Apoptosis-regulating Proteins Research Articles

Multiple effects of Xihuang pill aqueous extract on the Hs578T triple-negative breast cancer cell line.

The management of triple-negative breast cancer (TNBC) is challenging due to the aggressive behavior, lack of therapeutic options and relatively poor prognosis. Xihuang pill (XHP) is a well-known Traditional Chinese Medicine with anticancer activity. The aim of the present study was to investigate whether the aqueous extract of XHP (AEXHP) has anti-proliferative activity against the Hs578T TNBC cell line, and to elucidate its molecular mechanisms of action. First, an MTT assay was used to evaluate the anti-proliferative activity of AEXHP on the Hs578T cell line; furthermore, the cell cycle distribution, mitochondrial membrane potential and apoptotic rate were determined by flow cytometry, and western blot analysis was used to assess the expression of apoptosis and cell cycle regulatory proteins to investigate the mechanisms of action. The results revealed that the cell viability was significantly inhibited by AEXHP in a dose- and time-dependent manner. Apoptosis and mitochondrial membrane potential loss were detected, and after treatment with 4, 8 and 12 mg/ml AEXHP for 24 h, cleaved caspase-3 was 1.70-, 1.81- and 1.84-fold of that of the control, while procaspase-3, procaspase-8, cleaved caspase-8, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and the Bcl-2/Bax ratio were not significantly affected. Cell cycle analysis revealed that treatment with AEXHP led to S-phase arrest of Hs578T cells. Furthermore, AEXHP treatment resulted in decreased expression of cyclin A and cyclin dependent kinase 2 (CDK2), and increased expression of cyclin E and p21Cip1, as compared to the control group. In conclusion, the viability of Hs578T cells was significantly inhibited by AEXHP in a dose- and time-dependent manner, the likely mechanisms of which being induction of apoptosis, probably via the intrinsic, Bcl-2-independent pathway, and cell cycle arrest in S phase due to decreased expression of cyclin A and CDK2, and increased expression of cyclin E and p21Cip1.

Differences in the mechanisms of action of BDE-47 and its metabolites on OVCAR-3 and MCF-7 cell apoptosis.

Data concerning possible carcinogenic action of polybrominated diphenyl ethers (PBDEs) in hormone-dependent tissues are limited. Our earlier studies showed that 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) stimulated OVCAR-3 and MCF-7 cell proliferation, while its hydroxylated metabolites (5-OH-BDE-47 and 6-OH-BDE-47) increased estrogen receptors protein expression and extracellular signal-regulated kinase 1/2 and protein kinase Cα phosphorylation in these cell lines. In addition to cell proliferative disorder, a failure in the regulation of apoptosis can also lead to the formation and development of tumors. Therefore, in the present study, we investigated the effect of BDE-47 and its metabolites (2.5-50 ng ml-1 ) on the expression of apoptosis regulatory genes and proteins, caspase-8 and -9 activity and DNA fragmentation induced by extracellular signal-regulated kinase inhibitor (PD098059) and protein kinase Cα inhibitor (Gӧ 6976) in ovarian (OVCAR-3) and breast (MCF-7) cancer cells. In OVCAR-3 cells, BDE-47 upregulated expression of most of the investigated genes and increased protein expression of tumor necrosis factor (TNF)-α, TNF receptor 1, caspase-6, Bcl-xl and caspase-8 activity. Whereas in MCF-7 cells, BDE-47 resulted in the downregulation of most of the investigated genes, and decreased caspase-8 and -9 activity. In both OVCAR-3 and MCF-7 cells, the expression of most of the investigated genes were downregulated by metabolites. Exposure of OVCAR-3 cells to 5-OH-BDE-47 corresponded with a decrease in the protein expression of caspase-6, caspase-9 and Bcl-xl and treatment with 6-OH-BDE-47 decreased Bcl-xl and TNF receptor 1 expression in OVCAR-3 cells and caspase-9 expression in MCF-7 cells. Hydroxylated metabolites of BDE-47 have strong inhibitory effects on apoptosis in ovarian and breast tumor cells and thus should be considered potential carcinogens in hormone-dependent cancers. Copyright © 2016 John Wiley & Sons, Ltd.

Pro-Apoptotic Activity of New Honokiol/Triphenylmethane Analogues in B-Cell Lymphoid Malignancies.

