Bcl-2 Phosphorylation Research Articles

RET Kinase-Regulated MicroRNA-153-3p Improves Therapeutic Efficacy in Medullary Thyroid Carcinoma.

Background: Medullary thyroid carcinoma (MTC) presents a disproportionate number of thyroid cancer deaths due to limited treatment options beyond surgery. Gain-of-function mutations of the human REarranged during Transfection (RET) proto-oncogene have been well-established as the key driver of MTC tumorigenesis. RET has been targeted by tyrosine kinase inhibitors (TKIs), such as cabozantinib and vandetanib. However, clinical results have been disappointing, with regular dose reductions and inevitable progression. This study aimed to identify RET-regulated microRNAs (miRNAs) and explore their potential as novel therapeutic targets. Methods: Small RNA sequencing was performed in MTC TT cells before and after RET inhibition to identify RET-regulated miRNAs of significance. In vitro gain-of-function studies were performed to investigate cellular and molecular effects of potential miRNAs on cell phenotypes. Systemic delivery of miRNA in MTC xenografts using EDV™ nanocells, targeted to epidermal growth factor receptor on tumor cells, was employed to assess the therapeutic potential and possible modulation of TKI responses. Results: The study demonstrates the tumor suppressive role of a specific RET-regulated miRNA, microRNA-153-3p (miR-153-3p), in MTC. Targeted intravenous delivery of miR-153-3p impeded the tumor growth in MTC xenografts. Furthermore, combined treatment with miR-153-3p plus cabozantinib caused greater growth inhibition and appeared to reverse cabozantinib resistance. Mechanistically, miR-153-3p targets ribosomal protein S6 kinase B1 (RPS6KB1) of mTOR signaling and reduced downstream phosphorylation of Bcl-2 associated death promoter. Conclusion: This study provides evidence to establish systemic miRNA replacement plus TKIs as a novel therapeutic for patients with metastatic, progressive MTC.

C-type lectin domain family 5, member A (CLEC5A, MDL-1) promotes brain glioblastoma tumorigenesis by regulating PI3K/Akt signalling.

ObjectivesGlioblastoma is the most common malignant glioma of all brain tumours. It is difficult to treat because of its poor response to chemotherapy and radiotherapy and high recurrence rate after treatment. The aetiology of glioblastoma is a result of disorders of multiple factors. Depending on cell signal transduction, these glioblastoma‐associated factors lead to cell proliferation, differentiation and apoptosis. Therefore, investigation of the potential factors which involved in the development of glioblastoma could provide a new target for the treatment of glioblastoma.Materials and methodsWe analysed the transcript expression of CLEC5A in glioblastoma by accessing The Cancer Genome Atlas (TCGA). qRT‐PCR was performed to detect the RNA expression of genes in cells and tissues, and Western blot was used to measure the protein levels (Cyclin D1, Bcl‐2, BAX, PCNA, MMP2, MMP9, Akt and Akt phosphorylation) in tissues and cells. Cell proliferation, migration, invasion, cycle and apoptosis were measured by CCK‐8, transwell and flow cytometry assays, respectively. Ki67 level and lung metastasis were determined by immunochemistry and H&E staining.ResultsIn this study, we found that CLEC5A was highly upregulated in glioblastoma compared to normal brain tissues, which had an opposite relation with the overall patient survival. Downregulation of CLEC5A could inhibit cell proliferation, migration and invasion via promoting apoptosis and G1 arrest. In contrast, overexpression of CLEC5A stimulated cell proliferation, migration and invasion. In addition, we found that CLEC5A level was positively correlated with Akt phosphorylation level. Akt inhibitor or agonist could reverse the modulation effects of CLEC5A in glioblastoma. Moreover, In vivo results suggested that inhibition of CLEC5A significantly reduced tumour size, weight, cell proliferation ability and lung metastasis via inhibition of phosphorylation Akt.ConclusionBoth in vitro and in vivo evidences supported that CLEC5A was involved in glioblastoma pathogenesis via regulation of PI3K/Akt pathway. Thus, CLEC5A might serve as a potential therapeutic target in the treatment of glioblastoma in the future.

Upregulated cyclin B1/CDK1 mediates apoptosis following 2-methoxyestradiol-induced mitotic catastrophe: Role of Bcl-XL phosphorylation

2-Methoxyestradiol is an endogenous nonpolar metabolite of 17β-estradiol with a strong antitubulin activity. Earlier we showed that 2-methoxyestradiol increases the level and activity of cyclin B1/CDK1, which subsequently induces mitotic prometaphase arrest. In the present study, we demonstrate that upregulation of cyclin B1/CDK1 is responsible for the increased phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-XL in 2-methoxyestradiol-induced, mitotically-arrested cancer cells. Additional analysis shows that only the increase in phosphorylation of Bcl-XL, but not Bcl-2, is associated with activation of the mitochondrial cell death pathway. We find that MAD2 is an important upstream mediator of the antitubulin function of 2-methoxyestradiol, resulting in activation of the MKK4-JNK1 pathway. JNK1 activation then leads to cyclin B1/CDK1 upregulation, which further increases Bcl-2 and Bcl-XL phosphorylation. Together, these results indicate that cyclin B1/CDK1 upregulation in cancer cells undergoing 2-methoxyestradiol-induced mitotic catastrophe causes apoptosis via Bcl-XL phosphorylation.

