Higher Antiproliferative Effect Research Articles

Inhibitory properties of camel whey protein hydrolysates toward liver cancer cells, dipeptidyl peptidase-IV, and inflammation

This report describes an investigation of camel whey protein hydrolysates (CWPH) produced by gastric and pancreatic enzymes for their in vitro antidiabetic, anticancer, and anti-inflammatory properties. Degree of hydrolysis (DH) ranged from 8.54 to 47.53%, with hydrolysates generated using chymotrypsin for 6 h displaying the highest DH. Reverse phase-HPLC analysis showed that α-lactalbumin underwent complete degradation, with no intact α-lactalbumin detected in CWPH. The CWPH displayed enhanced antidiabetic activity compared with intact whey proteins; with pepsin- and chymotrypsin-generated CWPH displaying greater inhibition of dipeptidyl peptidase IV (DPP-IV), α-glucosidase, and α-amylase compared with trypsin-generated CWPH. The highest antiproliferative effect was observed for CWPH generated by chymotrypsin for 3 h, with only 4.5 to 6.5% viable liver cancer cells (HepG2) remaining when tested at concentrations from 400 to 1,000 µg/mL. The highest anti-inflammatory activity was manifested by CWPH generated by pepsin at 6-h hydrolysis. We report enhanced antiproliferative, antidiabetic, and anti-inflammatory activities upon hydrolysis of camel whey proteins, indicating their potential utilization as bioactive and functional ingredients.

Synthesis of Oleanolic Acid Analogues and Their Cytotoxic Effects on 3T3 Cell Line.

Oleanolic acid (OA) is a known natural compound with many important biological activities. Thirteen oleanolic acid derivatives linked at C-3 and C-28 were synthesized and their structures were confirmed by 1H- and 13C NMR and mass spectral analyses. Among them, compounds 4, 6, 8-10, 12, 13 were synthesized for the first time. They were evaluated for their cytotoxic activity. They showed proliferative effect at low concentrations while cytotoxic effect was observed at high concentrations in a dose dependent manner. We have first synthesized compounds 1 and 2 from the reaction of methyl iodide and OA. Compound 1 was reduced with LiAlH4 to give compound 3, and compound 2 gave compound 9 with MOMBr as a new compound. The compound 10 was then obtained from the reduction of compound 9 with LiAlH4 as a new oleanolic acid derivative. A diol derivative 11 was synthesized from OA and LiAlH4 at the room temperature. Compound 4 was obtained from the reaction of compound 3 with CBr4 as a new analogue of OA, and the reduction of compound 4 afforded compound 5 as a known product. In addition, we synthesized compounds 6-8 from compound 3 using MsCl, MeI and p-nitrobenzoyl chloride, respectively, in good yields. Compounds 6 and 8 are new analogues of OA. The new compounds 12 and 13 were also synthesized starting from OA using with MOMBr and TBDMSiCl as the reagents. The all synthesized compounds were purified by using column chromatography and/or crystallization. In the present study, thirteen OA derivatives linked at C-28 and (or) C-3 were synthesized and evaluated for their cytotoxic activity on 3T3 cell lines which are the standard fibroblast cell lines, derived from Swiss albino mouse embryo tissue. 3T3 cell viability was observed at low concentrations of the tested triterpenoids while they displayed anti-proliferative effect at higher concentrations. Oleanolic acid 28-methyl ester (2) showed fairly different behavior from all the other compounds tested and found to be the least cytotoxic compound. However, at 200 µM concentration, it exhibited the same cytotoxicity with compounds 3, 9 and 10 around 58-59%. Among the tested 13 compounds, 7 exhibited the most drastic decline for the viability from 12,5 µM to 25 µM concentration. Compound 6 displayed the most cytotoxic effect, almost in all concentrations, particularly at 6.25 and 25 µM concentrations while the highest cytotoxic effect at 50 µM was observed for compound 11 among all the tested triterpenoids. As a result, all the tested OA derivatives showed proliferative effect at 1,56 µM although no proliferative effect was observed for OA. Moreover, OA exhibited higher cytotoxic effect than its derivatives, particularly at higher concentrations (50, 100, 200 µM) with an exception for compound 11. Because, the latter showed highest proliferative effect at lowest concentration, and highest anti-proliferative effect at highest concentration which surpassed all the OA derivatives.

Abstract 1890: Inecalcitol respectively induces or increases CD38 expression at the surface of CD38- or CD38+ AML cell lines representative of all 9 FAB subtypes except M6

