Expression Of Procaspase-3 Research Articles

Abstract LB075: Small molecule procaspase activating compound 1 (PAC-1) enhances CAR-T immunotherapy for solid tumors in syngeneic murine models

Abstract Chimeric antigen receptor (CAR)-T cell therapy has been shown to effectively treat solid tumors in syngeneic murine models, but antigen loss and T cell trafficking into the tumors pose major obstacles to its long-term efficacy. Here we explored a combination therapy against advanced stages of colorectal and ovarian syngeneic murine tumor models using a tumor-specific small molecule (PAC-1) that triggers the engagement of the host immune system and synergizes with a Tn-specific adoptively transferred 237 CAR-T immunotherapy. PAC-1 has been shown to induce apoptosis in tumor cells with high expression of procaspase 3, via chelation of inhibitory zinc and release of active executioner caspase 3. PAC-1 also recently completed a phase I trial (NCT02355535) and was shown to be well-tolerated in late-stage cancer patients. RNA sequencing of CT26 colorectal cells stimulated with PAC-1 for 24 hours revealed that PAC-1 induced global changes at the transcriptome level, up-regulating genes involved in immune activation and apoptosis, and down-regulating genes involved in DNA repair, cell cycle regulation, and myc-driven proliferation. Validation of RNAseq data was performed using a combination of RT-qPCR, Western blot, flow cytometry, and co-culture studies. A combination of transcriptome and exome sequencing analyses in murine colorectal cell line CT26 revealed that the PAC-1-mediated down-regulation of DNA repair machinery led to somatic alterations in the genome and a higher mutational load in tumor cells, subsequently presented as neo-antigens by antigen-presenting cells. As a result, host CD8+ cytotoxic T cells were able to infiltrate tumors and reduce the tumor burden. Tn-dependent CARs have been used recently to demonstrate the proof of concept in the targeting of cancer-specific Tn-antigens such as the Tn-MUC1 antigen in humans, and an analogous Tn-OTS8 target in mice. In our murine ID8 ovarian tumor model, untreated mice with disseminated tumors have a median survival of 71 days. As a single agent administered intraperitoneally, PAC-1 extended the median survival to 90 days (p<0.0001), while 237 CAR-T treated mice have a median survival of 103 days (p<0.0001). A combination of the two treatment modalities (PAC-1 administered before 237 CAR-T) further extended the median survival to 145 days (p=0.048). Administering PAC-1 after 237 CAR-T also modestly extended the median survival to 132 days (p=0.06). Taken altogether, our results demonstrate that PAC-1 engages the immune system to treat cancer by inducing expression of pro-inflammatory cytokines in tumor cells allowing the recruitment of CD8+ effector T cells to the tumor microenvironment. Combination strategies that boost T cell trafficking shows great promise to enhance the anti-tumor efficacy and feasibility of adoptive CAR-T therapy against solid tumors. Citation Format: Diana R. Ranoa, Yifei Kang, Jenny Drnevich, Gloria Rendon, Christopher J. Fields, Keith Bailey, Edward J. Roy, Timothy M. Fan, David M. Kranz, Paul J. Hergenrother. Small molecule procaspase activating compound 1 (PAC-1) enhances CAR-T immunotherapy for solid tumors in syngeneic murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB075.

Effect of the extra-nuclear cation on the cytotoxicity and mechanism of action of pyridine-2,6-dicarboxylate Ga(III) complexes

Two Ga(III) complexes (C1) and (C2) were prepared by the one-pot reaction of pyridine-2,6-dicarboxylic acid and aminopyridine derivatives with gallium(III) nitrate octahydrate. The compounds were characterized by single-crystal X-ray diffraction. The distorted octahedral geometry was confirmed by crystallographic data for both complexes. The study of the in vitro cytotoxicity of the compounds showed that the presence of different extra-nuclear cations can affect the cytotoxicity of the same anionic complexes. The most significant antiproliferative activity was observed for C1 (IC50 = 0.69 μM, MAE = 73.96%) and C2 (IC50 = 3.78 μM, MAE = 60.35%) (where MAE represents the maximal antiproliferative effect) against A431 cell line. The mechanistic study evidenced the same pathway for the death of A431 cells treated with the complexes, although the results for C2 were obtained at approximately five times the concentration of C1. According to the study, both complexes induced cell cycle arrest in G2/M phase in A431 cells by upregulating the levels of p21, p27, p-cdc25C, and p-cdc2 and downregulating the levels of cdc25C, cdc2, and cyclin B1. In addition, apoptosis via a caspase-dependent mitochondrial pathway was confirmed by a decrease in Bcl-2 family proteins and an increase in the expression of procaspase-9 and 3. Also, the complexes induced autophagic cell death by activating the RAGE /PI3KC3/Beclin 1 pathway in A431 cells. Data availabilityCCDC 874052 and 874055 contain the supplementary crystallographic data for C1 and C2, respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/services/structures?pid=ccdc:874052,874055&sid=CCDCManual, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223–336–033; or e-mail: deposit@ccdc.cam.ac.uk.

