Proteins Puma Research Articles

Myeloid Cell Leukemia 1 Inhibition: An in Silico Study Using Non-equilibrium Fast Double Annihilation Technology.

2017 , 57 , 1240 ]. As an important byproduct, this work constitutes hence one of the first and most challenging tryouts for the GAFF2 parameter set. Agreement with experimental measurements is found to be generally satisfactory, validating the GAFF2 parametrization of the ligands and foreseeing a possible role of FSDAM for industrial application in drug discovery.

Folding and binding pathways of BH3-only proteins are encoded within their intrinsically disordered sequence, not templated by partner proteins

Intrinsically disordered regions are present in one-third of eukaryotic proteins and are overrepresented in cellular processes such as signaling, suggesting that intrinsically disordered proteins (IDPs) may have a functional advantage over folded proteins. Upon interacting with a partner macromolecule, a subset of IDPs can fold and bind to form a well-defined three-dimensional conformation. For example, disordered BH3-only proteins bind promiscuously to a large number of homologous BCL-2 family proteins, where they fold to a helical structure in a groove on the BCL-2–like protein surface. As two protein chains are involved in the folding reaction, and the structure is only formed in the presence of the partner macromolecule, this raises the question of where the folding information is encoded. Here, we examine these coupled folding and binding reactions to determine which component determines the folding and binding pathway. Using Φ value analysis to compare transition state interactions between the disordered BH3-only proteins PUMA and BID and the folded BCL-2–like proteins A1 and MCL-1, we found that, even though the BH3-only protein is disordered in isolation and requires a stabilizing partner to fold, its folding and binding pathway is encoded in the IDP itself; the reaction is not templated by the folded partner. We suggest that, by encoding both its transition state and level of residual structure, an IDP can evolve a specific kinetic profile, which could be a crucial functional advantage of disorder.

Restoring PUMA induction overcomes KRAS-mediated resistance to anti-EGFR antibodies in colorectal cancer.

Intrinsic and acquired resistance to anti-EGFR antibody therapy, frequently mediated by mutant or amplified KRAS oncogene, is a significant challenge in the treatment of colorectal cancer (CRC). However, the mechanism of KRAS-mediated therapeutic resistance is not well understood. In this study, we demonstrate that clinically used anti-EGFR antibodies, including cetuximab and panitumumab, induce killing of sensitive CRC cells through p73-dependent transcriptional activation of the pro-apoptotic Bcl-2 family protein PUMA. PUMA induction and p73 activation are abrogated in CRC cells with acquired resistance to anti-EGFR antibodies due to KRAS alterations. Inhibition of aurora kinases preferentially kills mutant KRAS CRC cells and overcomes KRAS-mediated resistance to anti-EGFR antibodies in vitro and in vivo by restoring PUMA induction. Our results suggest that PUMA plays a critical role in meditating the sensitivity of CRC cells to anti-EGFR antibodies, and that restoration of PUMA-mediated apoptosis is a promising approach to improve the efficacy of EGFR-targeted therapy.

The p53 activator overcomes resistance to ALK inhibitors by regulating p53-target selectivity in ALK-driven neuroblastomas

Anaplastic lymphoma kinase (ALK) is an oncogenic receptor tyrosine kinase that is activated by gene amplification and mutation in neuroblastomas. ALK inhibitors can delay the progression of ALK-driven cancers, but are of limited use owing to ALK inhibitor resistance. Here, we show that resistance to ALK inhibitor in ALK-driven neuroblastomas can be attenuated by combination treatment with a p53 activator. Either ALK inhibition or p53 activator treatment induced cell cycle arrest, whereas combination treatment induced apoptosis, and prevented tumour relapse both in vitro and in vivo. This shift toward apoptosis, and away from cell-cycle arrest, in the presence of an ALK inhibitor and a p53 activator, is mediated by inhibition of the ALK–AKT–FOXO3a axis leading to a specific upregulation of SOX4. SOX4 cooperates with p53 to upregulate the pro-apoptotic protein PUMA. These data therefore suggest a novel combination therapy strategy for treating ALK-driven neuroblastomas.

Regulation of apoptosis by an intrinsically disordered region of Bcl-xL.

