Subdiploid DNA Content Research Articles

Enhanced anti-tumor efficacy and tumor accumulation of Tamoxifen through cRGD functionalized cholesterol based lipopolymeric nanoparticles for targeted breast cancer therapy

Targeting ligand mediated drug delivery systems hold promise for enhancing the anti-tumor efficacy and suppressing the off-target effects of chemotherapeutic molecules. The current study aims at the development of a peptide conjugated cholesterol based lipopolymeric nanoparticles for delivery of Tamoxifen (TMX). cRGD conjugated, cholesterol containing lipopolymers were successfully synthesized by maleimide-thiol coupling and characterized by 1H NMR. Emulsion-solvent evaporation technique was employed to formulate TMX loaded cRGD conjugated lipopolymeric nanoparticles which were further analyzed to determine the particle size, encapsulation efficiency and release kinetics and compared with TMX loaded mPEG-MTC-Chol nanoparticles. The cRGD conjugated lipopolymeric nanoparticles exhibited particle size ∼133.61 ± 4.70 nm with 43.86 ± 1.85 % encapsulation efficiency. Interestingly, the presence of cRGD greatly enhanced the cellular internalization and inhibitory activity of lipopolymeric nanoparticles in 4T1 cells with an IC50 value of ∼19.04 μM in comparison to TMX loaded mPEG-MTC-Chol NP (∼28.07 μM) Further, as evidenced by the rise in sub-diploid DNA content, TMX loaded cRGD-PEG-MTC Chol nanoparticles triggered apoptosis ∼2.34 fold greater than TMX loaded mPEG-MTC-Chol nanoparticles. Additionally, cRGD-PEG-MTC-Chol nanoparticles demonstrated longer half-life (∼1.84 fold) and improved bioavailibility (∼2.1 fold) and a ∼2 fold increment in drug concentration within the isolated tumor tissues when administered as cRGD-PEG-MTC-Chol NP vs mPEG-MTC-Chol NP in tumor bearing mice. This could be majorly attributed to integrin mediated receptor endocytosis which concurrently accounted for significant anti-tumor effects in EAC breast cancer mouse model.

Nanostructured lipid carriers as a novel tool to deliver sclareol: physicochemical characterisation and evaluation in human cancer cell lines

Sclareol (SC) is arousing great interest due to its cytostatic and cytotoxic activities in several cancer cell lines. However, its hydrophobicity is a limiting factor for its in vivo administration. One way to solve this problem is through nanoencapsulation. Therefore, solid lipid nanoparticles (SLN-SC) and nanostructured lipid carriers (NLC-SC) loaded with SC were produced and compared regarding their physicochemical properties. NLC-SC showed better SC encapsulation than SLN-SC and was chosen to be compared with free SC in human cancer cell lines (MDA-MB-231 and HCT-116). Free SC had slightly higher cytotoxicity than NLC-SC and produced subdiploid DNA content in both cell lines. On the other hand, NLC-SC led to subdiploid content in MDA-MB-231 cells and G2/M checkpoint arrest in HCT-116 cells. These findings suggest that SC encapsulation in NLC is a way to allow the in vivo administration of SC and might alter its biological properties

In vitro assessment of the cytotoxicity of Gallium(III) complexes with Isoniazid-Derived Hydrazones: Effects on clonogenic survival of HCT-116 cells

Cancer cells have high iron demand to mediate their rapid proliferation. Aiming to obtain cytotoxic compounds with potential iron metabolism disturbance in malignant cells, [Ga(HAPIH)(APIH)](NO3)2⋅2H2O (1) and [Ga(HPAmIH)(PAmIH)](NO3)2⋅2H2O (2) were synthesized with the iron chelators 2-acetylpyridine- and 2-pyridineformamide isonicotinoyl hydrazone (HAPIH and HPAmIH, respectively) and gallium(III), an iron(III) mimetic. The hydrazones and their gallium(III) complexes were assayed for their action against HL-60 (leukemia), MCF-7 (breast cancer), HCT-116 (colorectal carcinoma) and PC3 (prostate cancer) tumor cell lines, as well as non-malignant Human Embryonic Kidney293 (HEK-293) cells. HAPIH and its complex 1 were the most cytotoxic compounds, with HCT-116 being the most susceptible line (IC50 = 1.6 μM for HAPIH and 0.4 μM for complex 1). Moreover, the compounds proved to be at least 25-fold less toxic to HEK-293 than to the tumor cells. According to clonogenic survival assays, the relative number of colonies formed by HCT-116 cells was reduced by around 95% after pretreatment with HAPIH and 1 at their IC90 concentrations (18 μM and 4 μM, respectively). Both compounds were also able to inhibit the cell cycle and increased the subdiploid DNA content of HCT-116 after 24 h treatment, which suggests they possess a pro-apoptotic potential.