The JNK signaling pathway plays a key role in methuosis (non-apoptotic cell death) induced by MOMIPP in glioblastoma

BackgroundSynthetic indolyl- pyridinyl- propenones (IPPs) induce methuosis, a form of non-apoptotic cell death, in glioblastoma and other cancer cell lines. Methuosis is characterized by accumulation of cytoplasmic vacuoles derived from macropinosomes and late endosomes, followed by metabolic failure and rupture of the plasma membrane. However, not all IPPs that cause vacuolization are cytotoxic. The main goals of the present study were to identify key signaling pathways that contribute to methuosis induced by cytotoxic IPPs and to evaluate the anti-tumor potential of a prototype IPP in vivo.MethodsWe utilized metabolic flux analysis, glucose uptake, immunoblotting, and selective pharmacological inhibitors to compare the effects of closely related cytotoxic and non-cytotoxic IPPs in cultured glioblastoma cells. To determine whether the use of methuosis-inducing IPPs might be feasible in a therapeutic context, we quantified the distribution of our lead IPP compound, MOMIPP, in mouse plasma and brain, and tested its ability to inhibit tumor growth in an intracerebral glioblastoma xenograft model.ResultsThe cytotoxic IPP compound, MOMIPP, causes early disruptions of glucose uptake and glycolytic metabolism. Coincident with these metabolic changes, MOMIPP selectively activates the JNK1/2 stress kinase pathway, resulting in phosphorylation of c-Jun, Bcl-2 and Bcl-xL. At the same concentration, the non-cytotoxic analog, MOPIPP, does not activate these pathways. Pharmacologic inhibition of JNK activity promotes survival, even when cells are extensively vacuolated, but suppression of c-Jun transcriptional activity offers no protection. MOMIPP readily penetrates the blood-brain barrier and is moderately effective in suppressing progression of intracerebral glioblastoma xenografts.ConclusionsThe results suggest that interference with glucose uptake and induction of JNK-mediated phosphorylation of pro-survival members of the Bcl-2 family represent key events in the methuosis death process. In addition to providing new insights into the underlying molecular mechanism of methuosis, the results indicate that compounds of the cytotoxic IPP class may have potential for further development as therapeutic agents for brain tumors.

SET protein accumulation prevents cell death in head and neck squamous cell carcinoma through regulation of redox state and autophagy

Molecular alterations in cell death pathways and imbalances in regulators of up- or downstream signaling pathways can lead to resistance to cell death, which is one of the hallmarks of cancer. These signaling modifications are strategies that tumor cells use to resist chemotherapy and that contribute to the high recurrence rate of head and neck squamous cell carcinoma (HNSCC). The SET oncoprotein is a PP2A inhibitor that accumulates in HNSCC and represents a promising therapeutic target. Here we report the role that SET protein plays in resistance to death of two HNSCC cell lines: Cal 27 and HN13. SET protein regulated intracellular redox balance by controlling cellular localization of APE 1 – an endonuclease that is part of the SET complex and regulates antioxidant gene transcription. SET protein knockdown (siSET) associated with tert-butyl hydroperoxide-induced oxidative stress sensitized Cal 27 and HN13 cells to apoptosis via the extrinsic and intrinsic pathways, respectively. SET protein upregulated autophagy in HNSCC cells in a PP2A-dependent manner and apparently regulated ULK1 expression. The fact that siSET lowered Bcl-2 phosphorylation levels indicated that SET protein interfered with an alternative pathway that modulated autophagy in HNSCC cells. Overall, SET protein regulated intracellular redox state and sustained autophagy in HNSCC cells, which may explain resistance to death of HNSCC cells. Altogether, the findings reported herein support SET protein as therapeutic target for HNSCC.

Systems analysis of phosphorylation-regulated Bcl-2 interactions establishes a model to reconcile the controversy over the significance of Bcl-2 phosphorylation.

The biological significance of the multi-site phosphorylation of Bcl-2 at its loop region (T69, S70 and S87) has remained controversial for decades. This is a major obstacle for understanding apoptosis and anti-tumour drug development. We established a mathematical model into which a phosphorylation and de-phosphorylation process of Bcl-2 was integrated. Paclitaxel-treated breast cancer cells were used as experimental models. Changes in the kinetics of binding with its critical partners, induced by phosphorylation of Bcl-2 were experimentally obtained by surface plasmon resonance, using a phosphorylation-mimicking mutant EEE-Bcl-2 (T69E, S70E and S87E). Mathematical simulations combined with experimental validation showed that phosphorylation regulates Bcl-2 with different dynamics depending on the extent of Bcl-2 phosphorylation and the phosphorylated Bcl-2-induced changes in binding kinetics. In response to Bcl-2 homology 3 (BH3)-only protein Bmf stress, Bcl-2 phosphorylation switched from diminishing to enhancing the Bcl-2 anti-apoptotic ability with increased phosphorylation of Bcl-2, and the turning point was 50% Bcl-2 phosphorylation induced by 0.2μM paclitaxel treatment. In contrast, Bcl-2 phosphorylation enhanced the anti-apoptotic ability of Bcl-2 towards other BH3-only proteins Bim, Bad and Puma, throughout the entire phosphorylation procedure. The model could accurately predict the effects of anti-tumour drugs that involve the Bcl-2 family pathway, as shown with ABT-199 or etoposide.