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease classified into 9 FAB subtypes according to the origin and/or maturity of the malignant cells: M0 undifferentiated, M1 minimally mature myeloblastic, M2 mature myeloblastic, M3 promyelocytic, M4 myelomonocytic, M4eos eosinophilic, M5 monocytic, M6 erythrocytic and M7 megakaryocytic. Overcoming this heterogeneity by the induction of a common surface antigen which would already be the target of an existing immunotherapy would be of great potential therapeutic interest to standardize a common immunotherapy approach to AML. Inecalcitol, a vitamin D receptor agonist characterized by a high anti-proliferative effect and a low calcemic potential, has been shown to stimulate CD38 expression at the surface of the M3 subtype HL-60 cell line and of the M5 subtype U-937, MOLM-13 and THP-1 cell lines (ASH 2016, AACR 2017). We now report the effect of inecalcitol on AML cell lines representative of the 7 other FAB subtypes: KG-1 (M0), UOC-M1 (M1), Kasumi-1 (M2), OCI-AML2 (M4), EOL-1 (M4eos), HEL-92.1.7 (M6) and CMK (M7). Inecalcitol induced CD38 at the surface of CD38- cell lines (HL-60, U-937 and EOL-1) and strongly increased CD38 density at the surface of already CD38+ cell lines (KG-1, UOC-M1, Kasumi-1, OCI-AML2, MOLM-13, THP-1 and CMK) in a concentration-dependent manner, but remained without effect on CD38- HEL-92.1.7 cells. We have quantified the number of CD38 molecules at the surface of all cell lines (except HEL-92.1.7 which was unresponsive to inecalcitol) using the CellQuant calibrator (Biocytex) analysis by flow cytometry. In basal vehicle-treated condition, the number (in thousands, mean ± sem, n=2 or 3) of CD38 molecules per CD38+ cell were: Kasumi-1, 1.5 ± 1.0; MOLM-13, 2.5 ± 0.6; KG-1, 2.7 ± 0.9; THP-1, 7.0 ± 0.2; CMK, 12.1 ± 1.5; OCI-AML2, 16.1 ± 1.2. With 10nM inecalcitol, a maximal increase in CD38 density was usually obtained within 3 days, or after 7 days for KG-1 and CMK: Kasumi-1, 16.1 ± 3.3 (x11); MOLM-13, 43.3 ± 0.3 (x17); KG-1, 15.4 ± 3.7 (x5.7); THP-1, 17.9 ± 0.6 (x2.3); CMK, 15.4 ± 3.3 (x1.3); OCI-AML2, 73.5 ± 3.5 (x4.6). After 3 days of exposure to 10 nM inecalcitol, CD38 molecules appeared at the surface of initially CD38- cells and became quantifiable: U-937, 7.5 ± 0.4 (n=3); HL-60, 24.4 ± 4.9 (n=3) and EOL-1 ranged from 5.5 to 72.7 (n=2). In conclusion, after exposure to inecalcitol, CD38 appeared at the surface of CD38- AML cell lines and the density of CD38 increased at the surface of CD38+ AML cell lines, altogether representative of all 9 FAB classification subtypes M0 to M7, except M6 (erythrocytic) which represents 5% of all AML cases. These results suggest that treatment by inecalcitol may render 95% of AML patients sensitive to highly sensitive to anti-CD38 immunotherapy such as daratumumab. Citation Format: Enguerran Mouly, Cécile Planquette, Emilie Rousseau, Rémi Delansorne. Inecalcitol respectively induces or increases CD38 expression at the surface of CD38- or CD38+ AML cell lines representative of all 9 FAB subtypes except M6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1890.

PO-028 Effectiveness and molecular basis of CDK4/6 inhibition in combination with taxanes in pancreatic cancer

IntroductionPancreatic Ductal Adenocarcinoma (PDAC) is among the deadliest human cancers with a 5 year survival rate of less than 5% using the standard of care gemcitabine/nab-paclitaxel. The most frequently disrupted genes in PDACs are first K-RAS and second CDKN2A, which encodes the cyclin-dependent kinase (CDK)4/6 inhibitor p16. Recently, CDK4/6 inhibitors have been approved for breast cancer treatment, and preclinical assays for PDAC are giving promising results.Material and methodsPDAC Patient-Derived Xenografts (PDX) models and PDX-derived cell lines were used for in vivo and in vitro studies, respectively. Cellular studies were performed using proliferation and cell cycle assays in combination with flow cytometry, immunoblotting, fluorescence microscopy and live cell imaging techniques. Drug treatments were performed with the CDK4/6 inhibitor PD-0332991 (Palbociclib), and with Paclitaxel (Taxol) or Nab-Paclitaxel (Abraxane) for in vitro and in vivo studies, respectively.Results and discussionsTreatment of different PDX-derived cell lines with the combination of taxanes and CDK4/6 inhibitors resulted in a higher anti-proliferative effect than both drugs used as single agent. Cell cycle studies showed that inhibition of CDK4/6 prevented recovery from treatment with taxol. At the molecular level we found that the combined treatment induced a clear interruption in retinoblastoma pathway, even higher than CDK4/6 inhibition in monotherapy. Gene expression profiles comparing single versus combined treatment are currently being performed to further understand the molecular basis underlying the effectiveness of this type of treatment. Moreover, to assess the efficacy of this new combined treatment in vivo, we treated nine PDAC PDX models with PD-0332991 and nab-paclitaxel, following the same schedule. Importantly, eight of them presented an increased tumour growth inhibition in the combination with respect to the monotherapies.ConclusionAlthough the molecular mechanism underlying the effectiveness of this treatment is not completely understood yet, our data suggest a good therapeutic value for the combination of CDK4/6 inhibitors and taxanes in PDAC treatment.