Intrinsically disordered regions (IDRs) of proteins often regulate function upon posttranslational modifications (PTMs) through interactions with folded domains. An IDR linking two α-helices (α1–α2) of the anti-apoptotic protein, Bcl-xL, experiences several PTMs, which reduce anti-apoptotic activity. Here, we report that PTMs within the α1–α2 IDR promote its interaction with the folded core of Bcl-xL that inhibits the pro-apoptotic activity of two types of regulatory targets, BH3-only proteins and p53. This autoregulation utilizes an allosteric pathway where, in one direction, the IDR induces a direct displacement of p53 from Bcl-xL coupled to allosteric displacement of simultaneously bound BH3-only partners. This pathway operates in the opposite direction when the BH3-only protein PUMA binds to the BH3 binding groove of Bcl-xL, directly displacing other bound BH3-only proteins, and allosterically remodeling the distal site, displacing p53. Our findings show how an IDR enhances functional versatility through PTM-dependent, allosteric regulation of a folded protein domain.

The pro-apoptotic protein Bmf co-operates with Bim and Puma in neuron death induced by β-amyloid or NGF deprivation

The pro-apoptotic Bcl-2 homology 3 domain only (BH3-only) proteins are central regulators of cell death in various physiological and pathological conditions, including Alzheimer's disease (AD). Bcl-2 modifying factor (Bmf) is one such BH3-only protein that is implicated in various death paradigms such as anoikis, seizures, cancer and autoimmunity. It also co-operates with other BH3-only proteins such as Bim in various death paradigms. However, its role in neurodegeneration is under-investigated. Here, we report for the first time the essential role of Bmf and its co-operativity with direct activator BH3-only proteins Bim and Puma in neuron death induced by beta-amyloid (Aβ) toxicity or NGF deprivation. Oligomeric Aβ is main pathologic species in AD and NGF deprivation is relevant for both developmental as well as pathologic neuron death. We find that Bmf over-expression causes cell death and Bmf knockdown protects neurons against death evoked by Aβ or NGF deprivation. We also find that Bmf co-operates with other important BH3-only proteins such as Bim and Puma in neuron death induced by Aβ or NGF deprivation. Simultaneous knocking down of these molecules by their respective shRNAs provide enhanced protection against Aβ. Taken together, our results elucidate the essential role of Bmf and its co-operative effects with already known neuron death inducers, Bim and Puma, in neuron death evoked by Aβ treatment or NGF deprivation.

Evaluation of PUMA and NOXA expression as predictive biomarkers in prostate cancer.

28 Background: PUMA and NOXA are two pro-apoptotic members of the BH3-only subgroup of the BCL-2 family. These two proteins play a role in the initiation of apoptosis. The objective of this study is to analyse their expression by immunofluorescence, alone or in combination, in benign and tumor prostate tissues to determine if there is a correlation between their expression and patient biochemical recurrence (BCR). Methods: Biomarker antibodies were verified for specificity and optimized by western blot and with tissue microarrays (TMA) containing prostate cancer cell lines and cell line derived xenograft tissues. Subsequently, quantification of expression for both biomarkers was performed on six TMA generated from radical prostatectomy samples (285 patients). The TMA were constructed using two cores of benign adjacent to the tumor and two cores of tumor tissue from each patient. Analysis of biomarker expression was semi-automated using the VisiomorphDP software. To optimize the analysis, we developed 2 different immunofluorescence masks: a cocktail of anti-cytokeratin-8 and -18 antibodies to identify epithelial cells and a combination of anti-p63 and anti-cytokeratin high marker weight to discriminate benign glands within tumor cores. Correlation with patient clinical outcome was determined with SPSS V20 software. Results: There was no correlation of PUMA and NOXA expression and BCR in tumor cores and stroma. In contrast, in benign epithelial cells Kaplan-Meier analysis showed a significant association between an extreme (low or high) PUMA expression and BCR (Log rank = 11.349, p = 0.001). Further analysis revealed a significant association between high NOXA expression in benign epithelial cells and BCR (Log rank = 6.133, p = 0.013). The combination of extreme PUMA and high NOXA identified patients with a poor prognosis (Log rank = 16.041, p = 0.000). In a multivariate Cox regression model, PUMA and NOXA proteins were also identified as independent predictive biomarkers of BCR. Conclusions: By studying benign epithelial cells adjacent to the tumor we identified two potential biomarkers that discriminate high-risk patients, independent of Gleason score or pathologic stage.

