Genes Noxa Research Articles

Potential Anticancer Activity of the Parasol Mushroom, Macrolepiota procera (Agaricomycetes), against the A549 Human Lung Cancer Cell Line.

Mushrooms comprise an unlimited source of active compounds that have beneficial health effects without known negative side effects and can potentially be used as important therapeutic products against cancer, which is the leading cause of death worldwide. In this study we investigated the cytotoxic, antiproliferative, apoptotic, and anti-invasion effects of Macrolepiota procera, which is valued as an edible and medicinal mushroom, on A549 lung cancer cells. The cytotoxic effect of the M. procera extract was determined by using the XTT method. Total RNA was isolated from cells with TRI Reagent to determine the apoptotic effect of the extract, after which complementary DNA was synthesized. Expression profiles of the target genes were determined by quantitative reverse-transcriptase polymerase chain reaction, and protein changes were determined by using Western blotting. We used the TUNEL assay to evaluate the apoptotic effects of the M. procera extract. Effects of M. procera on cell invasion were investigated by using a Matrigel chamber assay. The half-maximal inhibitory concentration of the M. procera extract was determined to be 2 mg/mL against A549 lung cancer cells at 72 hours. According to our results, expression of Cyclin Dl, CDK4, CDK6, Bcl-2, Akt, and NOXA genes significantly decreased and that of Bax, Caspase-3, Caspase-9, PTEN, PUMA, p21, and p53 increased in cells from the dose group compared with their expression in control cells. According to the results of the TUNEL assay, 28 ± 3.6% of cells were apoptotic in the dose group. The M. procera extract also reduced invasion in A549 cancer cells. The results suggest that M. procera has an antiproliferative effect in a dose- and time-dependent manner.

Thiamine deficiency activates hypoxia inducible factor-1α to facilitate pro-apoptotic responses in mouse primary astrocytes.

Thiamine is an essential enzyme cofactor required for proper metabolic function and maintenance of metabolism and energy production in the brain. In developed countries, thiamine deficiency (TD) is most often manifested following chronic alcohol consumption leading to impaired mitochondrial function, oxidative stress, inflammation and excitotoxicity. These biochemical lesions result in apoptotic cell death in both neurons and astrocytes. Comparable histological injuries in patients with hypoxia/ischemia and TD have been described in the thalamus and mammillary bodies, suggesting a congruency between the cellular responses to these stresses. Consistent with hypoxia/ischemia, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α) under physiological oxygen levels. However, the role of TD-induced HIF-1α in neurological injury is currently unknown. Using Western blot analysis and RT-PCR, we have demonstrated that TD induces HIF-1α expression and activity in primary mouse astrocytes. We observed a time-dependent increase in mRNA and protein expression of the pro-apoptotic and pro-inflammatory HIF-1α target genes MCP1, BNIP3, Nix and Noxa during TD. We also observed apoptotic cell death in TD as demonstrated by PI/Annexin V staining, TUNEL assay, and Cell Death ELISA. Pharmacological inhibition of HIF-1α activity using YC1 and thiamine repletion both reduced expression of pro-apoptotic HIF-1α target genes and apoptotic cell death in TD. These results demonstrate that induction of HIF-1α mediated transcriptional up-regulation of pro-apoptotic/inflammatory signaling contributes to astrocyte cell death during thiamine deficiency.

LINCRNA-P21 IS REGULATED BY CYCLIC MECHANICAL STRETCH AND UPREGULATED IN AORTIC ANEURYSMS

Emerging evidence suggests that long noncoding RNAs (lncRNAs) may represent a cellular hub for coordination of cellular processes involved in cardiovascular health and disease. Alterations in the characteristics of vascular smooth muscle cells, triggered largely by mechanical stress, play a critical role in the pathophysiology of vascular diseases. We hypothesized that lncRNAs are involved in the pathogenesis of mechanical stretch-induced changes in human aortic smooth muscle cells (HASMCs). HASMCs were subjected to 20% elongation (1 Hz) for 24 h (Flexcell® FX4000™). Total RNA was extracted with TRIzol. LncRNAs were profiled using the Arraystar Human LncRNA Microarray V3.0. Human aneurysmal and non-aneurysmal aortic samples were collected from patients undergoing aortic arch repair and coronary artery bypass grafting, respectively. Aneurysmal and non-aneurysmal samples were also collected from the aorta of ApoE-/- mice after 4-week infusion of angiotensin II and saline, respectively. Gene expression was quantified via qRT-PCR. For knockdown studies, siLincRNA-p21 and scrambled control were transfected into HASMCs at 10 nM and 5nM final concentration, respectively, using Dharmafect 2 (Dharmacon). Apoptosis was assessed using Annexin V/PI double staining. Of the 30,586 human lncRNAs screened, 580 were differentially expressed (P < 0.05) in the stretched vs. static group. Human long intergenic non-coding RNA-p21 (lincRNA-p21) was significantly upregulated in stretched HASMCs (N = 3, P < 0.05) and human aneurysmal samples (N = 10, P < 0.05). The murine ortholog of lincRNA-p21 was upregulated in aortic tissues from angiotensin-II-infused ApoE-/- mice (N = 3, P < 0.05). Upon silencing of lincRNA-p21 in stretched HASMCs, there was a decrease in total apoptosis (N = 3, P < 0.05) and expression of the p53-downstream genes Bax, Puma, Noxa, and Mdm2 (N = 3, P < 0.05). Our data provides the first transcriptome profile of stretched HASMCs and implicates lincRNA-p21 as a mechanoresponsive regulator of aneurysm formation in mice and humans. This data provides novel insights into the regulatory switches governing aberrant VSMC remodeling, which may have importance in the pathogenesis of aneurysms and hypertension.

