Anti-apoptotic Protein Survivin Research Articles

Cell number in mesenchymal stem cell aggregates dictates cell stiffness and chondrogenesis

BackgroundAlthough mesenchymal stem/stromal cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we inquired if the initial number of cells forming the aggregate (aggregate cell number (ACN)) can impact chondrogenesis.MethodsAggregates of varying ACN were formed using well-established centrifugation approach. Progression of chondrogenic differentiation in the aggregates was assessed over 3 weeks in presence and absence of transforming growth factor-beta 1 (TGF-β1). Mechanical properties of the cells were characterized using high-throughput real-time deformability cytometry (RT-DC), and gene expression was analyzed using Affymetrix gene array. Expression of molecular markers linked to chondrogenesis was assessed using western blot and immunofluorescence.ResultsReducing ACN from 500 k to 70 k lead to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, which involves changes to β-catenin-dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. RT-DC analysis revealed that stiffness and size of cells within aggregates are modulated by ACN. A direct correlation between progression of chondrogenesis and emergence of stiffer cell phenotype was found. Affymetrix gene array analysis revealed a downregulation of genes associated with lipid synthesis and regulation, which could account for observed changes in cell stiffness. Immunofluorescence and western blot analysis revealed that increasing ACN upregulates the expression of lipid raft protein caveolin-1, a β-catenin binding partner, and downregulates the expression of N-cadherin. As a demonstration of the relevance of these findings in MSC-based strategies for skeletal repair, it is shown that implanting aggregates within collagenous matrix not only decreases the necessity for high cell numbers but also leads to marked improvement in the quality of the deposited tissue.ConclusionsThis study presents a simple and donor-independent strategy to enhance the efficiency of MSC chondrogenic differentiation and identifies changes in cell mechanics coincident with MSC chondrogenesis with potential translational applications.

The feline calicivirus leader of the capsid protein causes survivin and XIAP downregulation and apoptosis

Calicivirus infection causes intrinsic apoptosis, leading to viral propagation in the host. During murine norovirus infection, a reduction in the anti-apoptotic protein survivin has been documented. Here we report that in feline calicivirus infection, a downregulation of the anti-apoptotic proteins survivin and XIAP occur, which correlates with the translocation of the pro-apoptotic protein Smac/DIABLO from the mitochondria to the cytoplasm and the activation of caspase-3. Inhibition of survivin degradation by lactacystin treatment caused a delay in apoptosis progression, reducing virus release, without affecting virus production. However, the overexpression of survivin caused a negative effect in viral progeny production. Overexpression of the leader of the capsid protein (LC), but not of the protease-polymerase NS6/7, results in the downregulation of survivin and XIAP, caspase activation and mitochondrial damage. These results indicate that LC is responsible for the induction of apoptosis in transfected cells and most probably in FCV infection.

Deguelin induces apoptosis in colorectal cancer cells by activating the p38 MAPK pathway.

ObjectivesDeguelin, a rotenoid extracted from Mundulea sericea (Leguminosae), exhibits antitumor effects on several types of human cancers. Due to the limited studies of deguelin on colorectal cancer (CRC), the present study was designed to investigate the antitumor effect of deguelin and to explore the underlying mechanism in CRC.Materials and methodsCell viability was assessed by the cell counting kit-8 (CCK-8) assay, and cell apoptosis was determined by the annexin v-propidium iodide staining using flow cytometry and Western blot in CRC cell lines after incubation with deguelin. The antitumor effect of deguelin was further evaluated in tumor xenograft models. Moreover, SB203580, a specific inhibitor of p38 MAPK, was used to confirm the involvement of p38 MAPK pathway in deguelin-induced apoptosis.ResultsDeguelin significantly inhibited cell proliferation and induced apoptosis in CRC cell lines (SW620 and RKO) in a time-dependent and dose-dependent manner. Western blot analysis also showed that the expression of proapoptotic proteins (cleaved caspase 3 and cleaved PARP) was upregulated, while that of antiapoptotic proteins (Bcl-2 and survivin) was downregulated after deguelin treatment in CRC cell lines. Moreover, oral administration of deguelin significantly suppressed tumor growth and induced apoptosis in subcutaneous xenograft mouse models without obvious toxicity. Additionally, Western blot revealed that deguelin-induced apoptosis might be regulated by the p38 MAPK pathway and inhibition of p38 MAPK could attenuate deguelin-induced proliferative inhibition and apoptosis in CRC cells.ConclusionCollectively, these results demonstrated that deguelin inhibited CRC cell growth by inducing apoptosis via activation of p38 MAPK pathway.