Apoptosis induced by methanol extract of Potentilladiscolor in human mucoepidermoid carcinoma cells through STAT3/PUMA signaling axis.

Potentilla discolor has been used in traditional Chinese medicine for the treatment of hyperglycemia. However, the potential role of Potentilla discolor against cancer and its mode of action remain to be fully elucidated. The present study explored the apoptotic effect of methanol extract of Potentilla discolor (MEPD) in human mucoepidermoid carcinoma (MEC) cell lines of salivary glands. MEPD markedly suppressed the growth and induced apoptotic cell death in MC3 and YD15 cells. MEPD treatment significantly upregulated the expression of PUMA and reduced STAT3 phosphorylation. Overexpression of STAT3 partially recovered the growth of MEC cells inhibited by MEPD. In addition, dephosphorylation of STAT3 by cryptotanshinone (a potent STAT3 inhibitor) was sufficient to inhibit the growth of MEC cells and induce apoptosis via affecting PUMA protein. These results suggest that MEPD has a potential anticancer property via the STAT3/PUMA signaling axis in human MEC cells of salivary gland.

CDK9-mediated phosphorylation controls the interaction of TIP60 with the transcriptional machinery.

The acetyltransferase TIP60 is regulated by phosphorylation, and we have previously shown that phosphorylation of TIP60 on S86 by GSK-3 promotes p53-mediated induction of the BCL-2 protein PUMA. TIP60 phosphorylation by GSK-3 requires a priming phosphorylation on S90, and here, we identify CDK9 as a TIP60S90 kinase. We demonstrate that a phosphorylation-deficient mutant, TIP60S90A, exhibits reduced interaction with chromatin, histone 3 and RNA Pol II, while its association with the TIP60 complex subunit EPC1 is not affected. Consistently, we find a diminished association of TIP60S90A with the MYC gene. We show that cells expressing TIP60S90A, but also TIP60S86A, which retains S90 phosphorylation, exhibit reduced histone 4 acetylation and proliferation. Thus, our data indicate that, during transcription, phosphorylation of TIP60 at two sites has different regulatory effects on TIP60, whereby S90 phosphorylation controls association with the transcription machinery, and S86 phosphorylation is regulating TIP60 HAT activity.

Autophagy promotes escape from phosphatidylinositol 3-kinase inhibition in estrogen receptor-positive breast cancer.

Hyperactivation of the PI3K pathway has been implicated in resistance to antiestrogen therapies in estrogen receptor α (ER)-positive breast cancer, prompting the development of therapeutic strategies to inhibit this pathway. Autophagy has tumor-promoting and -suppressing roles and has been broadly implicated in resistance to anticancer therapies, including antiestrogens. Chloroquine (CQ) is an antimalarial and amebicidal drug that inhibits autophagy in mammalian cells and human tumors. Herein, we observed that CQ inhibited proliferation and autophagy in ER+ breast cancer cells. PI3K inhibition with GDC-0941 (pictilisib) induced autophagy. Inhibition of autophagy using CQ or RNA interference potentiated PI3K inhibitor-induced apoptosis. Combined inhibition of PI3K and autophagy effectively induced mitochondrial membrane depolarization, which required the BH3-only proapoptotic proteins Bim and PUMA. Treatment with GDC-0941, CQ, or the combination, significantly suppressed the growth of ER+ breast cancer xenografts in mice. In an antiestrogen-resistant xenograft model, GDC-0941 synergized with CQ to provide partial, but durable, tumor regression. These findings warrant clinical evaluation of therapeutic strategies to target ER, PI3K, and autophagy for the treatment of ER+ breast cancer.-Yang, W., Hosford, S. R., Traphagen, N. A., Shee, K., Demidenko, E., Liu, S., Miller, T. W. Autophagy promotes escape from phosphatidylinositol 3-kinase inhibition in estrogen receptor-positive breast cancer.

