought to investigate the effects of survivin knockdown and overexpression on the expression of Bad gene, cell cycle progression, and apoptosis of esophageal carcinoma cell. The mRNA expression levels of survivin and Bad were determined in EC tissue samples. The knockdown and overexpression experiments were performed in ECA109 and KYSE450 cells via transfection with survivin overexpression and shRNA plasmids. A Bad overexpression experiment was conducted to confirm the biological effect on knockdown of survivin via modulating Bad expression. RT-qPCR and Western blot analysis were used to detect mRNA and protein expression, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. The chromatin immunoprecipitation (ChIP) was conducted to determine the binding sites of survivin on the promoter of Bad gene. By analyzing the mRNA expression of survivin and Bad in 40 ESCC patient specimens, we found that the positive expression rate of survivin in tumor tissues (88%, 35/40) was remarkably high, compared with the distal nontumor tissues (48%, 19/40, p < 0.01). On the other hand, the positive expression rate of Bad in tumor tissues (70%, 28/40) was remarkably low, compared with the distal nontumor tissues (95%, 38/40, p < 0.01). Overexpression of survivin decreases Bad mRNA and protein expression and promotes transformation of cell cycle to S phase. Conversely, knockdown of survivin increases Bad mRNA and protein expression and induces cell cycle arrest and apoptosis. Bad overexpression inducing apoptosis of esophageal carcinoma cell shows the similar apoptotic effect with survivin knockdown. ChIP assays indicate that survivin directly binds to the Bad promoter region, diminishing the transcriptional activity of Bad. In conclusion, the result suggested that survivin regulates Bad gene expression by binding to its promoter and modulates cell cycle and apoptosis in esophageal carcinoma cell.

Contrasting role of autophagy in different types of cancer: A review toward biomarkers and therapeutic improvement

Autophagy, the major cellular pathway, is known not only for the degradation and recovering in mammalian cells but also it maintains the cellular energy homeostasis and produces the building blocks. However, if the process starts destroying the essential ones other than the bad proteins, then the normal cellular control will be lost, and here, we see similar kinds of phenomena in cancer. It has been reported by previous papers related to this field that the novel pathway has paradoxical role in cancer, as it acts as both tumor promoter and suppressor. Therefore, autophagy could provide new resources for the improvement of antitumor drugs in near future. Here, in this review, we will know about the autophagy process in brief. We will also try to understand the contrasting role of autophagy in different cancers types, its significance as prognostic markers and its therapeutic implication for cancer treatment in the near future.

Cardamonin inhibits the progression of oesophageal cancer by inhibiting the PI3K/AKT signalling pathway.

Background: Oesophageal cancer is the most common malignant tumour with a poor prognosis, and the current treatment methods are limited. Therefore, identifying effective treatment methods has become a research hotspot. Cardamonin (CAR) is a natural chalcone compound and has been reported to play an anticancer role in several cancers. However, its function in oesophageal cancer and the possible underlying mechanism are still unclear. The purpose of this study was to demonstrate the anticancer effect of CAR on oesophageal cancer in vivo and in vitro and to explore the underlying mechanism.Materials and Methods: MTT, crystal violet, and colony formation assays were used to detect oesophageal cancer cell proliferation. The effects of CAR on oesophageal cancer cell migration and invasion were detected by wound healing assay and Transwell assay. Hoechst 33258 staining and flow cytometry were used to detect cell apoptosis. Protein expression levels were detected by Western blot. A tumour xenograft model was established to further test the effect of CAR on the growth of oesophageal cancer in vivo.Results: The results showed that CAR inhibited the proliferation, migration, and invasion of oesophageal cancer cells in a concentration-dependent manner and induced apoptosis. Furthermore, the Western blot assay showed that CAR could suppress metastasis by inhibiting epithelial-mesenchymal transition (EMT) as indicated by downregulated expression of the mesenchymal markers N-cadherin and vimentin, the EMT transcription factor Snail, and matrix metalloproteinases (MMPs) and upregulated expression of the epithelial marker E-cadherin. CAR was associated with upregulation of the pro-apoptotic proteins Bax and Bad and downregulation of the anti-apoptotic protein Bcl-2 and triggered the mitochondrial apoptosis pathway, which in turn promoted caspase-3 activation and subsequent cleavage of PARP; however, the mitochondria-related apoptotic effects induced by CAR were blocked by caspase inhibitor Z-VAD-FMK pretreatment, which prevented programmed cell death triggered by CAR. In addition, CAR reduced the phosphorylation level of downstream effector molecules of phosphatidylinositol 3 kinase (PI3K) in a dose-dependent manner, and treatment with the PI3K agonist 740Y-P could partially reverse the anticancer effect of CAR, demonstrating that CAR played an antitumour role by inhibiting the PI3K/AKT signalling pathway in oesophageal cancer cells. Moreover, the EC9706 xenograft model further confirmed that CAR can significantly inhibit tumour growth in vivo.Conclusion: In summary, CAR exhibited a strong anticancer effect on human oesophageal cancer cells and promoted apoptosis by inhibiting the PI3K/AKT signalling pathway, suggesting that CAR can be used as new strategy for oesophageal cancer treatment.

