Specific Induction Of Apoptosis Research Articles

Novel engineered TRAIL-based chimeric protein strongly inhibits tumor growth and bypasses TRAIL resistance.

Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies.

Chitosan capped ZnO nanoparticles with cell specific apoptosis induction through P53 activation and G2/M arrest in breast cancer cells – In vitro approaches

Now a days the well-organized strategy to induce apoptosis in cancer chemotherapy is to produce anti-cancer agent without any side effects is in needy. Hence the present investigation was aimed to explore the anticancer potentials of Amorphophallus paeoniifolius reduced zinc nanoparticles capped with chitosan against MCF 7 cell line (breast cancer cells)and studied for its optical and surface charge properties. The size, shape, dispersion and uniform distribution of biosynthesized zincoxide nanoparticle was examined using Field emission scanning electron microscope (FESEM) and Transmission electron microscope (TEM) respectively. The spherical and cubic nanocrystals were found to be lethal against MCF 7 cells on MTT assay at dose dependant manner (20–80 μg/ml) whose IC50 value 42 μg/ml. Bright field light microscopic study showed the apoptotic morphology of treated and control MCF-7 cells. Fluorescence staining A/O:EB and DAPI methods further cleared the chromosome condensation, nuclear fragmentation and confirms the apoptosis induced by Ch-Ap-ZnONPS within IC50 concentrations. Significant cell cycle arrest at particular stage of G2/M was achieved with the nanocomplex treatment at dose dependant manner. Finally, it was observed that the apoptotic gens and protein expressions of MCF-7 cell line were up and down regulation with the treatment of Ch-Ap-ZnONPS when compared to normal cells.

PPAR-Induced Fatty Acid Oxidation in T Cells Increases the Number of Tumor-Reactive CD8+ T Cells and Facilitates Anti-PD-1 Therapy.

Although PD-1 blockade cancer immunotherapy has shown potential for a wide range of patients with cancer, its efficacy is limited, in part, due to the loss of effector cytotoxic T lymphocytes (CTLs) via terminal differentiation-induced apoptosis. We previously demonstrated that mitochondrial activation, by the agonists of peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1-α (PGC-1α)/transcription factor complexes, had synergistic effects with a PD-1-blocking monoclonal antibody in a mouse tumor model. In the current study, we examined the molecular mechanism of the synergistic effects of bezafibrate, an agonist of PGC-1α/ PPAR complexes, which enhanced the tumoricidal effects of PD-1 blockade. Bezafibrate activated CTL mitochondria and upregulated oxidative phosphorylation as well as glycolysis, resulting in more proliferation of naïve T cells and improved effector function in CTLs. Bezafibrate also increased fatty acid oxidation (FAO) and mitochondrial respiratory capacity, which supports the extra energy demands of cells in emergencies, allowing cell survival. Carnitine palmitoyl transferase 1 (Cpt1), which is needed for FAO, and Bcl2 were both upregulated. Cpt1 and Bcl2 can form a complex to prevent apoptosis of CTLs. Together, these results indicate that bezafibrate increases or maintains the number of functional CTLs by activating mitochondrial and cellular metabolism, leading in turn to enhanced antitumor immunity during PD-1 blockade. Cancer Immunol Res; 6(11); 1375-87. ©2018 AACR.

BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells

Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.

PIL6-TRAIL-engineered umbilical cord mesenchymal/stromal stem cells are highly cytotoxic for myeloma cells both in vitro and in vivo

