Dose-dependent Antiproliferative Effect Research Articles

Improving the bioactivity of Zn(ii)-curcumin based complexes

New Zn(II)-curcumin based heteroleptic complexes (1-5) have been synthesized and fully characterized, with the aim to improve the bioactivity of the precursor derivative [(bpy-9)Zn(curc)Cl] (A), a potentially intercalating antitumor agent recently reported. Some structural changes have been made starting from the reference complex A, in order to introduce new functionalities, such as electrostatic and/or covalent interactions. In particular, keeping the same N,N chelating ligand, namely bpy-9, two completely different Zn(II) species have been obtained: a tetracoordinated Zn(II) cation with tetrafluoroborate as counterion (1) and a dimeric neutral complex in which the sulfate anion acts as a bridging group through two Zn(II) centres (2). Moreover, by changing the N,N chelating unit, [(L(n))Zn(curc)Cl] complexes (3-5), in which the Zn(II) ion shows the same pentacoordination seen in the precursor complex A, have been obtained. The antitumour activity of all new Zn(II) complexes was tested in vitro against the human neuroblastoma cell line SH-SY5Y in a biohybrid membrane system and the results indicate that all species exhibit strong cytotoxic activity. In particular the ionic tetrafluoroborate Zn(II) complex, 1, and the neutral phenanthroline based Zn(II) derivative, 4, show the strongest growth inhibition, being even more effective than the model complex A. Both complexes have a dose-dependent anti-proliferative effect on cells as demonstrated by the decrease of viability and the increase of Annexin V and PI-positive cells with the increase of their concentration. Cells treated with complexes 1 and 4 undergo apoptosis that involves the activation of JNK, caspase 3 and MMP changes. Finally, complex 1 is more effective in the induction of caspase-3 activation demonstrating its ability to trigger the execution-phase of cell apoptosis.

Pomegranate Juice Metabolites, Ellagic Acid and Urolithin A, Synergistically Inhibit Androgen-Independent Prostate Cancer Cell Growth via Distinct Effects on Cell Cycle Control and Apoptosis

Ellagitannins (ETs) from pomegranate juice (PJ) are bioactive polyphenols with chemopreventive potential against prostate cancer (PCa). ETs are not absorbed intact but are partially hydrolyzed in the gut to ellagic acid (EA). Colonic microflora can convert EA to urolithin A (UA), and EA and UA enter the circulation after PJ consumption. Here, we studied the effects of EA and UA on cell proliferation, cell cycle, and apoptosis in DU-145 and PC-3 androgen-independent PCa cells and whether combinations of EA and UA affected cell proliferation. EA demonstrated greater dose-dependent antiproliferative effects in both cell lines compared to UA. EA induced cell cycle arrest in S phase associated with decreased cyclin B1 and cyclin D1 levels. UA induced a G2/M arrest and increased cyclin B1 and cdc2 phosphorylation at tyrosine-15, suggesting inactivation of the cyclin B1/cdc2 kinase complex. EA induced apoptosis in both cell lines, while UA had a less pronounced proapoptotic effect only in DU-145. Cotreatment with low concentrations of EA and UA dramatically decreased cell proliferation, exhibiting synergism in PC-3 cells evaluated by isobolographic analysis and combination index. These data provide information on pomegranate metabolites for the prevention of PCa recurrence, supporting the role of gut flora-derived metabolites for cancer prevention.

Interference of a novel indolylmaleimide with microtubules induces mitotic arrest and apoptosis in human progenitor and cancer cells

Indolylmaleimides display a broad spectrum of biological activity and offer great opportunity to influence several aspects of cell fate, as proliferation and differentiation. In this study we describe the effect of PDA-66, a newly synthesised indolylmaleimide, showing a strong dose dependent anti-proliferative effect on immortalised human progenitor and cancer cells. We demonstrated a highly depolymerizing effect on in vitro tubulin assembly and conclude that PDA-66 acts as microtubule destabilising agent. In addition we found that PDA-66 induces mitotic arrest of cells in the G2/M phase of the cell cycle. Subsequently cells undergo apoptosis, indicating the major mechanism of the anti-proliferative effect. To prove a potential anti-cancer activity of PDA-66 we examined the effect of PDA-66 on human SH-SY5Y neuroblastoma and A-459 lung cancer cells, showing a significant reduction in cancer cell proliferation in a dose dependent manner. Thus PDA-66 is a new anti-mitotic compound with an indole-core with the potential to be used for cancer therapy.