Targeting genotoxic and proteotoxic stress‐response pathways in human prostate cancer by clinically available PARP inhibitors, vorinostat and disulfiram

Castration-resistant prostate cancer (PCa) represents a serious health challenge. Based on mechanistically-supported rationale we explored new therapeutic options based on clinically available drugs with anticancer effects, including inhibitors of PARP1 enzyme (PARPi), and histone deacetylases (vorinostat), respectively, and disulfiram (DSF, known as alcohol-abuse drug Antabuse) and its copper-chelating metabolite CuET that inhibit protein turnover. Drugs and their combination with ionizing radiation (IR) were tested in various cytotoxicity assays in three human PCa cell lines including radio-resistant stem-cell like derived cells. Mechanistically, DNA damage repair, heat shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and immunoblotting. We observed enhanced sensitivity to PARPi/IR in PC3 cells consistent with lower homologous recombination (HR) repair. Vorinostat sensitized DU145 cells to PARPi/IR and decreased mutant p53. Vorinostat also impaired HR-mediated DNA repair, as determined by Rad51 foci formation and downregulation of TOPBP1 protein, and overcame radio-resistance of stem-cell like DU145-derived cells. All PCa models responded well to CuET or DSF combined with copper. We demonstrated that DSF interacts with copper in the culture media and forms adequate levels of CuET indicating that DSF/copper and CuET may be considered as comparable treatments. Both DSF/copper and CuET evoked hallmarks of UPR in PCa cells, documented by upregulation of ATF4, CHOP and phospho-eIF2α, with ensuing heat shock response encompassing activation of HSF1 and HSP70. Further enhancing the cytotoxicity of CuET, combination with an inhibitor of the anti-apoptotic protein survivin (YM155, currently undergoing clinical trials) promoted the UPR-induced toxicity, yielding synergistic effects of CuET and YM155. We propose that targeting genotoxic and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration-resistant PCa.

Paeonol attenuates isoflurane anesthesia-induced hippocampal neurotoxicity via modulation of JNK/ERK/P38MAPK pathway and regulates histone acetylation in neonatal rat

Objective: Volatile anesthetic such as isoflurane causes widespread neurodegeneration in the developing animal brains and also induces cognitive impairments. Paeonol is a plant-derived phenolic compound possessing numerous bioactive properties. The study investigates the neuroprotective effects of paeonol against isoflurane-induced neurodegeneration and cognitive disturbances in neonatal rats.Methods: Paeonol (50, 100, and 150 mg/kg body weight/day) was given orally to separate groups of neonatal rats from postnatal day 3 (P3) to P21 and were exposed to isoflurane (0.75%; 6 h) on P7.Results: Neuroapoptosis following isoflurane exposure was remarkably reduced by paeonol. Isoflurane-induced elevated cleaved caspase-3, Bad, and Bax expression, were down-regulated on paeonol administration. Paeonol significantly enhanced expression of antiapoptotic proteins (Bcl-2, Bcl-xL, xIAP, c-IAP-1, c-IAP-2, and survivin) and improved acetylation of HK39 and HK412. The expression of histone deacetylases (HDACs)—HDAC2 and HDAC-3 were down-regulated. Isoflurane-induced activation of JNK/p38MAPK signaling and suppressed ERK signaling and were effectively regulated by paeonol. General behavior and freezing responses of the rats were improved. Results of the Morris Water Maze tests revealed improved learning and memory retention on paeonol treatment.Conclusions: Paeonol effectively inhibited neuroapoptosis and improved isoflurane-induced cognitive dysfunctions via regulating histone acetylation and JNK/ERK1/2/p38MAPK signaling pathways.

Adaptation to chronic exposure to sepantronium bromide (YM155), a prototypical survivin suppressant is due to persistent DNA damage-response in breast cancer cells.

Sepantronium bromide (YM155), originally developed against the anti-apoptotic protein survivin, performed exceptionally well in pre-clinical and phase I clinical trials. However, in phase II trials of several cancer types including breast cancer it performed poorly. Additionally, no definitive correlation between survivin level and response to therapy was found. In an attempt to understand the true reason of the late-stage failure of this promising drug, we developed YM155-resistant MCF-7 breast cancer cell line and characterized side-by-side with the drug-naïve parental cell line. Chronic YM155 treatment resulted in downregulation of survivin expression yet triggered cellular responses typical of adaptation to persistent DNA damage. Lowering endogenous antioxidant glutathione level and activity of cell cycle check-point kinase restored YM155 activity. Thus, contrary to its development as a survivin suppressant, YM155 primarily acts as a chemotherapeutic drug causing oxidative stress-mediated DNA damage. Adaptation to long-term exposure to YM155 can be prevented and/or overcome by interfering with detoxification and DNA damage-response pathways. Finally, proteins associated with DNA damage-response pathway will be more appropriate as predictive biomarkers of YM155 in breast tumor cells.