PO-031 NAA induces antitumoral effects in BXPC3 pancreatic cancer cell line

IntroductionPancreatic Ductal Adenocarcinoma (PDAC) is among the deadliest human cancers with a 5 year survival rate of less than 5% using the standard of care gemcitabine/nab-paclitaxel. The most frequently disrupted genes in PDACs are first K-RAS and second CDKN2A, which encodes the cyclin-dependent kinase (CDK)4/6 inhibitor p16. Recently, CDK4/6 inhibitors have been approved for breast cancer treatment, and preclinical assays for PDAC are giving promising results.Material and methodsPDAC Patient-Derived Xenografts (PDX) models and PDX-derived cell lines were used for in vivo and in vitro studies, respectively. Cellular studies were performed using proliferation and cell cycle assays in combination with flow cytometry, immunoblotting, fluorescence microscopy and live cell imaging techniques. Drug treatments were performed with the CDK4/6 inhibitor PD-0332991 (Palbociclib), and with Paclitaxel (Taxol) or Nab-Paclitaxel (Abraxane) for in vitro and in vivo studies, respectively.Results and discussionsTreatment of different PDX-derived cell lines with the combination of taxanes and CDK4/6 inhibitors resulted in a higher anti-proliferative effect than both drugs used as single agent. Cell cycle studies showed that inhibition of CDK4/6 prevented recovery from treatment with taxol. At the molecular level we found that the combined treatment induced a clear interruption in retinoblastoma pathway, even higher than CDK4/6 inhibition in monotherapy. Gene expression profiles comparing single versus combined treatment are currently being performed to further understand the molecular basis underlying the effectiveness of this type of treatment. Moreover, to assess the efficacy of this new combined treatment in vivo, we treated nine PDAC PDX models with PD-0332991 and nab-paclitaxel, following the same schedule. Importantly, eight of them presented an increased tumour growth inhibition in the combination with respect to the monotherapies.ConclusionAlthough the molecular mechanism underlying the effectiveness of this treatment is not completely understood yet, our data suggest a good therapeutic value for the combination of CDK4/6 inhibitors and taxanes in PDAC treatment.

Glyceryl ester surfactants: Promising excipients to enhance the cell permeating properties of SEDDS

AimThe aim of the study is the evaluation of the impact of glyceryl ester surfactants on cell permeating properties of SEDDS (self-emulsifying drug delivery systems). MethodsSEDDS containing the glyceryl ester surfactants polyglyceryl-3-stearate (TGlysurf9), polyglyceryl-5-oleate (TGlysurf11.5) and glyceryl stearate citrate (TGlysurf12) were prepared and characterized regarding droplet size and zeta potential. Toxicity studies were performed on Caco-2 cells using resazuring assay. The formulations were loaded with fluorescein diacetate (FDA) and curcumin, and cell uptake studies on Caco-2 cells were performed. Cell uptake was visualized via real time live confocal microscopy. Cell permeability of the SEDDS was tested and trans-epithelial electrical resistance (TEER) measurements were performed. Furthermore, the anti-proliferative and anti-migration activity of curcumin loaded in the SEEDS was investigated. ResultsThe developed SEDDS (0.05% m/v) showed no cytotoxicity on Caco-2 cells after 3 h of incubation. Glyceryl esters-SEDDS showed a significant higher FDA and curcumin cell uptake than SEDDS without glyceryl ester surfactants (p < 0.05). TGlysurf9-SEDDS showed thereby the most pronounced permeation enhancing properties. TEER remained constant during the permeation study. Curcumin loaded in TGlysurf9-SEDDS exhibited 1.9-fold higher anti-proliferative effect than curcumin loaded in SEDDS without glyceryl ester surfactants. Furthermore, curcumin loaded in glyceryl ester-SEDDS inhibited Caco-2 cells migration to a higher extent than unloaded curcumin and curcumin loaded in SEDDS without the glyceryl ester surfactants. ConclusionsGlyceryl ester surfactants and in particular polyglyceryl-3-stearate might be a promising excipient for the formulation of SEDDS exhibiting enhanced cellular uptake and permeation enhancing properties.

Synthesis of steroidal imidazolidinthiones as potential apoptotic agents: Investigation by theoretical and experimental studies

New steroidal imidazolidinthione derivatives (4–6) were synthesized from steroidal thiosemicarbazones and dichloroethane. The synthesized compounds were characterized using spectral data analysis. Theoretical DFT involving B3LYP/6-31G∗∗ level of theory was employed to gain insights into the molecular structure of the target compounds. MEPS and FMO analysis were carried out. HOMO-LUMO energy gap was determined which helped to evaluate various global descriptors like hardness, chemical potential, electronegativity, nucleophilicity and electrophilicity index, etc. The calculated properties established that the synthesized products are more or less similar in their reactivity behaviour. To explore their biological potential, interaction studies of compounds (4–6) with DNA were carried out using various biophysical techniques. The compounds bind DNA preferentially through electrostatic and hydrophobic interactions with Kb of 3.21 × 103 M−1, 2.79 × 103 M−1 and 2.26 × 103 M−1, respectively indicating the higher binding affinity of compound 4 towards DNA. Gel electrophoresis of compound 4 demonstrated strong interaction during the concentration dependent cleavage activity with pBR322 DNA. It was observed that these steroidal imidazolidinthiones are minor groove binders of DNA which was validated using molecular docking studies. An in vitro cytotoxicity screening using MTT assay revealed that the compounds (4–6) exhibit potential toxicity against different human cancer cells. Highest antiproliferative effect was observed on HeLa cells by compound 4. The results suggested that compounds 4–6 cause apoptotic cell death by cleaving apoptotic protein caspase-3 and suppress anti-apoptotic protein Bcl-2 in HeLa cancer cells.