Apoptotic and anti-Warburg effect of Morusin via ROS mediated inhibition of FOXM1/c-Myc signaling in prostate cancer cells.

Though Morusin is known to induce apoptotic, antiprolifertaive, and autophagic effects through several signaling pathways, the underlying molecular mechanisms of Morusin still remain unclear until now. To elucidate antitumor mechanism of Morusin, cytotoxicity assay, cell cycle analysis, Western blotting, TUNEL assay, RNA interference, immunofluorescense, immunoprecipitation, reactive oxygen species (ROS) measurement, and inhibitor study were applied in this study. Morusin enhanced cytotoxicity, increased the number of TUNEL positive cells, sub-G1 population and induced the cleavages of PARP and caspase3, attenuated the expression of HK2, PKM2, LDH, c-Myc, and Forkhead Box M1 (FOXM1) along with the reduction of glucose, lactate, and ATP in DU145 and PC3 cells. Furthermore, Morusin disrupted the binding of c-Myc and FOXM1 in PC-3 cells, which was supported by String and cBioportal database. Notably, Morusin induced c-Myc degradation mediated by FBW7 and suppressed c-Myc stability in PC3 cells exposed to MG132 and cycloheximide. Also, Morusin generated ROS, while NAC disrupted the capacity of Morusin to reduce the expression of FOXM1, c-Myc, pro-PARP, and pro-caspase3 in PC-3 cells. Taken together, these findings provide scientific evidence that ROS mediated inhibition of FOXM1/c-Myc signaling axis plays a critical role in Morusin induced apoptotic and anti-Warburg effect in prostate cancer cells. Our findings support scientific evidence that ROS mediated inhibition of FOXM1/c-Myc signaling axis is critically involved in apoptotic and anti-Warburg effect of Morusin in prostate cancer cells.

Anti-Warburg effect via generation of ROS and inhibition of PKM2/β-catenin mediates apoptosis of lambertianic acid in prostate cancer cells.

To elucidate the underlying antitumor mechanism of lambertianic acid (LA) derived from Pinus koraiensis, the role of cancer metabolism related molecules was investigated in the apoptotic effect of LA in DU145 and PC3 prostate cancer cells. MTT assay for cytotoxicity, RNA interference, cell cycle analysis for sub G1 population, nuclear and cytoplasmic extraction, lactate, Glucose and ATP assay by ELISA, Measurement of reactive oxygen species (ROS) generation, Western blotting, and immunoprecipitation assay were conducted in DU145 and PC3 prostate cancer cells. Herein LA exerted cytotoxicity, increased sub G1 population and attenuated the expression of pro-Caspase3 and pro-poly (ADP-ribose) polymerase (pro-PARP) in DU145 and PC3 cells. Also, LA reduced the expression of lactate dehydrogenase A (LDHA), glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2 (PKM2) with reduced production of lactate in DU145 and PC3 cells. Notably, LA decreased phosphorylation of PKM2 on Tyr105 and inhibited the expression of p-STAT3, cyclin D1, C-Myc, β-catenin, and p-GSK3β with the decrease of nuclear translocation of p-PKM2. Furthermore, LA disturbed the binding of p-PKM2 and β-catenin in DU145 cells, which was supported by Spearman coefficient (0.0463) of cBioportal database. Furthermore, LA generated ROS in DU145 and PC3 cells, while ROS scavenger NAC (N-acetyl L-cysteine) blocked the ability of LA to reduce p-PKM2, PKM2, β-catenin, LDHA, and pro-caspase3 in DU145 cells. Taken together, these findings provide evidence that LA induces apoptosis via ROS generation and inhibition of PKM2/β-catenin signaling in prostate cancer cells.