Anlotinib induces hepatocellular carcinoma apoptosis and inhibits proliferation via Erk and Akt pathway

Although anlotinib, a multi-targeted receptor tyrosine kinase inhibitor has been reported have antitumor effects in many preclinical and clinical trials, little is known about its effect on hepatocellular carcinoma (HCC). Here, we have shown the antitumor effects of anlotinib on HCC. Data indicated that anlotinib application significantly inhibited HCC cell viability, proliferation, colony formation, and prompted apoptosis in vitro. Furthermore, animal experiments also illustrated that anlotinib alleviated HCC progression. Mechanically, we demonstrated that anlotinib treatment downregulated the anti-apoptotic protein Bcl-2 and Survivin, but upregulated pro-apoptotic molecule Bax, which accounts for its therapeutic effect on HCC. Pathway analysis has shown decreased phosphorylation levels of Erk and Akt. Together, this study suggests that anlotinib may have a direct antitumor progression effect on HCC by inhibiting Bcl-2 and Survivin expression, promoting Bax expression via inactivating Erk and Akt pathways and could be a promising agent treating HCC.

In silico analysis of molecular interactions between the anti-apoptotic protein survivin and dentatin, nordentatin, and quercetin

Survivin is a member of the inhibitor of apoptosis (IAP) family and is reportedly overexpressed in various types of human malignancies. Because the phytochemical compounds dentatin, nordentatin, and quercetin have demonstrated antiproliferative effects in various cancer cell lines, we compared their binding affinities for survivin in silico. Molecular docking analyses were performed using PyMol, Discovery Studio Biovia 2017, AutoDock Vina, and AutoDock Tools version 1.5.4. These computations indicated greater survivin binding affinity of quercetin (ΔG −7.0 kcal/mol) than nordentatin and dentatin (ΔG −6.5 and −5.5 kcal/mol, respectively), but suggest that all three compounds act as ligand inhibitors of survivin. The present data warrant validation using in vitro and in vivo assays.

Optical Biosensing System for the Detection of Survivin mRNA in Colorectal Cancer Cells Using a Graphene Oxide Carrier-Bound Oligonucleotide Molecular Beacon.

The anti-apoptotic protein survivin is one of the most promising cancer biomarkers owing to its high expression in human cancers and rare occurrence in normal adult tissues. In this work, we have investigated the role of supramolecular interactions between a graphene oxide (GO) nanosheet nanocarrier and a survivin molecular beacon (SurMB), functionalized by attaching fluorophore Joe and quencher Dabcyl (SurMB-Joe). Molecular dynamics simulations revealed hydrogen bonding of Joe moiety and Dabcyl to GO carriers that considerably increase the SurMB-GO bonding strength. This was confirmed in experimental work by the reduced fluorescence background in the OFF state, thereby increasing the useful analytical signal range for mRNA detection. A new mechanism of hairpin–hairpin interaction of GO@SurMB with target oligonucleotides has been proposed. A low limit of detection, LOD = 16 nM (S/N = 3), has been achieved for complementary tDNA using GO@SurMB-Joe nanocarriers. We have demonstrated an efficient internalization of SurMB-Joe-loaded GO nanocarriers in malignant SW480 cells. The proposed tunability of the bonding strength in the attached motifs for MBs immobilized on nanocarriers, via structural modifications, should be useful in gene delivery systems to enhance the efficacy of gene retention, cell transfection and genomic material survivability in the cellular environment.

Abstract 5803: Homoharringtonine, a natural protein synthesis inhibitor, inhibits growth of triple negative breast cancer in vitro and in vivo