Ethanol extract of Gleditsia sinensis thorn suppresses angiogenesis in vitro and in vivo

BackgroundGleditsia sinensis thorns have been widely used in traditional Korean medicine for the treatment of several diseases, including obesity, thrombosis, and tumor-related diseases. The aim of the study is to determine the antiangiogenic effect of Gleditsia sinensis thorns in vitro and in vivo in a bid to evaluate its potential as an anticancer drug.MethodsEthanol extract of Gleditsia sinensis thorns (EEGS) were prepared and used for in vitro and in vivo assays. In vitro antiangiogenic effect of EEGS was determined in HUVEC primary cells by cell migration and tube formation assays. In vivo antiangiogenic effect of EEGS was determined by measuring vessel formation and vascular endothelial cells migrating into the implanted matrigels in nude mice. The angiogenesis-related proteins of which expression levels were altered by EEGS were identified by proteomic analysis.ResultsEEGS exerted a dose-dependent antiproliferative effect on HUVEC cells without significant cytotoxicity. Angiogenic properties, such as cell migration and tube formation, were significantly inhibited by EEGS in a dose-dependent manner. New vessel formation was also suppressed by EEGS, as determined by the directed in vivo angiogenesis assays in nude mice. EEGS reduced the expression of proangiogenic proteins, endothelin 1 and matrix metallopeptidase 2, in HUVEC cells.ConclusionsOur findings suggest that EEGS can inhibit angiogenesis by down-regulating proangiogenic proteins, and therefore it should be considered as a potential anticancer drug targeting tumor-derived angiogenesis.

Upregulation of Focal Adhesion Kinase, a Potential Therapeutic Target, in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS).

Abstract 2827 IntroductionThe myelodysplastic syndromes (MDS) are a group of leukemia characterized by bone marrow failure and increased risk of transformation to acute myelogenous leukemia (AML). Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase (PTK), plays a key role in the integration of protein-mediated signal transduction. It is a critical mediator of the integrin signaling cascade, which modulates cell proliferation, apoptosis, adhesion, spreading and migration. FAK is upregulated in a wide variety of human cancers. It has been reported that expression of FAK in acute myeloid leukemia (AML) is associated with enhanced blast migration, increased cellularity and poor prognosis. Furthermore, FAK silencing inhibited leukemogenesis in BCR-ABL-transformed hematopoietic cells. FAK has been proposed a potential target in cancer therapy. In this study, we studied the expression patterns of FAK in 98 patients with MDS and performed preclinical studies of FAK inhibition in leukemia cell lines. MethodsCD34+ cells from 98 newly diagnosed patients with MDS and 5 normal donor bone marrow specimens were sorted with CD34 isolation kit from Miltenyi Biotec. Total cellular RNA was isolated using Trizol, cDNA was obtained by using TaqMan reverse transcription reagent (Applied Biosystems). For real-time PCR, FAK assay were purchased from Applied Biosystems and analyzed with an Applied Biosystems Prism 7500 Sequencing detection system. GAPDH was used as internal control. Immunohistochemistry was used to detect FAK protein level in cytospin from MDS CD34+ cells. FAK antibody was obtained from abcam. FAK inhibitor was purchased from Santa Cruz Biotechnology. ResultsFor the 98 MDS patients studied, 78% were older than 60 years; by IPSS score, 17 (17.3%) low risk, 35 (35.7%) INT-1, 24 (24.5%) INT-2, 10 (10.2%) high risk, 12 (12.2%) not available. By cytogenetics, diploid 44 (45%), 20q- 7 (7%), -5/5q- 7 (7%), -7/7q- 7 (7%), -5/5q- and -7/7q- 6 (6%), 8+ 6 (6%), IM 6 (6%), MISC 14 (14%). By real-time PCR, we observed FAK overexpression in 28% of MDS CD34+ cells (fold 2.2–26). We then analyzed FAK protein expression in 10 MDS CD34+ cell cytospin with either higher or lower RNA expression by QPCR using immunohistochemistry. The protein expression patterns were 100% consistent with RNA expression level. This result suggests that FAK expression is upregulated in MDS CD34+ cells. We then performed analysis of clinical associations between FAK expression and clinical characteristics. No association was observed in particular between FAK expression and survival. Because of the potential role of FAK as a therapeutic target, we then studied the effects of FAK inhibition in cell lines. We studied FAK expression level in leukemia cell lines of AML origin and found high protein expression level of FAK in AML leukemia cell line OCI-AML3 and KG1. We treated these cells with FAK inhibitor 14 and detected a dose dependent anti-proliferative effect on both cell lines. Using Annexin V - FITC analysis by flow cytometry, we found the FAK inhibition could induce apoptosis in both cell lines at concentrations of 10uM both at 24 hours and 48 hours. ConclusionsThis study shows that FAK is aberrantly expressed in MDS CD34+ cells, together with the anti-proliferative and apoptosis induction effect of FAK inhibitor, our study demonstrates that FAK may be a potential therapeutic target in MDS and AML patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Phytochemical profile and nutraceutical value of old and modern common wheat cultivars.