The Effects of Silica Nanoparticles on Apoptosis and Autophagy of Glioblastoma Cell Lines.

Silica nanoparticles (SiNPs) are one of the most commonly used nanomaterials in various medical applications. However, possible mechanisms of the toxicity caused by SiNPs remain unclear. The study presented here provides novel information on molecular and cellular effects of SiNPs in glioblastoma LBC3 and LN-18 cells. It has been demonstrated that SiNPs of 7 nm, 5–15 nm and 10–20 nm induce time- and dose-dependent cytotoxicity in LBC3 and LN-18 cell lines. In contrast to glioblastoma cells, we observed only weak reduction in viability of normal skin fibroblasts treated with SiNPs. Furthermore, in LBC3 cells treated with 5–15 nm SiNPs we noticed induction of apoptosis and necrosis, while in LN-18 cells only necrosis. The 5–15 nm SiNPs were also found to cause oxidative stress, a loss in mitochondrial membrane potential, and changes in the ultrastructure of the mitochondria in LBC3 cells. Quantitative real-time PCR results showed that in LBC3 cells the mRNA levels of pro-apoptotic genes Bim, Bax, Puma, and Noxa were significantly upregulated. An increase in activity of caspase-9 in these cells was also observed. Moreover, the activation of SiNP-induced autophagy was demonstrated in LBC3 cells as shown by an increase in LC3-II/LC3-I ratio, the upregulation of Atg5 gene and an increase in AVOs-positive cells. In conclusion, this research provides novel information concerning molecular mechanisms of apoptosis and autophagy in LBC3 cells.

Abstract 5178: CB002, a novel p53 pathway-restoring compound that induces apoptosis through the pro-apototic protein NOXA

Abstract P53 is a transcription factor that becomes stimulated upon genotoxic and cellular stress signals ultimately leading to the transcriptional activation of genes involved in processes such as DNA repair, cell cycle arrest, and apoptosis etc. TP53 is the most commonly mutated tumor suppressor and this is associated with cancer initiation, cancer progression and therapy resistance. P53-targeted therapy has proven to be challenging because direct functional restoration of p53 as a transcription factor has been difficult to achieve. We haven taken a different approach by investigating small molecules that functionally restore the p53-signaling pathway instead of direct p53 targeting. To date there is no approved drug that restores the p53 pathway in cancer cells with mutant p53. Therefore, we performed a high-throughput screen to identify such molecules. We identified a family of small molecules, CB002 and its structural analogs, as candidates for restoration of the p53 pathway in p53-null and mutant p53-expressing cancer cells. CB002 was specific in causing apoptosis in colorectal cancer cells and not in normal human fibroblasts cells as indicated by a Sub-G1 assay. Utilizing the xCELLigence RTCA DP system and CIM-Plate we show that CB002 treatment decreases the ability of colorectal cancer cells to migrate. The ability of CB002 to restore the p53 pathway was validated by studying the activation of p53 target genes p21, DR5, and NOXA in various cancer cell lines and expression of well-established apoptotic markers. Stable knockdown of NOXA completely abrogated the expression of cleaved PARP and reduced Sub-G1 cells, implicating the relevance of NOXA as a key mediator of cell death induction by CB002. We are currently exploring the mechanism by which NOXA induces apoptosis, as preliminary results suggest that degradation of Mcl-1 is not required for CB002 mediated cell death. In addition, we are evaluating the ability of CB002 structural analogs in decreasing the stability of mutant p53 using a larger panel of p53 mutants, as CB002 appears to target R175H p53 mutants. Our data supports the continued investigation of CB002 as a p53 pathway-restoring compound that may be further developed as a cancer therapeutic for p53-null tumors as well as tumors with p53 mutations. Citation Format: Liz J. Hernandez Borrero, Shengliang Zhang, David T. Dicker, Wafik S. El-Deiry. CB002, a novel p53 pathway-restoring compound that induces apoptosis through the pro-apototic protein NOXA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5178. doi:10.1158/1538-7445.AM2017-5178