Abstract Introduction: Triple negative breast cancer (TNBC) is a heterogeneous disease for which new therapeutic approaches are urgently needed. Homoharringtonine (HHT) is a protein synthesis inhibitor clinically approved in chronic myeloid leukemia. Its multi-factor mechanism of action includes downregulation of short-lived cellular proteins like Mcl-1. As increased protein synthesis lies behind several hallmarks of cancer present in TNBC, we investigated the effect of HHT on TNBC in vitro and in vivo. Methods: TNBC cell lines CAL-51, MDA-MB-231 and MDA-MB-468, representative of aggressive, BRCA1 non-mutated TNBC (Lehmann, J Clin Invest 2011) were used. HHT was provided by LeukePharma, Houston, TX, USA. Cell viability was assessed by sulphorhodamine B assay. Number of cells in different cell cycle phases or in apoptosis was determined by flow cytometry. Protein expression was evaluated by western blot. For in vivo experiments, xenografts of MDA-MB-231 were established in Swiss nu/nu mice, treated for 7 days by subcutaneous HHT (1 mg/kg, bi-daily), and analyzed on day 10. Evaluation of HHT on MDA-MB-468 and CAL-51 xenografts is ongoing. Results: Already after 24h of exposure to 100 ng/ml HHT (mean plasma HHT concentration after 5 mg/m2/day in humans (Sentenac S et al, Blood 2003)), viability of CAL-51 and MDA-MB-468 cells was strongly reduced (10.4±1.0% and 19.2±2.2% of control, respectively). This inhibition level was obtained in MDA-MB-231 cells only after 72h (18.7±4.8%). After 48h in 100 ng/ml HHT, MDA-MB-231 and -468 cells accumulated in the S phase of the cell cycle, whereas the CAL-51 accumulated in the G1/0 phase. This was associated with an increase of apoptosis in CAL-51 (42.0±2.4% vs 9.2±1.3%, untreated vs treated cells, respectively) and in MDA-MB-468 (24.2±2.8% vs 12.2±3.6%) but not in MDA-MB-231 (9.0±2.2% vs 3.6±1.7%). Concordantly, 100 ng/ml of HHT induced, after only 2h, a marked decrease of the level of anti-apoptotic proteins Mcl-1, Bcl-2, survivin and XIAP in CAL-51 and MDA-MB-468 cells, which preceded by 2h the reduction of caspase-3 level. No change in expression of those proteins was observed in MDA-MB-231 during all 48h of exposure to HHT. On the other side, the quantity of phosphorylated S6 increased in MDA-MB-231 but decreased in CAL-51 and MDA-MB-468 already after 24h of incubation with 100 ng/ml HHT. In vivo, HHT reduced growth of MDA-MB-231 xenografts for 36.5% (tumor volume on day 10: 230.4±27.7% mm3 vs 146.2±15.5% mm3, untreated vs treated, respectively). Conclusion: HHT exerts cytotoxic action on TNBC cell lines CAL-51 and MDA-MB-468 whereas its effect on MDA-MB-231 is dominantly cytostatic. Because of its capacity to rapidly reduce the levels of anti-apoptotic proteins, HHT is worth further studies as potential sensitizer of TNBC to chemo- and/or targeted therapy. Citation Format: Mohamad Yakhni, Arnaud Briat, Elisabeth Miot-Noirault, Florent Cachin, Frederique Penault-Llorca, Nina Radosevic-Robin. Homoharringtonine, a natural protein synthesis inhibitor, inhibits growth of triple negative breast cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5803.

Abstract 1863: Ursolic acid sensitizes rhTRAIL-resistant triple negative breast carcinoma to rhTRAIL-induced apoptosis

Abstract Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) possesses the ability to induce apoptosis in cancer cells independent of their p53 status while exhibiting minimal toxicity to normal, non-transformed cells, and thus, it is a promising anti-cancer therapeutic. However, rhTRAIL-induced apoptosis is not as effective in a majority of breast cancers due to the up-regulation of anti-apoptotic proteins, down-regulation of pro-apoptotic proteins, and/or down-regulation of death receptors (DRs) 4 and 5. A combinatorial approach of rhTRAIL with the “mother nature”-derived compound ursolic acid (UA) has been applied to sensitize rhTRAIL-resistant triple negative breast carcinoma. UA is derived from the leaves and berries of various plants and found in the coatings of fruits and does not exhibit toxicity to normal, non-transformed cells. UA has been revealed to possess the ability to up-regulate DR5 and diminish the expression of anti-apoptotic proteins survivin and FLIP in cancer cells and thereby, making UA an encouraging choice to be utilized as a sensitizing agent. The aim of this study was to determine the capacity of UA to sensitize rhTRAIL-resistant triple negative breast cancer (TNBC) BT-20 and HCC1937 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanisms for UA's sensitization. The combinatorial treatment of UA and rhTRAIL augmented the induction of apoptosis when compared to single agent UA and rhTRAIL treatments as detected by Annexin V/PI assays and through by the execution of the extrinsic pathway as marked by the activation of caspase 8, activation of the executioner caspases 3 and 7, and eventual PARP cleavage (a hallmark of apoptosis). The underlying mechanisms for UA's sensitization of rhTRAIL-resistant TNBCs were established to be through the down-regulation of the anti-apoptotic protein FLIP and through the up-regulation of DR4 and DR5. Overall, these findings reveal that UA is an efficacious sensitizing agent for rhTRAIL-resistant TNBCs. Citation Format: Jasmine Manouchehri, Michael Kalafatis. Ursolic acid sensitizes rhTRAIL-resistant triple negative breast carcinoma to rhTRAIL-induced apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1863.