Among health-promoting phytochemicals in whole grains, phenolic compounds have gained attention as they have strong antioxidant properties and can protect against many degenerative diseases. Aim of this study was to profile grain phenolic extracts of one modern and five old common wheat (Triticum aestivum L.) varieties and to evaluate their potential antiproliferative or cytoprotective effect in different cell culture systems.Wheat extracts were characterized in terms of antioxidant activity and phenolic composition (HPLC/ESI-TOF-MS profile, polyphenol and flavonoid contents). Results showed that antioxidant activity (FRAP and DPPH) is mostly influenced by flavonoid (both bound and free) content and by the ratio flavonoids/polyphenols. Using a leukemic cell line, HL60, and primary cultures of neonatal rat cardiomyocytes, the potential antiproliferative or cytoprotective effects of different wheat genotypes were evaluated in terms of intracellular reactive oxygen species levels and cell viability. All tested wheat phenolic extracts exerted dose-dependent cytoprotective and antiproliferative effects on cardiomyocytes and HL60 cells, respectively. Due to the peculiar phenolic pattern of each wheat variety, a significant genotype effect was highlighted. On the whole, the most relevant scavenging effect was found for the old variety Verna. No significant differences in terms of anti-proliferative activities among wheat genotypes was observed. Results reported in this study evidenced a correspondence between the in vitro antioxidant activity and potential healthy properties of different extracts. This suggests that an increased intake of wheat grain derived products could represent an effective strategy to achieve both chemoprevention and protection against oxidative stress related diseases.

Abstract A54: Potential mechanisms of resistance to targeted agents in human clear cell renal cell carcinoma

Abstract Targeted therapy with multiple receptor tyrosine kinase inhibitors (RTKI) has led to a substantial improvement in the standard of care for patients with advanced or metastatic clear cell renal cell carcinoma (CCRCC). We have previously reported that using a phosphoproteomics approach, Ras and Rab interactor 1 (RIN1) was identified as a potential target modulating the antiproliferative effects of sorafenib and sunitinib in a panel of human CCRCC (Proc. AACR 51: Abst. #1626, 2010). While both 10 μM sorafenib and 7.5 μM sunitinib were effective in down-regulating RIN1 protein levels in CCRCC following treatment for 48h, during subsequent recovery for 48h in drug free medium, recovery of RIN1 protein levels was observed in sorafenib but not sunitinib treated cells. mRNA expression profiling revealed that: (a) VEGF-A and HIF-1α levels were unaffected during 48h treatment with sorafenib or sunitinib or subsequent recovery; and (b) while HIF-2α levels were increased >2-fold following 48h exposure to sorafenib or sunitinib, during subsequent recovery in drug-free medium, the increased levels of HIF-2α persisted in cells treated with sorafenib but not sunitinib. Compared to CCRCC cells that were growth inhibited in vitro at concentrations of 5–10 μM sorafenib or sunitinib, in 27 patient CCRCC samples evaluated, the median mRNA levels of RIN1 was 7.2-fold lower in 22 samples (Range 1.6–22.3) and 2.2-fold higher in 5 samples (Range 1.2–27.1). Stable over-expression and down-regulation of RIN1 in CCRCC led to resistance or sensitivity respectively, to the anti-proliferative effects of sorafenib and sunitinib but not the mTOR inhibitor temsirolimus. In contrast, CCRCC cells adapted to proliferate in 4 μM everolimus exhibited a >3-fold increase in IC50, and cross-resistance to temsirolimus but not sorafenib or sunitinib. Evaluation in vitro of other targeted agents, demonstrated dose-dependent anti-proliferative effects with the PI3K/mTOR dual inhibitor BEZ235 (>80% growth inhibition at 10nM) but not the pan-PI3K inhibitor BKM120 (<30% growth inhibition at 10–500 nM) with CCRCC cells. The observed anti-proliferative effects of BEZ235 or BKM120 were comparable in CCRCC cells harboring the wild type or mutant von Hippel-Lindau gene. While BEZ235 (1–5 nM) or everolimus (100 nM) alone led to <40% growth inhibition, the combination significantly enhanced anti-proliferative effects (>85% growth inhibition) in CCRCC cells expressing resistance to everolimus. These results suggest the PI3K/mTOR dual inhibitor BEZ235 may possibly improve the treatment efficacy of mTOR inhibitors for CCRCC. The findings suggest RTKI and mTOR inhibitors have distinct mechanisms of resistance, and combinations may be useful to improve disease control of metastatic CCRCC.