Prostaglandin E2 Promotes Early Bone Marrow Reconstitution for Survival after Lethal Irradiation

Introduction: High dose total body irradiation (TBI) causes severe damage to the hematopoietic system, resulting in ablation of both red and white blood cells and a high probability of infection, hemorrhage, and death. This is known as the hematopoietic syndrome of the acute radiation syndrome (HS-ARS). TBI is used therapeutically, and incidental exposure through nuclear accidents or radical terrorism is a current threat for which preparation is critical. Safe, effective, and pragmatic agents against HS-ARS are needed. We have identified prostaglandin E2 (PGE2) as a promising medical countermeasure to promote hematopoietic recovery and increase survival from lethal irradiation. Here, using the PGE2 analog 16,16-dimethyl PGE2 (dmPGE2), pre-irradiation dosing strategies were assessed in survival studies with an established mouse model of HS-ARS, and time course analyses were performed to elucidate the early cellular events associated with PGE2 radioprotection.Methods: C57BL/6 mice (n = 20/group) were exposed to LD70/30 (872 cGy) or LD90/30 (904 cGy) and treated with one of the following four dmPGE2 dosing strategies: a single dose of 35 μg at time -30 min, -1 hr, or -3 hr, or a double-dose of 20 μg each at time -45 min and -15 min pre-irradiation. Thirty-day survival was evaluated. The double-dose regimen was further utilized to assess cellular effects over time following LD70/30. Peripheral blood (PB) and bone marrow (BM) cells were harvested on days 1, 2, 4, 7, and 10 post-irradiation (n = 3/group/day). BM was analyzed by flow cytometry with immunostaining for surface markers (Sca-1, c-Kit, CD150, CD48 and blood cell lineage markers), cell cycle (Hoechst/PyroninY), and apoptosis (AnnexinV/7-AAD). PB differentials were assessed by a veterinary hematology analyzer.Results: All dmPGE2 regimens conferred 100% survival to mice receiving LD70/30, and all regimens provided a distinct survival advantage to mice receiving LD90/30 with the double-dose strategy conferring 100% survival at LD90/30. The double-dose was then chosen to assess cellular effects of dmPGE2 throughout the critical 10-day time period following LD70/30. Within the BM, marker-defined HSC numbers were significantly preserved by day 1 post-irradiation compared to vehicle controls, with a drop to vehicle levels observed on day 2 only. Cell cycling frequency among primitive hematopoietic cells was also conserved on day 1, decreased on day 2, then restored to non-irradiated control levels by days 7-10, while that of the unprotected mice remained significantly lower. Differentiated BM cells (lineage marker-positive) were substantially less apoptotic by day 2 and, in significant contrast to the untreated mice, returned to control levels of basal apoptosis by days 7-10. Overall BM cell populations were found to be largely replenished within one week as compared to the vehicle-treated mice, which remained significantly depressed. Further, 73-88% of detectable BM cells from the untreated mice were non-viable by day 4 post-irradiation based on sub-diploid DNA content and did not recover through day 10; dmPGE2-treated mice maintained significantly greater BM cell viability at each time point, returning to only 17-26% sub-diploid by day 7 (non-irradiated controls demonstrated ~5% sub-diploid DNA content). A corresponding effect on PB resulted in significant rescue from neutropenia, anemia, and thrombocytopenia by or before day 10.Conclusions: A single treatment with PGE2 prior to lethal irradiation can facilitate virtually complete survival, though a double treatment regimen may be most effective; further work will test whether this may be primarily due to the combined higher dose or to the altered timing. This PGE2-mediated survival involves early protection of both primitive and differentiated hematopoietic BM cells, affecting cell number, cell cycling, and apoptosis. Early hematopoietic BM recovery subsequently rescues PB components, facilitating ultimate survival. With further study, PGE2 may serve to provide an effective radioprotectant with the potential to safeguard patients undergoing TBI therapy, as well as military personnel and first responders, from the risk of radiation-associated mortality. DisclosuresNo relevant conflicts of interest to declare.

Stress responses of human retinal pigment epithelial cells to glyoxal.

Intracellular formation of advanced glycation end products (AGEs) is a crucial pathological process in retinal diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Glyoxal is a physiological metabolite produced during formation of AGEs and has also been shown to derive from photodegraded bisretinoid fluorophores in aging retinal pigment epithelial (RPE) cells. Flow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of Nε-carboxymethyllysine (CML)-modifications of intracellular proteins (AGEs) and changes in the production of stress response proteins; or 2) vital staining to determine apoptosis rates (annexin V binding), formation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and changes in intracellular pH upon treatment of cells with glyoxal. The percentage of apoptotic cells was further quantified by flow cytometry after staining of fixed cells with propidium iodide to determine cells with a subdiploid (fragmented) DNA content. Apoptosis related activation of caspase 3 was determined by Western blotting. Glyoxal induced changes in VEGF-A165a mRNA expression and protein production were determined by real-time PCR and by flow cytometry after immunocytochemical staining. Increasing glyoxal concentrations resulted in enhanced formation of AGEs, such as CML modifications of proteins. This was associated with elevated levels of intracellular reactive oxygen species, a depolarized MMP, and a decreased intracellular pH, resulting in an increased number of apoptotic cells. Apoptosis related caspase 3 activation increased in a dose dependent manner after glyoxal incubation. In consequence, the cells activated compensatory mechanisms and increased the levels of the anti-oxidative and stress-related proteins heme oxygenase-1, osteopontin, heat shock protein 27, copper/zinc superoxide dismutase, manganese superoxide dismutase, and cathepsin D. Furthermore, VEGF-A165a mRNA expression and VEGF-A protein production were significantly increased after incubation with glyoxal in ARPE-19 cells. The glyoxal-induced oxidative stress and apoptosis in ARPE-19 cells may provide a suitable in vitro model for studying RPE cellular reactions to AGEs that occur in AMD or in DR.