Gfi1 Transcription Factor Is a Pro-Survival Molecule in Multiple Myeloma Cells

Rational: Multiple myeloma (MM) is the second most common hematologic malignancy and is incurable for most patients. We reported that MM cells induce expression of the transcriptional repressor, Growth independent factor 1 (Gfi1), in bone marrow stromal cells (BMSC) repressing Runx2 gene transcription, and results in prolonged suppression of osteoblast differentiation. Since Gfi1 is an anti-apoptotic factor in other hematologic malignancies, we hypothesized that Gfi1 has an important pro-survival role in MM cells by blocking apoptosis and can attenuate the pro-apoptotic effects of bortezomib.Methods: CD138+ cells isolated from MM patients, healthy donors and human MM cell lines (HMCLs) (H929, JJN3, and MM1.S) were tested for Gfi1 expression levels and the effects of Gfi1 knock down (KD) on MM cell survival by transduction with pLKO.1-puro lentivirus vectors encoding Gfi1 or non-mammalian shRNAs. HMCLs were treated with IL-6, S1P or TNFa or co-cultured with a human BMSC line (SAKA-T) to assess their effects on Gfi1 expression. The anti-apoptotic effects of Gfi1 overexpression (o/e) in MM1.S and H929 were tested by transduction with a pUC2 lentivirus encoding Gfi1 or with the empty vector followed by bortezomib (2 - 5nM) or vehicle (DMSO) treatment for 24 and 48 hours. MTT assays and cleaved caspase 3 protein levels were used to assess cell viability and cell death. Since acetylation of Gfi1 and p53 affects their activity and ability to bind each other, we also characterized HDAC inhibitors (HDACi)-induced changes on p53 enrichment at the Noxa, PUMA and p21 gene promoters by ChIP assays and the effects of acetylation of Gfi1 on its p53 binding capacity in MM cells.Results:We found that Gfi1 is highly expressed, at the mRNA and protein level, in CD138+ cells from MM patients and cell lines than CD138+ cells from normal donors. Gfi1 expression was further increased in MM cells by exogenous IL-6 (5ng/ml) and sphingosine-1-phosphate (S1P) (0.1 µM), but not by TNFα (10 ng/ml). KD of Gfi1 inhibited the growth and induced apoptosis of MM cells, as measured by increased mRNA levels of Bax, PUMA, Noxa, increased cleaved caspase 3 protein levels and decreased protein levels of Mcl-1 and c-Myc. Gfi1 (o/e) in MM cells conferred a survival advantage over their respective empty vector transduced controls as assessed by cell counts and MTT assays. Further, Gfi1 o/e protected MM cells from apoptosis induced by treatment with bortezomib as measured by MTT and cleaved caspase 3 protein levels. Since SphK1 activitycan also prevent apoptosis of MM cells, we next determined if Gfi1 regulated SphK1 in MM cells.CD138+ cells from MM patients had increased SphK1 mRNA levels compared with normal donors, and SphK1 mRNA levels and protein activity were further increased in MM cells by exogenous IL-6 and S1P. Co-culture of MM cells with BMSC also enhanced Gfi1, IL6 (3 fold) and SphK1 (2.5 fold) mRNA levels in MM cells. Importantly, Gfi1 KD in MM cells profoundly downregulated SphK1 mRNA levels and reduced expression of phospho-SphK1, suggesting that Gfi1 enhances MM growth in part via increasing expression and activity of SphK1. Gfi1’s inhibition of apoptosis resulted in part from binding of Gfi1 to p53, which blocked p53’s access to its pro-apoptotic target gene promoters. HDACi treatment resulted in acetylation of Gfi1 and inhibited Gfi1’s suppression of apoptosis by preventing Gfi1-p53 binding and subsequent enrichment of p53 at the Noxa, PUMA and p21 promoters in MM cells.Conclusion: Taken together, our results suggest that Gfi1 may act as a key regulator of MM growth and survival through its regulation of p53 and SphK1 activity, and that targeting Gfi1 may be a novel therapeutic strategy for MM patients. DisclosuresGiuliani:Celgene: Research Funding; Janssen: Research Funding. Roodman:Amgen: Consultancy.

LncRNA H19 confers chemoresistance in ERα-positive breast cancer through epigenetic silencing of the pro-apoptotic gene BIK.

Breast cancer is a common malignancy in women. Acquisition of drug resistance is one of the main obstacles encountered in breast cancer therapy. Long non-coding RNA (lncRNA) has been demonstrated to play vital roles in both development and tumorigenesis. However, the relationship between lncRNAs and the development of chemoresistance is not well established. In the present study, the high expression of lncRNA H19 was identified as a powerful factor associated with paclitaxel (PTX) resistance in ERα-positive breast cancer cells, but not in ERα-negative breast cancer cells. LncRNA H19 attenuated cell apoptosis in response to PTX treatment by inhibiting transcription of pro-apoptotic genes BIK and NOXA. H19 was further confirmed to suppress the promoter activity of BIK by recruiting EZH2 and by trimethylating the histone H3 at lysine 27. Interestingly, our data showed that lncRNA H19 was one of the downstream target molecules of ERα. Altered ERα expression may therefore change H19 levels to modulate the apoptosis response to chemotherapy in breast cancer cells. Our data suggest that the ERα-H19-BIK signaling axis plays an important role in promoting chemoresistance.

Changes in apoptosis-related gene expression and cytokine release in breast cancer cells treated with CpG-loaded magnetic PAMAM nanoparticles

CpG-oligodeoxynucleotide (CpG-ODN) can function as an immune adjuvant. Previously, we showed that stimulation of breast cancer cells with CpG-ODN conjugated with PAMAM dendrimer-coated magnetic nanoparticles (DcMNPs) has induced apoptosis. The aim of the current study was to evaluate the expression levels of some apoptosis-regulating genes in several human breast cancer cells treated with CpG/DcMNPs. Treated MDA-MB231 cells showed an increase in Noxa and Bax gene expression levels, whereas the expression level of Survivin decreased. Similarly, Noxa gene was overexpressed in treated MCF7 cells. In treated SKBR3 cells, a decline in the c-Flip mRNA level was determined. Furthermore, release of cytokines, IL-6, IL-10, and TNF-α, was determined in cell culture supernatants. CpG/DcMNP treatment leads to an increase in the release of IL-6 in MDA-MB231 and SKBR3 cells, whereas release of IL-10 and TNF-α did not change significantly. It is indicated that CpG-ODN may show its cytotoxic effect by regulating the expression of apoptosis-related genes and the release of cytokine in breast cancer cells.

The Evaluation and Comparison of Transcriptionally Targeted Noxa and Puma Killer Genes to Initiate Apoptosis Under Cancer-Specific Promoter CXCR1 in Hepatocarcinoma Gene Therapy.