Chloroquine in combination with aptamer-modified nanocomplexes for tumor vessel normalization and efficient erlotinib/Survivin shRNA co-delivery to overcome drug resistance in EGFR-mutated non-small cell lung cancer

Although novel molecular targeted drugs have been recognized as an effective therapy for non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) activating mutations, their efficacy fails to meet the expectation due to the acquired resistance in tumors. Up-regulation of the anti-apoptotic protein Survivin was shown to contribute to the resistance to EGFR tyrosine kinase inhibitors (TKI) in EGFR mutation–positive NSCLC. However, the unorganized tumor blood vessels impeded drug penetration into tumor tissue. The resulting insufficient intracellular drug/gene delivery in drug-resistant cancer cells remarkably weakened the drug efficacy in NSCLC. In this work, a multi-functional drug delivery system AP/ES was developed by using anti-EGFR aptamer (Apt)-modified polyamidoamine to co-deliver erlotinib and Survivin-shRNA. Chloroquine (CQ) was used in combination with AP/ES to normalize tumor vessels for sufficient drug/gene delivery to overcome drug resistance in NSCLC cells. The obtained AP/ES possessed desired physicochemical properties, good biostability, controlled drug release profiles, and strong selectivity to EGFR-mutated NSCLC mediated by Apt. CQ not only enhanced endosomal escape ability of AP/ES for efficient gene transfection to inhibit Survivin, but also showed strong vessel-normalization ability to improve tumor microcirculation, which further promoted drug delivery and enhanced drug efficacy in erlotinib-resistant NSCLC cells. Our innovative gene/drug co-delivery system in combination with CQ showed a promising outcome in fighting against erlotinib resistance both in vitro and in vivo. This work indicates that normalization of tumor vessels could help intracellular erlotinib/Survivin-shRNA delivery and the down-regulation of Survivin could act synergistically with erlotinib for reversal of erlotinib resistance in EGFR mutation-positive NSCLC. Statement of SignificanceNSCLC patients who benefited from EGFR-TKIs inevitably developed acquired resistance. Previous research focused on synthesis of new generation of molecular targeted drugs that could irreversibly inhibit EGFR with a particular gene mutation to overcome drug resistance. However, they failed to inhibit EGFR with other gene mutations. Activation of bypass signaling pathway and the changes of tumor microenvironment are identified as two of the mechanisms of acquired resistance to EGFR-TKIs. We therefore constructed multifunctional gene/drug co-delivery nanocomplexes AP/ES co-formulated with chloroquine that could target the both two mechanisms. We found that chloroquine not only enhanced endosomal escape ability of AP/ES for efficient gene transfection to inhibit Survivin, but also showed strong vessel-normalization ability to improve tumor microcirculation, which further promoted drug delivery into tumor tissue and enhanced drug efficacy in erlotinib-resistant NSCLC.

Expression of antiapoptotic proteins livin and survivin in pediatric AML patients, as prognostic markers

Objective: Survivin and livin are highly expressed in various malignancies and their expression levels may be related to unfavorable prognosis. The aim was to investigate the relationships of these two markers with some prognostic factors and with survival of the children with acute myeloid leukemia (AML). Methods: Livin and survivin expression was investigated quantitatively by immunohistochemistry staining technique in 43 primary formalin-fixed, paraffin-embedded bone marrow blocks in pediatric age group (<18 years). Results: Both survivin and livin were expressed in 81.4% of AML patients. Livin expression showed significant positive association with high level of primary WBC (p = .002). Survivin expression showed significant positive correlations with risk of relapse (p ≤ .001) and high level of primary WBC (p = .003). The relationship of overall survival (OS) of the patients with livin and survivin expression, were investigated separately in disease subtypes. Significant association was observed between survivin expression and shorter OS regardless of subtypes including acute promyelocytic (APL) (p = .01) and nonacute promyelocytic leukemia (non-APL) (p = .008). Also, significant association of livin expression with shorter OS was detected, but only in APL subgroup (p = .046). Nevertheless, in Cox regression model after adjusting for disease subtypes, stage and cytogenetics; survivin and livin showed no significant association with OS (p > .05). Conclusion: Livin and survivin showed significant associations with some poor prognostic factors of AML. Although survivin in both subtypes and livin in non APL subtype, showed a significant relationship with shorter OS, none of them was determined as independent prognostic factors. Further studies with larger sample size are suggested.