Honokiol and triphenylmethanes are small molecules with anti-tumor properties. Recently, we synthesized new honokiol analogues (HAs) that possess common features of both groups. We assessed the anti-tumor effectiveness of HAs in B-cell leukemia/lymphoma cells, namely in chronic lymphocytic leukemia (CLL) cells ex vivo and in pre-B-cell acute lymphoblastic leukemia (Nalm-6), Burkitt lymphoma (BL; Raji), diffuse large B-cell lymphoma (DLBCL; Toledo) and multiple myeloma (MM; RPMI 8226) cell lines. Four of these compounds appeared to be significantly active against the majority of cells examined, with no significant impact on healthy lymphocytes. These active HAs induced caspase-dependent apoptosis, causing significant deregulation of several apoptosis-regulating proteins. Overall, these compounds downregulated Bcl-2 and XIAP and upregulated Bax, Bak and survivin proteins. In conclusion, some of the HAs are potent tumor-selective inducers of apoptosis in ex vivo CLL and in BL, DLBCL and MM cells in vitro. Further preclinical studies of these agents are recommended.

Abstract 3824: Targeting MUC1-C oncoprotein inhibits AKT-S6K1-elF4A pathway regulated TIGAR translation in colorectal cancer

Abstract Colorectal cancer is third most common malignancy and is the second most common cause of cancer-related death. The MUC1 multi-domain oncoprotein is aberrantly overexpressed in colorectal cancer and has been linked to poor outcomes in this disease. However, it is not known if MUC1 is of functional significance to colorectal tumors. Here, we investigate the effects of the MUC1-C inhibitor (GO-203), which disrupts MUC1-C homo-oligomerization, on AKT-mTORC-S6K translation pathway in human colorectal cancer cells. The present study demonstrates that GO-203 treatment was associated with inhibition of AKT-S6K1 signaling in colorectal cancer cells. Targeting MUC1-C with GO-203 downregulates TP53-inducible glycolysis and apoptosis regulator (TIGAR) protein. We show that GO-203-induced suppression of TIGAR is mediated by inhibition of AKT and the downstream mTOR pathway. The results also demonstrate that targeting MUC1-C blocks eIF4A cap-dependent translation of TIGAR. TIGAR promotes the shunting of glycolytic intermediates into the pentose phosphate pathway and thus is of importance for maintaining redox balance. In concert with these results, GO-203-induced suppression of TIGAR was associated with decreases glutathione levels. GO-203 treatment also resulted increases in reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential. Consistent with these results, GO-203 inhibited the growth of colon cancer cells in vitro and xenografts in nude mice. These findings thus indicate that MUC1-C is a potential target for the treatment of colorectal cancer. Citation Format: Rehan Ahmad, Maroof Alam, Omar Al-Obaid, Masanori Hasegawa, Donald Kufe. Targeting MUC1-C oncoprotein inhibits AKT-S6K1-elF4A pathway regulated TIGAR translation in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3824.

Betulin induces reactive oxygen species-dependent apoptosis in human gastric cancer SGC7901 cells.

Betulin, an abundant natural compound, significantly inhibited the cell viability of advanced human gastric cancer SGC7901 cells. Mechanism study demonstrated that betulin induced apoptosis through mitochondrial Bax and Bak accumulation-mediated intrinsic apoptosis pathway. Downregulation of the anti-apoptosis proteins Bcl-2 and XIAP was involved during betulin-induced cell apoptosis. Reactive oxygen species (ROS) was generated in cells after betulin treatment in a time- and dose-dependent manner. Addition of antioxidant N-acetyl-L-cysteine (NAC) significantly attenuated betulin-induced ROS generation as well as Bcl-2 and XIAP downregulation. The mitochondrial accumulation of Bax and Bak, as well as caspase activity, was also remarkably inhibited by NAC treatment, indicating that ROS are important signaling intermediates that lead to betulin-induced apoptosis by modulating multiple apoptosis-regulating proteins in SGC7901 cells.

Molecular characterization of protein kinase C delta (PKCδ)-Smac interactions.