Abstract A020: 5-Azacytidine primes AML cells for venetoclax-induced apoptosis by upregulating NOXA and PUMA protein expression

Abstract Acute myeloid leukemia (AML) is a clonal hematologic malignancy characterized by complex heterogeneity and epigenetic features with an incidence of 20,830 new cases in 2015. Older patients, age ≥65 years, define a hard-to-treat population with a median overall survival (OS) of 2-8 months whose treatment options include induction chemotherapy, low-dose-Ara-C, best supportive care, and hypothemylating agents (HMAs). The HMA 5-Azacytidine (5-Aza) is an epigenetic targeting therapy that provides a median OS of 10.4 months in elderly AML patients (≥65 years of age; Dombret et al., 2015; Tamamyan et al., 2017). This antitumorigenic activity can be at least in part attributed to the induction of apoptosis since 5-Aza’s preclinical activity can be limited by members of the antiapoptotic BCL-2 protein family (Bogenberger et al., 2014). Venetoclax (ABT-199) is a highly selective, orally bioavailable BCL-2 inhibitor that is efficacious in numerous preclinical models of hematologic malignancies. A phase II clinical trial in heavily pretreated relapsed and refractory (R/R) AML patients has demonstrated venetoclax is active as a monotherapy (overall response rate (ORR) of 19%; Konopleva et al., 2016), providing the foundation for combinational studies with other antitumorigenic agents in AML. Early phase Ib clinical data in elderly AML patients (≥ 65 year of age) indicate that combining venetoclax with 5-Aza results in an ORR of 60-68% (Pratz et al., 2017). However, the underlying mechanism for the combinational activity observed between venetoclax and 5-Aza is unknown. Herein we report that 5-Aza upregulates the expression of the BH3-only proteins NOXA and PUMA in human AML cell lines. This is associated with an increase in the amount of NOXA and PUMA in complex with antiapoptotic BCL-2 proteins, serving to “prime” AML cells for induction of apoptosis by venetoclax treatment, and rationalizing the significant synergistic cell death observed when a panel of AML cell lines was treated with venetoclax and 5-Aza in vitro. Further, the venetoclax/5-Aza combination provided added benefit over either agent alone in two xenograft models of AML. Collectively these data provide a preclinical basis for an ongoing randomized phase III clinical trial evaluating venetoclax activity in combination with 5-Aza in treatment-naïve AML patients ineligible for standard induction therapy (M15-656, NCT02993523). Citation Format: Sha Jin, Julie Purkal, Relja Popovic, Yu Xiao, Larry Solomon, Erwin Boghaert, Joel Leverson, Darren C. Phillips. 5-Azacytidine primes AML cells for venetoclax-induced apoptosis by upregulating NOXA and PUMA protein expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A020.

BOK promotes chemical-induced hepatocarcinogenesis in mice

BCL-2-related ovarian killer (BOK) is a conserved and widely expressed BCL-2 family member with sequence homology to pro-apoptotic BAX and BAK, but with poorly understood pathophysiological function. Since several members of the BCL-2 family are critically involved in the regulation of hepatocellular apoptosis and carcinogenesis we aimed to establish whether loss of BOK affects diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. Short-term exposure to DEN lead to upregulation of BOK mRNA and protein in the liver. Of note, induction of CHOP and the pro-apoptotic BH3-only proteins PUMA and BIM by DEN was strongly reduced in the absence of BOK. Accordingly, Bok-/- mice were significantly protected from DEN-induced acute hepatocellular apoptosis and associated inflammation. As a consequence, Bok-/- animals were partially protected against chemical-induced hepatocarcinogenesis showing fewer and, surprisingly, also smaller tumors than WT controls. Gene expression profiling revealed that downregulation of BOK results in upregulation of genes involved in cell cycle arrest. Bok-/- hepatocellular carcinoma (HCC) displayed higher expression levels of the cyclin kinase inhibitors p19INK4d and p21cip1. Accordingly, hepatocellular carcinoma in Bok-/- animals, BOK-deficient human HCC cell lines, as well as non-transformed cells, showed significantly less proliferation than BOK-proficient controls. We conclude that BOK is induced by DEN, contributes to DEN-induced hepatocellular apoptosis and resulting hepatocarcinogenesis. In line with its previously reported predominant localization at the endoplasmic reticulum, our findings support a role of BOK that links the cell cycle and cell death machineries upstream of mitochondrial damage.