BackgroundMesenchymal/stromal stem cells (MSCs) are favorably regarded in anti-cancer cytotherapies for their spontaneous chemotaxis toward inflammatory and tumor environments associated with an intrinsic cytotoxicity against tumor cells. Placenta-derived or TRAIL-engineered adipose MSCs have been shown to exert anti-tumor activity in both in-vitro and in-vivo models of multiple myeloma (MM) while TRAIL-transduced umbilical cord (UC)-MSCs appear efficient inducers of apoptosis in a few solid tumors. However, apoptosis is not selective for cancer cells since specific TRAIL receptors are also expressed by a number of normal cells. To overcome this drawback, we propose to transduce UC-MSCs with a bicistronic vector including the TRAIL sequence under the control of IL-6 promoter (pIL6) whose transcriptional activation is promoted by the MM milieu.MethodsUC-MSCs were transduced with a bicistronic retroviral vector (pMIGR1) encoding for green fluorescent protein (GFP) and modified to include the pIL6 sequence upstream of the full-length human TRAIL cDNA. TRAIL expression after stimulation with U-266 cell conditioned medium, or IL-1α/IL-1β, was evaluated by flow cytometry, confocal microscopy, real-time PCR, western blot analysis, and ELISA. Apoptosis in MM cells was assayed by Annexin V staining and by caspase-8 activation. The cytotoxic effect of pIL6-TRAIL+-GFP+-UC-MSCs on MM growth was evaluated in SCID mice by bioluminescence and ex vivo by caspase-3 activation and X-ray imaging. Statistical analyses were performed by Student’s t test, ANOVA, and logrank test for survival curves.ResultspIL6-TRAIL+-GFP+-UC-MSCs significantly expressed TRAIL after stimulation by either conditioned medium or by IL-1α/IL-1β, and induced apoptosis in U-266 cells. Moreover, when systemically injected in SCID mice intratibially xenografted with U-266, those cells underwent within MM tibia lesions and significantly reduced the tumor burden by specific induction of apoptosis in MM cells as revealed by caspase-3 activation.ConclusionsOur tumor microenvironment-sensitive model of anti-MM cytotherapy is regulated by the axis pIL6/IL-1α/IL-1β and appears suitable for further preclinical investigation not only in myeloma bone disease in which UC-MSCs would even participate to bone healing as described, but also in other osteotropic tumors whose milieu is enriched of cytokines triggering the pIL6.

P53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation.

SummaryEmbryonic stem cells (ESCs) are fast proliferating cells capable of differentiating into all somatic cell types. In somatic cells, it is well documented that p53 is rapidly activated upon DNA damage to arrest the cell cycle and induce apoptosis. In mouse ESCs, p53 can also be functionally activated, but the precise biological consequences are not well characterized. Here, we demonstrated that doxorubicin treatment initially led to cell-cycle arrest at G2/M in ESCs, followed by the occurrence of massive apoptosis. Neither p53 nor its target gene p73 was required for G2/M arrest. Instead, p53 and p73 were fully responsible for apoptosis. p53 and p73 were also required for differentiation-induced apoptosis in mouse ESCs. In addition, doxorubicin treatment induced the expression of retinoblastoma protein in a p53-dependent manner. Therefore, both p53 and p73 are critical in apoptosis induced by DNA damage and differentiation.

Sugar-decorated mesoporous silica nanoparticles as delivery vehicles for the poorly soluble drug celastrol enables targeted induction of apoptosis in cancer cells

Cancerous cells have a rapid metabolism by which they take up sugars, such as glucose, at significantly higher rates than normal cells. Celastrol is a traditional herbal medicine known for its anti-inflammatory and anti-cancer activities. The poor aqueous solubility and lack of target selectivity of celastrol result in low therapeutic concentration of the drug reaching subcellular compartments of the target tissue, making it an interesting candidate for nanoparticulate delivery. The goal of this study was to utilize glucose as an affinity ligand decorated on mesoporous silica nanoparticles (MSNs), with the aim of delivering these celastrol-loaded MSNs with high specificity to cancer cells and inducing minimal off-target effects in healthy cells. MSNs were thus functionalized with sugar moieties by two different routes, either by conjugation directly to the MSN surface or mediated by a hyperbranched poly(ethylene imine), PEI layer; the latter to increase the cellular uptake by providing an overall positive surface charge as well as to increase the reaction sites for sugar conjugation. The effect of surface functionalization on the target-specific efficacy of the particles was assessed by analyzing the uptake in HeLa and A549 cells as cancer cell models, as compared to mouse embryonic fibroblasts (MEF) as a representative for normal cells. To this end a comprehensive analysis strategy was employed, including flow cytometry, confocal microscopy, and spectrophotometry. When the apoptotic effect of celastrol was evaluated, the anti-cancer activity of celastrol was shown to be significantly enhanced when it was loaded into the specifically designed MSNs. The particles themselves did not induce any toxicity, and normal cells displayed minimal off-target effects. In summary, we show that glucose-functionalized MSNs can be used as efficient carriers for targeted celastrol delivery to achieve specific induction of apoptosis in cancer cells.