The caspase-8 and procaspase-3 expression in gastric cancer and non-cancer mucosa in relation to clinico-morphological factors and some apoptosis-associated proteins

PurposeThe aim of the study was to assess the expression of caspase-8 and procaspase-3 proteins in gastric cancer (GC) cells and non-cancerous mucosa in relation to clinical and morphological characteristics of the tumor, postoperative survival as well as other apoptosis-related proteins. Materials and methodsThe study included 91 ​GC patients. Expression of the proteins was assessed using immunohistochemical method. ResultsPositive expression of procaspase-3 was found in all GC cells. A significant difference was found between high expression of this protein in cancer cells (70.3%) and non-cancerous mucosa (1.25%) (p ​≤ ​0.05). Caspase-8 expression was observed in 50.7% of GC cells and 46.7% of mucosa. Caspase-8 was more common in Lauren type II compared to Lauren type I cancer (p ​= ​0.009), while a statistically significant difference was reported between positive procaspase-3 expression and differentiation of GC (p ​= ​0.043) and Lauren's classification (p ​= ​0.028). We observed a significant positive correlation between the expression of caspase-8 and bcl-xl (p ​= ​0.030) as well as between the procaspase-3 and BID (p ​= ​0.026). Positive caspase-8 expression was associated with longer survival of GC patients (p ​≤ ​0.01). ConclusionsOur findings indicate the potential role of the analyzed proteins in GC pathogenesis. Positive expression of caspase-8 is associated with longer survival and better patient prognosis.

Erythropoietin ameliorates cognitive deficits by improving hippocampal and synaptic damage in streptozotocin-induced diabetic mice

Recent studies have shown that erythropoietin (EPO) is an effective neuroprotective and neurotrophic agent for neurological disorders, such as traumatic brain injury and Alzheimer's disease. However, the effectiveness of EPO administration against diabetic cognitive impairments has rarely been examined. In this study, we investigated the effects of EPO on streptozotocin (STZ)-induced male C57BL/6 J mice. Then, we sought to clarify the mechanisms of EPO-mediated neuroprotection in high-glucose (HG)-stimulated HT22 cells. In vivo, we found that STZ-induced diabetic mice showed impaired spatial learning and memory, which was alleviated by EPO treatment. EPO also significantly lowered elevated fasting blood glucose levels, improved pancreatic and hippocampal damage, and restored oxidative stress in the STZ-induced diabetic mice. In vitro, EPO markedly increased cell viability, restrained the expression of pro-apoptotic Bax, enhanced the expression of pro-caspase 3, anti-apoptotic Bcl-2, brain-derived neurotrophic factor (BDNF) and postsynaptic density 95 (PSD-95), and attenuated the upregulation of N-methyl-d-aspartic acid (NMDA) receptor subunits NR1, NR2A and NR2B in HG-induced HT22 cells. The protective effects of EPO was obviously abolished by treatment with an NMDA receptor agonist. Our findings revealed that EPO impedes hippocampal and synaptic damage and neuronal apoptosis by regulating BDNF and PSD-95 expression through NMDA receptors, thereby ameliorating cognitive impairments in mice with T1DM.

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Effect of Negative Regulation of LncRNA XIST to MiR-196b on the Biological Behavior of Acute Myeloid Leukemia Cells KG1a