BackgroundCancerous cells proliferate as fast as possible without a proper surveillance system. This rapid cell division leads to enormous mutation rates, which help a tumor establish.ObjectivesThis study evaluated the potential of inducing apoptosis using Noxa and Puma in a hepatocarcinoma cell line.MethodsThe current study generated two recombinant lentiviruses, pLEX-GCN and pLEX-GCP, bearing Noxa and Puma, respectively. Transduction of both genes to hepatocarcinoma (HepG2) was verified using fluorescent microscopic analysis, western blotting, and quantitative real-time polymerase chain reaction (PCR). To evaluate the potential of Noxa and Puma to initiate apoptosis, a caspase-9 real-time, MTT assay, and a 4’, 6-diamidino-2-phenylindole (DAPI) reagent were performed to stain apoptotic cells.ResultsThe data verified successful transduction to HepG2 and HEK293T. Higher relative expression of Noxa and Puma rather than the untransduced cell line showed these genes are expressed more in HepG2 in comparison to HEK293T. The results of the real-time PCR, MTT assay, and DAPI reagent illustrated that higher cells initiated apoptosis following Puma transduction rather than Noxa.ConclusionsIn this approach, the suicide gene was transferred to transformed cells and ignited apoptosis to exterminate them. Puma is a more potent killer gene and has higher capabilities to start intrinsic apoptosis pathway.

Zinc oxide nanoparticles-induced epigenetic change and G2/M arrest are associated with apoptosis in human epidermal keratinocytes.

As an engineered nanomaterial, zinc oxide nanoparticles (ZnO NPs) are used frequently in biological applications and can make contact with human skin. Here, we systematically investigated the effects of ZnO NPs on non-tumorigenic human epidermal keratinocytes, which were used as a test model for this in vitro study, at the epigenetic and molecular levels. Our results showed that ZnO NPs induced cell cycle arrest at the G2/M checkpoint before the viability of human epidermal keratinocytes was reduced, which was associated with the chromatin changes at the epigenetic level, including increased methylation of histone H3K9 and decreased acetylation of histone H4K5 accompanied by chromatin condensation at 24 hours. The mRNA expression of the methyltransferase genes G9a and GLP was also increased upon treatment with ZnO NPs, and the acetyltransferase genes GCN5, P300, and CBP were downregulated. Reactive oxygen species were found to be more abundant after treatment with ZnO NPs for 6 hours, and DNA damage was observed at 24 hours. Transmission electron microscopy and flow cytometry confirmed that ZnO NPs were absorbed into the cell when they were added to the medium. Apoptotic human epidermal keratinocytes were detected, and the expression of the proapoptotic genes Bax, Noxa, and Puma increased significantly, while the expression of the antiapoptotic gene Bcl-xl decreased 24 hours after exposure to ZnO NPs. These findings suggest that the ZnO NPs induced cell cycle arrest at G2/M, which was associated with epigenetic changes and accompanied by p53-Bax mitochondrial pathway-mediated apoptosis.

Abstract LB-121: DAXX is a novel Notch-1 gene target and biomarker of GSI-sensitivity in ER+ breast cancer

Abstract Breast cancer is a heterogeneous disease that is best treated based on the expressed biomarker profile. Currently, the major clinical challenges are drug resistance and metastatic spread. Cancer stem cells (CSCs) are believed to be responsible for drug resistance and disease progression despite therapy. We previously identified novel biomarkers that could potentially be used with novel therapeutic strategies that target CSCs. We demonstrated in a presurgical window biomarker study of 20 breast cancer patients with ER-positive disease that signaling through Notch receptors mediates expression of 18 genes. Twenty women with ER-positive tumors were treated with 14 days of ET (tamoxifen or letrozole), with the addition of the oral GSI MK-0752 on day 15 (3 days on, 4 days off, 3 days on). Definitive surgery was on day 25. Analysis of biomarkers using microarray and RT-PCR during ET combined with MK-0752 GSI revealed statistically significant modulation of 18 genes: pro-apoptotic DAXX and NOXA; a tumor suppressor LFNG; Notch signaling such as NOTCH1, NOTCH4, HEYL, HES1, and HEY2; as well as proliferation-associated transcripts MIK67, CCND1, CCNA2; stem cell markers RUNX1 and ALDH1; and novel genes such as RICTOR, RPTOR, MMP7, ADAM19, and PgR (Albain et al. Proc SABCS 2014). The goal of the current study was to identify whether Notch1 directly regulated the 18 identified genes. We measured binding of Notch1 to CBF-1 (CSL/RPBJκ) binding sites of the 18 genes using a Chromatin Immunoprecipitation assay (ChIP) to determine whether Notch1 directly regulates the 18 genes. We scanned and designed primers 5 kb upstream and downstream of transcription start sites of the 18 genes and demonstrated that Notch1 was recruited to CBF-1 sites of 16 out of 18 genes. Different distribution of Notch1 binding was observed across all of the genes. Classical Notch targets, HEY1 and HES1 were among the most responsive genes and used as positive controls. Notch1 was found to be highly enriched on CBF-1 regulatory elements for the DAXX and NOXA genes. Binding of Notch1 to DAXX promoter elements increased in response to estrogen deprivation and this increase was attenuated upon GSI treatment. The biological activity of most genes was measured using the mammosphere-forming assay as a surrogate for CSC survival. Estrogen deprivation increased mammosphere-forming efficiency of ER+ breast cancer cells more than 2 fold compared to estrogen treatment. GSI blocked mammosphere formation by 95%-98% in response to anti-estrogen treatment. DAXX expression, was found to be necessary for GSI-mediated blockade of mammosphere formation. ChIP and mammosphere forming data calculated by ANOVA were found to be statistically significant. These results demonstrated that Notch1 is a direct transcriptional regulator of 16 genes identified by the clinical trial and in particular, DAXX, a pro-apoptotic gene that could serve as a cancer stem cell biomarker for anti-Notch therapy in ER+ breast cancer. Supported by a grant from the Breast Cancer Research Foundation. Citation Format: Andrei Zlobin, Debra Wyatt, Jeffrey C. Bloodworth, Susan Hilsenbeck, Suzanne Fuqua, Lucio Miele, Kathy S. Albain, Clodia Osipo. DAXX is a novel Notch-1 gene target and biomarker of GSI-sensitivity in ER+ breast 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 LB-121.