CS-32 * SapC-DOPS INDUCES Smac-AND Bax-MEDIATED MITOCHONDRIAL APOPTOSIS IN NEUROBLASTOMAS

SapC-DOPS nanovesicles are a stable protein-lipid mixture assembled from saposin C (SapC) and dioleylphosphatidylserine (DOPS). It shows selective tumor-targeting activity in multiple animal tumor models and specific induction of apoptosis in various cancer cell lines. The preferential targeting is due to higher levels of exposed phosphatidylserine on the outer cell membrane of cancer cells. Importantly, induction of apoptosis by SapC-DOPS was shown to occur through intracellular ceramide elevation. Ceramide is known to be a tumor suppressor molecule capable of acting synergistically with Bax and regulating mitochondrial-mediated apoptosis. Effectors that mediate SapC-DOPS-induced apoptosis downstream of ceramide are currently unknown. Therefore, we hypothesized that mitochondria could play a major role during SapC-DOPS induced apoptotic cancer cell death. Cell death following SapC-DOPS treatment was assessed by several indices such as: G6PD release assay to estimate necrosis, DAPI staining and cell cycle analysis to estimate apoptosis and DNA fragmentation, JC-1 assay to estimate mitochondrial membrane potential (ΔΨM) and caspase-3 activation by Western blotting. SapC-DOPS treatment induced significant apoptosis as denoted by the appearance of a subG1 peak, loss of ΔΨM, mitochondrial release of Smac, Cyto c and AIF, as well as mitochondrial Bax translocation and oligomerization. ShRNA-mediated Smac knockdown and Bax inhibition with V5 peptide led to retention of ΔΨM and abrogation of apoptosis with decreased caspase-3 activation. Our results indicate that Smac and Bax play major roles in SapC-DOPS-induced mitochondria-mediated apoptotic pathway in human neuroblastoma cells

ET-26 * COMBINED TREATMENT WITH ABT263 AND GX15-070 YIELDS A SYNERGISTIC ANTIPROLIFERATIVE EFFECT IN GLIOBLASTOMA

BACKGROUND: Glioblastoma represents the most common primary brain tumor in adults and still remains to be among those human diseases that are related to the worst prognosis despite major effort in refining current treatment modalities. Multi-targeting is one of the current approaches that are undertaken to overcome this desperate situation. The members of the Bcl-2 family are commonly upregulated in glioblastoma. Unfortunately, inhibition of these proteins by existing inhibitors such as BH3-mimetics fall short of expectations due to high intrinsic levels of Mcl-1 in glioblastoma urging novel combination therapies involving Mcl-1 inhibitors such as GX15-070 to overcome this problem. METHODS: The IC50 was determined for GX15-070 and ABT263 in U87MG, U87-EGFRvIII, LN229 and T98G established glioblastoma cell lines as well as glioma stem-like cells GS9-6 and NCH644 after 72 h of continuous drug exposure. The antiproliferative effect of combining both agents was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assays. Moreover, cell death and specific induction of apoptosis were analyzed by staining for annexin V or propidium iodide prior to flow cytometric analysis. Soft agar assays were performed to examine antitumorigenic effects. RESULTS: Combined treatment with GX15-070 and ABT263 resulted in at least additive antiproliferative effects on all examined cell lines. On the cellular level we confirmed that this effect is due to induction of apoptosis. Moreover, we showed that combined treatment with both agents inhibited anchorage-independent growth in a cooperative manner. CONCLUSIONS: In conclusion, we present that combined treatment with the Bcl-2 inhibitor ABT263 and the Mcl-1 inhibitor GX15-070 results in a synergistic antiproliferative effect on established and glioma stem-like cells and therefore may represent a novel and promising therapeutic approach in this disease.