To investigate the effect and molecular mechanism of lncRNA X-inactive specific transcript (XIST) on the proliferation and apoptosis of acute myeloid leukemia cells KG1a. Forty-one patients with acute myeloid leukemia from January 2017 to May 2019 treated in Beijing Aerospace Center Hospital were collected, as well as 20 patients who conformed to the international standard of iron deficiency anemia as control group. KG1a cells were divided into pcDNA group, pcDNA-XIST group, pcDNA-XIST+miR-NC group, and pcDNA-XIST+miR-196b group. Real-time fluorescence quantitative PCR was used to detect the expressions of XIST and miR-196b, CCK-8 was used to detect cell activity, flow cytometry was used to detect cell cycle and apoptosis, Western blot method was used to detect the protein expressions of cleaved-caspase3, pro-caspase3, Bax, and Bcl-2, and dual luciferase report experiment was used to detect the targeting relationship between XIST and miR-196b. The expression level of lncRNA XIST in bone marrow cells in the AML group was significantly lower than that in the iron deficiency anemia group (P<0.001). Compared with pcDNA group, the expression level of lncRNA XIST, proportion of cells in G0/G1 phase, apoptosis rate, and the expression levels of cleaved-caspase3 and Bax in the pcDNA-XIST group of KG1a cells were significantly increased (all P<0.001), while the expression level of miR-196b, cell viability, the proportion of S-phase cells, and the expression levels of pro-caspase3 and Bcl-2 were significantly decreased (all P<0.001). Compared with pcDNA-XIST group, the cell activity, proportion of S-phase cells, and the expression levels of pro-caspase3 and Bcl-2 in the pcDNA-XIST+miR-196b group were significantly increased (all P<0.001), while the proportion of cells in the G0/G1 phase, apoptosis rate, and the expression levels of cleaved-caspase3 and Bax decreased (all P<0.001). Overexpression of lncRNA XIST can inhibit the proliferation of acute myeloid leukemia cells KG1a and promote apoptosis by down-regulating the expression of miR-196b.

Protective activity of tert-butylhydroquinone against oxidative stress and apoptosis induced by glutamate agonizts in R28 cells and mice retina.

Glutamate excitotoxicity can cause cell damage and apoptosis and play an important role in a variety of retinal diseases. Tertiary-butylhydroquinone (tBHQ) is an approved food-grade phenolic antioxidant with antioxidant activity in a variety of cells and tissues. We observed the protective effect of tBHQ on glutamatergic agonist-induced retina and explored its possible mechanism of action through in vitro cell experiments. The results showed that tBHQ had protective effects on NMDA-induced mouse retinal excitotoxicity and glutamate-induced excitotoxicity in rat retinal precursor cells (R28 cells). tBHQ reversed glutamate-induced apoptosis, production of intracellular reactive oxygen species, and reduction of mitochondrial membrane potential. Western blot analysis showed that tBHQ could increase the expression of procaspase-3, Bcl-2, AIF precursor, CAT, SOD2, Nrf2, NQO1, HO-1 and NF-κB in glutamate-treated cells, and decrease the expression of AIF cleavage products. Furthermore, we discovered that tBHQ activated müller glial cells. Based on these results, tBHQ may have antioxidant and anti-apoptotic properties, thus serving as a potential retinal protective agent. Its anti-oxidative stress effect was attributed to up-regulation of Nrf2, and its anti-apoptotic effect was related to its up-regulation of Bcl-2 expression and inhibition of mitochondria-dependent apoptosis.

Synergistic effect of obeticholic acid and fasting-mimicking on proliferative, migration, and survival signaling in prostate cancer

The systemic and resistant nature of the androgen-independent stage of prostate cancers makes it largely incurable even after intensive multimodal therapy. Apoptosis and epithelial-mesenchymal transition (EMT) are two fundamental events that are deeply linked to carcinogenesis. Hence, it is necessary to find a new combination of several therapies targeting apoptosis and EMT without causing side effects. Several recent studies have indicated that the Farnesoid X receptor is extensively associated with human tumorigenesis. The FXR agonist obeticholic acid (INT 747) has preliminarily exhibited a tumor suppressor potential. In this present study, we assess the potential synergism of FXR activation under nutrient deprivation in prostate cancer cell lines to investigate whether FXR activation enhances starvation-induced apoptosis in PC3 cells. In this study, PC-3 treatment with INT 747 significantly repressed cell proliferation and clonogenic potential. In addition, it significantly induced apoptosis of PC-3 cells and decreased their cancerogenic potential, as evaluated by annexin v apoptosis and transwell migration assay, respectively. The decreased expression of pro-caspase 3 by western blot analysis further confirmed INT 747-induced apoptosis. Furthermore, the fasting-mimicking diet (FMD) potentiated the antiproliferative, pro-apoptotic, and antimetastatic effects of INT 747. Mechanistically, these effects were mediated through the downregulation of cyclin D1 and upregulation of PTEN. In conclusion, INT 747 alone markedly decreases, and when combined with FMD abrogates the growth and migration of PC-3 cells.

The Antitumor Effect of Cinnamaldehyde Derivative CB-PIC in Hepatocellular Carcinoma Cells via Inhibition of Pyruvate and STAT3 Signaling.