Abstract 3660: ATF4 is a putative tumor suppressor gene in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) is derived from the calcitonin-producing C-cells of the thyroid gland. Oncogenic mutations of the RET proto-oncogene are found in all hereditary MTC and ∼45% of the sporadic cases. However, it is clear that additional genetic lesions contribute to the development of both RET-dependent and RET independent MTC. We analyzed 30 primary medullary thyroid cancer (MTC) tumors by comparative genomic hybridization (CGH) arrays (Agilent 244K) and a subset of those tumors by ultra-high-resolution SNP arrays (Affymetrix SNP6.0). A consistent finding with both arrays was a loss localized to 22q13.1 in 40% of tumors. This region contains the ATF4 gene and loss of this gene was validated by real time PCR. ATF4 is a central mediator of integrated stress response pathway and activates expression of a cohort of downstream target genes known to be involved in cell survival, apoptosis, autophagy and senescence. We hypothesize that the loss of ATF4 expression contributes to MTC progression through mechanisms involving aberrant proliferation and escape from apoptosis. We present several findings to support this. First, ATF4 protein levels were found downregulated in 50% MTC tumor tissues (n = 43) and low ATF4 expression was associated with poor overall survival in MTC patients (HR = 5.015, 95% CI: 1.38-10.64), P = 0.013). A negative correlation was observed between RET and ATF4 protein expression in MTC tumors (r = -0.89, R square = 0.799, p&amp;lt;0.0001). Second, we showed that targeted heterozygote and homozygous deletion of Atf4 in mice causes C cell hyperplasia, a precancerous lesion preceding MTC. Third, ATF4 overexpression inhibits proliferation and survival of MTC derived cell lines. ATF4 decreases RET protein levels by inducing ubiquitination and degradation of activated RET. In contrast, ATF4 depletion upregulates RET protein levels as well as RET autophosphorylation. Finally, combination treatment of tyrosine kinase inhibitor (sunitinib) and eeyarestatin that stimulates ATF4 transcription, induces apoptosis of the MTC cells in a synergistic manner through activation of ATF4 and its target genes NOXA and PUMA as well. These results suggest ATF4 as a potential tumor suppressor gene in MTC. We postulate that inactivation of ATF4 is a central impediment to induction of apoptosis in MTC tumors. Therefore, strategies to upregulate ATF4 during TKI therapy would render tumor cells exquisitely sensitive to stress induced apoptosis, offering a novel synergistic treatment. Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Elizabeth G. Grubbs, Michelle D. Williams, Steven I. Sherman, Robert F. Gagel. ATF4 is a putative tumor suppressor gene in medullary thyroid 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 3660.

17β-Estradiol Protects Skeletal Myoblasts From Apoptosis Through p53, Bcl-2, and FoxO Families.

17β-Estradiol (E2 ) protects several nonreproductive tissues from apoptosis, including skeletal muscle. Previously, we showed that E2 at physiological concentrations prevented apoptosis induced by H2 O2 in skeletal myoblasts, reverting PKCδ, JNK, and p66Shc activation and exerting a beneficial action over mitochondria. Since genomic actions underlying the regulation of nuclear gene transcription are a common property of this steroid, the present work characterizes the transcriptional activity modulated by E2 to exert its antiapoptotic effect. We report that E2 protects skeletal myoblasts against apoptosis induced by H2 O2 modulating p53 and FoxO transcription factors and then their target genes Bcl-2, Bim, Puma, PERP, and MDM2, without affecting Noxa gene. The results presented in this work support the notion that the transcription factors FoxO and p53 coordinate apoptosis in C2C12 cells, and deepens our knowledge about a putative molecular mechanism by which E2 exerts beneficial effects against oxidative stress in skeletal myoblasts. J. Cell. Biochem. 118: 104-115, 2017. © 2016 Wiley Periodicals, Inc.

A new semisynthetic 1-O-acetyl-6-O-lauroylbritannilactone induces apoptosis of human laryngocarcinoma cells through p53-dependent pathway

Initiation of apoptosis is an important event for chemoprevention and chemotherapy of cancer. Naturally derived products had drawn growing attention as lead compounds for anticancer drug discovery. ABL-L, a semisynthetic analogue of natural sesquiterpenoid 1-O-acetylbritannilactone (ABL) isolated from Inula britannica, showed stronger suppression against three solid tumor cell lines with 4–10 fold improvement than ABL. However, its molecular mechanism of cell death induction has still not been determined. The present study evaluated the anticancer efficacy of ABL-L and its biological activities mechanism on human laryngocarcinoma cells HEp-2 in vitro. We found that ABL-L-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest. Typical apoptotic morphological and biochemical features were also observed in treated cells. Furthermore, the levels of the anti-apoptotic Bcl-2, pro-caspase 3/8/9 and poly(ADP-ribose) polymerase PARP decreased, and the level of pro-apoptotic Bax increased. Involvement of the caspase-mediated apoptosis was confirmed using caspase inhibitor Z-VAD-FMK pretreatment. In addition, ABL-L induced a tumor suppressor p53 and its target genes expression p21, fas, noxa and puma. The results of p53 knockdown suggest that caspase-mediated apoptosis induced by ABL-L was in p53-dependent pathway on HEp-2 cells. Our data indicate that the cytotoxicity of the novel semisynthetic analogue ABL-L involved G1 cell cycle arrest and apoptosis via a p53-dependent, caspase-mediated pathway on human laryngocarcinoma cells.