Abstract 4745: Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance

Abstract Although docetaxel (DTX) remains a standard of care for advanced prostate cancer (PCa), limited long-term responses, side effects and resistant disease suggested the need of novel combination strategies. Increased expression of histone deacetylases (HDAC) and alteration of the mevalonate pathway (MVP) are common aberrations in PCa. In this study, we analyzed the antitumor effect of DTX in combination with valproic acid (VPA), an anticonvulsant with HDAC inhibitory activity, and simvastatin (SIM), a cholesterol-lowering drug inhibiting the rate-limiting enzyme of MVP HMG-CoA reductase, on androgen-dependent 22RV1 and LNCAP and androgen-independent PC3 and DU145 PCa cells, as well as on the highly aggressive SIM-resistant DU145R80 subline developed in our laboratory from DU145 cells (Milone MR et al. Cell Death Dis. 2013; Milone MR et al. Oncotarget 2014). We first showed a potent synergistic anti-proliferative effect of VPA/SIM combination, assessed by calculating combination index (CI) according to the method of Chou and Talalay, on all cell lines, including SIM-resistant cells, whatever schedule of administration (simultaneous vs sequential) we used, confirming our previous data on the combination between the HDAC inhibitor (HDACi) panobinostat and zoledronic acid, the latter also targeting the MVP pathway (Bruzzese F. et al, Cell Death Dis. 2013). Notably, exposure to triple combinations (VPA/SIM/DTX) resulted in a further strong synergistic anti-proliferative effect, with sequential exposure with 24h delay between VPA/SIM and DTX as the best schedule. The synergistic interaction of VPA/SIM and DTX combination involved apoptotic effect, measured by FACS analysis of sub-diploid DNA content and caspase 3/7 cleavage, and DNA damage induction, assessed by γH2AX expression. Synergistic effect of VPA/SIM and DTX combination was confirmed by soft agar clonogenic assay and by 3D culture on self-assembled PCa spheroids. Significantly, VPA/SIM combination was also able to revert DTX-resistance in DTX-resistant PC3 and DU145 sublines developed in our laboratory from the parental cells. All together these findings suggested that the combination of two safe generic drugs such as VPA and SIM can improve DTX efficacy, representing a novel therapeutic approach that warrant clinical investigation in advanced PCa patients. Our study also suggests a new strategy to overcome resistance to standard taxane-based therapy in PCa patients. Citation Format: Federica Iannelli, Rita Lombardi, Biagio Pucci, Maria Rita Milone, Chiara Ciardiello, Alessandra Leone, Elena Di Gennaro, Alfredo Budillon, Francesca Bruzzese. Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance. [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 4745.

Improved In Vitro Antileukemic Activity of All-Trans Retinoic Acid Loaded in Cholesteryl Butyrate Solid Lipid Nanoparticles.

All-trans retinoic acid, a hydrophobic drug, has become one of the most successful examples of differentiation agents used for treatment of acute promyelocytic leukemia. On the other hand, histone deacetylase inhibitors, such as cholesteryl butyrate, present differentiating activity and.can potentiate action of drugs such as all-trans retinoic acid. Solid lipid nanoparticles represent a promising alternative for administration of hydrophobic drugs such as ATRA. This study aimed to develop, characterize, and evaluate the cytotoxicity of all-trans retinoic acid-loaded solid lipid nanoparticles for leukemia treatment. The influence of in situ formation of an ion pairing between all-trans retinoic acid and lipophilic amines on the characteristics of the particles (size, zeta potential, encapsulation efficiency) was evaluated. Cholesteryl butyrate, a butyric acid donor, was used as a component of the lipid matrix. In vitro activity on cell viability and distribution of cell cycle phases were evaluated for HL-60, Jurkat, and THP-1 cell lines. The encapsulation efficiency of all-trans retinoic acid in cholesteryl butyrate-solid lipid nanoparticles was significantly increased by the presence of the amine. Inhibition of cell viability by all-trans retinoic acid-loaded solid lipid nanoparticles was more pronounced than the free drug. Analysis of the distribution of cell cycle phases also showed increased activity for all-trans retinoic acid-loaded cholesteryl butyrate-solid lipid nanoparticles, with a clear increase in subdiploid DNA content. The ion pair formation in SLN containing cholesteryl butyrate can be explored as a simple and inexpensive strategy to improve the efficacy and bioavail-ability of ATRA in the treatment of the cancer and metabolic diseases in which this retinoid plays an important role.