Sertad1 antagonizes iASPP function by hindering its entrance into nuclei to interact with P53 in leukemic cells

BackgroundAs the important suppressor of P53, iASPP is found to be overexpressed in leukemia, and functions as oncogene that inhibited apoptosis of leukemia cells. Sertad1 is identified as one of the proteins that can bind with iASPP in our previous study by two-hybrid screen.MethodsCo-immunoprecipitation and immunofluorescence were perfomed to identified the interaction between iASPP and Sertad1 protein. Westernblot and Real-time quantitative PCR were used to determine the expression and activation of proteins. Cell proliferation assays, cell cycle and cell apoptosis were examined by flow cytometric analysis.ResultsiASPP combined with Sertad1 in leukemic cell lines and the interaction occurred in the cytoplasm near nuclear membrane. iASPP could interact with Sertad1 through its Cyclin-A, PHD-bromo, C terminal domain, except for S domain. Overexpression of iASPP in leukemic cells resulted in the increased cell proliferation and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertad1 at the same time could slow down the cell proliferation, lead the cells more vulnerable to the chemotherapy drugs, the resistance to chemotherapeutic drug in iASPPhi leukemic cells was accompanied by Puma protein expression. Excess Sertad1 protein could tether iASPP protein in the cytoplasm, further reduced the binding between iASPP and P53 in the nucleus.ConclusionsSertad1 could antagonize iASPP function by hindering its entrance into nuclei to interact with P53 in leukemic cells when iASPP was in the stage of overproduction.

BRAF Inhibitors Amplify the Proapoptotic Activity of MEK Inhibitors by Inducing ER Stress in NRAS-Mutant Melanoma.

Purpose: NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF-MEK-ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected.Experimental Design and Results: BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant melanoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2α and upregulation of the ER stress-related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process.Conclusions: The data presented herein encourage further advanced in vivo and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma. Clin Cancer Res; 23(20); 6203-14. ©2017 AACR.

Ovatodiolide isolated from Anisomeles indica induces cell cycle G2/M arrest and apoptosis via a ROS-dependent ATM/ATR signaling pathways

Ovatodiolide was isolated from the traditional Chinese medicinal herb Anisomeles indica, possesses anti-bacterial and anti-inflammatory properties; however, the anti-cancer activity and its mechanisms have been limitedly reported. This study aimed to examine the effect and molecular action of ovatodiolide in lung cancer cells. Cell cycle distribution and reactive oxygen species (ROS) generation were measured by flow cytometry. Apoptosis was detected by propidium iodide/annexin V staining and TUNEL assay. DNA damage was investigated by comet assay and γ-H2AX staining. Caspase activity was determined using caspase fluorometric kits. Moreover, protein levels were examined by western blot. Ovatodiolide provoked reactive oxygen species generation and DNA damage, as well as inhibited cell growth and induced apoptosis in human lung cancer A549 and H1299 cell lines. DNA damage-related molecules, ATM/ATR and CHK1/CHK2 were activated by ovatodiolide. Moreover, ovatodiolide-mediated G2/M arrest was associated with the decrease of Cyclin B1 and CDC25C levels, and increase of p21WAF1/CIP1 expression. Additionally, ovatodiolide-triggered apoptosis was through both intrinsic and extrinsic pathways characterized by the elevating PUMA, Bax, and DR5 proteins, decreasing Bcl-2 and Mcl-1, and activating caspase-8, caspase-9 and caspase-3. Caffeine, an ATM/ATR inhibitor, rescued ovatodiolide-mediated cell cycle arrest and apoptosis, but not reactive oxygen species generation. Nevertheless, antioxidant N-acetyl-cysteine completely blocked ovatodiolide-mediated molecular events, G2/M arrest, and apoptosis. These observations suggest that ovatodiolide stimulates reactive oxygen species generation, causes oxidative stress and DNA damage; subsequently, provokes DNA damage signaling pathways, eventually leads to block cell cycle at G2/M phase and trigger apoptosis in lung cancer A549 and H1299 cells.

Plasmid pUM505 encodes a Toxin–Antitoxin system conferring plasmid stability and increased Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin–Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET-pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans, indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.