Upregulating the Expression of Survivin‐HBXIP Complex Contributes to the Protective Role of IMM‐H004 in Transient Global Cerebral Ischemia/Reperfusion

IMM-H004, a 3-piperazinylcoumarin compound derived from coumarin, has been proved effective against CA1 cell loss and spatial learning impairments resulting from transient global ischemia/reperfusion (TGCI/R), while the mechanism is still largely unknown. Here, we confirmed that treatment of rats with IMM-H004 immediately after TGCI/R ameliorated delayed neuronal death (DND) in the CA1 of hippocampus and cortex. Further study suggested that IMM-H004 contributed to the expression of antiapoptotic protein survivin through the activation of PI3K-dependent protein kinase B (PKB/Akt), which led to the phosphorylation of forkhead box O1 (FoxO1), then relieved the inhibiting effect on survivin promoter. Additionally, IMM-H004 also enhanced the expression of hepatitis B X-interacting protein (HBXIP), which formed a complex with survivin to prevent the activation of caspase death cascade, thereby halting apoptotic cell death. Finally, we injected a HBXIP siRNA into hippocampus and performed microelectroporation before ischemia/reperfusion, which abolished the protective effect of IMM-H004. Further study revealed that HBXIP maintained the high expression of Akt and survivin. Collectively, our findings demonstrated that DND after TGCI/R was alleviated by IMM-H004 through promoting the formation of survivin-HBXIP complex, which further emphasized the importance of endogenous protein involved in self-repair after stroke. Support or Funding Information This work was supported by the National Natural Science Foundation of China (81730096, U1402221), CAMS Innovation Fund for Medical Sciences (CIFMS) ( 2016-I2M-1-004), the Scientific Research Foundation of the Higher Education Institutions of Hunan Province (15K091), Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (BZ0150), PUMC Graduate Education and Teaching Reform Project (10023201600801). Schematic diagram of the neural protective role of IMMH004 after TGCI/R This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The heparan sulfate 3-O-sulfotransferases (HS3ST) 2, 3B and 4 enhance proliferation and survival in breast cancer MDA-MB-231 cells.

Heparan sulfate 3-O-sulfotransferases (HS3STs) catalyze the final maturation step of heparan sulfates. Although seven HS3ST isozymes have been described in human, 3-O-sulfation is a relatively rare modification, and only a few biological processes have been described to be influenced by 3-O-sulfated motifs. A conflicting literature has recently reported that HS3ST2, 3A, 3B and 4 may exhibit either tumor-promoting or anti-oncogenic properties, depending on the model used and cancer cell phenotype. Hence, we decided to compare the consequences of the overexpression of each of these HS3STs in the same cellular model. We demonstrated that, unlike HS3ST3A, the other three isozymes enhanced the proliferation of breast cancer MDA-MB-231 and BT-20 cells. Moreover, the colony forming capacity of MDA-MB-231 cells was markedly increased by the expression of HS3ST2, 3B and 4. No notable difference was observed between the three isozymes, meaning that the modifications catalyzed by each HS3ST had the same functional impact on cell behavior. We then demonstrated that overexpression of HS3ST2, 3B and 4 was accompanied by increased activation of c-Src, Akt and NF-κB and up-regulation of the anti-apoptotic proteins survivin and XIAP. In line with these findings, we showed that HS3ST-transfected cells are more resistant to cell death induction by pro-apoptotic stimuli or NK cells. Altogether, our findings demonstrate that HS3ST2, 3B and 4 share the same pro-tumoral activity and support the idea that these HS3STs could compensate each other for loss of their expression depending on the molecular signature of cancer cells and/or changes in the tumor environment.