Antitumor activity of ethanol extract from Hippophae rhamnoides L. leaves towards human acute myeloid leukemia cells in vitro.

We studied the effects of ethanol extract from Hippophae rhamnoides L. leaves on the growth and differentiation of human acute myeloid leukemia cells (KG-1a, HL60, and U937). The extract of Hippophae rhamnoides L. leaves inhibited cell growth depending on the cell strain and extract dose. In a high concentration (100 μg/ml), the extract also exhibited a cytotoxic effect on HL60 cells. Hippophae rhamnoides L. leaves extract did not affect cell differentiation and did not modify the differentiating effect of calcitriol, active vitamin D metabolite. Inhibition of cell proliferation was paralleled by paradoxical accumulation of phase S cells (synthetic phase) with a reciprocal decrease in the count of G1 cells (presynthetic phase). The extract in a concentration of 100 μg/ml induced the appearance of cells with a subdiploid DNA content (sub-G1 phase cells), which indicated induction of apoptosis. The antiproliferative effect of Hippophae rhamnoides L. extract on acute myeloid leukemia cells was at least partially determined by activation of the S phase checkpoint, which probably led to deceleration of the cell cycle and apoptosis induction.

Carnosic Acid Induces Apoptosis Through Reactive Oxygen Species-mediated Endoplasmic Reticulum Stress Induction in Human Renal Carcinoma Caki Cells.

Background:Carnosic acid, which is one of extract components of rosemary, has anti-inflammatory, anti-oxidant, and anti-cancer effects. However, the anti-cancer effect of carnosic acid in human renal carcinoma cells is unknown.Methods:Flow cytometry analysis was used to examine the effects of carnosic acid on apoptosis, and Asp-Glu-Val-Asp-ase activity assay kit was used to investigate the involvement of caspase activation. To determine protein expression of apoptotic and endoplasmic reticulum (ER) stress-related proteins, we used Western blotting. Intracellular accumulation of reactive oxygen species (ROS) was determined using the fluorescent probes 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA).Results:Carnosic acid induced sub-diploid DNA content, sub-G1, population and poly (ADP-ribose) polymerase (PARP) cleavage and activated caspase-3. A pan-caspase inhibitor, a benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone, markedly reduced apoptosis in carnosic acid-treated cells. Carnosic acid promoted intracellular ROS production, and pretreatment with the ROS scavengers (N-acetyl-L-cysteine and glutathione ethyl ester) inhibited carnosic acid-induced apoptosis. Furthermore, carnosic acid also induced expression of ER stress marker proteins, including activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein-homologous protein (CHOP), in a dose- and time-dependent manner. Down-regulation of ATF4 and CHOP by small interfering RNA (siRNA) markedly reduced carnosic acid-induced sub-G1 population and PARP cleavage. In addition, carnosic acid induced apoptosis in human breast carcinoma MDA-MB-361 and human hepatocellular carcinoma SK-HEP1 cells, but not in normal human skin fibroblast cells and normal mouse kidney epithelial TMCK-1 cells.Conclusion:Carnosic acid induced apoptosis through production of ROS and induction of ER stress in human renal carcinoma Caki cells.

Lactaptin Induces p53-Independent Cell Death Associated with Features of Apoptosis and Autophagy and Delays Growth of Breast Cancer Cells in Mouse Xenografts

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.

The Antiangiogenic Compound Aeroplysinin-1 Induces Apoptosis in Endothelial Cells by Activating the Mitochondrial Pathway

Aeroplysinin-1 is a brominated metabolite extracted from the marine sponge Aplysina aerophoba that has been previously characterized by our group as a potent antiangiogenic compound in vitro and in vivo. In this work, we provide evidence of a selective induction of apoptosis by aeroplysinin-1 in endothelial cells. Studies on the nuclear morphology of treated cells revealed that aeroplysinin-1 induces chromatin condensation and nuclear fragmentation, and it increases the percentage of cells with sub-diploid DNA content in endothelial, but not in HCT-116, human colon carcinoma and HT-1080 human fibrosarcoma cells. Treatment of endothelial cells with aeroplysinin-1 induces activation of caspases-2, -3, -8 and -9, as well as the cleavage of apoptotic substrates, such as poly (ADP-ribose) polymerase and lamin-A in a caspase-dependent mechanism. Our data indicate a relevant role of the mitochondria in the apoptogenic activity of this compound. The observation that aeroplysinin-1 prevents the phosphorylation of Bad relates to the mitochondria-mediated induction of apoptosis by this compound.