160: Unveiling a novel immunomodulatory role for interferon-β therapy in multiple sclerosis: The selective reduction of pathogenic memory B cells

Objective B cells are emerging as central players in Multiple sclerosis (MS) pathogenesis, thus we sought to evaluate whether IFN-β therapy may regulate B cell responses in Relapsing Remitting MS (RRMS) patients. Methods Apoptotic phenotype of naive and memory B cells was analyzed longitudinally in MS patients before and after therapy either by staining with Annexin-V and 7-actinomycin D or by evaluating intracellular Caspase 3 activation and Fas Receptor (Fas) expression. In vitro experiments were also conducted treating with an anti-Fas mAb PBMCs isolated from MS patients. Results Our results showed that IFN-β specifically targets the B cell compartment by reducing memory B cell frequency. Enhanced expression of Fas, Annexin-V and active Caspase 3 was found in memory B cells upon IFN-β therapy, suggesting a specific induction of apoptosis in this population also confirmed by in vitro experiments with anti-Fas mAb. Taking into account that memory B cells represent the major Epstein barr-virus (EBV) reservoir and that EBV represents one of the candidate for MS etiopathogenesis, the ability of IFN-β therapy to reduce the enhanced expression of the EBV latent LMP2A transcript found in MS patients as compared to healthy subjects, indicates that this treatment may also exert some antiviral effects in MS patients. Conclusions All our findings suggest that the specific reduction of memory B cells is a novel effect induced by IFN-β therapy, similarly to what found for the anti-CD20 mAb Rituximab. Thus, IFN-β therapy may play a dual role by exerting both immunomodulatory and anti-viral activities, modulating pathogenic B cell responses and controlling EBV infection in MS patients via the depletion of EBV-harboring cells. This work was supported by FISM Grant ( #2009/R/7 to EMC).

Cyclosporine A regulates pro-inflammatory cytokine production in ulcerative colitis.

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel diseases (IBD), which are defined as relapsing inflammations of the gastrointestinal tract. Cyclosporine A (CsA) is a potential rescue treatment to avoid colectomy in severe steroid-refractory UC patients. The molecular mechanism of action of CsA in UC is nevertheless still not well understood. The aim of this study was to investigate the effect of CsA on a possible modulation of cytokine production by peripheral blood mononuclear cells (PBMCs) of controls and patients with UC or CD. Upon CsA treatment, analyses of cytokine levels revealed a significant reduction of IL-13 expression in PBMCs from patients with UC, whereas other cytokine expression levels remained unaffected. To address the question whether CsA treatment impinges on the induction of cell death, apoptosis assays were performed using CD4(+) T cells from peripheral blood of patients suffering from either UC or CD. It became clear that CsA treatment resulted in a specific induction of apoptosis in samples from controls and patients with UC but not with CD. Apoptosis induction was not mediated via the mitochondrial apoptosis pathway. The present data support the concept that CsA treatment modulates pro-inflammatory cytokine production and T cell survival in UC via the induction of apoptosis and might therefore help to explain the clinical efficacy of CsA in patients with UC.

Mode of Action of Diterpene and Characterization of Related Metabolites from the Soft Coral, Xenia elongata

Chemical and biological investigation of the cultured marine soft coral Xenia elongata led to the isolation of two new diterpenes (2, 3). Their structures were elucidated using a combination of NMR and mass spectrometry. Biological evaluations and assessments were determined using the specific apoptosis induction assay based on genetically engineered mammalian cell line D3 deficient in Bak and Bax and derived from a mouse epithelial cell. The diterpenes induce apoptosis in low micromolar concentrations. The results indicate that the previously isolated compound (1) affects cell in a manner similar to that of HSP90 and HDAC inhibitors and in a manner opposite of PI3 kinase/mTOR inhibitors. Compound (3) inhibits selectively HDAC6 in high micromolar concentrations.

Chitosan-modified cobalt oxide nanoparticles stimulate TNF-α-mediated apoptosis in human leukemic cells

The objective of this study was to develop chitosan-based delivery of cobalt oxide nanoparticles to human leukemic cells and investigate their specific induction of apoptosis. The physicochemical properties of the chitosan-coated cobalt oxide nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, X-ray diffraction, and Fourier transform infrared spectroscopy. The solubility of chitosan-coated cobalt oxide nanoparticles was higher at acidic pH, which helps to release more cobalt ions into the medium. Chitosan-coated cobalt oxide nanoparticles showed good compatibility with normal cells. However, our results showed that exposure of leukemic cells (Jurkat cells) to chitosan-coated cobalt oxide nanoparticles caused an increase in reactive oxygen species generation that was abolished by pretreatment of cells with the reactive oxygen species scavenger N-acetyl-L-cysteine. The apoptosis of Jurkat cells was confirmed by flow-cytometric analysis. Induction of TNF-α secretion was observed from stimulation of Jurkat cells with chitosan-coated cobalt oxide nanoparticles. We also tested the role of TNF-α in the induction of Jurkat cell death in the presence of TNF-α and caspase inhibitors. Treatment of leukemic cells with a blocker had a greater effect on cancer cell viability. From our findings, oxidative stress and caspase activation are involved in cancer cell death induced by chitosan-coated cobalt oxide nanoparticles.