Cell growth inhibition and apoptosis in breast cancer cells induced by anti-FZD7 scFvs: involvement of bioinformatics-based design of novel epitopes.

FZD7 has a critical role as a surface receptor of Wnt/β-catenin signaling in cancer cells. Suppressing Wnt signaling through blocking FZD7 is shown to decrease cell viability, metastasis and invasion. Bioinformatic methods have been a powerful tool in epitope designing studies. Small size, high affinity and human origin of scFv antibodies have provided unique advantages for these recombinant antibodies. Two epitopes from extracellular domain of FZD7 were designed using bioinformatic methods. Specific anti-FZD7 scFvs were selected against these epitopes through panning process. The specificity of the scFvs was assessed by phage ELISA and the ability to bind to FZD7 expressing cell line (MDA-MB-231) was determined by flowcytometry. Antiproliferative and apoptotic effects of the scFvs were evaluated by MTT and Annexin V/PI assays. The effects of selected scFvs on expression level of Surivin, c-Myc and Dvl genes were also evaluated by real-time PCR. Results demonstrated selection of two specific scFvs (scFv-I and scFv-II) with frequencies of 35 and 20%. Both antibodies bound to the corresponding peptides and cell surface receptors as shown by phage ELISA and flowcytometry, respectively. The scFvs inhibited cell growth of MDA-MB-231 cells significantly as compared to untreated cells. Growth inhibition of 58.6 and 53.1% were detected for scFv-I and scFv-II, respectively. No significant growth inhibition was detected for SKBR-3 negative control cells. The scFvs induced apoptotic effects in the MDA-MB-231 treated cells after 48h, which were 81.6 and 74.9% for scFv-I and scFv-II, respectively. Downregulation of Surivin, c-Myc and Dvl genes were also shown after 48h treatment of cells with either of scFvs (59.3-93.8%). ScFv-I showed significant higher antiproliferative and apoptotic effects than scFv-II. Bioinformatic methods could effectively select potential epitopes of FZD7 protein and suggest that epitope designing by bioinformatic methods could contribute to the selection of key antigens for cancer immunotherapy. The selected scFvs, especially scFv-I, with high antiproliferative and apoptotic effects could be considered as effective agents for immunotherapy of cancers expressing FZD7 receptor including triple negative breast cancer.

The in vitro radiosensitizer potential of resveratrol on MCF-7 breast cancer cells

Radiation therapy is commonly applied in breast cancer (BC) patients. However, radioresistance and side effects are limiting factors of this practice. Therefore, studying substances that can enhance the radiation effect and, at the same time, protect normal cells is very relevant. Thus, the aim of this work was to assess the radiosensitizer effect of resveratrol (RV) on BC cells (MCF-7). A high cytotoxic and antiproliferative effect was observed in the treatment with 10 μM of RV + 3 Gy ionizing radiation (IR). Our results indicate that, 24 h after the exposition of cell cultures to RV + IR, an induction of necrosis/senescence has occurred. Furthermore, was observed the activation of extrinsic apoptosis pathway through a decrease of the Bax/Bcl-2 ratio and a high activity of caspase 8. Moreover, our data show that this treatment affected the oxidative cell metabolism, increasing oxidative protein, lipid and membrane damage and also acted to decrease the antioxidant enzymes activity. The antiproliferative effect on 72 h cultures may be associated with a high expression of p53 and an interruption of cell cycle in the S phase. Therefore, our results suggest that RV is a potential radiosensitizer of MCF-7 BC cells.

Algae from Portuguese Coast Presented High Cytotoxicity and Antiproliferative Effects on an In vitro Model of Human Colorectal Cancer.

Background:The marine environment has shown to be an interesting source of new antitumor agents, representing an important tool in cancer research.Objective:The aim of this study was to evaluate the antitumor activities of 12 algae from Peniche coast (Portugal) on an in vitro model of human colorectal cancer (Caco-2 cells).Materials and Methods:The antitumor potential was accessed by evaluating Caco-2 cell's viability and proliferation through the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide and calcein-AM methods.Results:The dichloromethane extracts of Asparagopsis armata and Sphaerococcus coronopifolius induced the highest decrease on cell's viability (1 mg/mL; 24 h), 98.96% ± 0.39% and 98.08% ± 0.89%, respectively, followed by the methanolic extracts of S. coronopifolius (96.47% ± 1.26%) and A. armata (92.68% ± 1.17%). Regarding cell proliferation, the highest decrease of Caco-2 cell's proliferation (1 mg/mL; 24 h) was induced by the dichloromethane extract of A. armata (100% ± 0.48%), S. coronopifolius (99.04 ± 0.51%), and Plocamium cartilagineum (95.05% ± 1.19%). The highest potency was shown by the dichloromethane extract of S. coronopifolius in both, cytotoxicity and antiproliferative tests, with an IC50 of 21.3 and 36.5 μg/mL, respectively.Conclusion:The extracts of A. armata and S. coronopifolius are promising sources of new bioactive molecules with application in cancer therapeutics.SUMMARY Algae from Peniche coast (Portugal) revealed to be a promising source of new bioactive compounds with potential application on cancer therapeutics. Abbreviations Used: MTT: 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide; DMSO: Dimethyl sulfoxide; FBS: Fetal bovine serum; MEM: Minimum Essential Medium; SEM: Standard error of the mean;SP : Sulfated polysaccharides

Combination of etoposide and fisetin results in anti-cancer efficiency against osteosarcoma cell models.