Though cinnamaldehyde derivative (CB-PIC), a major compound of cinnamon, is known to have anticancer activity, its underlying mechanism is not fully understood. In the present study, the anticancer mechanism of CB-PIC was investigated in human hepatocellular carcinoma cells (HCCs) in association with signal transducer and activator of transcription 3 (STAT3) signaling. CB-PIC exerted cytotoxicity in HepG2 and Huh7 cells. CB-PIC increased the sub G1 population and attenuated the expression of pro-poly (ADP-ribose) polymerase (PARP) and pro-Caspase3 in HepG2 and Huh7 cells. Interestingly, CB-PIC significantly abrogated the expression of a glycolytic enzyme pyruvate kinase M2 (PKM2) in HepG2 cells more than in LNCaP, A549, and HCT-116 cells. Consistently, CB-PIC reduced the expression of hexokinase 2 (HK2) and PKM2, along with a reduced production of lactate in HepG2 and Huh7 cells. Notably, CB-PIC suppressed the phosphorylation of STAT3 in HepG2 and Huh7 cells and conversely STAT3 depletion enhanced the capacity of CB-PIC to suppress the expression of HK2, PKM2, and pro-caspase3 and to reduce the viability in Huh7 cells. Furthermore, CB-PIC activated the phosphorylation of AMPK and ERK and suppressed expression of IL-6 as STAT3-related genes in HepG2 and Huh7 cells. Conversely, pyruvate treatment reversed the inhibitory effect of CB-PIC on p-STAT3, HK2, PKM2, and pro-PARP in Huh7 cells. Overall, there findings suggest that CB-PIC exerts an apoptotic effect via inhibition of the Warburg effect mediated by p-STAT3 and pyruvate signaling.

Induction of Human Oral Squamous Carcinoma Apoptosis by Derris scandens Benth and Elephantopus scaber Linn Extracts

D scandens ( Derris scandens Benth.) and E scaber ( Elephantopus scaber Linn.) contain flavonoids and phenolic acids, which have antitumor activity in various cancer cell lines. Oral cancer is among the most common cancers in Southeast Asia, and the survival rate remains low. Thus, this study screened 2 ethanolic plant extracts for cytotoxicity on the oral human squamous carcinoma cell line (HSC-2), and compared the mechanisms of action. Extracts of D scandens and E scaber showed cytotoxicity against HSC-2 cells in a dose-dependent manner. Observation of nuclear morphology by Hoechst 33342 staining revealed chromatin condensation. Apoptosis was confirmed by Annexin V-FITC staining and cell sorting (fluorescence-activated cell sorting) analysis. We demonstrated that cancer apoptosis was accompanied by changes in the expression of procaspase 3 and that D scandens-mediated apoptosis in HSC-2 cells was potentiated by protein kinase B (Akt) and B-cell lymphoma-2 (Bcl-2), while E scaber apoptosis was mediated by mitogen-activated protein kinase (MAPK) pathways, involving stress-activated protein kinases/jun amino-terminal kinase (SAPK/JNK) and p38-MAPK. Further investigation into targets for apoptosis induction by these plant extracts may have potential in oral cancer therapy.

Suppression of the Proliferation of Huh7 Hepatoma Cells Involving the Downregulation of Mutant p53 Protein and Inactivation of the STAT 3 Pathway with Ailanthoidol.

Ailanthoidol (ATD) has been isolated from the barks of Zanthoxylum ailanthoides and displays anti-inflammatory, antioxidant, antiadipogenic, and antitumor promotion activities. Recently, we found that ATD suppressed TGF-β1-induced migration and invasion of HepG2 cells. In this report, we found that ATD exhibited more potent cytotoxicity in Huh7 hepatoma cells (mutant p53: Y220C) than in HepG2 cells (wild-type p53). A trypan blue dye exclusion assay and colony assay showed ATD inhibited the growth of Huh7 cells. ATD also induced G1 arrest and reduced the expression of cyclin D1 and CDK2. Flow cytometry analysis with Annexin-V/PI staining demonstrated that ATD induced significant apoptosis in Huh7 cells. Moreover, ATD increased the expression of cleaved PARP and Bax and decreased the expression of procaspase 3/8 and Bcl-xL/Bcl-2. In addition, ATD decreased the expression of mutant p53 protein (mutp53), which is associated with cell proliferation with the exploration of p53 siRNA transfection. Furthermore, ATD suppressed the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and the expression of mevalonate kinase (MVK). Consistent with ATD, the administration of S3I201 (STAT 3 inhibitor) reduced the expression of Bcl-2/Bcl-xL, cyclin D1, mutp53, and MVK. These results demonstrated ATD’s selectivity against mutp53 hepatoma cells involving the downregulation of mutp53 and inactivation of STAT3.