Mutations in the RNA Splicing Factor SF3B1 Promote Transformation through MYC Stabilization

Mutations in the RNA splicing factor SF3B1 are recurrent in CLL and myeloid neoplasms but their functional role in promoting tumorigenesis remain poorly understood. While SF3B1 mutations have been identified as promoting use of aberrant 3' splice sites (3'ss), consistent identification of mis-spliced transcripts and pathways that functionally link mutant SF3B1 to transformation remains elusive. Moreover, large-scale analyses of the impact of mutant SF3B1 on gene expression and gene regulatory networks, which may be distinct from aberrant splicing changes, remain to be performed. We therefore sought to elucidate the effects of SF3B1 mutations across hematopoietic malignancies and cancer lineages at the level of both mRNA splicing and expression. To this end, we collected RNA-seq data from 79 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations. The most frequent hotspot, K700E, was the most common mutation in CLL and breast cancers while mutations at position R625 were restricted to melanomas (Figure A, B).Regulatory network analysis of differentially expressed genes in SF3B1 mutated CLL identified MYC as the top master regulator (Figure C). MYC activation in SF3B1 mutated CLL was also verified by differential expression analyses (Figure D) and was common to SF3B1K700E mutant cancers while absent in cancers with mutations affecting R625. Taken together, these observations suggested that tumors harboring SF3B1K700E mutations activate the MYC transcriptional program.We next sought to verify the effects of c-Myc activation by mutant Sf3b1 in the B-cell lineage in vivo. We crossed Cd19-cre Sf3b1K700E/+ mice with Eμ-Myc transgenic mice to generate Cd19-cre+ control, Sf3b1K700E/+, Eμ-MycTg/+, and Sf3b1K700E/+Eμ-MycTg/+ double-mutant mice. While control or single mutant primary mice did not develop disease over one year, double-mutant mice developed a lethal B-cell malignancy. This effect was consistent in serial transplantation, where mice transplanted with double-mutant cells had shorter survival compared to single-mutant controls (Figure E). These data provide the first evidence that SF3B1 mutations contribute to tumorigenesis in vivo.To understand the molecular mechanism for MYC activation across SF3B1 mutant human and mouse cells, we analyzed RNA-seq data from CLL patients, isogenic Nalm-6 cells, and splenic B-cells from the mouse models. This revealed a significant overlap in aberrant (3'ss) events across SF3B1 mutant samples. Interestingly, mis-spliced events across mouse and human SF3B1K700E mutant samples identified aberrant 3'ss usage and decay of PPP2R5A (Figure F), a gene whose product has previously been shown to regulate c-MYC protein stability and the only gene whose aberrant splicing was most prominent in K700E compared with R625 mutant SF3B1. PPP2R5A is a subunit of the PP2A phosphatase complex that dephosphorylates Serine 62 (S62) of c-MYC, resulting in an unstable form of c-MYC that is a substrate for proteasomal degradation. Consistent with this, SF3B1K700E mutant cells exhibited dramatic increase in S62-phosphorylated c-MYC and increased stability of c-MYC protein. MYC expression, stability, and S62 phosphorylation could be abrogated in SF3B1 mutant cells by restoring PPP25RA expression. In addition to c-MYC S62 phosphorylation, PPP2R5A-containing PP2A reduced S70 phosphorylation of BCL2 (a modification important for apoptosis induction) in SF3B1 mutant cells. To functionally evaluate the importance of impaired PP2A enzymatic activity in SF3B1 mutant cells further, we assessed the therapeutic potential of the FDA-approved oral PP2A activator, FTY-720. SF3B1 mutant cells were more sensitive to FTY-720 treatment than SF3B1 WT counterparts, experiencing growth arrest at lower concentration (Figure G). Moreover, both S62-phosphorylated c-MYC and S70-phosphorylated BCL2 decreased in a dose-dependent manner upon treatment with FTY-720 (Figure H).Here through combined evaluation of the effects of the SF3B1 mutation on splicing, gene expression, and transcriptional networks across cancer types, we identify a novel mechanism by which mutant SF3B1-mediated alterations in RNA splicing contribute to activation of oncogenic MYC through effects on MYC proteolysis. Moreover, these data highlight a novel therapeutic approach targeting the impact of mutant SF3B1 on post-translational modification of MYC. DisclosuresMato:Janssen: Consultancy, Honoraria; Celgene: Consultancy; Prime Oncology: Speakers Bureau; TG Therapeutics: Research Funding; Regeneron: Research Funding; Abbvie: Consultancy; Sunesis: Honoraria, Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria, Research Funding.

Selenoprotein S protects against high glucose-induced vascular endothelial apoptosis through the PKCβII/JNK/Bcl-2 pathway.