A novel Ru(II) complex derived from hydroxydiamine as a potential antitumor agent: Synthesis and Structural Characterization

Abstract A novel Ru(II)-Schiff base complex has been synthesized by the interaction of ligand, 1,3-bis{[(E)-(2-chlorophenyl)methylidene]amino}-2-propanol, L with [RuCl2(PPh3)3]. The structure of ligand, L has been determined on the basis of X-ray diffraction while the geometry of the Ru(II) complex has been ascertained by FT-IR, 1H, 13 C{1H}, 31P{1H} NMR and UV–vis studies. The in vitro antitumor activity of these compounds was assessed by examining their ability to inhibit cell proliferation against human breast and pancreatic cancer cell lines. The results show a dose-dependent anti-proliferative effect and induction of apoptotic cell death in both the cancer cell lines, thus indicating pharmacological significance of Ru(II) complex against cancer.

Cinaciguat prevents neointima formation after arterial injury by decreasing vascular smooth muscle cell migration and proliferation

AimsVascular smooth muscle cell (VSMC) migration, proliferation and remodeling of the extracellular matrix contribute to lumen loss after arterial injury leading to restenosis. Several studies indicated the role of the cyclic guanosine monophosphate signaling in neointimal formation. Cinaciguat, the novel soluble guanylate cyclase activator, currently being in phase IIb clinical trial, has been shown to exert antiplatelet and anti-remodeling effects in animal models of vascular pathology. In this study we investigated the effects of cinaciguat on post-injury arterial stenosis. Methods and ResultsMale Sprague-Dawley rats (n=100) underwent endothelial denudation by wire injury of the right common carotid artery. Cinaciguat (10mg/kg/day orally) were administered to 50 rats (1-, 2-, 3-day and 1-, 3-week treatment time), while 50 rats received placebo. A 3-week treatment resulted in a significantly reduced vascular stenosis (17.53±10.84% in the treatment group vs. 43.25±30.83% in the control wire injury group) and neointima/media area ratio (0.45±0.32 in the treatment group vs. 1.09±0.69 in the control wire injury group). By using quantitative real-time PCR, Western blot and immunohistochemistry, matrix-metallopreoteinase-9 (MMP-9) was found to be upregulated in the control-injured carotids over the whole follow-up, and cinaciguat significantly decreased MMP-9 expression by 3weeks. As assessed by protein immunoblot, injury-induced local decrease of soluble guanylate cyclase β1 subunit could be recovered by cinaciguat. In vitro wound healing assay with VSMCs revealed dose-dependent antimigratory and antiproliferative effects of cinaciguat. Plasma level of cyclic guanosine monophosphate was significantly elevated after 3weeks of treatment. ConclusionOur results show that cinaciguat prevents injury-induced neointimal hyperplasia by decreasing VSMC migration and proliferation through the regulation of MMP-9.

Trans-Resveratrol Alters Mammary Promoter Hypermethylation in Women at Increased Risk for Breast Cancer