BackgroundProtein kinase C δ (PKCδ) is known to be an important regulator of apoptosis, having mainly pro- but also anti-apoptotic effects depending on context. In a previous study, we found that PKCδ interacts with the pro-apoptotic protein Smac. Smac facilitates apoptosis by suppressing inhibitor of apoptosis proteins (IAPs). We previously established that the PKCδ-Smac complex dissociates during induction of apoptosis indicating a functional importance. Because the knowledge on the molecular determinants of the interaction is limited, we aimed at characterizing the interactions between PKCδ and Smac.ResultsWe found that PKCδ binds directly to Smac through its regulatory domain. The interaction is enhanced by the PKC activator TPA and seems to be independent of PKCδ catalytic activity since the PKC kinase inhibitor GF109203X did not inhibit the interaction. In addition, we found that C1 and C2 domains from several PKC isoforms have Smac-binding capacity.ConclusionsOur data demonstrate that the Smac-PKCδ interaction is direct and that it is facilitated by an open conformation of PKCδ. The binding is mediated via the PKCδ regulatory domain and both the C1 and C2 domains have Smac-binding capacity. With this study we thereby provide molecular information on an interaction between two apoptosis-regulating proteins.

Hematologic malignancies: newer strategies to counter the BCL-2 protein.

BCL-2 is the founding member of the BCL-2 family of apoptosis regulatory proteins that either induce (pro-apoptotic) or inhibit (anti-apoptotic) apoptosis. The anti-apoptotic BCL-2 is classified as an oncogene, as damage to the BCL-2 gene has been shown to cause a number of cancers, including lymphoma. Ongoing research has demonstrated that disruption of BCL-2 leads to cell death. BCL-2 is also known to be involved in the development of resistance to chemotherapeutic agents, further underscoring the importance of targeting the BCL-2 gene in cancer therapeutics. Thus, numerous approaches have been developed to block or modulate the production of BCL-2 at the RNA level using antisense oligonucleotides or at the protein level with BCL-2 inhibitors, such as the novel ABT737. In this article, we briefly review previous strategies to target the BCL-2 gene and focus on a new approach to silence DNA, DNA interference (DNAi). DNA interference is aimed at blocking BCL-2 gene transcription. Evaluations of this technology in preclinical and early clinical studies are very encouraging and strongly support further development of DNAi as cancer therapeutics. A pilot phase II clinical trial in patients with relapsed or refractory non-Hodgkin lymphoma, PNT2258 demonstrated clinical benefit in 11 of 13 patients with notable responses in diffuse large B cell lymphoma and follicular lymphoma. By targeting the DNA directly, the DNAi technology promises to be more effective compared with other gene-interference strategies that target the RNA or protein but leaves the dysregulated DNA functional.

Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividualex vivoapoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p= 0.0067 andp= 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p= 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.

A comparative proteomics study of a synovial cell line stimulated with TNF-α.

To elucidate the pathogenesis of rheumatoid arthritis (RA), we used proteomic analysis to determine the protein profile in a synovial cell line, MH7A, established from patients with RA. Proteins were extracted from MH7A cells that were or were not stimulated with tumor necrosis factor‐α (TNF‐α), and then analyzed on a liquid chromatography/mass spectrometry system equipped with a unique long monolithic silica capillary. On the basis of the results of this proteomic analysis, we identified 2650 proteins from untreated MH7A cells and 2688 proteins from MH7A cells stimulated with TNF‐α. Next, we selected 269 differentially produced proteins that were detected only under TNF‐α stimulation, and classified these proteins by performing gene ontology analysis by using DAVID as a functional annotation tool. In TNF‐α‐stimulated MH7A cells, we observed substantial production of plasminogen‐activator inhibitor 2 and apoptosis‐regulating proteins such as BH3‐interacting domain death agonist, autophagy protein 5, apolipoprotein E, and caspase‐3. These results indicate that the upregulation of plasminogen‐activator inhibitor 2 and apoptosis‐regulating proteins in synovial cells in response to TNF‐α stimulation might represent a predominant factor that contributes to the pathogenesis of RA.

Expression of Apoptosis-regulating Proteins Bcl-2 and Bax in Lymph Node Aspirates from Dogs with Lymphoma.