Facio-Genital Dysplasia-5 Regulates Matrix Adhesion and Survival of Human Endothelial Cells

The function of the endothelial cell (EC)-enriched Rho family guanine nucleotide exchange factor, facio-genital dysplasia-5 (FGD5), is poorly understood. We sought to determine whether FGD5 regulates endothelial cytoskeletal reorganization and angiogenesis. We observed that FGD5 is expressed in primary human EC isolated from sites across the vasculature. Inhibition of FGD5 expression using RNA interference decreased the protein by ≈70%. In 3-dimensional vascular endothelial growth factor-stimulated angiogenesis in vitro, FGD5-deficient endothelial sprout protrusion was markedly blunted versus nonsilenced controls. FGD5 knockdown impaired adhesion to fibronectin and collagen IV and remodeling of matrix adhesion complexes. Similarly, monolayer electric impedance was decreased, and impedance increased at a slower rate after seeding FGD5-deficient cells versus controls, reflecting decreased EC spreading. Further, FGD5 plays a role in cell survival, because expression of cleaved caspase-3 was increased in FGD5-deficient EC after loss of cell-matrix contacts, and proapoptotic tumor necrosis factor-α stimulation elicited EC with subdiploid DNA content among FGD5-deficient EC. Mechanistically, the phosphatidylinositol 3-kinase/Akt pathway that regulates both adhesive and survival signal transduction pathways requires FGD5. Vascular endothelial growth factor-stimulated Akt phosphorylation and downstream forkhead box protein-O1 inactivation is inhibited by FGD5 loss. FGD5 regulates endothelial adhesion, survival, and angiogenesis by modulating phosphatidylinositol 3-kinase signaling.

Abstract 3738: A rationale for combining the targeted investigational agents TAK-733, a MEK1/2 inhibitor, with alisertib (MLN8237), an Aurora A kinase inhibitor, for cancer therapy

Abstract MEK1/2 kinases have diverse functions in cell proliferation and survival, including the regulation of cell cycle transitions between the G1, S, and G2 phases and mediation of DNA damage response pathways. Therefore, inhibition of MEK1/2 may facilitate the antiproliferative effects of other therapies that disrupt cell cycle progression leading to DNA damage, including antimitotic therapies. AAK is a serine/threonine protein kinase essential for normal mitotic progression. Inhibition of AAK leads to inappropriate progression through mitosis in the presence of misaligned chromosomes resulting in aneuploidy and DNA damage, which leads to apoptosis or senescence. Here, we evaluated the combination of the allosteric MEK1/2 inhibitor TAK-733 with alisertib, the selective AAK inhibitor. Proliferation assays in multiple tumor cell lines showed a concentration-dependent decrease in the IC50 of TAK-733 and alisertib with increasing concentrations of the second agent, suggesting increased anti-proliferative activity of these agents when combined. This result was corroborated by measuring changes in cell confluence over time, a surrogate for proliferation rate, using an IncuCyte live-cell imaging system. The decreased proliferation rate of the combination was due in part to induction of apoptosis, as demonstrated by Annexin V and sub-diploid DNA content analysis. Cell cycle progression differences of the TAK-733/alisertib combination relative to the single agents were assessed using time-lapse video microscopy by measuring the time to the first mitotic event after drug addition, time spent in mitosis, and time between mitotic events. Of these, the only observed difference in cell-cycle progression time in the A2780 ovarian carcinoma cell line was the time between mitotic events, which was 10 hours longer for the combination relative to single agent alisertib. Moreover, the combination of TAK-733 and alisertib significantly reduced the ability of the cells to re-enter the cell cycle after one mitosis compared to either single agent; 30% for the combination, compared to 90% for TAK-733 and 76% for alisertib. Flow cytometry DNA content analysis showed that TAK-733 suppressed the formation of >4N/tetraploid cells induced by alisertib alone in multiple cell lines. This observed decrease in the proportion of >4N cells is greater than the decrease that could be extrapolated solely from the cell cycle-slowing effect of the combination, suggesting that TAK-733 reduces the proliferative potential of the alisertib-generated aneuploid cells via cell-cycle arrest and apoptosis. Collectively, these findings demonstrate an added benefit of TAK-733 over alisertib alone and provide a pre-clinical rationale for further investigation of the combination of TAK-733 and alisertib for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3738. doi:1538-7445.AM2012-3738

FGD5 deficiency confers defective matrix adhesion and survival among endothelial cells