Trans-resveratrol, present in high concentration in the skin of red grapes and red wine, has a dose-dependent antiproliferative effect in vitro, prevents the formation of mammary tumors, and has been touted as a chemopreventive agent. Based upon in vitro studies demonstrating that trans-resveratrol downregulates the expression of 1) DNA methyltransferases and 2) the cancer promoting prostaglandin (PG)E2, we determined if trans-resveratrol had a dose-related effect on DNA methylation and prostaglandin expression in humans. Thirty-nine adult women at increased breast cancer risk were randomized in double-blind fashion to placebo, 5 or 50 mg trans-resveratrol twice daily for 12 wk. Methylation assessment of 4 cancer-related genes (p16, RASSF-1α, APC, CCND2) was performed on mammary ductoscopy specimens. The predominant resveratrol species in serum was the glucuronide metabolite. Total trans-resveratrol and glucuronide metabolite serum levels increased after consuming both trans-resveratrol doses (P < .001 for both). RASSF-1α methylation decreased with increasing levels of serum trans-resveratrol (P = .047). The change in RASSF-1α methylation was directly related to the change in PGE2 (P = .045). This work provides novel insights into the effects of trans-resveratrol on the breast of women at increased breast cancer risk, including a decrease in methylation of the tumor suppressor gene RASSF-1α. Because of the limited sample size, our findings should be validated in a larger study.

Characterisation of antioxidant and antiproliferative acidic polysaccharides from Chinese wolfberry fruits

Abstract Wolfberry fruit polysaccharides (WFPs) were isolated by hot-water extraction and ethanol precipitation. With HPLC analysis, WFPs were for the first time identified as acidic polysaccharides with galacturonic acid being the main component monosaccharide (24.9%), followed by galactose (21.3%), arabinose (18.5%), and glucose (15.9%), accounting for up to 80.6% of the molar percentage. WFPs exhibited a high antioxidant activity and a dose-dependent antiproliferative effect, with IC50 values of 134.9, 70.1 and 138.4 μg/mL against A549, MCF-7 and LoVo cancer cells after 48 h of incubation as estimated by MTT assay, respectively. Flow cytometry analysis showed that WFPs exerted a stimulatory effect on apoptosis of MCF-7 cells, and induced the cell-cycle arrest at the G0/G1 phase, with the observation of intracellular ROS production and DNA damage. The present study demonstrated that these polysaccharides might have the potential to provide significant natural defence against human cancer.

Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia

Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL.

The Relationships Between Biological Activities and Structure of Flavan-3-Ols

Flavan-3-ols are involved in multiple metabolic pathways that induce inhibition of cell proliferation. We studied the structure-activity relationship of gallic acid (GA) and four flavan-3-ols: epigallocatechin gallate (EGCG), epigallocatechin (EGC), catechin (C), and epicatechin (EC). We measured the cell viability by the MTT assay and we determined the concentration of testing compound required to reduce cell viability by 50% (IC50). All tested compounds showed a dose-dependent and time-dependent inhibitory antiproliferative effect on Hs578T cells; IC50 values varying from the 15.81 to 326.8 μM. Intracellular ROS (reactive oxygen species) were quantified using a fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA). Only the treatment with 10 μM EGC and EGCG was able to induce a significant decrease of ROS concentration and increased levels of ROS were registered for 100 μM EGCG, EGC and GA. Flavans-3-ols and GA induced apoptosis in a dose- and time-dependent manner, which indicated that the induction of apoptosis mediated their cytotoxic activity at least partially. The galloylated catechins have shown a stronger antiproliferative activity and apoptotic effect than the one produced by non galloylated catechins. The galloylated flavan-3-ols are potential therapeutic agents for patients with triple negative breast cancer via induction of apoptosis.

Treatment with Nutlin-3, a Small-Molecule Antagonist of MDM2, in Combination with Low Doses of Pegylated Interferon Alpha 2a Targets Hematopoietic Progenitor Cells From Polycythemia Vera (PV) Patients