BackgroundDysregulated apoptosis is a hallmark of tumorigenesis, and is also involved in resistance to cytotoxic treatment, and might be relevant in lymphoma in dogs.Hypothesis/ObjectivesThat Bcl‐2/Bax expression patterns differ between lymphoma immunophenotypes, and that Bcl‐2/Bax ratio is correlated with prognosis.AnimalsFifty‐five client‐owned dogs with multicentric lymphoma and 5 healthy dogs.MethodsProspective, case–control study. We compared 3 methods (flow cytometry, qRT‐PCR, Western blot) for Bcl‐2 and Bax quantification in a subset of dogs. The effect of time on Bcl‐2/Bax ratios measured by flow cytometry was assessed in lymphoma cell lines. Immunophenotype and Bcl‐2/Bax expression by flow cytometry were determined in LN aspirates from all dogs with multicentric lymphoma compared to healthy dogs. Progression‐free survival (PFS) was retrospectively evaluated in a group of dogs all receiving similar treatment.ResultsBcl‐2/Bax ratios remain consistent for at least 5 days after sample collection. Bcl‐2/Bax ratio was higher in dogs with T‐cell lymphoma (TCL; median 0.97, range 0.37–1.36) compared to B‐cell lymphoma (BCL; median 0.36, range 0.07–1.45) (P < .0001) and normal dogs (median 0.36, range 0.21–0.48) (P = .0006), respectively. Dogs with Bcl‐2/Bax ratios higher than the median of the group experienced a median PFS of 101 days and dogs with ratios equal and lower than the median had PFS of 130 days (P = .19).Conclusions and clinical importanceHigher intrinsic resistance to apoptosis following cytotoxic treatment might contribute to the less favorable prognosis associated with multicentric TCL in dogs. Whether Bcl‐2/Bax will be helpful to identify canine BCL and TCL with more aggressive and more indolent behavior, respectively, should be evaluated in larger prospective clinical studies.

Caspase activity and apoptotic signaling in proliferating C2C12 cells following cisplatin or A23187 exposure.

Investigating cell death signaling using cell culture is commonly performed to examine the effects of novel pharmaceuticals or to further characterize discrete cellular signaling pathways. Here, we provide data regarding the cell death response to either cisplatin or A23187 in sub-confluent C2C12 cells, by utilizing several concentrations and incubation times for each chemical. These data include an assessment of the activation of the proteolytic enzymes caspase-3, caspase-8, caspase-9, calpain, and cathepsin B/L. Additionally, the expression of the apoptosis-regulating proteins Bax, Bcl2, and p53 are presented.

Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes.

The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B’s proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis.

Effects of iron overload condition on liver toxicity and hepcidin/ferroportin expression in thalassemic mice

AimsAlthough iron-overload conditions can be found in β-thalassemic patients, resulting in cellular damage, particularly in the liver, the mechanism for this iron-mediated hepatic injury specifically in β-thalassemic (HT) mice is unclear. This study aimed to investigate the roles of L-type calcium channels (LTCC), T-type calcium channels (TTCC) and divalent metal transporter1 (DMT1) in iron-mediated hepatic injury in HT mice. Main methodsIron chelator deferoxamine (DFO), LTCC blocker, TTCC blocker and DMT1 blocker were used to determine the roles of these channels regarding liver iron accumulation, apoptosis and iron regulatory protein expression in HT mice. Key findingsTTCC and DMT1 blockers and DFO decreased liver iron and malondialdehyde (MDA) in HT mice indicating their antioxidant effects, whereas LTCC blocker produced no decrease in liver iron or MDA. However, only DFO decreased liver apoptosis through the reduced Bax/Bcl-2 ratio in wild type (WT) mice. The levels of iron regulatory hormone hepcidin were markedly higher in HT mice even before iron loading while ferroportin levels did not alter. Each of the pharmacological interventions increased ferroportin protein back to normal levels only in WT while HT mice showed no difference. SignificanceThalassemic mice have different hepcidin/ferroportin and apoptotic protein expression as a defense mechanism to iron-overload compared with those in WT mice. DFO was the most effective intervention in preventing liver apoptosis under iron-overload conditions in WT but did not have the same effect in HT mice.

Interaction of antihistaminic drugs with human translationally controlled tumor protein (TCTP) as novel approach for differentiation therapy.

Translationally controlled tumor protein (TCTP) represents an exquisite target for cancer differentiation therapy, because it was most strikingly down-regulated in tumor reversion experiments. Since TCTP is identical with the histamine releasing factor, antihistamic drugs may inhibit TCTP. Indeed, antihistaminics, such as promethazine, thioridazine, perphemazine and chlorpromazine reveal antiproliferative effects. The aim of this investigation was to study antihistaminic drugs as new TCTP inhibitors to inhibit tumor growth. Levomepromazine and buclizine showed higher in silico binding affinities to TCTP among 12 different antihistaminic compounds including the control drugs, promethazine and hydroxyzine by using Autodock4 and AutodockTools-1.5.7.rc1. Recombinant human TCTP was codon-optimized, expressed in E. coli and purified by chitin affinity chromatography. For experimental validation of in silico data, we applied microscale thermophoresis. Levomepromazine bound with a Kd of 57.2 μM (p < 0.01) and buclizine with a Kd of 433μM (p < 0.01) to recombinant TCTP. Both drugs inhibited MCF-7 breast cancer cell growth in resazurin assays. TCTP expression was down-regulated after treatment with the two drugs. Cell cycle was arrested in the G1 phase without apoptosis as confirmed by the expression of cell cycle and apoptosis-regulating proteins. Annexin V-PI staining and Trypan blue exclusion assay supported that the two drugs are cytostatic rather than cytotoxic. Induction of differentiation with two drugs was detected by the increased appearance of lipid droplets. In conclusion, levomepromazine and buclizine inhibited cancer cell growth by binding to TCTP and induction of cell differentiation. These compounds may serve as lead compounds for cancer differentiation therapy.