English

Cancer is one of the major public health problems of the 21st century. Its incidence is increasing in both developed and low-income countries. This trend is justified by the change in people&#39;s lifestyles. Cancer of the prostate is one of major cancer types affecting male population. The present study focused on the use of methanolic extract of Striga hermonthica (Delile) Benth. to evaluate total phenolics and flavonoids contents, antioxidant activities, anti-proliferative and anti-migratory effects, using ATCC prostate cancer cell lines (22RV1, DU145, C4-2 and RWPE-1). Total phenolics and flavonoids content were estimated using spectrophotometric methods. Antioxidant potential was evaluated using three methods while cytotoxicity assay, anti-migratory assay and western blot analysis were used for the determination of extracts anti-proliferative activity on prostate cancer cells. Therefore, to assess apoptosis induction in prostate cancer cell lines, the effect of S. hermonthica (Delile) Benth. methanolic extract was investigated on procaspase-3, procaspase-8, and cyclin-B2 expression. A range of half-dilution concentrations beginning from 500 &micro;g/mL have been prepared to determine the IC50 in the various tests. Antioxidant compounds were shown to be promising biomolecules, with particular interest in cancer management. Among plant derivative compounds, phenolics and derived molecules have endorsed antioxidant capacities and potential anti-proliferative activities. The best inhibition was obtained on C4-2 cells with an IC50 = 65.76 &micro;g/mL and a selectivity index of 2.35. The results obtained from this study could partially justify the traditional indications of this plant in the treatment of prostate cancer. Key words: Plant derived products, anti-tumoral, prostate cancer, antioxidants.

COMMENTARY: Direct Indexing: A Point Solution for Global Crises

Background: Canine melanoma is the most common type of tumor in dogs. We investigated the effects of canine interferon-beta (cIFN-β)-overexpressing adipose tissue-derived mesenchymal stem cells (cATMSCs) on apoptosis and proliferation of canine melanoma cells. Materials and Methods: Expression of IFN-β in cATMSCs was confirmed using reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assays. Flow cytometry was performed for cell-cycle analysis and apoptotic cell quantification of LMeC (melanoma) cells. Protein expression of cyclin D1, procaspase-3, activated caspase-3, and Bcl-2 homologous antagonist killer (Bak) was evaluated by western blot analysis. Results: Decreased proportions of cells in S- and G₀/G₁ phases were observed in parallel with decreased cyclin D1 expression in LMeC cells treated with cIFN-β-cATMSC-conditioned media. Protein expression of active forms of caspase 3 and Bak increased in response to treatment with cIFN-β-cATMSC-conditioned media. Conclusion: IFN-β overexpression by cATMSCs was associated with pro-apoptotic and growth-inhibitory effects on canine melanoma cells. The antitumor effects of these cells have therapeutic potential for the treatment of canine melanoma.

BMP-6 Attenuates Oxygen and Glucose Deprivation-Induced Apoptosis in Human Neural Stem Cells through Inhibiting p38 MAPK Signaling Pathway