Osteosarcoma chemotherapy is often limited by chemoresistance, resulting in poor prognosis. Combined chemotherapy could, therefore, be used to prevent resistance to chemotherapeutics. Here, the effects of fisetin on osteosarcoma cells were investigated, as well as cytostatic potential in combination with the anti-cancer drug etoposide. For this, different osteosarcoma cell lines were treated with fisetin, with etoposide and with respective combinations. Fisetin was associated with decrease in colony formation in Saos-2 and in U2OS cells but not in MG-63 cells. Notwithstanding, upon evaluation of cellular growth by crystal violet assay, MG-63 and Saos-2 cells showed decreased cell proliferation at 40 and 20µM fisetin, respectively. Depending on the relative concentrations, fisetin:etoposide combinations showed negative-to-positive interactions on the inhibition of cell proliferation. In addition, fisetin treatment up to 50µM for 48h resulted in G2-phase cell cycle arrest. Regardless of the combination, fisetin:etoposide increased % cells in G2-phase and decreased % cells in G1-phase. In addition, mixtures with more positive combined effects induced increased % cells in S-phase. Compared to etoposide treatment, these combinations resulted in decreased levels of cyclins B1 and E1, pointing to the role of these regulators in fisetin-induced cell cycle arrest. In conclusion, these results show that the combination of fisetin with etoposide has higher anti-proliferative effects in osteosarcoma associated with cell cycle arrest, allowing the use of lower doses of the chemotherapeutic agent, which has important implications for osteosarcoma treatment.

Abstract 1591: Inhibition of prostate tumor growth by an extract from the muscadine grape

Abstract Despite decades of advancements, cancer remains the second leading cause of mortality in the United States with an estimated 1.7 million new cases and over 600,000 deaths predicted for 2016, indicating a clear need for more effective chemotherapeutic and chemopreventive agents. While natural products derived from plants have served as an abundant source of anticancer agents, it is predicted that less than 2 percent of the plant species with medicinal value have been investigated. Muscadine grape (Vitis rotundifolia) skin and/or seed extracts are a popular nutraceutical supplement due to the high anti-oxidant, anti-inflammatory, and anti-proliferative effects of a distinct phytochemical composition of polyphenols and proanthocyanidins. The current study examines the effects of a proprietary muscadine grape extract formulation (MGE; Piedmont Research &amp; Development Corp.) on the proliferation of prostate cancer cells and tumors. MGE inhibited the growth of human LnCaP prostate cancer cells, in both a time- and dose-dependent manner, as quantified using an IncuCyte Zoom Imaging system. Cell growth was reduced by 36.8% after a 5-day treatment with 20 µg/mL MGE compared to untreated cells (n = 6, p &amp;lt; 0.001). Athymic mice (male, 15-20 g, 5-6 weeks of age) were injected subcutaneously on the lower flank with 1.6 x 106 LNCaP cells, to assess the effect of the MGE in vivo. When the tumors reached 100 mm3, the mice were randomized and MGE was added to the drinking water at a concentration of 0.2 µg phenolics/mL (1.0 mg phenolics/mouse/day for a 25 g mouse); control mice drank untreated water. After 5 weeks, the mice were sacrificed and the tumors were weighed. The volume of prostate tumors growing in the flank of nude mice drinking water with MGE was markedly reduced compared to the size of the tumors from control mice [252.6 ± 61.73 mm3 (MGE) versus 765.2 ± 101.8 mm3 (control); n=6, p&amp;lt;0.001). Further, the weight of prostate tumors from mice drinking MGE was also significantly decreased [0.96 ± 0.14 g (control) as compared to 0.41 ± 0.11 g (MGE)], a reduction of 57% (p &amp;lt; 0.05). Five microns tumor sections were incubated with an antibody to CD34 and tumor vessels were identified by positive immunoreactivity and morphology. MGE reduced the number of blood vessels/field from 10.25 ± 0.9 to 7.10 ± 0.6 (p &amp;lt; 0.05), suggesting that the extract inhibits angiogenesis. Vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) were measured by qPCR, to determine the effect of the extract on angiogenic factors. MGE reduced VEGF mRNA from 1.02 ± 0.06 to 0.72 ± 0.07 relative units (p &amp;lt; 0.05) and PLGF mRNA from 1.03 ± 0.11 to 0.63 ± 0.07 relative units (p &amp;lt; 0.05), providing additional evidence of an anti-angiogenic effect of the extract. Collectively, these results are the first to demonstrate that a novel MGE formulation reduces prostate tumor growth in a mouse model, by inhibiting angiogenesis, suggesting that MGE may be an effective treatment for prostate cancer. Citation Format: Patricia E. Gallagher, E. Ann Tallant. Inhibition of prostate tumor growth by an extract from the muscadine grape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1591. doi:10.1158/1538-7445.AM2017-1591

Augmented Anticancer Effects of Cantharidin with Liposomal Encapsulation: In Vitro and In Vivo Evaluation.