Sensitive detection and apoptotic cell death induction of adult T-cell leukemia/lymphoma (ATL) cells with photodynamic actions

Adult T cell leukemia/lymphoma (ATL) is an aggressive malignant disease of CD4 positive T lymphocytes caused by infection with human T cell leukemia virus type I (HTLV-I). HTLV-1 causes ATL in 3-5% of infected individuals after a long latent period of 40 to 60 years. The acute and lymphoma types are aggressive ATL characterized by resistance to chemotherapy and a poor prognosis. Leukemia/lymphoma cells and rapidly proliferating cells preferentially accumulate endogenous photosensitizer protoporphyrin IX (PpIX) when supplemented with 5-aminolevulinic acid (ALA). Treatment with 1mM ALA for 48h induced 10 to 100 times accumulation of PpIX in ATL leukemic cell lines and HTLV-I (+) T cell lines than that in healthy PBMCs. Specific induction of apoptosis was observed after 10 min light exposure (28 mW/cm 2 ) using Na-Li lamp in ATL leukemic cell lines and HTLV-I (+) T cell lines. ATL patient PBMC specimen showed strong accumulation of PpIX with the treatment of ALA compared to the healthy donor and HTLV-I carrier PBMCs, which could be useful for the diagnostic purposes and monitoring the patient status with high sensitivity. Photodynamic therapy is potentially hopeful treatment especially for lymphoma type ATL as a relatively selective, minimal or no scarring, non-invasive, safe, simultaneous and repeatable multiple lesions treatable modality.

Maintenance Of Bone Marrow Residency and Self-Renewal Of Leukemic Stem Cells By Cdc42 Gtpase

The leukemic stem cell (LSC) depends on specific interactions with extracellular matrix, soluble factors, and cellular components of the microenvironment, or niche. These interactions promote LSC self-renewal and survival, thus contributing to chemoresistance and treatment failure. Understanding the signaling pathways that promote LSC maintenance in response to niche interactions may reveal novel targets for therapy. Recent studies indicate a critical role for the small Rho GTPase, Cdc42, in the maintenance of normal hematopoietic stem and progenitor cells (HSPCs). Cdc42 coordinates actin cytoskeleton organization, adhesion, migration, self-renewal, cell polarity, proliferation, and survival of normal HSPCs in response to niche signaling through multiple cell surface receptors, including CXCL12/CXCR4, SCF/KIT, and fibronectin/integrin. Cdc42 activity is increased in both murine and human models of MLL-AF9 (MA9) acute myeloid leukemia (AML). Cdc42 expression is also increased in human patient AML samples across cytogenetic subtypes, compared to normal hematopoietic cell subsets, in analysis of curated datasets in the HemaExplorer database. In earlier work, we have shown that Cdc42 inhibition leads to peripheral mobilization of leukemia cells out of the marrow niche (Blood 114, 13). In the present study, we investigate whether Cdc42 inhibition also disrupts intrinsic LSC self-renewal. To interrogate Cdc42 in LSC self-renewal, MA9 cell lines were established following transduction of bone marrow HSPCs harvested from tamoxifen-inducible Cdc42 knockout mice, with Cre null donors as controls. Upon tamoxifen (TAM) treatment, Cdc42KO-MA9 cells had decreased CFU and small, diffuse colony morphology. Mice transplanted with untreated MA9 cells were divided to receive injections of TAM vs control. The Cdc42KO-MA9 cohort remains alive at over 180 days post-transplant, whereas vehicle control mice died of AML with latency similar to Cre null MA9 cell recipients (p<0.005). In vivo deletion of Cdc42 from MA9 leukemia in secondary recipients prolonged disease latency (p<0.005). AML cells recovered from vehicle control mice showed decreased growth in culture, reduced CFU content, and increased apoptosis following treatment with TAM to delete Cdc42. Cdc42KO-MA9 leukemia cells also had higher side scatter and Gr-1 expression, and decreased c-Kit, suggesting differentiation. These data indicate that Cdc42 is required for murine LSC maintenance. We used Tet-inducible shRNA targeting of Cdc42 in human cell lines expressing MA9 and mutant NRas (MA9/NRas). Cdc42 knockdown reduced MA9/NRas colony-forming ability, blocked actin polymerization and migration in response to CXCL12, and induced apoptosis. MA9/NRas cells co-expressing inducible Cdc42 shRNA and luciferase were transplanted into NSGS mice on doxycycline chow to induce knockdown. Bioluminescence imaging showed delayed AML progression in the knockdown group compared to non-targeting shRNA and regular chow controls. Thus, Cdc42 deficiency in human MA9 LSC reproduces the phenotype seen in the mouse genetic model. We used a novel small-molecule Cdc42-activity specific inhibitor, CASIN, to test pharmacologic inhibition of Cdc42 in AML. Consistent with knockdown data, in vitro CASIN treatment blocked MA9 cell colony-forming ability, actin polymerization, and migration. CASIN treatment led to specific induction of apoptosis in MA9 cells, while normal human umbilical cord blood CD34+ cells showed no significant toxicity in the dose range tested. Together, these studies show that Cdc42 signaling is critical to intrinsic LSC self-renewal and engagement of the niche, and Cdc42 inhibition represents a rational therapeutic principle to target LSC maintenance. Disclosures:No relevant conflicts of interest to declare.