Synthesis and biological evaluation of indole-based UC-112 analogs as potent and selective survivin inhibitors

The anti-apoptotic protein survivin is highly expressed in cancer cells but has a very low expression in fully differentiated adult cells. Overexpression of survivin is positively correlated with cancer cell resistance to chemotherapy and radiotherapy, cancer cell metastasis, and poor patient prognosis. Therefore, selective targeting survivin represents an attractive strategy for the development of anticancer therapeutics. Herein, we reported the extensive structural modification of our recently discovered selective survivin inhibitor UC-112 and the synthesis of thirty-three new analogs. The structure-activity relationship (SAR) study indicated that replacement of the benzyloxy moeity in UC-112 with an indole moiety was preferred to other moieties. Among these UC-112 analogs, 10f, 10h, 10k, 10n showed the most potent antiproliferative activities. Interestingly, they were more potent against the P-glycoprotein overexpressing cancer cell lines compared with the parental cancer cell lines. Mechanistic studies confirmed that new analogs maintained their unique selectivity against survivin among the IAP family members. In vivo study using 10f in a human A375 melanoma xenograft model revealed that it effectively inhibited melanoma tumor growth without observable acute toxicity. Collectively, this study strongly supports the further preclinical development of selective survivin inhibitors based on the UC-112 scaffold.

YXQ-EQ Induces Apoptosis and Inhibits Signaling Pathways Important for Metastasis in Non-Small Cell Lung Carcinoma Cells

Background/Aims: Lung cancer is one of the most prevalent malignancies in the world. The 5-year survival rate for non-small cell lung cancer (NSCLC) patients is only approximately 15%, with metastasis as the primary cause of death. This study was aimed to investigate cytotoxic effect of external qi of Yan Xin Qigong (YXQ-EQ) toward human lung adenocarcinoma A549 cells as well as its effect on signaling pathways promoting migration, invasion and epithelial-to-mesenchymal transition (EMT) in A549 cells. Methods: Cytotoxic effect of YXQ-EQ was evaluated using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] and cologenic assays. Apoptosis of treated cells was determined by Annexin V/propidium iodide staining and flow cytometry analysis, while cell migration and invasion were determined using transwell assays and EMT was assessed by morphological changes in cells. Protein expression and phosphorylation were examined by immunoblot analyses. Results: YXQ-EQ induced apoptosis in A549 cells, resulting in a pronounced reduction in viability and clonogenic formation. This was associated with inhibition of phosphorylation of AKT and ERK1/2 and reduced expression of anti-apoptotic proteins BCL-xL, XIAP and survivin. Furthermore, YXQ-EQ inhibited EGF/EGFR signaling and EGF mediated migration and invasion of A549 cells. While TGF-β1 induced phosphorylation of SMAD2/3 and EMT in A549 cells, YXQ-EQ suppressed TGF-β/SMAD signaling and induced cell death in these cells in the presence of TGF-β1. Conclusion: Our findings suggest that YXQ-EQ could exert anti-lung cancer effects via inhibiting signaling pathways that are important for NSCLC cell survival and NSCLC metastasis.

Niclosamide Inhibits Cell Growth and Enhances Drug Sensitivity of Hepatocellular Carcinoma Cells via STAT3 Signaling Pathway.

Aims: Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor worldwide, with a high mortality rate at advanced stages. In this study, we investigated the effect of niclosamide on cell growth and drug sensitivity in human HCC and elucidated the underlying mechanism.Methods: Three human HCC cell lines (HepG2, QGY-7703 and SMMC-7721) were used to evaluate the effect of niclosamide. Cell proliferation was measured by MTT assay and colony formation assay. Assessment of apoptosis was evaluated by flow cytometry and Hoechst staining. The mRNA and protein levels were analyzed by real-time PCR and western blot, respectively.Results: Niclosamide suppressed cell viability, inhibited clone formation, and induced cell apoptosis in HCC cells dose- and time-dependently. Furthermore, niclosamide synergized with cisplatin to promote the apoptosis of HCC cells. With niclosamide treatment, phospho-STAT3 (Y705) was inactivated and the downstream antiapoptotic proteins Mcl-1 and survivin were downregulated at both mRNA and protein levels in HCC cells.Conclusion: Niclosamide has effective function in anti-HCC and may be a single or combined drug treatment for HCC and acts via the STAT3 signaling pathway.