Abstract 796▪FN2▪This icon denotes a clinically relevant abstractThe chronic myeloproliferative neoplasms (MPN) originate at the level of the hematopoietic stem cell (HSC). The prolonged and frequently unpredictable natural history of the MPNs has limited the use of many therapeutic approaches some of which are associated with unacceptable toxicities. IFNα therapy reverses morphological marrow abnormalities, eliminates cytogenetic abnormalities, reduces the JAK2V617F allele burden and re-establishes polyclonal hematopoiesis in patients with PV. Prolonged IFNα therapy is, however also frequently difficult due to drug related toxicity. We hypothesized that the addition of a non-genotoxic therapeutic agent to lower doses of IFNα might be better tolerated and be effective in eliminating the PV clone. The tumor suppressor, p53 gene plays an important role in the control of DNA repair, cell cycling, and apoptosis and is frequently inactivated in many cancers. The cellular level of p53 is controlled by a master regulator, MDM2. Nutlin-3, a small-molecule antagonist of MDM2 which activates the p53 pathway in cancer cells with wild-type p53 and is currently being evaluated in clinical trials. In MPN, p53 mutations and heterozygous deletions have been identified in a limited number of patients undergoing transformation to acute leukemia. We have previously reported that IFNα acts through p38 MAP kinase (Lu M, et al. Exp Hematol. 2010;38:472-80) while others have shown that activating this pathway leads to increased p53 (Sancéau J, Oncogene. 2000;19:3372-83). We hypothesized that targeting alternative pathways which can up-regulate p53 might serve as a novel therapeutic strategy for treating MPN patients. In this study, the therapeutic effects of nutlin-3 combined with Pegylated interferon alpha 2a on PV and normal CD34+ cells were investigated. FACS and western blot analysis demonstrated that the p53 protein levels in PV CD34+ cells were lower than normal CD34+ cells. CD34+ cells isolated from 8 PV patients and 7 normal donors were exposed to low doses of Peg IFNa 2a (200 U/ml) and/or nutlin-3 (200 nM) for 4 days and the number of hematopoietic progenitor cells (HPC) assayed. A dose dependent anti-proliferative effect of nutlin-3 on PV CD34+ cells was documented. PV CD34+ cells treated with nutlin-3 at doses ranging from 100 nM to 1000 nM, led to a progressively greater inhibition of colony formation from 10% to 80%. Treatment of normal CD34+ cells with 200 nM of nutlin-3 did not affect BFU-E or CFU-GM numbers, but inhibited PV CFU-GM by 20% and BFU-E by 50%. Simultaneous treatment with low doses of both nutlin-3 and Peg IFNa 2a inhibited PV CFU-GM by 55% and BFU-E by 90%, while normal cord blood CFU-GM and BFU-E were inhibited by 18% and 22%, respectively. This inhibition of PV HPC was far greater than that observed following the treatment with either agent alone. The combination of drugs also further decreased the proportion of JAK2V617F positive HPCs, as compared to CD34+ cells treated with Peg IFNa 2a alone (between 12.5% and 30% greater reduction). In addition, in a case of JAK2V617F negative myelofibrosis with a deletion of the long arm of chromosome 20 del(20) (q11–q12), Peg IFNα 2a and Nutlin-3 treatment of CD34+ cells cultured in the presence of SCF, TPO, IL-3 and FLT3 ligand reduced the number of cytogenetically abnormal total cells to 1% as compared to 15% observed following Peg IFNa 2a treatment alone. These data suggest that Peg IFNα 2a combined with Nutlin-3 treatment can substantially reduce the number of malignant MPN HPCs. The mechanism by which nutlin-3 and Peg IFNa 2a act on PV HPC was explored by FACS and Western Blot analysis using a JAK2V617F positive erythroleukemia cell line (HEL cells) and K562 cells. Peg IFNa 2a and nutlin-3 treatment each individually increased the p53 protein level and nuclear translocation in HEL but not in K562 cells. A further increase in p53 was observed when HEL cells were exposed to the combination of Peg IFNa 2a and nutlin-3. These data suggest that Peg IFNa 2a leads to increased p53 production while nutlin-3 results in reduced p53 degradation thereby providing a means of up regulating wild type p53 in PV cells. It is anticipated that the completion of these studies will provide a rationale for a phase I clinical trial evaluating the effects of nutlin3 alone or in combination with low doses of IFNα for the treatment of MPN patients. Disclosures:No relevant conflicts of interest to declare.

Differential effect of atorvastatin and tacrolimus on proliferation of vascular smooth muscle and endothelial cells