FGD5 is a putative Rho family guanine nucleotide exchange factor enriched in vascular endothelial cells (EC). We observed FGD5 expression in human EC and seek to determine FGD5 function. Inhibition of FGD5 expression using RNAi decreased the protein by ~70%. In 3D angiogenesis in vitro, FGD5‐knockdown (kd) EC had 45 +/− 8% reduced sprout outgrowth and sprout length versus non‐silenced (NS) controls. We examined FGD5‐kd EC adhesion to matrix. FGD5‐kd cell adhesion to fibronectin was reduced by 28 +/− 4% vs. NS. Similarly, monolayer electrical impedance was decreased after seeding FGD5‐kd cells versus controls, and impedance increased at slower rate (4.6 +/− 0.6 siNS vs. 2.4 +/− 0.5 siFGD5 Ohm/min) reflecting decreased EC spreading. Apoptosis was examined by measurement of cleaved caspase‐3 expression and subdiploid DNA content. After pro‐apoptotic cyclohexamide and tumor necrosis factor‐α stimulus, FGD5‐kd markedly increased apoptosis (54 +/− 4% vs 34 +/− 8% EC display active caspase‐3 and 54 +/− 4% vs 29 +/− 3% EC have subdipolid DNA among siFGD5 vs siNS‐treated cells respectively). These results indicate that FGD5 in EC is involved in adhesion, survival, and angiogenesis.

Formation of ion pairing as an alternative to improve encapsulation and anticancer activity of all-trans retinoic acid loaded in solid lipid nanoparticles

This work aims to develop solid lipid nanoparticles (SLNs) loaded with retinoic acid (RA) to evaluate the influence of two lipophilic amines, stearylamine (SA) and benethamine (BA), and one hydrophilic, triethylamine (TA), on drug-encapsulation efficiency (EE) and cytotoxicity in cancer cell lines. The SLNs were characterized for EE, size, and zeta potential. The mean particle size decreased from 155 ± 1 nm (SLNs without amine) to 104 ± 4, 95 ± 1, and 96 ± 1 nm for SLNs prepared with SA, BA, and TA, respectively. SA-RA-loaded SLNs resulted in positively charged particles, whereas those with TA and BA were negatively charged. The EEs were significantly improved with the addition of the amines, and they increased from 36% ± 6% (without amine) to 97% ± 2%, 90% ± 2%, and 100% ± 1% for SA, TA, and BA, respectively. However, stability studies showed higher EE for BA-RA-loaded SLNs than TA-RA-loaded SLNs after 30 days. The formulations containing SA loaded or unloaded (blank SLNs) with RA were cytotoxic in normal and cancer cell lines. In contrast, the blank SLNs containing TA or BA did not show cytotoxicity in human breast adenocarcinoma cells (MCF-7), while RA-loaded SLNs with the respective amines were significantly more cytotoxic than free RA. Furthermore, the cytotoxicity of BA-RA-loaded SLNs was significantly higher than TA-RA-loaded SLNs. These findings are in agreement with the data obtained in the evaluation of subdiploid DNA content and cell-cycle analysis, which showed better anticancer activity for BA-RA-loaded SLNs than TA-RA-loaded SLNs and free RA. Taken together, these findings suggest that the BA-RA-loaded SLN formulation is a promising alternative for the intravenous administration of RA in the treatment of cancer.

Potentiation of Apoptosis in MDS/AML by Combination of Azacitidine and the EGFR-Tyrosine Kinase Inhibitor (TKI) erlotinib