Background and ObjectivesNeural stem cells (NSCs) remain in the mammalian brain throughout life and provide a novel therapeutic strategy for central nervous system (CNS) injury. Bone morphogenetic protein-6 (BMP-6) had shown a protective effect in different types of cells. However, the role of BMP-6 in NSCs is largely unclear. The present study was aimed to investigate whether BMP-6 could protect human NSCs (hNSCs) against the oxygen and glucose deprivation (OGD)-induced cell death.Methods and ResultsUpon challenge with OGD treatment, cell viability was significantly decreased in a time-dependent manner, as indicated by the CCK-8 assay. BMP-6 could attenuate the OGD-induced cell injury in a dose-dependent manner and decrease the number of TUNEL-positive cells. Moreover, BMP-6 markedly weakened the OGD-induced alterations in the expression of procaspase-8/9/3 and reversed the expression of cleaved-caspase-3. Interestingly, noggin protein (the BMP-6 inhibitor) attenuated the neuroprotective effect of BMP-6 in cultured hNSCs. Furthermore, the p38 MAPK signaling pathway was activated by OGD treatment and BMP-6 markedly inhibited the phosphorylation of p38 in a concentration-dependent manner. Pretreatment with noggin abolished the effect of BMP-6 on p38 activation. SB239063, a selective p38 inhibitor, exerted similar effects with BMP-6 in protecting hNSCs against the OGD-induced apoptosis. These results indicated that blocking the phosphorylation of p38 might contribute to the neuroprotective effect of BMP-6 against the OGD-induced injury in hNSCs.ConclusionsThese findings suggested that BMP-6 might be a therapeutic target in the OGD-induced cell death, which provides a novel therapeutic strategy for enhancing host and graft NSCs survival in hypoxic-ischemic brain injury.

Suppression of phosphoinositide 3‐kinase/phosphoinositide‐dependent kinase‐1/serum and glucocorticoid‐induced protein kinase pathway

In the current study, the pivotal roles of serum and glucocorticoid-induced protein kinase (SGK1) and NF-kB related signalings known as prognostic biomarkers in cervical cancers were explored in the antitumor effect of a ginseng saponin metabolite compound K (CK) in HeLa and SiHa cervical cancer cells. CK exerted significant cytotoxicity, induced sub-G1 accumulation, and attenuated the expression of proPoly (ADP-ribose) polymerase (pro-PARP) and Pro-cysteine aspartyl-specific protease (pro-caspase3) in HeLa cells more than in SiHa cells. CK inhibited phosphorylation of SGK1 and its upstream genes, phosphoinositide 3-kinases (PI3K), and phosphoinositide-dependent kinase-1 (PDK1) in HeLa cells. In addition, CK suppressed the phosphorylation of SGK1, NF-κB, and inhibitor of kappa B (IκB) and also NF-κB target genes such as X-linked inhibitor of apoptosis protein and B-cell lymphoma 2 (Bcl-2) in HeLa cells. Notably, Immunoprecipitation revealed that SGK1 binds to PI3K or PDK1 and also CK disturbed the binding between SGK1 and PI3K or PDK1 in HeLa cells. Furthermore, PI3K inhibitor LY294002 decreased expression of PI3K, p-PDK1, p-SGK1, and pro-caspase3 and SGK1 inhibitor GSK650394 also reduced expression of NF-κB and pro-caspase3 just like CK in HeLa cells. Overall, these findings suggest that CK induces apoptosis via suppression of PI3K/PDK1/SGK1 and NF-κB signaling axis.

MiR-124-3p Down-Regulation Influences Pancreatic-β-Cell Function by Targeting Secreted Frizzled-Related Protein 5 (SFRP5) in Diabetes Mellitus

Diabetes mellitus (DM) is a complex metabolic disease characterized by hyperglycemia, insulin resistance and pancreatic β-cell dysfunction. There are evidences showed that microRNAs (miRNAs) play important roles in DM. The purpose of our study was to determine the role of miR-124-3p in DM. Quantitative reverse transcription PCR (qRT-PCR) was applied to measure the level of miR- 124-3p in peripheral blood from healthy control patients and DM patients. Then we explored the effects of miR-124-3p inhibitor on the secretion of insulin of pancreatic β-cells. Moreover, we determined the effects of miR-124-3p inhibitor on the apoptosis and viability of pancreatic β-cells through flow cytometry and MTT assay. And we also used western blotting to detect the protein expression of cleaved-caspase3/pro-caspase3, and the activity of caspase3 was detected. In addition, we confirmed the direct target of miR-124-3p using Dual luciferase reporter assay. Our data showed that in the blood of DM patients, SFRP5 was significantly reduced, while miR-124-3p was increased significantly. Furthermore, we found that down-regulation of miR-124-3p increased total insulin content in INS-1 cells, enhanced insulin secretion in INS-1 cells. Furthermore, we revealed that miR-124-3p inhibitor enhanced INS-1 cell viability, decreased apoptosis of INS-1 cells, increased pro-caspase3 expression, decreased cleaved-caspase3 expression and caspase3 activity. In addition, we proved SFRP5 was a direct target of miR-124-3p in pancreatic β-cells. Moreover, SFRP5-siRNA reversed all the effects of miR-124-3p knockdown on pancreatic β-cells.