BAD inactivation exacerbates rheumatoid arthritis pathology by promoting survival of sublining macrophages.

The resistance of synovial sublining macrophages to apoptosis has a crucial role in joint inflammation and destruction in rheumatoid arthritis (RA). However, the underlying mechanism is incompletely understood. Here we report that inactivation of the pro-apoptotic BCL-2 family protein BAD is essential for survival of synovial sublining macrophage in RA. Genetic disruption of Bad leads to more severe joint inflammation and cartilage and bone damage with reduced apoptosis of synovial sublining macrophages in collagen-induced arthritis (CIA) and TNFα transgenic (TNF-Tg) mouse models. Conversely, Bad3SA/3SA mice, in which BAD can no longer be inactivated by phosphorylation, are protected from collagen-induced arthritis. Mechanistically, phosphorylation-mediated inactivation of BAD specifically protects synovial sublining macrophages from apoptosis in highly inflammatory environment of arthritic joints in CIA and TNF-Tg mice, and in patients with RA, thereby contributing to RA pathology. Our findings put forward a model in which inactivation of BAD confers the apoptosis resistance on synovial sublining macrophages, thereby contributing to the development of arthritis, suggesting that BAD may be a potential therapeutic target for RA.

Preclinical Efficacy and Biological Effects of Venetoclax and Ixazomib in Combination in Lymphoma Cells