PEGylated liposomes have received much attention as pharmaceutical carriers to deliver chemotherapeutic agents for therapeutic purpose. The aim of this study was to prepare and characterize PEGylated liposome of cantharidin and investigate its therapeutic effect on human hepatocellular carcinoma treatment in vitro and in vivo. Liposomal cantharidin was evaluated for their anticancer effects in vitro using human hepatocellular carcinoma HepG2 cells and in vivo using HepG2-bearing nude mice compared to free drug. PEGylated liposome of cantharidin had a particle size of 129.9 nm and a high encapsulation efficacy of approximately 88.9%. The liposomal cantharidin had a higher anti-proliferative effect vis-à-vis free cantharidin in inducing G2/M cell cycle arrest and apoptosis. Liposomal cantharidin killed more HepG2 cancer cells at the same concentration equivalent to free cantharidin. Further study in vivo also showed that liposomal cantharidin achieved a higher tumor growth inhibition efficacy than free drug on hepatocellular carcinoma. As our study exhibited enhanced cytotoxicity against HepG2 cells and augmented tumor inhibitory effects in vivo, the results validate the potential value of cantharidin-liposome in improving the therapeutic efficacy of cantharidin for liver cancer.

Anticancer and Cytotoxic Activities of [Cu(C6H16N2O2)2][Ni(CN)4] and [Cu(C6H16N2O2)Pd(CN)4] Cyanidometallate Compounds on HT29, HeLa, C6 and Vero Cell Lines.

In cancer, apoptosis relevant proteins-such as CaM kinase, Bcl-2 or P53, topoisomerase I, cell migration feature and DNA/BSA-macromolecules represent significant targets for current chemotherapeutics. We recently reported two coordination compounds-[Cu(C6H16N2O2)2][Ni(CN)4] (1) and [Cu(C6H16 N2O2)Pd(CN)4] (2)-together with their IR spectra, magnetic properties, thermal analyses and crystal structures. Herein, we describe the ability of these complexes to induce apoptosis in relevant proteins and stimulate topoisomerase I activity, cell migration velocity and DNA/BSA binding properties. The in vitro antiproliferative effects and cell toxicity of both compounds were investigated through pharmacological measurement techniques, and interactions between both compounds and CT-DNA/BSA were studied with UV-Vis spectroscopy and fluorescence spectroscopy. Results &amp; Conclusion: Studies on cells revealed that 2 (i) demonstrated a high antiproliferative effect, which was higher toward HeLa and C6 cancer cells than toward healthy Vero cells; (ii) impaired the migration of HeLa cells; (iii) altered the P53-Bcl-2 ratio in favor of apoptosis; (iv) strongly bound to DNA/BSA macromolecules; and (v) inhibited human topoisomerase I and KpnI or BamHI restriction endonucleases. In conclusion, this preliminary information demonstrates that 2 may represent a promising antiproliferative agent and a potential candidate for a therapeutic approach against HeLa.

Cytotoxic and apoptotic effects of different extracts of Artemisia biennis Willd. on K562 and HL-60 cell lines.

Objective(s):Artemisia is a genus of herbs and small shrubs forms an important part of natural vegetation in Iran. It has been reported that several Artemisia species possess anti-proliferative effects. Considering the value of this genus in anti-cancer researches we have chosen Artemisia biennis for cytotoxic and mechanistic studies.Materials and Methods:In this study we have investigated the cytotoxic and apoptotic effects of petroleum ether, dichloromethane, ethyl acetate, ethanol, and ethanol: water (1:1 v/v) extracts of A. biennis Willd. on two cancer human cell lines (K562 and HL-60) and J774 as normal cells.Results:CH2Cl2 extract was found to have the highest anti-proliferative effect on cancer cells. IC50 values obtained in AlamarBlue® assay for CH2Cl2 extract were 64.86 and 54.31 µg/ml on K562 and HL-60 cells respectively. In flow cytometry histogram of the cells treated with CH2Cl2 extract, sub-G1 peak was induced. DNA fragmentation, increased in the level of Bax and cleavage of PARP protein all showed the induction of apoptosis with CH2Cl2 extract after 48 hr contact with cells.Conclusion:The results can corroborate the cytotoxic and apoptotic effects of the CH2Cl2 extract of A. biennis on the K562 and HL-60 cancer cell lines.

Copper(ii) complexes of functionalized 2,2':6',2''-terpyridines and 2,6-di(thiazol-2-yl)pyridine: structure, spectroscopy, cytotoxicity and catalytic activity.