Although considered promising for use in drug-eluting stents (DES), tacrolimus failed clinically. Tacrolimus inhibits growth factor production but can also act as a growth factor on vascular smooth muscle cells (VSMC). This unexpected proliferative stimulus could reverse the beneficial effects of the drug on restenosis. We hypothesized that tacrolimus' association with statins, which lower cholesterol and impair cell proliferation, could restore tacrolimus' beneficial effect by abrogating the aberrant proliferative stimulus. Additionally, since maintenance of endothelial function represents a challenge for new-generation DES, we investigated the combined effect of tacrolimus and atorvastatin on endothelial cells. Human VSMC and umbilical vein endothelial cells (HUVEC) were incubated with 100 nM tacrolimus and increasing doses of atorvastatin (0-3.0 μM). Atorvastatin plus tacrolimus dose-dependently inhibited VSMC proliferation. The percentage of cells incorporating 5-bromo-2'-deoxyuridine (BrdU) in their DNA was 49 ± 14% under basal conditions, 62 ± 15% (P = 0.01) with tacrolimus, 40 ± 22% with 3 μM atorvastatin, and 30 ± 7% (P < 0.05) with 3 μM atorvastatin plus tacrolimus. Atorvastatin downregulated β-catenin, Erk1 and Erk2, and cyclin B in tacrolimus-stimulated VSMC. In contrast, atorvastatin plus tacrolimus did not affect proliferation of endothelial cells. The percentage of HUVEC incorporating BrdU in their DNA was 47 ± 8% under basal conditions, 58 ± 6% (P = 0.01) with tacrolimus, 45 ± 4% with 3 μM atorvastatin, and 49 ± 1% with 3 μM atorvastatin plus tacrolimus. Both agents stimulated endoglin production by HUVEC. Taken together, these results suggest that, when combined with tacrolimus, atorvastatin exerts a dose-dependent antiproliferative effect on VSMC. In contrast, atorvastatin acts in concert with tacrolimus in HUVEC to stimulate production of endoglin, a factor that has an important role in endothelial repair. Our study supports the conclusion that prevention of postcoronary in-stent restenosis and late thrombosis may benefit of concomitant association of tacrolimus and high doses of atorvastatin.

Enhanced Drug Loading on Magnetic Nanoparticles by Layer-by-Layer Assembly Using Drug Conjugates: Blood Compatibility Evaluation and Targeted Drug Delivery in Cancer Cells

Drug targeting using magnetic nanoparticles (MNPs) under the action of an external magnetic field constitutes an important mode of drug delivery. Low cargo capacity, particularly in hydrophobic drugs, is one limitation shown by MNPs. This article describes a simple strategy to enhance the drug-loading capacity of MNPs. The approach was to use polymer-drug conjugates to modify MNPs by layer-by-layer assembly (LbL). Curcumin (CUR) has shown remarkably high cytotoxicity toward various cancer cell lines. However, the drug shows low anticancer activity in vivo because of its reduced systemic bioavailability acquired from its poor aqueous solubility and instability. To address this issue, we synthesized cationic and anionic CUR conjugates by anchoring CUR onto poly(vinylpyrroidone) (PVP-Cur) and onto hyaluronic acid (HA-Cur). We used these oppositely charged conjugates to modify MNPs by layer-by-layer (LbL) assembly. Six double layers of curcumin conjugates were constructed on positively charged amino-terminated magnetic nanoparticles, TMSPEDA@MNPs. Finally, HA was coated onto the outer surface to form HA (HA-Cur/PVP-Cur)(6)@MNPs. Cellular viability studies showed the dose-dependent antiproliferative effect of HA (HA-Cur/PVP-Cur)(6)@MNPs in two cancer cell lines (glioma cells and Caco-2 cells). HA (HA-Cur/PVP-Cur)(6)@MNPs exhibited more cytotoxicity than did free curcumin, which was attributed to the enhanced solubility along with better absorption via hyaluronic acid receptor-mediated endocytosis. Flow cytometry showed enhanced intake of the modified MNPs by cells. Confocal microscope images also confirmed the uptake of HA (HA-Cur/PVP-Cur)(6)@MNPs with greater efficacy. Thus, the strategy that we adopted here appears to have substantial potential in carrying enhanced payloads of hydrophobic drugs to specified targets.

Chemical composition and anti-proliferative and anti-inflammatory effects of the leaf and whole-plant samples of diploid and tetraploid Gynostemma pentaphyllum (Thunb.) Makino

Leaf and whole-plant samples of the diploid and tetraploid Gynostemma pentaphyllum (GP) were investigated and compared for their chemical compositions, and their potential anti-proliferative and anti-inflammatory effects. The highest levels of total flavonoids and phenolics were observed in the diploid leaf botanical (2L3) at 36.84mg rutin equiv/g and 41.15mg gallic acid equiv/g, respectively. The diploid leaf sample (2L2) had the highest amount of rutin and quercetin contents of 77.7μmol quercetin equiv/g. The tetraploid whole-plant botanical (4L3) had the highest total saponin content of 227.1mg gypenoside equiv/g. Extracts from all tested GP samples showed time- and dose-dependent antiproliferative effects in HT-29 cells, and the diploid leaf samples had the overall highest inhibitory activity. These extracts had different order of antiproliferative properties in the LNCaP cells, suggesting the potential selective inhibition of GP extracts against different types of cancer cells and the effect of the cell model in screening and evaluation of antiproliferative components. In addition, the diploid leaf extracts showed the strongest inhibitory effects on the expression of TNF-α, IL-6 and COX-2 mRNA at final concentrations of 0.2 and 1mg botanical equiv/ml media. The results from this study will be used to develop new nutraceutical products from G. pentaphyllum.