Abstract 2790 Background:TKIs were initially developed as targeted therapies that would solely interfere with a “specific” aberrant signaling pathway in malignant cells. However, we and others showed that the EGFR TKI erlotinib (Erlo) has in vivo and in vitro efficacy in MDS and AML (Boehrer et al., Blood, 2008). We also previously observed, in a preliminary study, the potentiation of apoptosis upon combination of Erlo with azacitidine (Aza), but not with decitabine, in HL-60 cells and in cells from a few (five) MDS/AML patients (ASH 2010, 974). We decided to expand this pre-clinical study to define the potential interest of combining different TKI (Dasatinib (Dasa), Sorafenib (Sora) or Erlo) with Aza, now a reference first line treatment in higher risk MDS (Lancet Oncol, 2009). Methods:Erlo (10μM), Dasa (500nM), or Sora (5μM) were combined to Aza (1μM) and apoptosis over-time (24, 48 and 72h) quantified by FACS analysis following DioC3(6)/PI staining in different MDS/AML-derived cell lines (MOLM-13, SKM-1, MV4-11, Kasumi-1). Quantification of apoptosis at 48h or 72h was recapitulated ex vivo in CD34+ cells from patients with MDS (n=12), AML (n=14) or AML post MDS (n=5). For each single drug, as well as the respective combinationsErlo, Aza and Erlo+Aza, the capacity to induce cell cycle arrest (PI staining), cytotoxicity (MTT assay) and decrease of proliferation (Click-it EdU Assay) was assessed concomitantly. Differentiation was assessed by staining with CD11b on day 3 (FACS), evaluation of ROS production at 24/48h by FACS staining with CM-DFCDA (oxidative stress indicator) and HE (Hydroethidine). Functional relevance of apoptosis-related signaling pathways was determined by co-incubation of Aza (and drugs combinations with biochemical inhibitors against MAPK: JNK (SP600125, 10μM), p-38MAPK (SB203580, 10μM) and MEK (U0126, 5μM). Immunoblot analyses of caspase-3, PARP, Mcl-1 and Bcl-xl were performed at 24h and 48h. Results:Whereas co-incubation of Dasa or Sora with Aza did not increase the degree of apoptosis observed with Aza alone, combination of Erlo with Aza had synergistic effects already observed at 24h, an effect that increased over-time (SKM-1 72h (mean PI+ cells, n=3), Erlo: 15%, Aza: 40%, Erlo+Aza: 81% - MOLM-13 72h, Erlo: 21%, Aza: 25%, Erlo+Aza: 64%). The % of cells with intact metabolic activity at 72h (determined by the MTT assay using 2.5μM Erlo and 0.5μM Aza) decreased from 83% (Erlo) and 79% (Aza) to 23% (Erlo+Aza) in SKM-1 (similar results for MOLM-13 cells). To determine if Erlo also impacts on apoptosis in CD34+ cells from patients with MDS or AML, we screened 31 samples and observed a synergistic effect in 5/31 samples (2/12 MDS, 2/14 LAM, 1/5 AML post MDS) and an additive effect in 8/31 samples. Induction of apoptosis was not preceded by differentiation (no increase in CD11b) or production of ROS. On the other hand, analysis of cell cycle distribution at 24h showed that apoptosis was accompanied by an increase in G0/G1 arrest and a decrease in the % of cells in S phase upon incubation with the combination of Erlo+Aza (G0/G1 (mean, n=2) in SKM-1, DMSO: 51%, Erlo: 52%, Aza: 61%, Erlo+Aza: 78%, G0/G1 in MOLM-13, DMSO: 40%, Erlo: 51%, Aza: 38%, Erlo+Aza: 65%) confirmed by the study of newly synthesized DNA before induction of apoptosis (15h) with an alternative BrdU test (Click-EdU assay). Indeed, in SKM-1 cells, proliferation was decreased by 20% upon Erlo or Aza and by 80% with the association. Moreover, cell cycle arrest was associated with an increase in the % of apoptotic cells with a sub-diploid DNA content (about 10% for Erlo or Aza and 32% for Erlo+Aza in SKM-1. Immunoblot analyses confirmed an increase in cleaved PARP and caspase-3 upon incubation with Aza +Erlo as compared to the single agents, as well as a decrease of the anti-apoptotic protein Mcl-1 and Bcl-xl. Incubation of Aza with SP600125 induced the same extent of apoptosis observed with Erlo+Aza, whereas no effect was observed with the p-38MAPK inhibitor SB203580 or the MEK inhibitor U0126. In addition, Erlo decreased phosphorylation of JNK on Thr183/Tyr185, suggesting a potential implication of the JNK pathway in the mechanism of apoptosis. Conclusions:In this study, Erlo was the only TKI tested able to increase sensitivity towards Aza in MDS/AML cell lines and in patient-derived cells. These results suggest a potential clinical interest of combining Aza to Erlo in MDS/AML. Disclosures:Fenaux:Celgene: Honoraria, Research Funding.

USP1 Deubiquitinates ID Proteins to Preserve a Mesenchymal Stem Cell Program in Osteosarcoma

Inhibitors of DNA binding (IDs) antagonize basic-helix-loop-helix (bHLH) transcription factors to inhibit differentiation and maintain stem cell fate. ID ubiquitination and proteasomal degradation occur in differentiated tissues, but IDs in many neoplasms appear to escape degradation. We show that the deubiquitinating enzyme USP1 promotes ID protein stability and stem cell-like characteristics in osteosarcoma. USP1 bound, deubiquitinated, and thereby stabilized ID1, ID2, and ID3. A subset of primary human osteosarcomas coordinately overexpressed USP1 and ID proteins. USP1 knockdown in osteosarcoma cells precipitated ID protein destabilization, cell-cycle arrest, and osteogenic differentiation. Conversely, ectopic USP1 expression in mesenchymal stem cells stabilized ID proteins, inhibited osteoblastic differentiation, and enhanced proliferation. Consistent with USP1 functioning in normal mesenchymal stem cells, USP1-deficient mice were osteopenic. Our observations implicate USP1 in preservation of the stem cell state that characterizes osteosarcoma and identify USP1 as a target for differentiation therapy.

Ascorbic acid has superior antiviral and antiproliferative effects over IFN-alpha in HAM/TSP PBMC ex vivo