Neural-Induced Human Adipose Tissue-Derived Stem Cells Conditioned Medium Ameliorates Rotenone-Induced Toxicity in SH-SY5Y Cells.

Parkinson’s disease (PD) is an age-related neurodegenerative disease (NDD) characterized by the degenerative loss of dopaminergic neurons in the substantia nigra along with aggregation of α-synuclein (α-syn). Neurogenic differentiation of human adipose-derived stem cells (NI-hADSCs) by supplementary factors for 14 days activates different biological signaling pathways. In this study, we evaluated the therapeutic role of NI-hADSC-conditioned medium (NI-hADSC-CM) in rotenone (ROT)-induced toxicity in SH-SY5Y cells. Increasing concentrations of ROT led to decreased cell survival at 24 and 48 h in a dose- and time-dependent manner. Treatment of NI-hADSC-CM (50% dilution in DMEM) against ROT (0.5 μM) significantly increased the cell survival. ROT toxicity decreased the expression of tyrosine hydroxylase (TH). Western blot analysis of the Triton X-100-soluble fraction revealed that ROT significantly decreased the oligomeric, dimeric, and monomeric phosphorylated Serine129 (p-S129) α-syn, as well as the total monomeric α-syn expression levels. ROT toxicity increased the oligomeric, but decreased the dimeric and monomeric p-S129 α-syn expression levels. Total α-syn expression (in all forms) was increased in the Triton X-100-insoluble fraction, compared to the control. NI-hADSC-CM treatment enhanced the TH expression, stabilized α-syn monomers, reduced the levels of toxic insoluble p-S129 α-syn, improved the expression of neuronal functional proteins, regulated the Bax/Bcl-2 ratio, and upregulated the expression of pro-caspases, along with PARP-1 inactivation. Moreover, hADSC-CM treatment decreased the cell numbers and have no effect against ROT toxicity on SH-SY5Y cells. The therapeutic effects of NI-hADSC-CM was higher than the beneficial effects of hADSC-CM on cellular signaling. From these results, we conclude that NI-hADSC-CM exerts neuroregenerative effects on ROT-induced PD-like impairments in SH-SY5Y cells.

Bile Acid Toxicity and Protein Kinases.

If the bile acids reach to pathological concentrations due to cholestasis, accumulation of hydrophobic bile acids within the hepatocyte may result in cell death. Thus, hydrophobic bile acids induce apoptosis in hepatocytes, while hydrophilic bile acids increase intracellular adenosine 3',5'-monophosphate (cAMP) levels and activate mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways to protect hepatocytes from apoptosis.Two apoptotic pathways have been described in bile acids-induced death. Both are controlled by multiple protein kinase signaling pathways. In mitochondria-controlled pathway, caspase-8 is activated with death domain-independent manner, whereas, Fas-dependent classical pathway involves ligand-independent oligomerization of Fas.Hydrophobic bile acids dose-dependently upregulate the inflammatory response by further stimulating production of inflammatory cytokines. Death receptor-mediated apoptosis is regulated at the cell surface by the receptor expression, at the death-inducing signaling complex (DISC) by expression of procaspase-8, the death receptors Fas-associated death domain (FADD), and cellular FADD-like interleukin 1-beta (IL-1β)-converting enzyme (FLICE) inhibitory protein (cFLIP). Bile acids prevent cFLIP recruitment to the DISC and thereby enhance initiator caspase activation and lead to cholestatic apoptosis. At mitochondria, the expression of B-cell leukemia/lymphoma-2 (Bcl-2) family proteins contribute to apoptosis by regulating mitochondrial cytochrome c release via Bcl-2, Bcl-2 homology 3 (BH3) interacting domain death agonist (Bid), or Bcl-2 associated protein x (Bax). Fas receptor CD95 activation by hydrophobic bile acids is initiated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) signaling. However, activation of necroptosis by ligands of death receptors requires the kinase activity of receptor interacting protein1 (RIP1), which mediates the activation of RIP3 and mixed lineage kinase domain-like protein (MLKL). In this chapter, mainly the effect of protein kinases signal transduction on the mechanisms of hydrophobic bile acids-induced inflammation, apoptosis, necroptosis and necrosis are discussed.