Vascular endothelial apoptosis is closely associated with the pathogenesis and progression of diabetic macrovascular diseases. Selenoprotein S (SelS) participates in the protection of vascular endothelial and smooth muscle cells from oxidative and endoplasmic reticulum stress-induced injury. However, whether SelS can protect vascular endothelium from high glucose (HG)-induced apoptosis and the underlying mechanism remains unclear. The present study preliminarily analyzed aortic endothelial apoptosis and SelS expression in diabetic rats in vivo and the effects of HG on human umbilical vein endothelial cell (HUVEC) apoptosis and SelS expression in vitro. Subsequently, SelS expression was up- or downregulated in HUVECs using the pcDNA3.1-SelS recombinant plasmid and SelS-specific small interfering RNAs, and the effects of high/low SelS expression on HG-induced HUVEC apoptosis and a possible molecular mechanism were analyzed. As expected, HG induced vascular endothelial apoptosis and upregulated endothelial SelS expression in vivo and in vitro. SelS overexpression in HUVECs suppressed HG-induced increase in apoptosis and cleaved caspase3 level, accompanied by reduced protein kinase CβII (PKCβII), c-JUN N-terminal kinase (JNK), and B-cell lymphoma/leukemia-2 (Bcl-2) phosphorylation. In contrast, inhibiting SelS expression in HUVECs further aggravated HG-induced increase in apoptosis and cleaved caspase3 level, which was accompanied by increased PKCβII, JNK, and Bcl-2 phosphorylation. Pretreatment with PKC activators blocked the protective effects of SelS and increased the apoptosis and cleaved caspase3 level in HUVECs. In summary, SelS protects vascular endothelium from HG-induced apoptosis, and this was achieved through the inhibition of PKCβII/JNK/Bcl-2 pathway to eventually inhibit caspase3 activation. SelS may be a promising target for the prevention and treatment of diabetic macrovascular complications.

6-MOMIPP, a novel brain-penetrant anti-mitotic indolyl-chalcone, inhibits glioblastoma growth and viability.

3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (6-MOMIPP) is a novel indole-based chalcone that disrupts microtubules. The present study aims to define the mechanism through which 6-MOMIPP induces cell death and to evaluate the efficacy of the compound in penetrating the blood-brain barrier and inhibiting growth of glioblastoma xenografts. The effects of 6-MOMIPP were evaluated in cultured U251 glioblastoma cells, using viability, flow cytometry, and tubulin polymerization assays. Scintillation proximity and tubulin crosslinking methods were used to identify the binding site of 6-MOMIPP on tubulin, and western blots were performed to define the signaling pathways that contribute to cell death. LC/MS assays were used to study the pharmacokinetic behavior of 6-MOMIPP in mice. Subcutaneous and intracerebral xenograft models were utilized to assess the effects of 6-MOMIPP on growth of U251 glioblastoma in vivo. The findings indicate that 6-MOMIPP targets the colchicine site on β-tubulin. At concentrations ≥ 250nm, 6-MOMIPP induces mitotic arrest, caspase activation and loss of cell viability. Cells are protected by caspase inhibitors, pointing to an apoptotic mechanism of cell death. Loss of cell viability is preceded by activation of Cdk1(Cdc2) and phosphorylation of Bcl-2 and Bcl-xL. Inhibition of both events with a Cdk1 inhibitor prevents cell death. 6-MOMIPP has broad activity against the viability of multiple glioblastoma, melanoma and lung carcinoma cell lines. Viability of normal cells, including differentiated neurons, is not significantly affected at a drug concentration (1µM) that reduces viability in most cancer lines. Pharmacokinetic studies in mice show that concentrations of 6-MOMIPP in the brain mirror those in the plasma, indicating that 6-MOMIPP readily penetrates the blood-brain barrier. Studies with mice bearing human U251 glioblastoma xenografts demonstrate that 6-MOMIPP is effective in suppressing growth of subcutaneous and intracerebral tumors without causing general toxicity. The results indicate that 6-MOMIPP is anovel microtubule disruptor that targets the colchicine binding site on β-tubulin to induce mitotic arrest and cell death. The ability of 6-MOMIPP to penetrate the blood-brain barrier and inhibit growth of glioblastoma xenografts suggests that it warrants further preclinical evaluation as potential small-molecule therapeutic that may have advantages in treating primary and metastatic brain tumors.

129 - Bcl-2 phosphorylation permits a conducive pro-oxidant milieu for cancer cell survival and progression

An increased intracellular reactive oxygen species (ROS) level has been documented to provide a pro-oxidant milieu suitable for cell survival and proliferation. Nevertheless, an increase that overwhelms the antioxidant or detoxification system would induce cellular oxidative damage or cell death. In the case of carcinogenesis, it is imperative for cancer cells to maintain a conducive pro-oxidant milieu for their continual progression. To that end, our previous work has demonstrated that the GTPase Rac1 could interact with the anti-apoptotic Bcl-2 protein to induce a pro-oxidant milieu for cancer cell survival and proliferation. Keeping in mind that 1) a heightened ROS level would require a heightened antioxidant defence and that 2) the anti-apoptotic Bcl-2 protein also possesses an antioxidant role, we therefore question if Rac1-induced pro-oxidant milieu would mandate an enhanced Bcl-2 activation to protect cancer cells against the detrimental effects of heightened ROS level. Indeed, we show here that the constitutively active Rac1 is capable of elevating ROS level for an enhanced proliferation while keeping the antioxidant Bcl-2 activated through a sustained Bcl-2 phosphorylation. Pharmacological or genetic manipulation to reduce Bcl-2 phosphorylation reciprocally elevates ROS level, DNA damage and cell death. We further demonstrate that the effect of Rac1 to sustain Bcl-2 phosphorylation is its redox-dependent function in inhibiting the assembly of protein phosphatase PP2A. Importantly, our in vitro data are further corroborated by findings derived from primary lymphoma samples, where an elevated Rac1 protein level is significantly correlated to an enhanced Bcl-2 phosphorylation, which in turn is significantly associated with lower DNA damage and higher disease severity. Collectively, our findings demonstrate that active or overexpressing Rac1 cancer cells are capable of regulating the redox environment in favour of survival and progression by manipulating the phosphorylation status of the oncoprotein Bcl-2.