Abstract 08: Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies

Abstract The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) is FDA approved for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). However, IBR responses are incomplete in majority of cases, and not durable in MCL and high-risk CLL. We hypothesize that de novo resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be cotargeted with BTK to achieve deeper and durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generates synergistic cytotoxicity with IBR in MCL lines (Axelrod et al. Leukemia 2014;28:407-10). Recently, we extended these findings to circulating leukemic B cells in CLL, MCL (Jayappa et al. ASH 2016), and follicular lymphoma (FL) patient PBMCs treated ex vivo. Based on these results we initiated a phase I/Ib trial for IBR+VEN combination in MCL. Our ex vivo cytotoxicity studies showed a surprisingly high frequency of de novo resistance to IBR+VEN. Resistance did not correlate with previous therapy or clinical characteristics of patients. However, FACS analysis of primary CLL cells showed that cells displaying an “activation” phenotype (CD5+/CD19+/CD69+) were less sensitive to cytotoxicity of IBR+VEN, suggesting that cellular activation by extrinsic factors could generate drug resistance. To test this, we cocultured or preincubated CLL patient PBMCs with various stromal cells, cytokines, and other agonists and found that soluble CD40L, IL10, and CpG oligodeoxynucleotides (ODN) generated variable resistance to IBR+VEN in CLL samples, with CpG ODN, a specific TLR9 agonist, being the most effective. The combination of CpG ODN, sCD40L, and IL10 (“agonist mix”) generated near-complete resistance to IBR+VEN in most CLL(N=22)/MCL(N=7)/FL(N=4) patient PBMCs. The agonist mix treatment significantly upregulated nuclear localization of RelA and RelB proteins in CLL/MCL/FL cells analyzed by ImageStream, suggesting activation of NF-kB signaling. Agonist mix treatment also induced the NF-kB dependent overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, and inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame this drug resistance (Jayappa et al. ASH 2016). To further explore the role of TLR9 and other innate immune receptors in resistance, we performed IBR+VEN cytotoxicity analysis in CLL/MCL patient PBMCs cocultured with agonists of various TLRs and NOD1/2 receptors. Preliminary results showed that TLR9 agonist CpG ODN generated significant resistance to IBR+VEN in most CLL/MCL samples, and agonists of TLR1/2, TLR7, and NOD1/2 induced a moderate level of resistance in a MCL sample. The CpG ODN treatment induced a significant increase in NF-kB signaling measured by increased nuclear localization of RelB in CLL patient samples. To determine if soluble agonists in the extranodal macroenvironment of patients could generate NF-kB dependent resistance to IBR+VEN, we analyzed drug cytotoxicity in CLL(N=6) and MCL(N=1) patient PBMCs cultured with autologous plasma. We found that preincubation with autologous plasma induced variable levels of resistance to IBR+VEN in CLL/MCL samples, but did not induce resistance to a NF-kB inhibitor BMS-345541. In conclusion, soluble factors in the patient nodal/extranodal macroenvironment, particularly a TLR9 agonist, generate resistance to IBR+VEN by activating NF-kB signaling and overexpression of multiple anti-apoptotic proteins. The same resistance mechanism, based on innate immune signaling, occurs in three mature B-cell malignancies, CLL, MCL and FL, that differ in etiology and clinical course. The signaling pathway responsible for resistance contains new targets for therapeutic intervention with potential to prevent or reverse resistance to IBR and VEN in these and perhaps other cancers as well. Citation Format: Kallesh D. Jayappa, Craig A. Portell, Vicki L. Gordon, Timothy P. Bender, Michael E. Williams, Michael J. Weber. Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 08.

SOCS1 Gene Therapy Improves Radiosensitivity and Enhances Irradiation-Induced DNA Damage in Esophageal Squamous Cell Carcinoma.

STAT3 has been implicated recently in radioresistance in cancer. In this study, we investigated the association between STAT3 and radioresistance in esophageal squamous cell carcinoma (ESCC). Strong expression of activated phospho-STAT3 (p-STAT3) was observed in 16/22 ESCC patients with preoperative chemoradiotherapy (CRT), compared with 9 of 24 patients with surgery alone, where the prognosis of those with CRT was poor. Expression of p-STAT3 and the antiapoptotic proteins Mcl-1 and survivin was strongly induced in ESCC cells by irradiation. Ectopic STAT3 expression increased radioresistance, whereas expression of the STAT3 negative regulator SOCS1 via an adenoviral vector improved radioresponse. Inhibiting the STAT3-Mcl-1 axis by SOCS1 enhanced DNA damage after irradition and induced apoptosis. Combining SOCS1 with radiotherapy enhanced antitumor responses in a murine xenograft model compared with the individual therapies. Tumor repopulation occurred transiently after treatment by irradiation but not the combination SOCS1/radiotherapy. Tumors subjected to this combination expressed high levels of γH2AX and low levels of Ki-67, which was maintained after cessation of treatment. Overall, we demonstrated that inhibiting the STAT3-Mcl-1 signaling axis by ectopic SOCS1 improved radiosensitivity by inducing apoptosis and enhancing DNA damage after radiotherapy, offering a mechanistic rationale for a new ESCC treatment. Cancer Res; 77(24); 6975-86. ©2017 AACR.