Enhanced Sampling of Intrinsic Structural Heterogeneity of the BH3-Only Protein Binding Interface of Bcl-xL

Antiapoptotic Bcl-xL plays central roles in regulating programed cell death. Partial unfolding of Bcl-xL has been observed at the interface upon specific binding to the pro-apoptotic BH3-only protein PUMA, which in turn disrupts the interaction of Bcl-xL with tumor suppressor p53 and promotes apoptosis. Previous analysis of existing Bcl-xL structures and atomistic molecular dynamics (MD) simulations have suggested that substantial intrinsic structure heterogeneity exists at the BH3-only protein binding interface of Bcl-xL to facilitate its conformational transitions upon binding. In this study, enhanced sampling is applied to further characterize the interfacial conformations of unbound Bcl-xL in explicit solvent. Extensive replica exchange with solute tempering (REST) simulations, with a total accumulated time of 16 μs, were able to cover much wider conformational spaces for the interfacial region of Bcl-xL. The resulting structural ensembles are much better converged, with local and long-range structural features that are highly consistent with existing NMR data. These simulations further demonstrate that the BH3-only protein binding interface of Bcl-xL is intrinsically disordered and samples many rapidly interconverting conformations. Intriguingly, all previously observed conformers are well represented in the unbound structure ensemble. Such intrinsic structural heterogeneity and flexibility may be critical for Bcl-xL to undergo partial unfolding induced by PUMA binding, and likely provide a robust basis that allows Bcl-xL to respond sensitively to binding of various ligands in cellular signaling and regulation.

Apoptosis induced by caffeic acid phenethyl ester in human oral cancer cell lines: Involvement of Puma and Bax activation

ObjectiveCaffeic acid phenethyl ester (CAPE), a natural honeybee product exhibits a spectrum of biological activities including antimicrobial, anti-inflammatory, antioxidant and antitumor actions. The purpose of this research was to investigate the anticancer potential of CAPE and its molecular mechanism in human oral cancer cell lines (YD15, HSC-4 and HN22 cells). DesignTo determine the apoptotic activity of CAPE and identify its molecular targets, trypan blue exclusion assay, soft agar assay, Western blot analysis, DAPI staining, and live/dead assay were performed. ResultsCAPE significantly suppressed transformation of neoplastic cells induced by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) without inhibiting growth. CAPE treatment inhibited cell growth, increased the cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and augmented the number of fragmented nuclei in human oral cancer cell lines. CAPE activated Bax protein causing it to undergo a conformational change, translocate to the mitochondrial outer membrane, and oligomere. CAPE also significantly increased Puma expression and interestingly Puma and Bax were co-localized. ConclusionOverall, these results suggest that CAPE is a potent apoptosis-inducing agent in human oral cancer cell lines. Its action is accompanied by up-regulation of Bax and Puma proteins.

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death.

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway.

Propofol-induced neurotoxicity in hESCs involved in activation of miR-206/PUMA signal pathway

Studies in developing animals have demonstrated that when anesthetic agents, such as propofol, are early administered in life, it can lead to neuronal cell death and learning disabilities. However, the mechanisms causing these effectsremains unknown. A recent report found that propofol could significantly upregulat miR-206 expression in the human ASCs. miR-206 could also induce apoptosis in human malignant cancers. Therefore, in this study, we hypothesized that propofol induces neurotoxicity in human embryonic stem cells (hESCs). hESCs were exposed to propofol (50μM) for 6hr and cell death was assessed using TUNEL staining, and cleaved caspase-3 expression. miR-206 was knocked down using antagomir. PUMA was knocked down using a small interfering RNA. microRNA-206 (miR-206) expression was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). PUMA protein expression was detected using western blot assay. hESCs exposed to propofol showed a significant increase in TUNEL positive cells and cleaved caspase-3 expression, followed by the upregulation of miR-206 and PUMA expression. Targeting PUMA inhibits propofol-induced cell apoptosis; miR-206 knockdown decreased propofol-induced cell apoptosis, cleaved caspase-3 and PUMA expression. Propofol induce s cell death in hESC-derived neurons via activation of miR-206/PUMA signal pathway.