Introduction. Bcl-2 family proteins comprise anti-apoptotic and pro-apoptotic proteins. Interaction between these proteins, as well as severe regulation of their expression, mediates cell survival and can quickly induce cell death. Venetoclax is Bcl-2-targeting that has shown preclinical and clinical activity in hematologic malignancies. Due to the development of resistance and the loss of dependence on the target protein, the monotherapy may be insufficient for maximal effectiveness. To circumvent the resistance mechanisms, many preclinical studies have shown that combination of venetoclax with other agents may represent a more effective therapeutic strategy. Ubiquitin-proteasome signaling pathway is a potential target that plays an important role in the proteolysis of key regulatory proteins. Proteasome inhibitors include ixazomib that inhibits cell growth and induces apoptosis in hematological malignancies cells resistant to conventional therapies and bortezomib. Objective: To analyze the preclinical efficacy and associated biological effects of venetoclax combined with ixazomib in a panel of lymphoma cell lines with diverse expression levels of Bcl-2 and other Bcl-2 family proteins. Methods: 12 lymphoma cell lines including FL (RL, WSU-NHL, Karpas422), MCL (Jeko1, Granta519), DLBCL (OCI-LY3, OCI-LY18), CTCL (Hut-78), ALCL (Karpas299), HL (L1236, L540), CLL (Mec1) and two MCL primary patient samples were exposed to venetoclax (0.01 - 8 µM) and ixazomib (10 - 2000 nM) alone for 24 - 72 hours to calculate IC50. Subsequently, lymphoma cells were exposed to venetoclax (0.015 - 25 nM) in combination with ixazomib (0.015 - 0.5 nM) for 24 hours. Cell viability was determined by MTT. Coefficient of synergy (combination index - CI) was calculated using CalcuSyn. Cell cycle and induction of apoptosis were evaluated by flow cytometry and changes in Bcl-2 family members, caspase activation and AKT phosphorylation were determined by western blotting. Results. In vitro, venetoclax and ixazomib alone induced cell death in a dose- and time-dependent manner against lymphoma cell lines. The IC50 is between 0.5 and 8 µM for venetoclax and between 12 and 1250 nM for ixazomib. The combination of venetoclax (0.03, 0.06, 12.5, 25 nM) with ixazomib (0.03, 0.06, 0.25, 0.5 nM) produced a synergistic effect (CI &amp;lt; 1) after 24 h of treatment in the most lymphoma cells lines leading to inhibition of cell growth and induction of apoptosis between 26 % and 59 % accompanied by increased with cleavage of caspases-3, -9 and PARP. We observed an additive effect (CI = 1) in Jeko1 (MCL) and MEC1 cells (CLL) and antagonist effect (CI &amp;gt; 1) Hut-78 cells (CTCL). Synergistic effect has been seen in two MCL primary patient samples (CI = 0.5 - 0.7). In sensitive lymphoma cells, the combination abrogated colony formation in the methylcellulose medium. When lymphoma cell lines were co-cultured with mesenchymal stromal cells with both drugs we observed a decrease of cell viability and a fraction of apoptotic cells indicating that drug combination may overcome the tumor promoting effects of stromal cells. The apoptosis induced in FL and Granta519 cells (MCL) by drug combination was accompanied by partial downregulation of Bcl-2 and strong upregulation of Bax, Bad, Bim and Noxa proteins. Jeko-1 cells were less sensitive to venetoclax-ixazomib combination-induced apoptosis. Western blot analysis showed a differential expression of Bcl-2, Mcl-1 and Bcl-XL proteins in FL, MCL and HL cell lines. Jeko-1 cells showed a normal expression of Bcl-2 and Mcl-1 proteins and high Bcl-xL protein level. Co-expression of related anti-apoptotic Bcl-2 family proteins could limit activity of treatment. Combined treatment induced G0/G1 cell cycle arrest and increased the sub-G1 population that was linked by the upregulation of p27 and p21. In addition, in RL, WSU-NHL and Granta519, enhanced cell death is associated with AKT inactivation and with a reduction of p-4EBP1, leading to decreased levels of c-MYC. Conclusion. Venetoclax exhibits strong synergistic activity with ixazomib in lymphoma cells. Studies are still ongoing and signaling pathways that promote the combination of venetoclax with ixazomib are to be analyzed. These data offer a rationale to continue exploring venetoclax-ixazomib combination and suggest that suppression of Bcl-2 family protein driven survival signaling may be one important mechanism for combination synergy. Disclosures No relevant conflicts of interest to declare.

Deficits of urothelial cell proliferation, cytoskeleton, and barrier function protein expressions in patients with recurrent and persistent urinary tract infections.

Recurrent urinary tract infection (rUTI) is a common infectious disease in women. This study investigated the urothelial cell proliferation, the cytoskeleton, barrier proteins, and inflammatory protein expression in women with rUTIs. Female patients with recurrent or persistent UTIs were recruited. Bladder mucosal specimens were investigated by Western blot and immunohistochemical staining for the urothelial cytoskeleton proteins cytokeratin 5 (CK5), CK14, and CK20; proteins involved in cellular proliferation, including CD34, sonic hedgehog (SHH), and tumor protein 63 (TP63); barrier proteins zonula occludens 1 (ZO-1) and E-cadherin; inflammatory proteins p38 and tryptase; and proapoptotic proteins Bcl2-associated agonist of cell death protein (BAD), Bcl2-associated X protein (BAX), and caspase-3. Women with stress urinary incontinence without bladder symptoms served as controls. Bladder specimens from 18 recurrent UTI patients with rUTIs and 12 persistent UTIs, and 17 controls were analyzed, and protein expressions were compared between the three groups. Cell proliferation protein expression for CD34, SHH, and TP63 was significantly lower in the urothelium of patients with rUTIs than in controls. Expression of CK5 increased, whereas CK20 decreased significantly in rUTIs compared with those of controls. Apoptotic proteins BAD, BAX, and caspase-3 were significantly higher in patients with rUTIs. However, barrier proteins ZO-1 and E-cadherin, and tryptase were not significantly lower in patients with rUTIs. Deficits in expression of proteins involved in urothelial cell proliferation, cytoskeleton, and barrier function were noted in patients with rUTIs. These urothelial deficits may be due to deficient proliferation and differentiation resulting in inadequate urothelial barrier function and further in rUTIs.