IFN-alpha and high dose ascorbic acid (AA) have a modest clinical benefit in HAM/TSP (Nakagawa, 1996). We investigated the effect of ex vivo and in vitro AA and IFN-alpha treatment on HAM/TSP PBMC and HTLV-1-infected cell lines, respectively. We treated cells for 48-72h ex vivo and in vitro with low-high (10-100µg/ml) dose of AA and 1000U/ml of IFN-alpha and quantified lymphoproliferation by [3H]thymidine incorporation, tetraploid DNA content and PCNA expression (flow cytometry). Viral expression was measured at the RNA (tax, LTR) and protein (Tax, p19) level by RT-PCR, Western blot and ELISA, respectively. Apoptosis was quantified by subdiploid DNA content (flow cytometry). Th1/Th2/Th17 cytokines were quantified by cytometric bead array. AA induced a dramatic 95% decrease (control 3689±755 cpm vs. AA 121±52 cpm, p=0.001) in spontaneous, virus-driven lymphoproliferation and a decrease in tax and LTR transcription in HAM/TSP PBMC. In addition, AA decreased the exacerbated ex vivo IFN-gamma production in HAM/TSP PBMC. In HTLV-1 infected cell lines (MT-2 and MT-4), AA induced a dose-dependent increase in DNA fragmentation (p=0.02, p=0.005), paralleled by a decrease in PCNA (p=0.003), as well as p19 (p<0.001) and Tax levels. These effects appear virus-specific, since high-dose (100µg/ml) AA did not exert a significant antiproliferative or pro-apoptotic effect on PBMC of normal donors. On top, AA displayed a superior antiproliferative, antiviral and immunomodulatory effect over IFN-alpha in both cell lines and HAM/TSP PBMC. Considering the selective antiproliferative and antiviral effects of AA ex vivo and in vitro, the therapeutic potential of combination therapy with high dose AA in HAM/TSP should be further explored.

Abstract 197: Multi-cellular tumor spheroids in vitro model reveals a critical role of ERBB4 in survival of osteosarcoma

Abstract Multi-cellular tumor spheroids (MCTS) model is an in vitro model holding an intermediate complexity between monolayer culture and solid tumor. It is cell-cell adhesion dependent and closely resembles avascular tumor regions and micrometastasis in pro-angiogenesis stage. Osteosarcoma (OS) is the most common malignant bone tumor with high incidence of early lung metastasis; therefore, multi-cellular tumor spheroid can be used to model its in vivo behaviors. Our group previously reported the expression profile of ERBB family in osteosarcoma tissue samples and cell lines. To further study ERBB pathways in osteosarcoma tumor spheroids, we generated OS tumor spheroids by plating cells on non-adherent poly-HEMA pre-coated dishes. Under anchorage-independent conditions, OS cells spontaneously generate multi-cellular spheroids to acquired anoikis resistant by cell-cell adhesion; blocking the formation of spheroids will result rapid cell death and increased PARP cleavage. MCTSs showed lower proliferation rate than monolayer culture, and they held a chemo-resistant phenotype, as shown by proliferation assay and subdiploid DNA content. Surprisingly, ERBB4 expression was up-regulated in non-adherent tumor spheroids compared with monolayer culture (1.2, 2.9, 6.3 fold respectively in 3 cell lines) while EGFR and ERBB2 did not. With knockdown of ERBB4 expression by shRNA, OS cells became less tolerant to anoikis and serum starvation, as shown by PARP cleavage and subdiploid DNA content. Moreover, knockdown of ERBB4 increased the chemo-sensitivity of OS cells against multiple chemotherapy including cisplatin, doxorubicin, and methotrexate (71%, 71%, 60% in survival compared to scramble control). In conclusion, our data showed that ERBB4 contributes to the tumorigenicity of osteosarcoma and plays a protective role against multiple exogenous apoptotic stimuli including matrix detachment, nutrition insufficiency and cytotoxic reagents. Although wildly expressed in osteosarcoma, the role of ERBB4 in tumor biology is largely unknown. These findings indicate that ERBB4 is a key factor regulating survival pathway of OS under various cellular stress condition. Thus, in primary osteosarcoma and metastatic lesion, ERBB4 might be a potential therapeutic target to facilitate multi-modal therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 197. doi:10.1158/1538-7445.AM2011-197

Ikarugamycin induces DNA damage, intracellular calcium increase, p38 MAP kinase activation and apoptosis in HL-60 human promyelocytic leukemia cells

Ikarugamycin (IKA) is an antibiotic with strong antiprotozoal and cytotoxic activity. The purpose of our work was to provide insight into the mechanism of action characterizing the cytotoxic effect of IKA in HL-60 leukemia cells in order to evaluate its potential as an antineoplastic agent. Cell viability was reduced in response to IKA (IC 50 of 221.3 nM), while the amount of HL-60 cells with a subdiploid DNA content increased significantly after 24 h. Apoptotic cell death was confirmed by the cleavage of caspase-9, -8 and -3 using immunoblotting. Single cell gel electrophoresis pointed to an early genotoxic effect. Monitoring of intracellular calcium ([Ca 2+] i) levels by flow cytometric analysis of Fluo-3-AM fluorescence indicated an increase in cytosolic calcium that correlated with the cleavage of caspases. In addition, IKA triggered the activation of p38 MAP kinase which was partly dependent on elevated [Ca 2+] i concentrations and contributed to caspase activation. The data demonstrate that IKA induced apoptosis in HL-60 cells through genotoxicity and caspase activation which was in part correlated to an increase in intracellular calcium levels and activation of p38 MAP kinase.