Propranolol potentiates the anti-angiogenic effects and anti-tumor efficacy of chemotherapy agents: implication in breast cancer treatment

Recent clinical evidence revealed that the use of beta-blockers such as propranolol, prior to diagnosis or concurrently with chemotherapy, could increase relapse-free and overall survival in breast cancer patients. We therefore hypothesized that propranolol may be able to increase the efficacy of chemotherapy either through direct effects on cancer cells or via anti-angiogenic mechanisms. In vitro proliferation assay showed that propranolol (from 50-100 μM) induces dose-dependent anti-proliferative effects in a panel of 9 human cancer and "normal" cell lines. Matrigel assays revealed that propranolol displays potent anti-angiogenic properties at non-toxic concentrations (less than 50 μM) but exert no vascular-disrupting activity. Combining chemotherapeutic drugs, such as 5-fluorouracil (5-FU) or paclitaxel, with propranolol at the lowest effective concentration resulted in synergistic, additive or antagonistic effects on cell proliferation in vitro depending on the cell type and the dose of chemotherapy used. Interestingly, breast cancer and vascular endothelial cells were among the most responsive to these combinations. Furthermore, Matrigel assays indicated that low concentrations of propranolol (10 - 50 μM) potentiated the anti-angiogenic effects of 5-FU and paclitaxel. Using an orthotopic xenograft model of triple-negative breast cancer, based on s.c injection of luciferase-expressing MDA-MB-231 cells in the mammary fat pad of nude mice, we showed that propranolol, when used alone, induced only transient anti-tumor effects, if at all, and did not increase median survival. However, the combination of propranolol with chemotherapy resulted in more profound and sustained anti-tumor effects and significantly increased the survival benefits induced by chemotherapy alone (+19% and +79% in median survival for the combination as compared with 5-FU alone and paclitaxel alone, respectively; p less than 0.05). Collectively our results show that propranolol can potentiate the anti-angiogenic effects and anti-tumor efficacy of chemotherapy. The current study, together with retrospective clinical data, strongly suggests that the use of propranolol concurrently with chemotherapy may improve the outcome of breast cancer patients, thus providing a strong rationale for the evaluation of this drug combination in prospective clinical studies.

Synthesis and Biochemical Evaluation of Δ2-Isoxazoline Derivatives as DNA Methyltransferase 1 Inhibitors

A series of Δ(2)-isoxazoline constrained analogues of procaine/procainamide (7a-k and 8a-k) were prepared and their inhibitory activity against DNA methyltransferase 1 (DNMT1) was tested. Among them, derivative 7b is far more potent in vitro (IC(50) = 150 μM) than other non-nucleoside inhibitors and also exhibits a strong and dose-dependent antiproliferative effect against HCT116 human colon carcinoma cells. The binding mode of 7b with the enzyme was also investigated by means of a simple competition assay as well as of docking simulations conducted using the recently published crystallographic structure of human DNMT1. On the basis of the findings, we assessed that the mode of inhibition of 7b is consistent with a competition with the cofactor and propose it as a novel lead compound for the development of non-nucleoside DNMT inhibitors.

Inhibitory effects of Broccolini leaf flavonoids on human cancer cells

Broccolini (Brassica oleracea Italica × Alboglabra) is a hybrid between broccoli and Gai Lan, also known as Chinese broccoli and Chinese kale. The aim of this study was to assess the antitumor activity of Broccolini leaf flavonoids (BLF). Cell growth inhibition was evaluated using a standard colorimetric MTT assay, cellular morphology was observed using phase contrast microscopy and flow cytometry was introduced to further investigate cells apoptosis effect. The results showed that BLF possess a dose-dependent antiproliferative effects on four human cancer cell lines (SW480, HepG2, Hela, and A549) and apoptosis induction activity on SW480 cell line. Thus, the hybrid species Broccolini could be considered as a functional vegetable with potential in assisting for the treatment of four human cancers examined here.