Enhanced cytotoxicity of optimized liposomal genistein via specific induction of apoptosis in breast, ovarian and prostate carcinomas

Clinical use of genistein against cancer is limited by its extremely low aqueous solubility, poor bioavailability and pharmacokinetics. Based on structural analogy with steroidal compounds, liposomal vehicle compositions were designed and optimized for maximum incorporation of genistein’s flavonoid structure. Model conventional and stealth liposomes of genistein (GenLip) – incorporating unsaturated phospholipids and cholesterol – have demonstrated enhanced drug solubilization (over 350-folds > aqueous drug solution), shelf-life stability, and extended release profile. Owing to effective cellular delivery, preservation of genistein’s antioxidant activity was confirmed through marked neutralization of peroxides via GenLip, in both quantitative and microscopic fluorescent-probe oxidation assays. Furthermore, significant broad-spectrum anticancer efficacy of GenLip, in murine and human cancer cell lines (p < 0.05–0.001), was achieved in a concentration and time-dependent manner – approx. 5–7 lower IC50 values versus all non-incorporated drug controls. Indicative of key pro-apoptotic activity, GenLip produced DNA laddering, with 1/3 of free drug solution content, and resulted in the highest induction level of P53-independent apoptotic pathway markers, compared to all treatments, in our assays (namely, mitochondrial polarization, and caspase-3/7 enzymes). Our proof-of-principle pharmaceutical design of genistein-loaded liposomes shows optimal loading capacity and physico-chemical properties, which improved cellular delivery and specific pro-apototic effectiveness of incorporated drug, against various cancers.

Antitumor effect of 1, 8-cineole against colon cancer

Several essential oils possess pharmacological effects. Among the various constituents of essential oils, 1, 8-cineole has been shown to possess pharmacological effects such as anti-bacterial and anti-inflammatory effects. The effect of 1, 8-cineole on human colorectal cancer cells, however, has not reported previously. In this study, we have investigated the anti-proliferative effect of 1, 8-cineole on human colon cancer cell lines HCT116 and RKO by WST-8 and BrdU assays. The cytotoxicity of 1, 8-cineole was investigated by LDH activity and TUNEL staining. The mechanism of apoptosis by 1, 8-cineole was determined by western blot analyses. In in vivo study, RKO cells were injected into the SCID mice and the effect of 1, 8-cineole was investigated. Specific induction of apoptosis, not necrosis, was observed in human colon cancer cell lines HCT116 and RKO by 1, 8-cineole. The treatment with 1, 8-cineole was associated with inactivation of survivin and Akt and activation of p38. These molecules induced cleaved PARP and caspase-3, finally causing apoptosis. In xenotransplanted SCID mice, the 1, 8-cineole group showed significantly inhibited tumor progression compared to the control group. These results indicated 1, 8-cineole suppressed human colorectal cancer proliferation by inducing apoptosis. Based on these studies 1, 8-cineole would be an effective strategy to treat colorectal cancer.