Six new copper(ii) complexes with 2,2':6',2''-terpyridine (4'-Rn-terpy) [1 (R1 = furan-2-yl), 2 (R2 = thiophen-2-yl), and 3 (R3 = 1-methyl-1H-pyrrol-2-yl)] and 2,6-di(thiazol-2-yl)pyridine derivatives (Rn-dtpy) [4 (R1), 5 (R2), and 6 (R3)] have been synthesized by a reaction between copper(ii) chloride and the corresponding ligand. The complexes have been characterized by UV-vis and IR spectroscopy, and their structures have been determined by X-ray analysis. The antiproliferative potential of copper(ii) complexes of 2,2':6',2''-terpyridine and 2,6-di(thiazol-2-yl)pyridine derivatives towards human colorectal (HCT116) and ovarian (A2780) carcinoma as well as towards lung (A549) and breast adenocarcinoma (MCF7) cell lines was examined. Complex 1 and complex 6 were found to have the highest antiproliferative effect on A2780 ovarian carcinoma cells, particularly when compared with complex 2, 3 with no antiproliferative effect. The order of cytotoxicity in this cell line is 6 > 1 > 5 > 4 > 2 ≈ 3. Complex 2 seems to be much more specific towards colorectal carcinoma HCT116 and lung adenocarcinoma A549 cells. The viability loss induced by the complexes agrees with Hoechst 33258 staining and typical morphological apoptotic characteristics like chromatin condensation and nuclear fragmentation. The specificity towards different types of cell lines and the low cytotoxic activity towards healthy cells are of particular interest and are a positive feature for further developments. Complexes 1-6 were also tested in the oxidation of alkanes and alcohols with hydrogen peroxide and tert-butyl-hydroperoxide (TBHP). The most active catalyst 4 gave, after 120 min, 0.105 M of cyclohexanol + cyclohexanone after reduction with PPh3. This concentration corresponds to a yield of 23% and TON = 210. Oxidation of cis-1,2-dimethylcyclohexane with m-CPBA catalyzed by 4 in the presence of HNO3 gave a product of a stereoselective reaction (trans/cis = 0.47). Oxidation of secondary alcohols afforded the target ketones in yields up to 98% and TON = 630.

Bromelain-Functionalized Multiple-Wall Lipid-Core Nanocapsules: Formulation, Chemical Structure and Antiproliferative Effect Against Human Breast Cancer Cells (MCF-7).

This study was conducted a promising approach to surface functionalization developed for lipid-core nanocapsules and the merit to pursue new strategies to treat solid tumors. Bromelain-functionalized multiple-wall lipid-core nanocapsules (Bro-MLNC-Zn) were produced by self-assembling following three steps of interfacial reactions. Physicochemical and structural characteristics, in vitro proteolytic activity (casein substrate) and antiproliferative activity (breast cancer cells, MCF-7) were determined. Bro-MLNC-Zn had z-average diameter of 135nm and zeta potential of +23mV. The complex is formed by a Zn-N chemical bond and a chelate with hydroxyl and carboxyl groups. Bromelain complexed at the nanocapsule surface maintained its proteolytic activity and showed anti-proliferative effect against human breast cancer cells (MCF-7) (72.6 ± 1.2% at 1.250μgmL-1 and 65.5 ± 5.5% at 0.625μgmL-1). Comparing Bro-MLNC-Zn and bromelain solution, the former needed a dose 160-folds lower than the latter for a similar effect. Tripan blue dye assay corroborated the results. The surface functionalization approach produced an innovative formulation having a much higher anti-proliferative effect than the bromelain solution, even though both in vitro proteolytic activity were similar, opening up a great opportunity for further studies in nanomedicine.