IKBKE Impacts Radiation Resistance Of Glioblastoma Through AKT/GSK3β Pathway

Explore the effect of IKBKE on the radiation resistance of glioblastoma cell lines and its potential molecular mechanism. We used sh-IKBKE lentivirus and IKBKE overexpression lentivirus to construct U87-MG-shIKBKE cell, U251-shIKBKE cell and U118-oeIKBKE cell. In vivo experiments were performed to verify the difference in tumor growth rate between IKBKE-silent mice and IKBKE-expressing mice after radiotherapy, and the differences in γ-H2AX and Bad expression between the groups were confirmed by immunohistochemical staining. In vitro experiments, we analyzed the potential molecular mechanism. 1. After U87-MG-shIKBKE cell , U251-shIKBKE cell and 118 cell receiving radiotherapy, apoptosis inhibitory protein Bcl2 decreased significantly, apoptotic promoting protein Bad increased significantly, colony forming ability was greatly weakened, the number of γ-H2AX focal points and γ-H2AX protein expression level decreased significantly than U87-MG, U251, and U118-oeIKBKE cell respectively. 2. Comparing with U87-MG cell inoculation group mice, the tumor growth of the U87-MG-shIKBKE cell inoculation group mice was significantly reduced, and the γ-H2AX expression in IHC analysis of U87-MG-shIKBKE cell inoculation group mice was significantly increased. 3. We confirmed that high-expressing IKBKE could promote the phosphorylation of AKT serine 473, AKT threonine 308 and GSK-3β serine 9 after receiving radiotherapy by Immunoblot experiments. 4. The direct interaction between IKBKE and AKT was confirmed by Co-Immunoprecipitation experiments. 5. Rescue experiment demonstrated AZD5363 achieved lowest expression level of apoptosis-inhibiting protein Bcl2 and highest expression of γ-H2AX in U87-MG cells and U118 cells receiving radiotherapy. The IKBKE overexpression plasmid achieved highest expression level of Bcl2 and lowest expression of γ-H2AX in U87-MG cells and U118 cells receiving radiotherapy. After using AZD5363 and IKBKE overexpression plasmid, the expression of Bcl2 and γ-H2AX was moderate. 6. AZD5363 achieved highest apoptosis rate of U87-MG cells and U118 cells receiving radiotherapy. IKBKE overexpression plasmid achieved lowest apoptosis rate of U87-MG cells and U118 cells receiving radiotherapy. AZD5363 and IKBKE overexpression plasmid achieved moderate apoptosis rate of U87-MG cells and U118 cells receiving radiotherapy. 1. In vitro experiments, IKBKE significantly inhibited the apoptosis of U87-MG, U251 and U118 cells receiving radiotherapy.

Torin2 overcomes sorafenib resistance via suppressing mTORC2-AKT-BAD pathway in hepatocellular carcinoma cells

BackgroundSorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma (HCC). However, resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib. As the activation of AKT/mTOR played a pivotal role in sorafenib resistance, we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells. MethodsThe sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines. The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method. Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting. ResultsSorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells, which underscores the significance of AKT activation. Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction. Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis, leading to the dephosphorylation of Ser136 in BAD protein, and increased the expression of total BAD, which contributed to the apoptosis in sorafenib-resistant HCC cells. ConclusionsIn this study, Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenib-resistant HCC cells, via the suppression of mTORC2-AKT-BAD pathway. Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.