Role of Gfi1 Transcription Factor in Myeloma Cells Growth and Survival

RationalMultiple myeloma (MM) is a plasma cell malignancy that is the second most common hematologic malignancy and is currently incurable for the overwhelming majority of patients. We reported that MM cells induce expression of the transcriptional repressor, Growth independent factor 1 (Gfi1), in bone marrow stromal cells (BMSC) that results in prolonged suppression of osteoblast differentiation by repressing transcription of the Runx2 gene. Since Gfi1 is an anti‐apoptotic factor in other hematologic malignancies, we hypothesized that Gfi1 has an important pro‐survival role in MM cells by blocking apoptosis and regulating MM cell growth and survival.MethodsCD138+ cells isolated from MM patients and healthy donors and human MM cell lines (H929, JJN3, U266, RPMI‐1640 and MM1.S) were tested for Gfi1 expression levels and the effects of Gfi1 knock down (KD) on MM cell survival by transduction with pLKO.1‐puro lentivirus vectors encoding Gfi1 or non‐mammalian shRNAs. Since acetylation of Gfi1 and p53 can affect their activity and ability to bind each other, we also characterized HDAC inhibitor (HDACi)‐induced changes in p53 enrichment at the Noxa, PUMA and p21 gene promoters by ChIP assays and the effects on acetylation of Gfi1 on its p53 binding capacity in MM cells treated with trichostatin A (5 μM) and nicotinamide (10 mM). Gfi1 was immune‐precipitated followed by western blotting with anti‐acetylated lysine or anti‐p53.ResultsWe found that Gfi1 is highly expressed, at the mRNA and protein level, in CD138+ cells from MM patients and MM cell lines compared with CD138+ cells from normal donors. Gfi1 expression was further increased in MM cells by exogenous IL‐6 (5ng/ml) and sphingosine‐1‐phosphate (S1P) (0.1 μM), but not by TNFα (10 ng/ml). KD of Gfi1 inhibited the growth and induced apoptosis of MM cells, as measured by increased mRNA levels of Bax, PUMA, Noxa, increased cleaved caspase 3 protein levels and decreased protein levels of Mcl‐1 and c‐Myc. Gfi1's inhibition of apoptosis resulted in part from binding of Gfi1 to p53 which blocked p53's access to its pro‐apoptotic targets promoters. HDACi treatment inhibited Gfi1's suppression of apoptosis by acetylation of Gfi1 at the K292 residue, which prevented Gfi1‐p53 binding and subsequent enrichment of p53 at Noxa, PUMA and p21 promoters in MM cells. Since SphK1 activity can also prevent apoptosis of MM cells, we next determine if Gfi1 regulated SphK1 in MM cells. CD138+ cells from MM patients showed increased levels of SphK1 mRNA compared with normal donors and SphK1 mRNA levels and protein activity were further increased in MM cells by exogenous IL‐6 and S1P. Further, direct co‐culture of MM cells with BMSC enhanced Gfi1, IL6 (3 fold) and SphK1 (2.5 fold) mRNA levels in MM cells. Importantly, Gfi1 KD in MM cells profoundly downregulated SphK1 mRNA levels and reduced expression of phospho‐SphK1, suggesting that Gfi1 enhances MM growth in part via increasing expression and activity of SphK1.ConclusionTaken together, our results suggest that Gfi1 may act as a key regulator of MM growth and survival through its regulation of p53 and SphK1 activity, and that targeting Gfi1 may be a novel therapeutic strategy for MM patients.Support or Funding InformationVA Merit Review funds to GDR

Generation of Reactive Oxygen Species via NOXa Is Important for Development and Pathogenicity of Mycosphaerella graminicola

The ascomycete fungus Mycosphaerella graminicola (synonym Zymoseptoria tritici) is an important pathogen of wheat causing economically significant losses. The primary nutritional mode of this fungus is thought to be hemibiotrophic. This pathogenic lifestyle is associated with an early biotrophic stage of nutrient uptake followed by a necrotrophic stage aided possibly by production of a toxin or reactive oxygen species (ROS). In many other fungi, the genes CREA and AREA are important during the biotrophic stage of infection, while the NOXa gene product is important during necrotrophic growth. To test the hypothesis that these genes are important for pathogenicity of M. graminicola, we employed an over-expression strategy for the selected target genes CREA, AREA, and NOXa, which might function as regulators of nutrient acquisition or ROS generation. Increased expressions of CREA, AREA, and NOXa in M. graminicola were confirmed via quantitative real-time PCR and strains were subsequently assayed for pathogenicity. Among them, the NOXa over-expression strain, NO2, resulted in significantly increased virulence. Moreover, instead of the usual filamentous growth, we observed a predominance of yeast-like growth of NO2 which was correlated with ROS production. Our data indicate that ROS generation via NOXa is important to pathogenicity as well as development in M. graminicola.

Silica nanoparticles induce endoplasmic reticulum stress response and activate mitogen activated kinase (MAPK) signalling.