151 - Rac1-induced ROS is implicated in the sustained activation of anti-apoptotic protein Bcl-2 in cancer cells

The anti-apoptotic protein Bcl-2 is strongly associated with the development of therapy resistance in a host of hematopoietic and other cancers. To that end, the post-translational modification of Bcl-2, in particular its mono- or multi-site phosphorylation, have been reported to impact its apoptosis inhibitory activity. While multi-site phosphorylation compromises its death inhibitory activity, our recent work has highlighted the critical involvement of serine 70 phosphorylation (S70pBcl-2) in stabilizing its anti-apoptotic activity. S70pBcl-2 is a function of either an amplification of the activities of various kinases involved in this post-translational modification or a loss of function of the phosphatase regulating this modification. The pleiotropic phosphatase, PP2A, is specifically involved in the dephosphorylation of Bcl-2 at S70. In our search to identify redox dependent pathways impacting the sustained activation of Bcl-2 via S70P, we provided evidence that redox modification of the B56δ sub-unit of PP2A resulted in the sustained S70P of Bcl-2, thereby promoting its death inhibitory activity in cancer cells. Interestingly, here we present substantial evidence for the existence of a similar redox-dependent regulation of Bcl-2 phosphorylation in cells expressing GTP-bound active Rac1. We show that active Rac1 promotes sustained S70pBcl2 levels in cancer cells via physical interaction with Bcl-2, thereby permitting Rac1-induced production of superoxide (O2–) in close proximity to inactivate PP2A holoenzyme assembly and specific activity. Corroborating this, pharmacological inhibition of the interaction with BH3 mimetics such as HA14-1 or ABT199 as well as scavengers of O2– such as Tiron reduced S70pBcl2. Importantly, the interaction between Rac1 and Bcl-2 to promote S70pBcl2 as well as the oxidative inhibition of PP2A are re-capitulated in primary cells derived from lymphoma patients. Collectively, our findings demonstrate a novel redox mechanism of active Rac1 in sustaining S70pBcl2 in cancer cells.

Monoamine oxidase-A promotes protective autophagy in human SH-SY5Y neuroblastoma cells through Bcl-2 phosphorylation

Monoamine oxidases (MAOs) are located on the outer mitochondrial membrane and are drug targets for the treatment of neurological disorders. MAOs control the levels of neurotransmitters in the brain via oxidative deamination and contribute to reactive oxygen species (ROS) generation through their catalytic by-product H2O2. Increased ROS levels may modulate mitochondrial function and mitochondrial dysfunction is implicated in a vast array of disorders. However, the downstream effects of MAO-A mediated ROS production in a neuronal model has not been previously investigated.In this study, using MAO-A overexpressing neuroblastoma cells, we demonstrate that higher levels of MAO-A protein/activity results in increased basal ROS levels with associated increase in protein oxidation. Increased MAO-A levels result in increased Lysine-63 linked ubiquitination of mitochondrial proteins and promotes autophagy through Bcl-2 phosphorylation. Furthermore, ROS generated locally on the mitochondrial outer membrane by MAO-A promotes phosphorylation of dynamin-1-like protein, leading to mitochondrial fragmentation and clearance without complete loss of mitochondrial membrane potential. Cellular ATP levels are maintained following MAO-A overexpression and complex IV activity/protein levels increased, revealing a close relationship between MAO-A levels and mitochondrial function. Finally, the downstream effects of increased MAO-A levels are dependent on the availability of amine substrates and in the presence of exogenous substrate, cell viability is dramatically reduced.This study shows for the first time that MAO-A generated ROS is involved in quality control signalling, and increase in MAO-A protein levels leads to a protective cellular response in order to mediate removal of damaged macromolecules/organelles, but substrate availability may ultimately determine cell fate. The latter is particularly important in conditions such as Parkinson's disease, where a dopamine precursor is used to treat disease symptoms and highlights that the fate of MAO-A containing dopaminergic neurons may depend on both MAO-A levels and catecholamine substrate availability.

Cytoprotective effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) is mediated by the inhibition of BAK-dependent mitochondrial apoptosis pathway.