Cardioprotective effect of the TD anti-apoptotic peptide: Study of the mechanisms of action

Apoptosis is a main contributor of reperfusion injury after myocardial infarction. In a previous study, we evidenced the cardioprotective effect of a synthetic peptide (TD) able to interfere with FAS-DAXX interaction specifically activated during reperfusion-induced apoptosis. The aim of our study was to study the mechanisms of action of the TD anti-apoptotic peptide. Hearts from C57Bl6 mice were subjected ex vivo (Langendorff) to 40 minute-ischemia (coronary ligation) and 1 hour reperfusion (IR protocol). TD peptide (1 μM) was perfused during reperfusion. At the end of the protocol, a proteomic analysis by western-blot was realized in TD- versus non-treated IR hearts. Upon TD treatment, the downstream activation of the JNK/caspase 3 pathway was down-regulated. A decrease in both the expression of BAD protein and the level of activated caspase 9, both mediators of the intrinsic pathway, was also observed. TD treatment was able to decrease the FADD-caspase 8 extrinsic pathway probably due to its impact on DAXX-FADD cooperativity within the DISC. Of great interest, TD peptide treatment was not associated neither with a deleterious reduction of DAXX protein level already described to trigger unexpected pro-apoptotic effects (siRNA DAXX) nor with a switch to the necroptotic pathway (RIP1-RIP3-MLKL). Protein levels of HSP70, a hallmark of proteotoxic stress, were also reduced. Interestingly, the anti-apoptotic TD treatment was also related to an activation of the Reperfusion Injury Salvage Kinase (RISK) pathway including ERK1/2 and PI3kinase-AKT. TD peptide treatment appears as a particularly relevant therapeutic strategy to target reperfusion-induced apoptotic pathways. Targeting FAS:DAXX interaction from the extrinsic pathway also indirectly reduced the activation of the intrinsic (JNK-BAX crosstalk) cascade thus allowing a complete inhibition of reperfusion injury with a single pharmacological administration.

Multiple signaling pathways converge on proapoptotic protein BAD to promote survival of melanocytes.

Melanocyte survival is mediated by diverse signaling pathways. However, the molecular mechanisms they use and molecules that they target are incompletely understood. Here, we show that melanocyte survival is mediated by diverse, nonredundant signaling pathways, including ERK1/2, AKT, PKA, and PKC. Each of these pathways is exerting prosurvival effects by phosphorylating the BAD. While Ser112-BAD phosphorylation is regulated by pERK, pPKA and pPKC, Ser136 and Ser155 phosphorylation are exclusively controlled by pAKT and pPKA, respectively. Inhibition of these pathways individually resulted in only modest apoptosis; however, most significant apoptosis, as a result of BAD dephosphorylation, was seen when all pathways were inhibited concurrently. BAD phosphorylation was essential for survival of melanocytes as cells expressing phosphorylation-deficient BAD were not rescued by any of the identified pathway. Furthermore, melanocytes became insensitive to kinase inhibitor-induced apoptosis when BAD expression was knocked down by BAD-shRNA. Overexpression of BAD in melanocytes stimulated faster apoptosis in response to kinase inhibitors. Taken together, our results show that BAD is acting as a convergence point for diverse survival pathways in melanocytes. Understanding the molecular mechanisms of melanocyte survival provides fundamental new insights into physiological mechanisms involved in the development of various melanocyte pathologies such as melanoma and vitiligo.

The synthesis and anti‐tumour properties of novel 4-substituted phthalazinones as Aurora B kinase inhibitors

A series of novel 4-substituted phthalazinones as Aurora B kinase inhibitors was synthesized and evaluated the anti-proliferative activities against A549, HCT116, MCF-7 and HepG2 cells. 1-(4-(2-((4-Oxo-3,4-dihydrophthalazin-1-yl)amino)ethyl) phenyl)-3-(3-(trifluoromethyl)phenyl)urea (17b) exhibited the most potent anti-proliferative activity against HCT116 cells with IC50 value of 4.35 ± 1.21 μM, as well as the moderate Aurora B inhibitory activity with the IC50 value of 142 nM. Furthermore, 17b inhibited the phosphorylation of Aurora B on Thr232, leading to cell cycle arrest in the G2/M phase by down-regulating the expression of CyclinB1 and Cdc2 proteins, and apoptosis by up-regulating the expression of BAD and Bax proteins in HCT116 cells. In addition, a docking study revealed that 17b could form key hydrogen bonds with Ala173, Glu171 and Glu177 in Aurora B. All the results reveal that 17b is worthy of further development as an Aurora B kinase inhibitor.