Humans may be exposed to engineered silica nanoparticles (SiO2-NPs) but potential adverse effects are poorly understood, in particular in relation to cellular effects and modes of action. Here we studied effects of SiO2-NPs on cellular function in human hepatoma cells (Huh7). Exposure for 24h to 10 and 50μg/ml SiO2-NPs led to induction of endoplasmic reticulum (ER) stress as demonstrated by transcriptional induction of DNAJB9, GADD34, CHOP, as well as CHOP target genes BIM, CHAC-1, NOXA and PUMA. In addition, CHOP protein was induced. In addition, SiO2-NPs induced an inflammatory response as demonstrated by induction of TNF-α and IL-8. Activation of MAPK signalling was investigated employing a PCR array upon exposure of Huh7 cells to SiO2-NPs. Five of 84 analysed genes, including P21, P19, CFOS, CJUN and KSR1 exhibited significant transcriptional up-regulation, and 18 genes a significant down-regulation. Strongest down-regulation occurred for the proto-oncogene BRAF, MAPK11, one of the four p38 MAPK genes, and for NFATC4. Strong induction of CFOS, CJUN, FRA1 and CMYC was found after exposure to 50μg/ml SiO2-NPs for 24h. To analyse for effects derived from up-regulation of TNF-α, Huh7 cells were exposed to SiO2-NPs in the presence of the TNF-α inhibitor sauchinone, which reduced the induction of the TNF-α transcript by about 50%. These data demonstrate that SiO2-NPs induce ER stress, MAPK pathway and lead to inflammatory reaction in human hepatoma cells. Health implications of SiO2-NPs exposure should further be investigated for a risk assessment of these frequently used nanoparticles.

The First-in-Class Alkylating Histone-Deacetylase Inhibitor (HDACi) Fusion Molecule Edo-S101 Exerts Potent Preclinical Activity Against Tumor Cells of Hodgkin Lymphoma (HL) Including Bendamustine-Resistant Clones

Background: The highly unfavorable outcome of patients with recurrent HL, who progress after stem cell transplantation or are ineligible for such procedure make the development of new active agents an impellent medical need in this clinical setting. EDO-S101 is fusion molecule combining the DNA damaging effects of bendamustine (BDM) with the pan-histone deacetylase (HDAC) inhibitor, vorinostat. Given that BDM and HDAC inhibitors are active agents in recurrent HL we investigated the preclinical activity of EDO-S101 in this malignancy.Methods: We assessed the patterns of EDO-S101 cytotoxicity (0.39 to 50 µmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its regulatory effects on genes involved in DNA-damage/repair response, apoptosis and cell cycle checkpoints. As a further model we exploited an L1236 cell clone (R100) selected for resistance (R) to BDM through continuous exposure to increasing concentrations of the agent. R100 cells display a growth pattern indistinguishable from parental L1236 cells when cultured in the presence of BDM (100 µmol/L). Clonal identity of R100 cells with parental L1236 was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions.Results: EDO-S101 induced a significant time- and dose-dependent inhibition of growth and survival in all HL cell lines. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 1.88 µmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 2.06, 2.53, 2.26 and 16.2 µmol/L, respectively. These values were about 10-fold lower than the IC50 of BDM in the same cell lines. While exposure of L1236 cells to EDO-S101 caused cell accumulation in S-phase, qRT-PCR disclosed that cell death was mainly dependent on triggering of apoptosis, as shown by the early (24 hrs) and sustained (48 hrs) upregulation of NOXA, p21 and p27 genes. Data were confirmed by the significant increase (>150%) of Annexin V-expressing L1236 cells. In contrast, expression levels of PLK1, AKA and cyclin B1 genes remained unchanged or were increased. This excluded induction of the mitotic catastrophe (MC) as a major determinant of cytotoxic activity for EDO-S101 in L1236 cells. Exposure to EDO-S101 induced a strong DNA stress/repair response as shown by the activation of pATR/pATM and increase of the downstream DNA damage checkpoint proteins pCHK1-/-2 and CCNB1, along with the upregulation of the EXO1 gene. Most intriguingly, BDM-resistant L1236 cells (R100) were highly sensitive to EDO-S101, with an IC50 of only 4.56 µmol/L, but less responsive to vorinostat (IC50: 6.17 µmol/L) than parental L1236 cells (IC50: 0.58 µmol/L). Differently from native cells, EDO-S101 induced a late downregulation of transcripts for PLK1 and AKA genes and of cyclin B1 gene and protein in R100 cells, along with the early induction of NOXA and p21, but not p27 genes. In both L1236 and R100 cells, expression of MC-genes was unaffected by exposure to vorinostat. This suggests a more complex mechanism for EDO-S101 in BDM-resistant HL cells involving activation of the both apoptotic and MC pathways. Notably, we documented that EDO-S101 corrected the constitutive ATM/ATR unbalance of R100 cells by triggering the early (24 hrs) upregulation of ATR and a late (48 hrs) downregulation of ATM transcripts and proteins, along with increased levels of EXO1 and MGMT at 24 hrs. Vorinostat induced a similar effect. Finally, while baseline expression levels of HDAC isoforms were comparable among HL cell lines, EDO-S101 caused a significant (>40%) late downregulation of transcripts for all HDAC isoforms (HDAC-1 to -8) in R100 cells but only of HDAC-6 in native L1236. This pattern diverged from results obtained in both L1236, i.e. increase of all HDAC isoform transcripts except HDAC-6, and R100 cells, i.e. upregulation of all isoforms and reduction of HDAC-6, with vorinostat and BDM as single agents.Conclusions: We have described for the first time that EDO-S101 is effective in preclinical models of HL including cells resistant to BDM. The combined functions of in one molecule of a bifunctional alkylator and panHDAC inhibitor confer this agent unique antitumor property different from both of its single drug components. Following a strong DNA damage response, triggering of apoptosis and/or MC may take place in HL cells according to their sensitivity status to BDM. A phase 1/2 study in recurrent HL, including patients pretreated with BDM, is next to be launched. DisclosuresMehrling:4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Pinto:Takeda, Celgene, Roche, TEVA: Honoraria; Takeda: Research Funding.