Inecalcitol Induces CD38 Expression in Multiple Myeloma and Myeloid Cell Lines

Targeting CD38 has proven to be an effective strategy for the treatment of relapsed refractory multiple myeloma (MM), with the recent approval of the first therapeutic monoclonal antibody directed against human CD38: daratumumab (Sanchez L et al., 2016, J Hematol Oncol; van de Donk NWC et al., 2016, Immunol Rev). Interestingly, the pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) as compared to patients who achieved less than PR (Nijhof IS et al., 2016, Blood). Therefore, increasing the levels of CD38 expression is the next logical pharmacological step to enhance the efficacy of anti-CD38 therapy.In this context, we tested inecalcitol (INE: 14epi-, 19nor-, 23yne-, 1,25dihydroxy-cholecalciferol) on the MM.1S cell line (from ATCC, CRL-2974) in culture and found a reproducible concentration-dependent increase in CD38 expression levels after 3 days of treatment in 4 independent experiments. CD38 levels were measured at the surface of the cells by flow cytometry (ACEA NovoCyte flow cytometer) after one hour of incubation at 4°C with a fluorescent anti-human CD38 mouse FITC antiboby (eBioscience, 11-0389) or with the matched isotype control mouse IgG1 K (eBioscience, 11-4714). More than a 2-fold increase in labelling intensity was already observed with 0.1 nM INE and a maximal 5-fold stimulation was achieved at 3 nM INE. The half-maximal effective concentration (EC50) of INE was 0.47 ± 0.23 nM (n=4).Next, we tested INE on L-363 cells, another MM cell line, purchased from DSMZ (ACC 49). In a single experiment, the L-363 cells were also shown to be responsive to INE, but about 10 times less sensitive than the MM.1S cells: a similar maximal 5-fold increase in CD38 levels on cell surface was also reached but at the much higher concentration of 30 nM INE and the threshold 2-fold stimulation of fluorescent labelling was obtained with 1 nM INE. The EC50 of INE in this single experiment was 4 nM.In normal cell culture conditions, both MM cell lines showed a mix of CD38-positive (CD38+) and CD38-negative (CD38-) cells, as illustrated by the distribution spectrum of intensities of individual cell labelling. Without any treatment by INE, the CD38+/CD38- ratio was higher among the MM.1s cells (80/20) than the L-363 cells (30/70). The effect of INE resulted in both a higher proportion of CD38+ cells (>93% CD38+ MM.1S cells treated from 0.3 nM and higher INE concentrations; only 58% CD38+ L-363 cells at 3 nM INE), and a higher mean intensity of individual cell labelling.Finally, we studied the HL-60 and U-937 cells classically described as representative of myeloid hematological malignancies. In normal culture conditions, both cell lines were CD38-. A concentration-dependent increase in CD38 expression levels on the surface of U-937 cells (from ATCC, CRL-1593,2) was obtained after 3 days of treatment with INE in the range of 0.1 to 10 nM in two independent experiments (EC50 = 0.47 nM and 0.76 nM). A concentration-dependent increase in CD38 expression levels on the surface of HL-60 cells (from ECACC, 98070106) was obtained after 3 days of treatment with INE in the range of 0.1 to 3 nM in two independent experiments (EC50s = 0.33 nM and 0.38 nM). All HL-60 or U-937 cells were CD38+ after 3 days of treatment with INE 0.3 nM or 1 nM, respectively.Inecalcitol is a vitamin D receptor agonist already characterized by a high anti-proliferative effect and a low calcemic potential (Okamoto R et al., 2012, Int J Cancer; Ma Y et al., 2013, Cell Cycle), allowing its administration at high oral doses to human cancer patients (Medioni J et al, 2014, Clin Cancer Res). The present findings suggest that INE could be tested in MM patients to increase the percentage of CD38+ MM cells and the density of CD38 at their surface, thereby potentially increasing the clinical response to anti-CD38 antibodies such as daratumumab. Furthermore, HL-60 and U-937 cells, all initially CD38-, became CD38+ after 3 days of in vitro treatment by INE, suggesting that CD38- myeloid hematological malignancies could potentially represent new indications for daratumumab when used after "priming" or in combination with INE. DisclosuresBenjamin:Hybrigenics: Employment, Equity Ownership. Planquette:Hybrigenics: Employment, Equity Ownership. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: Inventor, but no royalties.

Wnt signaling inhibitor FH535 selectively inhibits cell proliferation and potentiates imatinib-induced apoptosis in myeloid leukemia cell lines.

Wnt signaling pathway plays a major role in leukemogenesis of myeloid leukemia. Aberrancy in its regulation results in hyperactivity of the pathway contributing to leukemia propagation and maintenance. To investigate effects of Wnt pathway inhibition in leukemia, we used human leukemia cell lines (i.e., K562, HL60, THP1, and Jurkat) and several Wnt inhibitors, including XAV939, IWP2 and FH535. Our results showed that leukemia cell lines (>95% cells) had increased endogenous levels of β-catenin as compared to mononuclear cells from healthy donors (0%). Among the tested inhibitors, FH535 demonstrated a markedly suppressive effect (IC50=358nM) on mRNA levels of β-catenin target genes (LEF1, CCND1, and cMYC). In addition, FH535 significantly potentiated imatinib-induced apoptosis. Evaluation of erythrocyte and megakaryocyte lineage using flow cytometry demonstrated that the potentiation mechanism is independent of the developmental stage, and is more likely due to crosstalk between other pathways and β-catenin. FH535 also displayed antiproliferative properties in other cell lines used in this study. In summary, FH535 showed significantly high antiproliferative effects at submicromolar dosages, and additionally enhanced imatinib-induced apoptosis in human leukemia cell lines. Our results highlight its potential antileukemic promise when used in conjunction with other conventional therapeutic regimens.

English

We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for L-asparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant L-asparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 312.8 U/mg. Kinetic parameters, Km, Vmax, Kcat and Kcat/Km of purified enzyme were found to be 0.5 mM, 500 U/mg, 14.9  103 s-1, and 29.9  103 mM-1s-1, respectively. Temperature and pH optimum were observed at 45oC and pH 7.5, respectively. The enzyme exhibited about 20 and 60% retention of activity following 100 min incubation at 55 or 40°C, respectively. The activity of enzyme was inhibited by EDTA, Hg2+, Cu2+, Ni2+, and enhanced by Mg2+. Detergents (Tween 20, Tween 80, Triton X-100, and Triton X-114) decreased enzyme activity. DTT and DMSO at appropriate concentrations enhanced enzyme activity. In vitro anti-cancer activity was performed using different tumor cell lines. Concentration of recombinant L-asparaginase at 50 µg/ml inhibited 45.32, 48.22, 53.68, 51.22% with HL-60, P388, P3X63Ag8, SP2/0-Ag14 cell lines. Recombinant L-asparaginase was expressed successfully in Escherichia coli with high expression level, had a high specific activity and antiproliferative effect on several tumor cell lines. Key words: Characterization, Erwinia chrysanthemi, L-asparaginase, purification, tumor cell line.