Relative cytotoxic potencies and cell death mechanisms of α1 -adrenoceptor antagonists in prostate cancer cell lines.

Some α1 -adrenoceptor antagonists possess anti-cancer actions that are independent of α1 -adrenoceptors and the aim of these studies was to assess the relative cytotoxic potencies of α1 -adrenoceptor antagonists and the mechanisms involved in these actions. PC-3 and LNCap human prostate cancer cells were exposed to α1 -adrenoceptor antagonists (0.01-100 μM) and cell survival assessed after 24-72 hr. The levels of apoptosis, autophagy and stress related proteins were also determined. The relative cytotoxic potency order was prazosin = doxazosin > terazosin = silodosin = alfuzosin > tamsulosin on both cell types, but LNCaP cells were significantly more sensitive to these effects than PC-3 cells. Prazosin and doxazosin increased levels of apoptotsis and autophagy in both cell lines, and activated EphA2 receptors in PC-3 cells. Autophagy contributed to survival of LNCaP, but promoted cell death in PC-3 cells. Treatment with prazosin (30 μM) altered the expression of several cell stress-related proteins: elevating phospho-p38α and reducing S6 kinase in both cell lines. Surprisingly some proteins were differentially affected in the two prostate cancer cell lines: Akt and p27 increasing and HIF-1α decreasing in LNCap cells but not PC-3, while ADAMTS1 was increased in PC-3 cells only. Prazosin and doxazosin demonstrated cytotoxic actions on both castration-resistant PC-3 and androgen-sensitive LNCap prostate cancer cells. The mechanisms involved included changes in a number of proliferation and apoptosis regulatory proteins. The role of autophagy depended on the cell type, but contributed to cell death in PC3 cells.

Abstract P5-04-02: The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation

Abstract Purpose: Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are the two most aggressive types of breast cancer whose molecular mechanisms remain unclear, representing a challenge for the development of effective targeted therapies. Single agent targeted therapies are of limited effectiveness in these types of breast cancer. Therefore, we aim to identify new combination therapeutic candidates for these aggressive diseases by comprehensive genomic screening. Experimental Design: We screened kinome siRNA libraries with the mitogen/extracellular signal-regulated kinase (MEK) inhibitor [pimasertib], and genome-wide functional mRNA expression with the histone deacetylase (HDAC) type I inhibitor [entinostat] in TNBC and IBC cell lines. We evaluated the relationship between targets of interest and breast cancer patient survival using the IBC consortium database composed of breast cancer patient samples with clinical follow up. After identifying the targets, we assessed the combinational synergistic effect and its mechanism via cytotoxicity assay, flow cytometry, anchorage-independent growth, quantitative real-time polymerase chain reaction, small interfering RNA, western blotting, and mammary fat pad xenograft mouse models. Results: We identified that knock-down of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, enhanced the anti-proliferative effect of pimasertib. We observed that entinostat induced the expression of Noxa, a pro-apoptotic BH3-only member of the Bcl-2 family that is known to bind and degrade Mcl-1. Interestingly, in a breast cancer patient cohort (N = 389), high Mcl-1/low Noxa co-expression was associated with poorer survival outcomes than low Mcl-1/high Noxa co-expression (P = 0.0038). We found that combination with pimasertib and entinostat enhanced the inhibition of tumor cell proliferation (P &amp;lt; 0.001) compared with entinostat or pimasertib alone. We also observed significant in vivo tumor growth inhibition in both IBC (SUM190, P &amp;lt; 0.0001) and TNBC (SUM149, P &amp;lt; 0.05) xenograft models. Specifically, TNBC and IBC cell lines that overexpressed Noxa after treatment with entinostat were observed to be selectively sensitive to combination treatment with pimasertib. The synergistic antitumor activity of the entinostat- pimasertib combination was due to increased expression of Noxa, which induced the degradation of Mcl-1, resulting in the induction of mitochondrial cell death. Conclusion: Our data provide evidence that entinostat has enhanced antitumor effect in combination with pimasertib, resulting in the induction of apoptosis by Noxa-mediated Mcl-1 degradation. These findings provide a novel preclinical rationale for developing a clinical trial based on combinatorial HDAC and MEK inhibition therapy for TNBC and IBC with high Mcl-1 expression. Citation Format: Torres-Adorno AM, Lee JJ, Kogawa T, Bartholomeusz C, Pitner MK, Ordentlich P, Lim B, Tripathy D, Ueno NT. The histone deacetylase inhibitor entinostat enhances the efficacy of the MEK inhibitor pimasertib against aggressive types of breast cancer through Noxa-mediated myeloid cell leukemia 1 degradation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-02.