Abstract C178: TAS4464, a novel NEDD8 activating enzyme inhibitor, potently induces cell death via both intrinsic and extrinsic apoptotic pathways in acute myeloid leukemia

Abstract BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by numerous genetic mutations and chromosomal abnormalities. Current chemotherapies have shown limited efficacy in AML patients, most of whom develop chemoresistance and relapse repeatedly. Because the expression of anti-apoptotic proteins reportedly correlates with poor prognosis in AML, the modulation of apoptosis-related proteins emerges as a promising treatment strategy. Apoptosis occurs mainly via two signaling pathways. In the mitochondria-mediated intrinsic apoptotic pathway, the release of cytochrome c triggers caspase-9 activation, which subsequently activates effector caspases (e.g., caspase-3). In the death receptor-mediated extrinsic apoptotic pathway, the binding of death receptor ligands to their receptors activates caspase-8 and, in turn, effector caspases, as in the intrinsic pathway. NEDD8 activating enzyme (NAE) inhibitors, which act on the NEDD8 conjugation pathway, induce quantitative changes in the substrates of E3 cullin-RING ligases (CRLs) and cause apoptosis in AML. Here, we describe the mechanism by which our highly potent and selective NAE inhibitor TAS4464 promotes apoptosis in AML. MATERIALS AND METHODS: Intracellular ATP levels were measured to assess in vitro cell growth. The effects of TAS4464 on NEDD8 conjugation, the protein levels of CRL substrates, and the activation of various caspases were evaluated by means of Western blotting. The transcription levels of the Noxa and c-FLIP genes were assessed by using qRT-PCR. The antitumor activity and in vivo pharmacodynamic activity of TAS4464 were evaluated in a THP-1 xenograft model. RESULTS: TAS4464 led to cell growth arrest and cell death in AML cell lines with various genetic backgrounds, including the MLL-AF9 fusion gene and FLT3-ITD mutation. TAS4464 activated both caspase-9 (intrinsic apoptotic pathway) and caspase-8 (extrinsic apoptotic pathway); combined treatment with inhibitors of these caspases markedly diminished the TAS4464-associated induction of apoptosis. By analyzing apoptosis-related factors in each apoptotic pathway, we found that TAS4464 increased the pro-apoptotic factor Noxa protein (intrinsic apoptotic pathway) and decreased the anti-apoptotic factor c-FLIP protein (extrinsic apoptotic pathway) with changes evident at the mRNA transcriptional level. In addition, treatment with TAS4464 led to accumulation of the CRL substrate c-Myc, and c-Myc knockdown by siRNA ablated the TAS4464-induced decrease in cFLIP protein. Intravenous administration of TAS4464 on day 1, 3 and 5 for 3 weeks resulted in tumor growth inhibition (TGI) of 100% including complete response in human THP-1 xenograft model, compared with twice-weekly administration of cytarabine that resulted in a TGI of only 6%. Pharmacodynamics analysis revealed that TAS4464 inhibited cullin neddylation and induced activation of caspases in THP-1 xenografted tumor. CONCLUSION: TAS4464 exerts a strong apoptosis-inducing effect in AML cell lines via both intrinsic and extrinsic apoptotic pathway by modulating apoptosis-related proteins. In addition, TAS4464 demonstrates marked antitumor activity in the THP-1 xenograft model. Given its potent preclinical activities, TAS4464 is a promising agent for treating AML. Citation Format: Hiroaki Ochiiwa, Chihoko Yoshimura, Hiromi Muraoka, Keiji Ishida, Tomonori Haruma, Shingo Tsuji, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel NEDD8 activating enzyme inhibitor, potently induces cell death via both intrinsic and extrinsic apoptotic pathways in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C178.

Overexpression of iASPP-SV in glioma is associated with poor prognosis by promoting cell viability and antagonizing apoptosis

Inhibitor of apoptosis-stimulating protein of p53 (iASPP), encoded by PPP1R13L gene, is often overexpressed in human cancers. From the PPP1R13L gene, at least two isoforms, iASPP-L and iASPP-SV, are produced through alternative splicing. However, the role of these isoforms in glioma is still elusive. In this study, we examined the expression of iASPP-SV in astrocytic glioma tissues with different grades and normal human cerebral tissues. The result showed a higher messenger RNA (mRNA) expression level of iASPP-SV in astrocytic glioma patients with World Health Organization (WHO) grade II to IV in comparison to the normal controls. Additionally, mRNA expression level of iASPP-SV was gradually increased with the raise of the grade in glioma. High mRNA expression level of iASPP-SV was significantly associated with malignant WHO grades (P < 0.001). The protein expression level of iASPP-SV was consistent with the mRNA expression level. The Kaplan-Meier analysis revealed that high iASPP-SV mRNA expression significantly affected overall survival and progression-free survival (both P < 0.001). Furthermore, multivariate analysis indicated that the mRNA expression of iASPP-SV was an independent prognostic marker in glioma (P < 0.001). To further explore the role of iASPP-SV in glioma, U87 cells were transfected with iASPP-SV by lentivirus and then treated with temozolomide (TMZ). Overexpression of iASPP-SV promoted the cell viability and downregulated the expression of pro-apoptosis genes (Bax, Puma, p21, and Noxa) to inhibit apoptosis induced by TMZ. Our study provides the first evidence that high iASPP-SV expression may be a novel prognostic factor and therapeutic target for glioma.