Dual PI3K/mTOR Inhibitor NVP-BEZ235 Sensitizes Docetaxel in Castration Resistant Prostate Cancer

Effective therapeutic strategies that can achieve long-term improvement in patients with castration resistant prostate cancer are urgently needed. We recently reported that the activated PI3K/Akt/mTOR signaling pathway induced by docetaxel explains resistance to docetaxel in castration resistant prostate cancer. In this study we explored the efficacy of NVP-BEZ235, a dual PI3K and mTORC1/2 inhibitor, for docetaxel resistant castration resistant prostate cancer. We used the 2 human castration resistant prostate cancer cell lines C4-2 and C4-2AT6. At our laboratory C4-2AT6 cells were established from C4-2 under androgen ablated treatment for 6 months. We investigated the efficacy of NVP-BEZ235 monotherapy and NVP-BEZ235 combined with docetaxel in vitro and in vivo. Increased phosphorylated Akt in C4-2AT6 cells was significantly inhibited by NVP-BEZ235 in a dose and time dependent manner. WST cell proliferation assay results in C4-2AT6 cells revealed that combined administration of NVP-BEZ235 and docetaxel had significant, synergistically greater cytotoxicity than NVP-BEZ235 or docetaxel monotherapy. Combined NVP-BEZ235 (40 mg/kg) and docetaxel (4 mg/kg) in vivo in a castrated mouse xenograft model inhibited C4-2AT6 tumor growth to a greater degree than in the monotherapy groups. Also, NVP-BEZ235 showed significant efficacy with docetaxel at a low concentration in vivo, suggesting that NVP-BEZ235 effectively decreased resistance to docetaxel. Results suggest that inhibition of the PI3K/Akt/mTOR signaling pathway by NVP-BEZ235 can overcome docetaxel resistance in human castration resistant prostate cancer. Our findings provide a molecular basis for the clinical use of combined administration of NVP-BEZ235 and docetaxel in patients with castration resistant prostate cancer.

Highlights of This Issue

Therapeutics targeting Her2 have shown impressive clinical activity, although there are resistance mechanisms leading to treatment failure. Granzyme B (GrB) is an intensely cytotoxic protein used by NK and CTL to kill target cells. Cao and colleagues fused GrB to the anti-Her2 scFv 4D5. This construct showed specific apoptosis induction and cytotoxicity to Her2 cells in vitro through a defined, multimodal mechanism and was active against 1apatanib, Herceptin-resistant, and MDR-expressing cells. Tumor-targeted GrB localized in tumor xenografts and resulted in tumor suppression. These studies suggest that tumor-directed GrB constructs have excellent potential as novel therapeutic agents.Drug resistance is a major cause of treatment failure in ovarian cancer, emphasizing the need for drugs with novel mechanism of action. Xu and colleagues identified gp130 as an important drug target in ovarian cancer and have developed a first-in-class small-molecule gp130 inhibitor. SC144 can be used as a new tool to interrogate the gp130-implicated signaling pathways affecting several malignancies. More significantly, SC144 shows efficacy in in vivo models of human ovarian cancer and displays no significant off-target toxicity in normal tissues. Our study offers a new approach to treat diseases with altered gp130 signaling, including multiple types of cancers.ATM signals DNA double strand breaks (DSB) to cell cycle checkpoints and for repair. Defects in ATM confer radiosensitivity and chemosensitivity, making ATM an attractive target for anticancer chemotherapy. Batey and colleagues have identified a potent ATM inhibitor, KU59403, that sensitizes human tumor cells to radiation and topoisomerase poisons in a p53-independent manner. Importantly, KU59403 is the first ATM inhibitor to demonstrate good tissue distribution and chemosensitization of human tumor xenografts without major toxicity, providing the first advanced pre-clinical proof-of-principle data to justify developing ATM inhibitors for clinical use.Alternate angiogenic pathways and/or hypoxia-induced eptithelial-to-mesenchymal transition (EMT) may facilitate resistance to anti-VEGF therapy. Kutluk Cenik and colleagues investigated the efficacy and biological effects of BIBF 1120 (nintedanib), a balanced inhibitor of VEGF, PDGF, and FGF pathways in xenografts of lung and pancreatic cancer. BIBF 1120 potently reduced tumor vascular density and function and inhibited primary tumor growth and metastasis. Despite induction of hypoxia, chronic BIBF 1120 did not enhance EMT or tumor invasiveness, suggesting a balanced multitarget strategy may combat the development of resistance. Clinical studies of BIBF 1120 are underway.