The inhibitory mechanism of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) against apoptosis induced by the microtubule-damaging agents (MDAs), nocodazole (NOC) and 2-methoxyestradiol (2-MeO-E2), or a DNA-damaging agent (DDA), camptothecin (CPT) were investigated in human Jurkat T cell clones (J/Neo and J/BCL-XL cells). Treatment of J/Neo cells with NOC, 2-MeO-E2, or CPT caused cytotoxicity and apoptotic DNA fragmentation but these events were significantly attenuated in the presence of CMEP-NQ. Although not only MDA (NOC or 2-MeO-E2)-induced mitotic arrest, CDK1 activation, and BCL-2, BCL-XL and BIM phosphorylation, but also DDA (CPT)-induced S-phase arrest and ATM-CHK1/CHK2-p53 pathway activation were not or were barely affected in the presence of CMEP-NQ, the levels of anti-apoptotic BAG3 and MCL-1, which were markedly downregulated after MDA- or DDA-treatment, were rather elevated by CMEP-NQ. Under the same conditions, MDA- or DDA-induced mitochondrial apoptotic events including BAK activation, mitochondrial membrane potential (Δψm) loss, caspase-9 activation, and PARP cleavage were significantly inhibited by CMEP-NQ. While MDA- or DDA-induced sub-G1 peak and Δψm loss were abrogated in J/BCL-XL cells, MDA-induced mitotic arrest and DDA-induced S-arrest were more apparent in J/BCL-XL cells than in J/Neo cells. Simultaneously, the induced cell cycle arrest in J/BCL-XL cells was not significantly disturbed by CMEP-NQ. MDA- or DDA-treatment caused intracellular reactive oxygen species (ROS) production; however, MDA- or DDA-induced ROS production was almost completely abrogated in J/BCL-XL cells. MDA- or DDA-induced ROS production in J/Neo cells was significantly suppressed by CMEP-NQ, but the suppressive effect was hardly observed in J/BCL-XL cells. Together, these results show that CMEP-NQ efficiently protects Jurkat T cells from apoptotic cell death via the elevation of BAG3 and MCL-1 levels, which results in the inhibition of intrinsic BAK-dependent mitochondrial apoptosis pathway, as does the overexpression of BCL-XL.

Antimalarial Dihydroartemisinin triggers autophagy within HeLa cells of human cervical cancer through Bcl-2 phosphorylation at Ser70

BackgroundAs an effective antimalarial medicine, Dihydroartemisinin (DHA) has therapeutic potential on human cervical cancer. However, its working mechanism has not been elucidated. PurposeThis study aimed to investigate the reversal effect of DHA on human cervical cancer HeLa cells, and explored its mechanism of action in vitro and in vivo. Study design/methodsThe effect and mechanism of DHA on HeLa cells was examined by using CCK-8 assay, flow cytometry, transmission electron microscopy, immunofluorescence, and Western blot analysis in human hepatocellular carcinoma cells. ResultsIn this study, it was confirmed that DHA had statistically equivalent anti-tumor efficiency in HeLa cells with a clinical chemotherapeutic agent of cisplatin. Meanwhile, DHA triggered autophagy, where LC3B-II expression was dose-dependently increased. Further, it was revealed that DHA promotes reactive oxygen species (ROS) generation, with DNA double-strand breaks (DSB) damage, as up-regulation of γH2AX protein and foci formation. Interestingly, we firstly demonstrated that DHA induced autophagy through promotion of the phosphorylation of Bcl-2 (Ser70), independent of the phosphorylated JNK1/2 (Thr183/Tyr185). Moreover, DHA-treated HeLa cells displayed an increase in the pro-autophagic protein Beclin-1 with downregulated the phospho-mTOR (Ser2448). Furthermore, upregulated pro-apoptotic protein Bak-1, but not Bax, suggesting Bak-1 is included in DHA-induced autophagy. ConclusionTherefore, DHA upregulates the phosphorylation of Bcl-2 (Ser70) and mTOR (Ser2448) and induces autophagic cell death in Hela cells. This study provided a mechanism to support DHA, an autophagy inducer, as a potential therapeutic agent for human cervical cancer.

Camel whey protein enhances lymphocyte survival by modulating the expression of survivin, bim/bax, and cytochrome C and restores heat stress-mediated pathological alteration in lymphoid organs.

Objective(s):Heat stress (HS) is a catastrophic stressor that dampens immunity. The current study investigates the effect of dietary administration with camel whey protein (CWP) on apoptotic pathway caused by HS. Materials and Methods:Forty-five male mice were divided into three groups: a control group; HS group; and HS mice that were orally supplemented with CWP (CWP-HS group). Results:We found that reactive oxygen species (ROS), pro-inflammatory cytokines (IL-6), and C reactive protein (CRP) were elevated in the HS group along with a significant increase of caspase-9 and -3 and decrease of total antioxidant capacity (TAC). HS mice revealed impaired phosphorylation of Bcl-2 and Survivin, as well as increased expression of Bax, Bim and cytochrome C. Additionally, we observed an aberrant distribution of HSP-70 expressing lymphocytes in the spleen and thymus of HS mice. Moreover, histopathological examination showed alterations on the architectures of immune organs. In comparison with CWP-HS group, we found that CWP restored the levels of ROS, IL-6, TAC and CRP induced by HS. Furthermore, CWP restored the expression of Bcl-2/Bax, improved the histopathological changes in immune organs and HSP-70 distribution in the spleen and thymus. Conclusion:Our findings revealed the possible ameliorative role of CWP supplementation against damages induced by exposure to HS.