Quercetin and vitamin E attenuate Bonny Light crude oil-induced alterations in testicular apoptosis, stress proteins and steroidogenic acute regulatory protein in Wistar rats

Studies have shown the reproductive effects of Bonny Light crude oil (BLCO) via the mechanism of oxidative stress and testicular apoptosis. We investigated the protective role of quercetin and vitamin E on BLCO-induced testicular apoptosis. Experimental rats were divided into four groups of four each. Animals were orally administered 2 ml/kg corn oil (control: group 1), BLCO-800 mg/kg body weight + 10 mg/kg quercetin (group 2), BLCO-800 mg/kg body weight + 50 mg/kg vitamin E (group 3) and BLCO-800 mg/kg body weight only (group 4) for 7 d. Protein levels of caspase 3, FasL, NF-kB, steroidogenic acute regulatory protein and stress response proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunofluorescence staining was used to quantify the expression of caspase 3, FasL and NF-kB. Apoptosis was quantified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptosis-related proteins by 50% and above after 7 d following BLCO exposure and a concomitant increase in expression of caspase 3, FasL and NF-kB expression by immunofluorescence staining. Apoptosis showed a significant increase in TUNEL positive cells. Co-administration with quercetin or vitamin E reversed BLCO-induced apoptosis and levels of stress protein, relative to control. These findings suggest that quercetin and vitamin E may confer protection against BLCO-induced testicular oxidative stress-related apoptosis.

Histone modifications as regulators of life and death in Saccharomyces cerevisiae.

Apoptosis or programmed cell death is an integrated, genetically controlled suicide program that not only regulates tissue homeostasis of multicellular organisms, but also the fate of damaged and aged cells of lower eukaryotes, such as the yeast Saccharomyces cerevisiae. Recent years have revealed key apoptosis regulatory proteins in yeast that play similar roles in mammalian cells. Apoptosis is a process largely defined by characteristic structural rearrangements in the dying cell that include chromatin condensation and DNA fragmentation. The mechanism by which chromosomes restructure during apoptosis is still poorly understood, but it is becoming increasingly clear that altered epigenetic histone modifications are fundamental parameters that influence the chromatin state and the nuclear rearrangements within apoptotic cells. The present review will highlight recent work on the epigenetic regulation of programmed cell death in budding yeast.

Identification of key regulators and their controlling mechanism in a combinatorial apoptosis network: a systems biology approach.

An experimentally validated set of apoptosis-regulatory proteins was subjected to network analysis, depicting a scale-free hierarchical fractal network. The power-law distribution of the various topological properties of the network revealed the fractal nature of the network, a signature of self-organization of the network where the network maintained the democratic constitution of nodes at various levels and showed the absence of the centrality-lethality control system. Even though network breakdown under the absence of the centrality-lethality rule of hub removal did not happen, the change in the topological properties of the network could be observed. Depending on the amount of change observed in topological properties, we identified a few proteins (hubs) which could be functionally important in the combinatorial apoptosis gene regulatory network. The crosstalk of these important proteins within the network along with functional modules probably tries to maintain the structural features of the network. NFKB1 was found to be the most efficient signal transducer followed by SP1. In addition, hsa-let-7a controlled the modules independently, revealing its importance in the incoherent and coherent types of feed forward loop motif analysis. The sub-modules and sub-sub-modules predicted bi-fan and multi-layer-perceptron motifs, suggesting the role of multifunctional signals in regulating apoptosis. Finally, SP1, NFKB1 and hsa-let-7a were observed to regulate apoptosis by influencing motifs, signal transduction, and module regulation, which was validated through the removal of hubs, signifying their biological importance in association with cancers.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.