Bcl-xL Protects From UPR-Associated Apoptosis During Plasma Cell Differentiation

Abstract 3288Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. As part of the program that allows for B cell differentiation to a plasma cell, a required signal includes the activation of an unfolded protein response (UPR). However unlike stress-induced activation of the UPR, induction of apoptosis does not occur, suggesting that compensatory survival signals are also activated during plasma cell differentiation. The compensatory survival pathways are less defined and require further research. Therefore we employed a model of plasma cell differentiation to better define the survival signaling during this process. The murine B cell lymphoma cell line, Bcl1 can be stimulated to secrete immunoglobulin using IL-5 and LPS. To determine the effects of exogenous ER stress on plasma cell differentiation, we treated the cells with the inhibitor of N-linked glycosylation, tunicamycin, for 5 hours prior to the differentiation signal. The 5 hour pulse of tunicamycin was sufficient to induce significant apoptosis in undifferentiated cells or cells treated with IL-5, resulting in 78% and 74% cell death respectively by 24 hours post treatment. However, if LPS was included in the differentiation stimulus the cells were able to differentiate into IgM-secreting plasma cells with similar kinetics as cells differentiated in the absence of tunicamycin pretreatment. Thus LPS-induced differentiation is sufficient to block ER stress-induced cell death. Since these cells also activate a UPR during differentiation, we hypothesized that part of the differentiation program included protection from UPR-associated cell death. To investigate this effect, we first examined the levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1 during plasma cell differentiation. We found that differentiation induced Bcl-xL and caused the loss of Mcl-1. From this data we hypothesized that the differentiation of these cells resulted in Bcl-xL dependence during plasma cell differentiation. To test this we used ABT-737, which selectively blocks the binding pocket of Bcl-xL and Bcl-2 but not Mcl-1 and kills cells that are dependent on Bcl-2 or Bcl-xL. Undifferentiated Bcl1 cells were insensitive to ABT-737 with an IC50 > 2μM. However ABT-737 sensitized LPS-treated Bcl1 cells to tunicamycin pretreatment resulting in 89% death in 24 h compared to 23% in untreated cells. These data suggest that the induction of Bcl-xL is responsible for the survival of cells undergoing ER stress. Most importantly, cells treated with LPS and IL-5 for differentiation became sensitive to ABT-737 with 59% cell death versus 26% in untreated cells, thus demonstrating that during plasma cell differentiation, cells switch to a Bcl-xL-dependent state. To determine the molecular basis for these findings we investigated the effects of ABT-737 on the expression levels of Bcl-2 proteins as well as the effects of differentiation on their interactions. ABT-737 did not induce changes in the expression of Bcl-2 family proteins. However, co-immunoprecipitation demonstrated a shift in Bim binding from Mcl-1 in untreated cells to Bcl-xL in differentiating cells. This latter finding is consistent with a shift from Mcl-1 dependence to Bcl-xL during plasma cell differentiation. To validate these data, primary C57BL/6 splenocytes were isolated, depleted of non-B cells and subsequently stimulated with IL-4 and LPS to differentiate into plasmablasts. Realtime qPCR showed an increase in Bcl-xL mRNA and loss of Mcl-1 and Bcl-2 mRNA in both the primary B cells and the Bcl1 cell line. Western blotting of primary B cell lysates also showed an increase in Bcl-xL protein and loss of Bcl-2 and Mcl-1 protein. Together these data indicate that during plasma cell differentiation the cell enters a Bcl-xL-dependent state that protects against differentiation-induced apoptosis. Disclosures:No relevant conflicts of interest to declare.