Cytotoxic Constituents from the Sclerotia of Poria cocos against Human Lung Adenocarcinoma Cells by Inducing Mitochondrial Apoptosis.

Previous studies have revealed the antitumor potential of Poria cocos Wolf against a broad spectrum of cancers. However, the biological activity of P. cocos against lung cancer, which is known as the leading cause of cancer mortality worldwide, and its underlying chemical and molecular basis, remain to be investigated. We aimed to evaluate the in vitro cytotoxicity of P. cocos toward human lung adenocarcinoma cells with different p53 statuses, to identify the bioactive constituents of P. cocos, and explicate the molecular mechanisms underlying the cytotoxicity of these constituents in human lung adenocarcinoma cells. An EtOH extract of the sclerotia of P. cocos exhibited cytotoxicity toward four human lung cancer cell lines: A549, H1264, H1299, and Calu-6, regardless of their p53 status. Chemical investigation of the extract resulted in the isolation of two triterpenoids, dehydroeburicoic acid monoacetate (1) and acetyl eburicoic acid (4); a sterol, 9,11-dehydroergosterol peroxide (2); and a diterpenoid, dehydroabietic acid (3). All of the isolated compounds were cytotoxic to the lung adenocarcinoma cell lines, exhibiting IC50 values ranging from 63.6 μM to 171.0 μM at 48 h of treatment. The cytotoxicity of the extract and the isolated compounds were found to be mediated by apoptosis, and accompanied by elevated Bax expression and/or Bcl-2 phosphorylation along with caspase-3 activation. Our data demonstrate that the sclerotium of P. cocos and its four bioactive constituents (1–4) exert cytotoxicity against human lung adenocarcinoma cells, regardless of their p53 status, by inducing apoptosis associated with mitochondrial perturbation, and proposing the potential to employ P. cocos in the treatment of lung cancer.

Inhibition of ERK-Drp1 signaling and mitochondria fragmentation alleviates IGF-IIR-induced mitochondria dysfunction during heart failure

Mitochondrial dysfunction is a major contributor to myocyte loss and the development of heart failure. Myocytes have quality control mechanisms to retain functional mitochondria by removing damaged mitochondria via specialized autophagy, i.e., mitophagy. The underlying mechanisms of fission affect the survival of cardiomyocytes, and left ventricular function in the heart is poorly understood. Here, we demonstrated the direct effect and potential mechanisms of mitochondrial functional defects associated with abnormal mitochondrial dynamics in heart failure. We observed that IGF-IIR signaling produced significant changes in mitochondrial morphology and function; such changes were associated with the altered expression and distribution of dynamin-related protein (Drp1) and mitofusin (Mfn2). IGF-IIR signaled extracellular signal-regulated kinase (ERK) activation to promote Drp1 phosphorylation and translocation to mitochondria for mitochondrial fission and mitochondrial dysfunction. Moreover, IGF-IIR signaling triggered Rab9-dependent autophagosome formation by the JNK-mediated phosphorylation of Bcl-2 at serine 87 and promoted ULK1/Beclin 1-dependent autophagic membrane formation. Excessive mitochondrial fission by Drp1 enhanced the Rab9-dependent autophagosome recognition and engulfing of damaged mitochondria and eventually decreased cardiomyocyte viability. Therefore, these results demonstrated the connection between Rab9-dependent autophagosomes and mitochondrial fission in cardiac myocytes, which provides a potential therapeutic strategy for treating heart disease.

Cross-talk between Bcl-2 and IKK in 4-hydroxynonenal-induced apoptosis

Apoptosis of macrophage foam cells loaded with modified/oxidized lipids is implicated in destabilization of advanced atherosclerotic plaques in humans. Concentration of HNE, main aldehydic product of plasma LDL peroxidation, elevates in atherosclerotic lesions as well as in cultured cells under oxidative stress. Although this reactive aldehyde has been shown to promote apoptosis with the involvement of p38 MAPK and JNK in various mammalian cell lines, roles of B-cell lymphoma 2 (Bcl-2) family proteins remain to be deciphered. We demonstrated that HNE-induced apoptosis was accompanied by concurrent downregulations of antiapoptotic Bcl-x(L) and Mcl-1 as well as upregulation of proapoptotic Bak. Furthermore, phoshorylation of Bcl-2 at Thr56, Ser70, and probably more phosphorylation sites located on N-terminal loop domain associated with HNE-induced apoptosis in both U937 and HeLa cells while ectopic expression of a phospho-defective Bcl-2 mutant significantly attenuated apoptosis. In parallel to this, HNE treatment caused release of proapoptotic Bax from Bcl-2. Pharmacological inhbition of Inhibitory kappa B kinase (IKK) inhibited HNE-induced Bcl-2 phosphorylation. Similarly, silencing IKKα and -β both ended up with abrogation of Bcl-2 phosphorylation along with attenuation of apoptosis. Moreover, both IKKα and -β coimmunoprecipitated with Bcl-2 and in vitro kinase assay proved the ability of IKK to phosphorylate Bcl-2. In view of these findings and considering HNE inhibits DNA-binding activity of nuclear factor-κB (NF-κB) through prevention of IκB phosphorylation/ubiquitination/proteolysis, IKK appears to directly interfere with Bcl-2 activity through phosphorylation in HNE-mediated apoptosis independent of NF-κB signaling.

BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation

BackgroundAutophagy is a conserved catabolic process with complicated roles in tumor development. Bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor (TGF-β) family of regulatory proteins, plays a crucial role in human malignancies. However, whether BMP4 contributes to the regulation of autophagy in hepatocellular carcinoma (HCC) progression remains elusive.MethodsFunctional analysis of BMP4 on HCC proliferation and autophagy was performed both in vitro and in vivo in HepG2 and HCCLM3 cells. Autophagic activity was estimated by Western blot for autophagic marker proteins and by transmission electron microscopy (TEM). Transfection of mRFP-GFP-LC3 adenovirus was applied to observe autophagic flux and high content screening was used for quantification. The signaling pathway of BMP4-regulated HCC proliferation and autophagy was investigated by Western blot.ResultsBMP4 treatment promoted HCC cells proliferation and induced autophagy. The in vivo xenograft model supported that BMP4 overexpression promoted the growth of HCC cells and autophagy induction while BMP4 knockdown exerted the opposite effect. 3-MA pre-treatment or knockdown of Beclin-1 (BECN1) blocked HCC autophagy by decreasing the expression of LC3-II and subsequently attenuated BMP4-induced autophagy and cells proliferation enhanced by BMP4 in vitro and in vivo. Mechanistic study revealed that the induction of autophagy by BMP4 was mediated through activating the JNK1/Bcl2 pathway. Furthermore, the JNK1 inhibitor and knockdown of JNK1 could attenuate autophagy induced by BMP4 and eliminated BMP4-promoted HCC cells growth.ConclusionsBMP4 promoted HCC proliferation by autophagy activation through JNK1/Bcl-2 signaling.

Abstract 5442: Dose-dependent toxicity of FTY720, a sphingosine-1-phosphate receptor agonist, on double hit lymphoma cells via PP2A-mediated dephosphorylation of c-myc and bcl-2

Abstract High grade B cell lymphoma with translocations involving MYC and rearrangements involving BCL2 and/or BCL6 define an aggressive subtype of diffuse large B cell lymphoma (DLBCL) with poor prognosis and high rates of relapse. The standard treatment for DLBCL is not considered effective and targeted molecular therapy has yet to be identified for this disease. The mechanisms that make this type of lymphoma particularly aggressive and refractory to treatment include cell growth signals from the c-myc transcriptional regulator working together with the anti-apoptotic activity of the bcl-2 protein. Protein Phosphatase 2A (PP2A) is a ubiquitous cellular enzyme that acts as a tumor suppressor through dephosphorylation of specific cellular phosphoproteins. In fact, PP2A has been shown to dephosphorylate bcl-2 at ser70 leading to inhibition of its function. Further, PP2A-mediated dephosphorylation of c-myc at ser62 promotes its degradation. In many cancers, PP2A activity is inhibited due to overexpression of binding proteins such as SET and CIP2A. Small molecules can act as PP2A activators by releasing PP2A from these inhibitory binding proteins. In the present study we investigated the efficacy of FTY720, a high-affinity agonist of the sphingosine 1-phosphate receptor-1 and PP2A activator, on the double hit lymphoma derived cell line, WSU DLCL2. WSU DLCL2 cells were treated with various concentrations of FTY720, a derivative of myriocin, a fungal metabolite of the Chinese herb Iscaria sinclarii known to promote PP2A activation. Cell viability and PP2A activity were measured 24 hours after FTY720 treatment. In addition, cell lysates were assayed by Western blot using antibodies to detect PP2A subunits, as well as total and phosphorylated c-myc and bcl-2 proteins. A dose-dependent decrease in cell viability was observed over the range of FTY720 concentrations from 1 to 10 micromolar with concomitant increase in in vitro PP2A activity. Western blots showed decreases in c-myc protein levels with FTY720 treatment as well as decreases in ser70 phosphorylation levels of bcl-2 and ser62 phosphorylation levels of c-myc. The results indicate that PP2A activation via FTY720, a small molecule activator of PP2A, leads to a dose-dependent induction of cell death in double hit lymphoma. The data are consistent with an underlying cell death mechanism that involves the targeted dephosphorylation of c-myc and bcl2. Taken together, these data suggest that FTY720 may act as a targeted treatment with therapeutic potential as a single agent or as a combination with other therapies in the treatment of this subtype of lymphoma. There is also the potential of a therapeutic role in other lymphoma subtypes with poor prognosis including transformed follicular lymphoma and relapsed burkitt's lymphoma that rely individually on bcl-2 and c-myc, respectively, for their growth. Citation Format: Geoffrey P. Shouse, Rosalia de Necochea-Campion, Saied Mirshahidi, Chien-Shing Chen, Kimberly J. Payne. Dose-dependent toxicity of FTY720, a sphingosine-1-phosphate receptor agonist, on double hit lymphoma cells via PP2A-mediated dephosphorylation of c-myc and bcl-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5442.